Adgrg6/Gpr126 is required for compact wall integrity and establishing trabecular identity during cardiac trabeculation

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: major_revision

Abstract
How adhesion G protein-coupled receptors (aGPCRs) control development remains unclear. aGPCR Adgrg6/Gpr126 has been associated with heart trabeculation. Defects in this process cause cardiomyopathies and cardiac dysfunction. How cardiomyocytes attain trabecular identity is poorly understood. Here, we show that different domains of Gpr126 distinctly regulate compact wall integrity and trabecular identity. Maternal zygotic (MZ) gpr126stl47 early truncation mutants exhibit hypotrabeculation, whereby N-cadherin distributes randomly along apical/basal/lateral membranes of compact layer cardiomyocytes. In contrast, zygotic and MZ gpr126st49 mutants, expressing a N-terminal fragment lacking the GPS motif (NTFΔGPS), exhibit a multilayered ventricular wall containing polarized cardiomyocytes with normal N-cadherin localization and increased Notch activity. Notably, endocardially expressed gpr126 C-terminal fragment (CTF) reinstates trabeculation in gpr126st49 mutants. Collectively, our data reveal domain-specific roles of Gpr126 during trabeculation, whereby the NTF is required for maintaining cell-cell adhesion and compact wall integrity, whereas the CTF is essential to provide trabecular identity.

Introduction
Adhesion G protein-coupled receptors (aGPCRs) are required for regulation of various biological and developmental processes1. Accordingly, mutations in aGPCRs have been associated with a wide variety of human diseases1–3. Moreover, ~34% of all FDA-approved drugs target 108 members of the GPCR superfamily, highlighting the translational accessibility of GPCRs as therapeutic targets4. Thus, exploring the roles of understudied GPCRs offers significant unexplored therapeutic opportunities.

aGPCRs are characterized by a seven-transmembrane (7TM) domain and a long extracellular domain (ECD) consisting of various adhesion domains. They are autoproteolytically cleaved in the endoplasmic reticulum within a conserved GPCR Autoproteolysis Inducing (GAIN) domain into a C-terminal fragment (CTF) containing the 7TM domain and an N-terminal fragment (NTF) containing the ECD5,6. Subsequently, NTF and CTF are transported to the plasma membrane, where they can reconstitute to form a receptor7,8. The signaling mechanisms of aGPCRs and their domain-specific functions remain poorly understood. Various models of receptor activation have been proposed9–11 while there are also reports that aGPCRs use their ECDs to mediate functions in a 7TM-independent manner12.

Adgrg6/Gpr126 has been reported to be required for the development of several tissues/organs, including the peripheral nervous system, intervertebral disc, ear, and placenta13–17. Several genome-wide Association and RNA-sequencing (RNA-Seq) studies have associated variants in the human GPR126 locus with diseases like adolescent idiopathic scoliosis, intellectual disability, severe arthrogryposis multiplex congenita, periodontitis, chronic obstructive pulmonary disease (COPD), and various human cancers18–21. Notably, the cellular basis for these Gpr126-linked syndromes is not well understood.

In the heart, Gpr126 is known to be transiently expressed in the endocardium in embryonic mice (E9.5 to E13.5)22,23. gpr126 is also expressed in the heart of zebrafish embryos13,22, but the cellular origin of expression was not determined. Global depletion of Gpr126 affects cardiac trabeculation in mice17,22,24 and zebrafish22. Waller-Evans et al. described the first Gpr126 knockout (KO) mouse line, which was characterized by mid-gestation lethality exhibiting circulatory failure like congestion, edema, and internal hemorrhage with secondary defects in trabeculation and a normally developed placenta24. Subsequently, two different Gpr126 KO mouse models were reported exhibiting embryonic lethality at E11.5 associated with hypotrabeculation, myocardial wall thinning, arrhythmia, bradycardia, and mitochondrial defects22,25. Morpholino-mediated gpr126 knockdown in zebrafish also resulted in hypotrabeculation, and rescue experiments using Gpr126-NTFΔGPS (NTFΔGPS) suggested that NTFΔGPS is a stable, secreted protein sufficient for heart development22. It was further reported that miR-27a/b is a post-transcriptional regulator of Gpr126 and modulation of gpr126 expression in zebrafish by inducing or inhibiting miR-27b resulted in defective trabeculation26. However, in a recent study utilizing a new Gpr126 KO mouse line, it has been proposed that placental defects are the primary cause of heart abnormalities17. The authors also did not observe any trabeculation defects in newly created gpr126 zebrafish mutants, gpr126bns341 (predicted early truncation) and gpr126bns342 (promoter-less allele)17.

Cardiac trabeculation is a crucial process during ventricular chamber development where the cardiomyocytes in the heart wall organize themselves to form two distinct layers, an outer compact wall and an inner trabecular layer. This process depends on communications between the epicardium, myocardium, and endocardium27,28. There are several signaling pathways/molecular regulators important for cardiac trabeculation. First, endocardial Notch signaling, a critical early regulator of trabeculation29–31, which in zebrafish is activated in the endocardium at 24 hpf and becomes restricted to atrioventricular valves and the outflow tract by 72 hpf32, regulates Neuregulin (Nrg1 in mice / nrg2a in zebrafish) in the endocardium which mediates ErbB2 signaling in the myocardium33–38. Second, cardiomyocyte crowding triggers their delamination during which adherens junctions between cardiomyocytes are remodeled39. N-cadherin, the major component of cardiac adherens junctions, re-localizes from the lateral to the basal domain in compact wall cardiomyocytes. In contrast, trabecular cardiomyocytes exhibit a punctate N-cadherin distribution on their entire surface39. The first trabeculae appear in the outer curvature of the ventricle at 60 hpf. Third, these delamination events in trabecular cardiomyocytes activate Notch signaling in their neighboring compact wall cardiomyocytes40–42, which subsequently inhibit their delamination42 by suppressing the actomyosin machinery and preserving myocardial wall architecture42. The first appearance of Notch activity in ventricular cardiomyocytes overlaps with the onset of trabeculation (60 hpf). Notch-negative cardiomyocytes depolarize and delaminate, adopting a trabecular identity. In contrast to endocardial Notch signaling, the genetic network underlying myocardial Notch-mediated fate specification remains poorly understood.

In this study, we utilized zebrafish gpr126 genetic models with domain-specific loss and gain of function approaches as well as rescue experiments, to assess the role of Gpr126 in zebrafish heart development. Our data suggest that both the NTF and CTF of Gpr126 are required for trabeculation. The NTF contributes to maintaining compact wall integrity at the onset of trabeculation by maintaining cell-cell junctions. The CTF subsequently confers trabecular identity to cardiomyocytes through modulation of myocardial Notch activity. Altogether, our results provide new insights into the domain-specific functions of Gpr126 and shed light on the regulation of the mechanisms underlying Notch-mediated fate specification during trabeculation.

Results
The stl47 and st49 mutant alleles produce truncated versions of Gpr126
In order to understand the role of Gpr126 as well as its NTF and CTF, the zebrafish gpr126 mutant lines gpr126stl4743 and gpr126st4915, which have been previously characterized for peripheral nervous system defects and semicircular canal morphogenesis defects, were analyzed. The stl47 allele carries a 5+3 insertion deletion (indel) at Asparagine 68, which produces a frameshift-induced stop codon in the CUB domain, predicting a truncated Gpr126 protein of 96 amino acids (Fig. S1a, b)43. The st49 allele carries a point mutation that converts a tyrosine-encoding codon into a stop codon near the GPS motif; as such, gpr126st49 mutant alleles could encode a truncated Gpr126 protein of 782 amino acids corresponding to the NTF without the GPS motif (NTFΔGPS) (Fig. S1a, b)15. Both mutations result in a puffy ear phenotype caused by abnormal semicircular canal morphogenesis, confirming previously published data (Fig. S1c)43. qPCR analysis revealed no differences in gpr126 mRNA levels in hearts obtained from 96 h post fertilization (hpf) wild type (WT), gpr126stl47, and gpr126st49 embryos (Fig. S1d). These data suggest that these mutant gpr126 mRNAs are not subjected to nonsense-mediated RNA decay, confirming previous data for gpr126st4915.

Thus far, it has not been tested whether the predicted truncated proteins are produced from the gpr126stl47 and gpr126st49 alleles. As no working anti-Gpr126 antibodies are currently available, we ectopically expressed zebrafish full-length gpr126 cDNA as well as gpr126 cDNA containing the stl47 or st49 mutation in the human retinal pigment epithelial cell line ARPE-19, which is routinely used for protein expression studies. The encoded proteins have an N-terminal HA-tag and a C-terminal GFP-tag (Fig. 1a). Immunofluorescence and Western blot (WB) analyses showed that WT and mutant forms of Gpr126 were expressed (Figs. 1 and S2). Notably, for the mutant forms, although the N-terminal HA signal was present, no C-terminal GFP signal was detected (Fig. 1b, c), indicating that the mutations resulted in truncated forms of Gpr126. These data were supported by WB analyses. Anti-GFP antibodies detected a band of ~70 kDa in the WT-transfected cells, which corresponds to Gpr126-CTF-GFP (69 kDa) (Fig. 1d). No bands were detected in cells expressing the mutant forms. Anti-HA antibodies detected two bands of ~90 and ~100 kDa in the WT-transfected cells, which correspond to HA-NTFΔGPS (91 kDa) (Fig. 1d). Similarly, a band of ~90 kDa was detected in cells expressing the st49-mutant form, which is predicted to encode the NTFΔGPS. In cells expressing the stl47-mutant form, a band of ~10 kDa was detected, the size of which corresponds to the predicted truncated Gpr126 protein containing the signal peptide and the partial CUB domain (11 kDa).

WT Gpr126 localized to the plasma membrane 24h post-transfection (Fig. 1b). In contrast, the mutant Gpr126 forms failed to localize to the membrane (Fig. 1b), and in a minority of transfected cells, cytoplasmic expression was detected (Fig. S2a, b). Notably, previously we have shown that ectopic expression of NTFΔGPS in HeLa as well as H9c2 cells results in a stable, secreted protein22. Thus, the failure to localize to the membrane and the reduced number of HA-positive cells (Fig. S2a, b) could be explained by the loss of the 7TM domain in both truncated forms. To confirm that a stable, secreted NTFΔGPS was produced by the gpr126st49 cDNA, the lysates and culture medium from ARPE-19 cells transfected with WT or St49 constructs were analyzed by WB (Fig. S2c). These data revealed that the culture medium contained NTF/NTFΔGPS. Moreover, treating the cells with brefeldin A, which blocks the trans-Golgi secretory pathway44,45, significantly decreased the amount of NTF/NTFΔGPS in the medium. Given that transient overexpression of proteins can lead to protein artifacts, we generated stable ARPE-19 lines expressing WT as well as St49 and Stl47 mutants of Gpr126 for a more reliable analysis (Fig. 1e). We observed membrane localization of WT Gpr126, while the mutant forms failed to localize to the membrane. In addition, there was no detectable cytoplasmic accumulation of the mutant forms, indicating that the truncated forms are not retained in the cytoplasm. WB analyses of the lysates and culture medium from the stable lines, with and without treatment with brefeldin A, confirmed that NTFΔGPS is secreted in the St49 overexpressing cells (Fig. 1f). These data suggest that gpr126st49 mutants express and secrete NTFΔGPS, which previously has been reported to be sufficient for heart development22.

Altogether, these data demonstrate that expressing zebrafish gpr126 cDNA containing the stl47 or st49 mutation in ARPE-19 cells results in the production of the predicted truncated forms of Gpr126: the zebrafish mutant gpr126stl47 expresses the first 96 amino acids of Gpr126 containing a part of the CUB domain, whereas gpr126st49 expresses secreted NTFΔGPS.

Maternal zygotic gpr126stl47 mutants exhibit hypotrabeculation
Previous reports aiming to define the role of Gpr126 during heart development are conflicting17,22,24. To determine whether Gpr126 is required for heart development, the gpr126stl47 line was crossed with the reporter line Tg(myl7:EGFP-hsa.HRAS) in which GFP is localized at the cardiomyocyte plasma membrane. In vivo confocal imaging of the hearts of WT siblings showed clear luminal projections into the ventricle, forming a complex network of trabeculae at 96 and 120 hpf (Fig. 2a, c). The analysis of zygotic (Z) gpr126stl47 mutant hearts revealed, at 120 hpf, no obvious gross morphological defects or trabeculation defects as compared to WT siblings (Fig. 2a, b). These data suggested initially that Gpr126 is not required for heart development in zebrafish. However, the lack of a phenotype in Z gpr126stl47 mutant hearts might be explained by compensation through maternal deposition of WT mRNA or protein into the egg.

Notably, it has previously been reported that gpr126stl47 maternal zygotic (MZ) mutants exhibit a more severe phenotype during Schwann cell development43. It has been described that 96% of aGPCR have at least some levels of maternal expression46. The qPCR data for Gpr126 show detectable expression at 3 hpf, suggesting the presence of maternal mRNA46. To confirm the maternal deposition of gpr126 RNA, we performed in situ hybridization in embryos prior to zygotic genome activation to visualize the maternal transcript and observed that WT as well as the MZ gpr126stl47 and MZ gpr126st49 mutants display gpr126 expression at 2 hpf (Fig. S3a).

Unlike Z mutants, MZ mutants have no maternal deposition of WT mRNA or protein into the egg and exhibited more pronounced defects in the expression of myelin basic protein than Z mutants43. We observed trabeculation defects in MZ gpr126stl47 mutants at 96 and 120 hpf (Fig. 2c): at 120 hpf, we observed in 16.7% (5 out of 30) of mutants that trabeculation was absent and the ventricular wall was single layered; in 26.7% (8 out of 30) of mutants, hypotrabeculation was detected; and in 20% (6 out of 30) of mutants abnormal multilayered ventricular walls were observed (Fig. 2d). Thus, in total 63.4% (19 out of 30) of the MZ gpr126stl47 mutants exhibited trabeculation defects, whereas 36.6% exhibited normal trabeculation. Note, a pericardial oedema was sporadically observed in MZ gpr126stl47 mutants, as previously reported43. Embryos with strong pericardial oedema were excluded from this study. We did not observe any other gross morphological defects in the MZ gpr126stl47 mutant larvae or developmental delays compared with WT larvae (Fig. S3b). For further characterization, we analyzed MZ animals and their WT control by IMARIS-based 3D surface reconstruction of the ventricular chambers at 120 hpf (Fig. 2e). In WT larvae, distinct muscular ridges in the ventricular lumen could be visualized. In contrast, MZ gpr126stl47 mutants exhibited a smooth ventricular wall with no trabecular projections.

These data indicate that maternal contribution of Gpr126 rescues the zygotic loss in around two-third of gpr126stl47 mutants, indicating incomplete penetrance, and that Gpr126 is required for cardiac chamber development in zebrafish.

Zygotic and maternal zygotic gpr126st49 mutants exhibit inner cardiac wall multilayering
Morpholino-mediated knockdown experiments, including rescue experiments, previously suggested that the NTFΔGPS is sufficient for trabeculation and heart development22. However, in vivo confocal imaging of zygotic gpr126st49 hearts revealed that a majority of mutant hearts exhibited a multilayered ventricular wall (60%, Fig. 2a, b). The analysis of MZ gpr126st49 mutants recapitulated the multilayered phenotype (66.67%, 20 out of 30) observed in the Z gpr126st49 mutants (Fig. 2c, d), with a similar phenotypic penetrance and expressivity. In addition, 6.67% (2 out of 30) of the mutants exhibited hypotrabeculation. IMARIS-based 3D surface reconstruction revealed a thick compact wall with few or no trabeculae in MZ gpr126st49 mutant hearts (Fig. 2e). In contrast, no obvious defect in the endocardium or the cardiac jelly between the endocardium and the myocardium was detected at 72 and 96 hpf, when analyzing MZ gpr126stl47 and gpr126st49 mutants in the background of Tg(myl7:mKATE-CAAX); Tg(krt4:GFP)47, which label the myocardium and the endocardium, respectively (Fig. S4). The only apparent difference is that the endocardium is not folded as normal, which appears to be due to the lack or a reduced number of trabeculae. These data suggested that either NTFΔGPS exhibits a dominant effect or that the CTF is required in conjunction with the NTF for proper heart development. To exclude the possibility that the multilayering phenotype of gpr126st49 hearts is due to a dominant effect of NTFΔGPS, we analyzed the Z and MZ heterozygotes of the mutants and observed that the heterozygotes of both mutants do not exhibit any obvious trabeculation defects (Fig. S5), indicating that NTFΔGPS exhibits no dominant effect.

As our new data appeared to be in contrast to our previous findings, we revisited our previously published data regarding overexpressing NTFΔGPS in gpr126 morphants22. While previously the focus was only on the “reappearance of trabeculae”, a closer look revealed that the data could also be interpreted as hypertrabeculation or multilayering (note, the analysis was limited to 80 hpf due to the morpholino-based approach). Therefore, we analyzed myocardial-specific (myl7-driven) stable expression of NTFΔGPS in WT and MZ gpr126stl47 mutants as well as mosaic expression in WT larvae, as our previous data indicate that NTF acts as a secreted molecule on cardiomyocytes22. Notably, overexpression of NTFΔGPS in MZ gpr126stl47 mutants led to a multilayering phenotype at 120 hpf, similar to MZ gpr126st49 mutants, while WT larvae overexpressing NTFΔGPS exhibited normal trabeculation (Fig. S6a, b). Mosaic overexpression of NTFΔGPS by injecting single-cell-stage Tg(myl7:EGFP-hsa.HRAS) embryos with a myl7:gpr126NTF-p2a-tdTomato plasmid revealed that mosaic NTFΔGPS overexpression in cardiomyocytes leads to local multilayering at 96 hpf in 62% of the injected embryos (5 out of 8) (Fig. S6c). The tdTomato-overexpressing cardiomyocytes adhere together along with tdTomato-negative cardiomyocytes, while the rest of the ventricular wall is monolayered.

These data suggest that gpr126st49 mutants express a functional NTFΔGPS and that the NTF is not sufficient for proper heart development, revising our previous conclusion22.

Gpr126 mutants do not exhibit modified exon usage
Alternative splicing is a common feature of adhesion GPCRs48 and the variable penetrance of trabeculation phenotypes in the gpr126 mutants could be a result of compensatory mechanisms through splice isoforms. To exclude the possibility that alternatively spliced mRNAs result in a functional protein compensating for the truncated non-functional transcripts, we performed differential exon usage (DEU) analysis of 72 hpf whole larvae (hereafter referred to as “full body DEU analysis”) as well as 96 hpf larval hearts (hereafter referred to as “heart-specific DEU analysis”) for MZ gpr126stl47, MZ gpr126st49 and their WT littermates to determine the presence of alternatively spliced gpr126 isoforms. The gpr126stl47 and gpr126st49 mutants introduce a stop codon in Exon 3 and Exon 16, respectively (Fig. S7a)15,43,49. Full body DEU analysis between WT and mutants highlighted exon counting bin (B) 006 as differentially used (p-adjusted = 0.001; Fig. S7b). This exon counting bin corresponds to part of exon 4 of the canonical transcript isoform adgrg6-204 (Table S1). Notably, the exon usage plot suggested lower usage for MZ gpr126st49. However, RT-PCR analysis utilizing RNA isolated from 72 hpf WT and MZ gpr126st49 larvae with primers spanning B004 to B007 did not confirm DEU analysis results, as no additional band (lacking B006) was detected (Fig. S7c). These RT-PCR data suggest there is no compensation through alternatively spliced isoforms in the MZ gpr126st49 mutants.

Similarly, heart-specific DEU analysis did not show any significant difference in the use of exon counting bins between WT and mutants, further supporting that alternative splicing in the heart is not compensating for the predicted non-functional transcripts. However, different from what was observed in the full body DEU analysis, heart-specific DEU results showed lower use of B008, corresponding to exon 6 of the canonical transcript isoform, in all samples, i.e., both WT and mutants (Fig. S7d). RT-PCR using full-body and heart-specific RNA from WT samples confirmed DEU analysis results, showing that the predominant gpr126 isoform in the heart lacks B008 (Fig. S7e), validating DEU analysis for alternative splicing assessment. These findings are consistent with the known canonical transcript structure of gpr126, as B002 (belonging to an alternative exon 1 in adgrg6-201, Table S1) could not be detected, and align with previously described splice isoforms that exclude Exon 24 of adgrg6-204, reflected in the lower usage of B028 (Table S1) in both full body and heart-specific samples. Notably, skipping of exon 6 results in deletion of 23 aa in zebrafish, affecting gpr126 protein conformation and signaling50. Consistently, the construct used in this study for rescuing NTF function lacks exon 6.

Taken together, our results suggest that alternative splicing in the heart is not compensating for the predicted non-functional transcripts.

Rescue experiments reveal that Gpr126-CTF is required for cardiac trabeculation
Our data indicate that the observed phenotype in gpr126st49 mutants is due to the lack of the CTF and not due to a dominant effect of the expressed NTFΔGPS. If this assumption is correct, then it should be possible to rescue the gpr126st49 mutant phenotype by CTF overexpression. Therefore, we first assessed whether gpr126 is, like in mice, expressed in endocardial cells of the zebrafish heart. We performed fluorescence-activated cell sorting (FACS) for endocardial cells and cardiomyocytes using isolated cells from extracted hearts of Tg(myl7:EGFP-hsa.HRAS); Tg(kdrl:nls-mCherry) larvae at 48 and 96 hpf. Endocardial and myocardial enrichment was confirmed by the relative expression levels of kdrl and myl7, respectively. qPCR analysis showed that in zebrafish hearts, gpr126 is enriched in endocardial cells compared to cardiomyocytes (Fig. 3a). This result was confirmed by RNAscope analysis (Fig. S8). Therefore, we generated a stable endothelial-specific transgenic line, Tg(fli1a:gpr126CTF-p2a-tdTomato) (Fig. 3b), that overexpresses a protein that is post-translationally cleaved into tdTomato and Gpr126-CTF, to perform rescue experiments in gpr126st49 mutants. The CTF domain is tagged with HA at its N-terminus after the signal peptide and FLAG-tagged at the C-terminus. In addition, a P2Y N-terminus peptide was fused to the Gpr126 CTF to facilitate plasma membrane localization of the receptor (Fig. 3b)9. Anti-FLAG whole mount immunostaining of 72 hpf Tg(fli1a:gpr126CTF-p2a-tdTomato) embryos showed that the CTF is localized to the cell membrane in endocardial as well as vascular endothelial cells (Fig. 3c). Tg(fli1a:gpr126CTF-p2a-tdTomato) larvae did not show any gross morphological defects (Fig. S9a). The analysis of 96 hpf Tg(fli1a:gpr126CTF-p2a-tdTomato) larval hearts showed normal trabeculation and exhibited no obvious defects (Fig. S9b).

To determine whether CTF overexpression in the endocardium rescues the multilayered ventricular wall in MZ gpr126st49 mutants, Tg(fli1a:gpr126CTF-p2a-tdTomato) were crossed with gpr126st49 mutants in the background of Tg(myl7:EGFP-hsa.HRAS). The analysis of 120 hpf MZ gpr126st49; Tg(fli1a:gpr126CTF-p2a-tdTomato); Tg(myl7:EGFP-hsa.HRAS) hearts revealed that ectopic CTF expression, identified by tdTomato-positive endocardium, reversed the multilayering phenotype in MZ gpr126st49 mutant hearts, resulting in the formation of trabecular projections into the ventricular lumen (Fig. 3d, e). MZ gpr126st49 mutants overexpressing CTF exhibited clear muscular ridges in the ventricular chamber, while MZ gpr126st49 mutants exhibited a thick, compact wall in the absence of mature trabecular ridges (Fig. 3f), as described above (Fig. 2e). These data show that endocardial expression of Gpr126-CTF rescues the multilayering phenotype observed in gpr126st49 mutants, indicating that NTFΔGPS in gpr126st49 mutants represents a fully functional NTF.

G proteins can signal through cAMP accumulation25,51 and it has been shown that treatment with the adenylyl cyclase activator forskolin (25 µM) can rescue the myelination defects and puffy ear phenotype of gpr126st49 mutants but not the radial sorting defects of gpr126stl47 mutants43. A phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX, 100 µM), which raises cAMP levels independently of adenylyl cyclase activity, was used to rescue ear defects in other mutant gpr126 alleles13. Therefore, we tested whether forskolin or IBMX treatment could also rescue the heart phenotype in gpr126st49 mutants. We confirmed that these treatments rescued the puffy ear phenotype (Fig. S10a). Analysis of the hearts revealed that both treatments could partially rescue the multilayering phenotype in MZ gpr126st49 mutants (Fig. S10b, upper panels). However, both treatments also blocked trabeculation, both in MZ gpr126st49 mutant hearts and in WT hearts (Fig. S10b, lower panels). Thus, it remains unclear whether Gpr126 acts through cAMP activation to promote trabeculation.

Collectively, our data suggest that endocardial-specific Gpr126-CTF signaling is required for cardiac trabeculation in zebrafish.

Gpr126-NTF is sufficient to maintain cell-cell adhesion and compact wall integrity
Compact wall integrity is required for proper initiation of trabeculation52. As N-cadherin is the major component of cardiac adherens junctions39, we determined whether N-cadherin localization in compact wall cardiomyocytes prior to trabeculation is affected in the two mutants. gpr126stl47 and gpr126st49 mutants were crossed in the background of TgBAC(cdh2:cdh2-EGFP) and analyzed at 72 hpf. Single-plane confocal images of WT hearts showed that N-cadherin specifically localizes laterally in the membrane of compact layer cardiomyocytes with some expansion to the basal side (Y-shape pattern) (Figs. 4, and S11a) as previously published39. Maximum intensity projection images confirmed the lateral localization of N-cadherin in WT hearts (Figs. 4, and S11a). In contrast, in MZ gpr126stl47 mutant hearts, N-cadherin was distributed randomly, still laterally at the junctions but also at the apical and/or basal membrane of the compact layer cardiomyocytes, which was also obvious in maximum intensity projections (Fig. 4). The maximum intensity projection images exhibit an organized distribution of N-cadherin in WT hearts; however, in the MZ gpr126stl47 mutant hearts, the N-cadherin distribution is haphazard and misorganized (Fig. S12). Notably, in the MZ gpr126st49 mutants, which express NTFΔGPS, N-cadherin was localized in a WT-like pattern at the junctions (laterally) of compact layer cardiomyocytes (Figs. 4 and S11a). Overexpression of CTF alone in MZ gpr126stl47 mutants did not rescue the compact wall integrity defects (Fig. S11b), further indicating the requirements of NTF to mediate cardiomyocyte adhesion. These data indicate that the NTFΔGPS is sufficient for the proper localization of adherens junctions in cardiomyocytes of the compact wall prior to trabeculation.

Gpr126-CTF is required to establish trabecular identity of cardiomyocytes
The compact layer cardiomyocytes exhibit apicobasal polarity, while trabecular layer cardiomyocytes are depolarized41. The observed multilayering phenotype of MZ gpr126st49 mutants raised the question whether the inner layer of cardiomyocytes consists of depolarized cardiomyocytes, indicating hypertrabeculation, or whether they consist of polarized cardiomyocytes caused by abnormal multilayering of the compact ventricular wall. To determine the apicobasal polarity of cardiomyocytes, MZ gpr126st49 mutants were crossed into the background of Tg(myl7:EGFP-Podxl); Tg(myl7:mKATE-CAAX), in which the cardiomyocyte membrane is labeled with mKATE and the apical membrane of polarized cardiomyocytes is marked by the apical protein podocalyxin fused with EGFP. In 96 hpf WT siblings, EGFP-podocalyxin is localized at the apical/abluminal side of compact layer cardiomyocytes and around the entire membrane of delaminated, trabecular cardiomyocytes, indicating their polarized and depolarized states, respectively (Fig. 5a). In contrast, in multilayered hearts of MZ gpr126st49 animals, cardiomyocytes of both the outer layer as well as the inner layers were mainly polarized (Fig. 5a). Quantification revealed that the percentage of depolarized cardiomyocytes in the inner layers of the ventricles was significantly reduced from 94.7% ± 1.7% in WT to 16.6% ± 2.6% in MZ gpr126st49 mutants (p < 0.001; WT: n = 6 hearts; MZ gpr126st49: n = 12 hearts; per heart 15-25 cardiomyocytes were counted) (Fig. 5b). These data indicate that the cardiomyocytes in multilayered hearts of MZ gpr126st49 mutants fail to depolarize and consequently cannot seed the trabecular layer.

During depolarization, compact layer cardiomyocytes remodel their adherens junctions, which involves the relocalization of N-cadherin from the lateral to the basal membrane. To test whether adherens junction remodeling is affected in MZ gpr126st49 mutant hearts, N-cadherin localization was analyzed at 96 hpf, when multilayering was observed. In WT hearts, the compact layer cardiomyocytes showed basolateral distribution of N-cadherin, and trabecular layer cardiomyocytes had a punctate distribution (Fig. 5c), as previously described39. In contrast, in the multilayered MZ gpr126st49 mutant hearts, N-cadherin was localized at the lateral side of the compact layer and inner-layered cardiomyocyte wall (Figs. 5d and S11c). These data suggest that cardiomyocytes in the multilayered wall of gpr126st49 mutants fail to attain trabecular identity due to defects in remodeling adherens junctions and cardiomyocyte polarity defects.

It has been demonstrated that Notch signaling is activated during trabeculation upon cardiomyocyte delamination in a subset of compact wall cardiomyocytes to inhibit their delamination40–42. As MZ gpr126stl47 mutants are characterized by hypotrabeculation, no change in myocardial Notch activity is expected. In contrast, we hypothesized that failure of gpr126st49 mutant cardiomyocytes to attain trabecular identity might be due to enhanced myocardial Notch activity. To test this hypothesis, gpr126stl47 and gpr126st49 mutants were crossed in the background of Tg(TP1:Venus-PEST), in which the Notch-responsive TP1 promoter drives expression of Venus-PEST, a fluorescent protein with a short half-life (~2 h). During trabeculation in WT larvae, Notch signaling can be detected in the atrioventricular canal (AVC), the outflow tract and a few cardiomyocytes in the compact layer of the myocardium (Fig. 6a). Analysis of the mutant hearts at 96 and 120 hpf indicated that Notch activity was not altered in MZ gpr126stl47 mutants (Fig. 6a). In contrast, Notch activity in the heart was markedly increased in MZ gpr126st49 mutants. Quantitative analysis of the Venus fluorescence intensity normalized to the signal in AVC valves confirmed that myocardial Notch activity was significantly increased in MZ gpr126st49 mutants (Fig. 6b). Notably, NTFΔGPS overexpression in MZ gpr126stl47 mutants, which led to myocardial multilayering mimicking MZ gpr126st49 (Fig. 4b, c), also resulted in a substantial increase of Notch activity at 120 hpf (Fig. 6c, d). On the other hand, overexpression of CTF in the MZ gpr126st49 mutants, which restored normal trabeculation, also normalized myocardial Notch activity (Fig. 6e, f). In contrast, overexpression of NTFΔGPS and CTF in the WT background did not lead to any substantial changes in myocardial Notch activity (Fig. S13a, b). Additionally, overexpression of CTF in MZ gpr126stl47 mutants failed to alter Notch activity (Fig. S13c, d).

Collectively, these data suggest that CTF signaling is required to inhibit myocardial Notch activity to allow cardiomyocytes to attain trabecular identity.

Methods
All research presented here complies with all relevant ethical regulations approved by the Tierschutzkommision (Animal Welfare Commission) of the REGIERUNG VON UNTERFRANKEN (local government, protocols: AZ.55.2.2-2532.2-1145-13 and AZ.I/39-1/FN003) and conforms to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

Zebrafish maintenance
Embryonic and adult zebrafish were raised and maintained in Tecniplast aquaculture main and stand-alone systems. The water temperature was maintained at 28.5 °C and the pH and conductivity were set at 7–7.5 and 450–500 µS. The light-dark cycle consisted of half an hour of gradual onset of light (8 am to 8:30 am) with 13.5 h of uninterrupted light and half an hour of gradual onset of darkness with 11.5 h of complete darkness. Zebrafish were fed three times a day, depending on age, with rotifers and granular food. Health monitoring was performed at least once per year.

Zebrafish mutant and transgenic lines
In this study, the following transgenic and mutant lines were used: Tg(myl7:EGFP-hsa.HRAS)s88373, Tg(−0.2myl7:EGFP-podocalyxin)bns1041, Tg(myl7:mKate-CAAX)sd11, abbreviated as myl7:mKate-CAAX74, TgBAC(cdh2:cdh2-eGFP,crybb1:ECFP)zf517, abbreviated as TgBAC(cdh2:cdh2-GFP)75, Tg(EPV.Tp1-Mmu.Hbb:Venus-Mmu.Odc1)s940, abbreviated as TP1:VenusPest76, gpr126stl4743, gpr126st4915, Tg(krt4:GFP)sqet3347, Tg(fli1:gpr126-p2a-tdTomato), Tg(myl7:gpr126NTF-p2a-tdTomato), and Tg(fli1:tdTomato-p2a-gpr126CTF).

Genotyping and matings of gpr126 mutants
stl47 were genotyped as previously described43. Briefly, the primers listed in Table S2 were used to amplify a 498 bp product from genomic DNA. Subsequently, the PCR products were digested with Mfe1 restriction enzymes. As the Mfe1 recognition site is disrupted in the mutant, only the WT PCR product is cleaved into 257 bp and 241 bp fragments. For st49, the primers listed in Table S2 were used to amplify a 604-base pair (bp) product from genomic DNA. The point mutation in st49 introduces a MaeIII recognition site, and the restriction digestion of the PCR product yields 437-bp and 167-bp fragments. The 604-bp product is retained in the digested WT DNA. For generating maternal zygotic (MZ) animals, homozygous gpr126stl47 or gpr126st49 mutant females were crossed with their heterozygous male counterparts. The WT siblings from heterozygous crosses were grown to adulthood, and their progeny were used as controls for MZ experiments and were referred to as WT cousins.

RNA isolation from zebrafish larvae hearts and quantitative PCR
Larval zebrafish hearts were isolated from WT and mutant embryos in the background of Tg(myl7:EGFP-hsa.HRAS) at 120 hpf following the protocol used by Burns and MacRae77. Larvae (n=200) were first anesthetized using 0.04% tricaine methanesulfonate (MS-222) and collected into 1.5 ml microcentrifuge tubes. The larvae were washed three times with ice-cold embryo disruption medium (EDM) and were resuspended in 1.25 ml EDM. Hearts were then fragmented from the larvae by mechanical disruption using a 5 ml syringe and a 1½ inch long 19-gauge needle. This was done by collecting the contents of the tube in the syringe through the needle and expelling it back into the microcentrifuge tube 30 times at the rate of 1 s per syringe motion. Fragmented larvae were filtered through a 105 µm nylon mesh, and the flow-through was collected in a 30 mm petri-dish. The flow-through was then applied to a 40 µm nylon mesh, which was then inverted, and the retained material was collected with EDM in a 30 mm petri dish. The beating hearts were identified with their EGFP expression under the Zeiss stereoscope and were collected into a 1.5 ml microcentrifuge tube containing RNAlater. All these steps were performed on ice. The hearts were then pelleted for 10 min at 10,000 x g. The supernatant was discarded, and TRIzol was added to the pellet, and RNA was isolated using RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions. For production of cDNA, 500 ng of RNA was reverse transcribed using Oligo (dT) 12-18mer primers and M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Cat# 280250139) according to the manufacturer’s instructions. qPCR assays were performed in experimental triplicates using SYBR Green (Bio-Rad) in a CFX Connect™ Real-Time PCR (Bio-Rad). gpr126 expression values were normalized using the housekeeping gene rpl13a and fold changes were calculated using the 2-ΔΔCt method. The primers for gpr126 amplifies part of exon 3 and 4 and the sequences used for qPCR can be found in Table S2.

RNAscope assay
Zebrafish embryos were fixed overnight at 48 and 96 hpf in 4% PFA in PBS at RT. A series of increasing methanol concentrations (25%, 50%, 75%, 2×100%) in 0.1% PBST was used to dehydrate the embryos stepwise in 10 min washes. The embryos were then rehydrated with decreasing methanol concentrations (75%, 50%, and 25%) and subjected to the RNAscope-based target retrieval by incubating the embryos in 1X target retrieval solution (Advanced Cell Diagnostics) at 100 °C for 15 min. Protease digestion of embryos using RNAscope Protease Plus (Advanced Cell Diagnostics) was performed for 15 min for 48 hpf embryos and 30 min for 96 hpf embryos at 40 °C by floating the tubes in a water bath. The probe hybridization and staining were performed using RNAscope Multiplex Fluoroscent v2 Assay kit (Advanced Cell Diagnostics), The target probe was hybridized at 40 °C for 2 h followed by washing in 1X wash buffer for 10 min. For RNA detection, the amplification steps were performed by incubation with the different amplifier solutions in a water bath at 40 °C followed by washing in 1X wash buffer for 10 min. The signal was then developed by incubating the embryos in HRP-C2 solution, followed by incubation with the fluorescent dye Opal 570, and then incubating in HRP blocker solution. All these steps were performed in 40 °C water bath for 15 min each, followed by washing with 1X wash buffer for 10 min twice. After the final wash, the embryos were immunostained by directly proceeding to the blocking step in the whole-mount immunostaining protocol.

Whole-mount immunostaining of zebrafish embryos
Embryos were treated with PTU at 24 hpf to avoid pigmentation. The embryos were anaesthetized with 0.4% (w/v) tricaine to stop the heartbeat, just before fixation, to prevent the heart from collapsing during fixation. 10 embryos were fixed overnight in 4% paraformaldehyde in 2 ml tubes. The fixative was washed three times using PBST. The embryos were permeabilized using proteinase-K (3 µg/ml) in PBST for 1 h. This treatment was followed by washes with PBDT buffer (PBS, 1% BSA, 1% DMSO, 0.5% Triton X-100), and blocking was performed for 1 h with 10% sheep serum in PBDT. Primary antibody (Mouse anti-FLAG M2, Sigma Aldrich, F3165, 1:500) incubation was performed in a 4 °C nutator in PBDT buffer with 1% sheep serum (gentle shaking). After washing with PBDT, the embryos were incubated in secondary antibody (donkey anti-mouse conjugated to Alexa Fluor-488, -594, and -647, Life Technologies, 1:500) in PBDT buffer with 1% sheep serum for 2 h at RT. This step was followed by 15 min incubation in DAPI (2 µg/ml) and final washes with PBST.

Plasmid constructs
For overexpression of zebrafish gpr126 and its mutant forms in mammalian cell lines, constructs were created using site-directed mutagenesis. HA-gpr126 fragment was amplified by PCR from pcDps-HA-gpr126-FLAG gifted by Ines Liebscher and cloned into the mammalian expression vector pAcGFP to create HA-Gpr126-GFP construct. Inserts with the stl47 and st49 mutations were generated through PCR by introducing complementary mutations in the primers, as listed in Table S2. The inserts with the mutations were assembled using the NEBuilder HiFi assembly master mix (New England Biolabs, Cat# E2621L) according to the manufacturer’s instructions, and transformed into competent bacteria. To generate the fli1a:gpr126-p2a-tdTomato and fli1a:gpr126CTF-p2a-tdTomato plasmids, sequences encoding the Gpr126 and Gpr126-CTF were PCR amplified. The amplified coding sequences followed by p2a-tdTomato were cloned downstream of the endothelial-specific fli1a promotor into a Tol2-enabled vector using the Cold Fusion Cloning kit (System Biosciences, Cat# MC010B-1). The CTF construct was designed with the signal peptide followed by Gpr126-CTF tagged with HA at the N-terminus and FLAG at the C-terminus9. In addition, P2Y N-terminus peptide was fused to the Gpr126 CTF to facilitate the plasma membrane localization of the receptor12. The NTF construct used was NTFΔGPS as described before22.

ARPE19 cells and plasmid transfection
The ARPE-19 cell line (ATCC, CRL-2302) was cultured in Dulbecco’s modified Eagle’s medium/Nutrient mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Plasmid transfection into ARPE-19 cells was carried out with 500 ng DNA per well of a 24-well plate using 1 µl Lipofectamine LTX (Thermo Fisher Scientific, Cat# 15338100)) according to the manufacturer’s instructions. Transfection complexes were formed by incubating DNA with Lipofectamine LTX in Opti-MEM for 20 min at room temperature. For WB analysis, transfection was performed in 6-well plates, and the volumes were adjusted by using fourfold amounts of reagents.

Production of lentiviral vectors
The lentiviral packaging plasmid psPAX2 and the VSV-G envelope plasmid pMD2.G were gifts from Didier Trono (psPAX2: Addgene plasmid #12260; http://n2t.net/addgene:12260; RRID: Addgene_12260; pMD2.G: Addgene plasmid #12259; http://n2t.net/addgene:12259; RRID: Addgene_12259). The transfer plasmid pLenti CMVtight Blast DEST (w762-1) was a gift from Eric Campeau (w762-1: Addgene plasmid #26434; RRID: Addgene_26434). The coding sequences of HA-Gpr126-GFP wt and mutants were cloned into w762-1 by NEBuilder® HiFi DNA Assembly according to the manufacturer’s instructions using the primers indicated in Table S2. To produce lentiviral vectors, psPAX2, pMD2.G, and the desired transfer plasmid were transfected in a 1:1:2 ratio into HEK293T (ATCC, CRL-3216) cells using PEI MAX (Polysciences, Cat# 24765-1). Supernatant containing lentiviral vectors was harvested 72 h after transfection, filtered through a 0.45 µm filter, and aliquots were snap frozen.

Generation of stable ARPE-19 cell lines
Lentiviral vector aliquots were rapidly thawed at 37 °C and diluted in growth medium containing 10 µg/ml Polybrene (Sigma-Aldrich, Cat# 107689). Cells were transduced overnight in 6-well plates using 1 ml of diluted lentiviral vector. The following morning, medium was refreshed, and cells were selected with 10 µg/ml of blasticidin for transgene integration 72 h after transduction.

Heart-specific RNA-sequencing
Larval hearts were collected from the gpr126 mutants and their WT cousins. 30 hearts from one clutch were obtained and pooled per biological replicate (4 biological replicates for each genotype). RNA was isolated using miRNeasy micro Kit (Qiagen) with on-column DNase digestion (RNase-Free DNase Set, Qiagen) to avoid contamination by genomic DNA. RNA and library preparation integrity were verified with LabChip Gx Touch (PerkinElmer). RNA amounts were normalized, and 1 ng of total RNA was used as input for SMART-Seq® HT Kit following manufacture’s protocol (Takara Bio). Sequencing was performed on the NextSeq 500 platform (Illumina) using a P3 flowcell with 75bp single-end setup. The resulting raw reads were assessed for quality, adapter content, and duplication rates with FastQC (Andrews S. 2010, FastQC: a quality control tool for high throughput sequence data. Available online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Trimmomatic v0.3978 was employed to trim reads after a quality drop below a mean of Q18 in a window of 5 nucleotides. Only reads above 30 nucleotides were employed for further analyses. Trimmed and filtered reads were aligned versus the zebrafish genome version danRer11 (ensembl release 99) using STAR v2.7.3a79 with the parameter “--outFilterMismatchNoverLmax 0.1” to increase the maximum ratio of mismatches to mapped length to 10%.

Full body RNA-sequencing
Full-length RNA was isolated from zebrafish larvae using the RNeasy mini kit (Qiagen) combined with on-column DNase digestion (RNase-Free DNase Set, Qiagen) to avoid contamination by genomic DNA. Library preparation was performed relying on the Illumina Stranded mRNA Prep kit (Illumina) using as input 200 ng of RNA for each sample. Sequencing was performed on a NovaSeq 6000 platform (Illumina) (paired-end; 2×159bp). Conversion of reads to fastq format, and demultiplexing were performed with bcl2fastq v2.17. Raw reads were analyzed relying on the nf-core RNA-Seq pipeline v3.980 (https://doi.org/10.5281/zenodo.1400710) using default parameters. In short, reads were adapter- and quality-trimmed using Trim Galore v0.6.7 (Babraham Bioinformatics) and mapped to the Ensembl danio rerio genome assembly GRCz11 (release 109)81 using STAR v2.7.10a79 to generate BAM files.

Differential exon usage analysis
Full body and heart-specific differential exon usage (DEU) analyses were performed in R v4.2.2 (https://www.R-project.org) relying on the DEXSeq package v1.44.082,83 following the official documentation (Appendix section 10.2 (https://bioconductor.riken.jp/packages/3.16/bioc/vignettes/DEXSeq/inst/doc/DEXSeq.html). First transcript annotations from the Ensembl danio rerio gene annotation file release 109, with coordinates of exon boundaries, were extracted and stored in a TxDb object using the function makeTxDbFromGFF() from the GenomicFeatures R package v1.50.484. The TxDb object was provided as input to the GenomicFeatures function exonicParts() to create non-overlapping exon counting bins. To this aim, the argument “linked.to.single.gene.only” was set to TRUE. Counting of read fragments overlapping with the exon counting bins was performed relying on summarizeOverlaps() from the R package GenomicAlignments v1.34.184 using the count method “Union”. For heart-specific DEU analysis, the function was run with the arguments “nter.feature=FALSE”, “singleEnd=TRUE”, “ignore.strand=TRUE”, and “fragments=FALSE”. Instead, for full body DEU analysis, the following parameters were set: “singleEnd=FALSE”, “inter.features=FALSE”, and “fragments=TRUE”. After read counting a DEXSeqDataSet object was built by calling DEXSeqDataSetFromSE() on the output of summarizeOveralps(). The estimateSizeFactors() and estimateDispersion() functions were used to estimate size factors and dispersions, respectively. Ultimately, DEU was performed through the DEXSeq function testForDEU() and differences in DEU were extracted using the function DEXSeqResults(). The plots of exon usage coefficient estimates were generated relying on the plotDEXSeq() function from DEXSeq, setting “expression=F” and “splicing=T”. An exon counting bin was defined as differentially used if its adjusted p-value was lower than 0.05.

RT-PCR for validating DEU analysis
PCR was carried out using 10 ng of cDNA with RedTaq DNA polymerase master mix from Genaxxon (Ulm, Germany). Primers used are provided in Table S2.

Immunofluorescence
For detection of HA-tag via immunofluorescence staining, cells were rinsed once with D-PBS and were fixed with 4% formaldehyde/PBS for 10 min at room temperature. Formaldehyde-fixed cells were permeabilized for 10 min with 0.5% Triton X-100/PBS. Prior to antibody staining, samples were blocked for 20 min using 5% BSA in 0.2% Tween-20 in PBS. The primary antibody (Rabbit anti-HA, Abcam, ab9110) was diluted (1:500) in the blocking reagent and incubated with the sample overnight at 4 °C in a humidified chamber. After removal of primary antibody solution and three 5 min washes with 0.1% NP40/PBS, samples were incubated for 60 min with fluorophore-coupled secondary antibody (donkey anti-rabbit conjugated to Alexa Fluor-594, and -647, Life Technologies) diluted in blocking reagent (1:500). DNA was visualized with 0.5 µg/ml DAPI (4′,6′-diamidino-2-phenylindole) in 0.1% NP40/PBS. After DAPI staining, cover slips were rinsed once with Millipore-filtered water and then mounted using Fluoromount-G mounting medium.

Western blotting
Transfected ARPE19 cells were washed with ice-cold PBS and extracted in RIPA buffer supplemented with EDTA-free protease inhibitor cocktails (cOmplete, Roche #11873580001). After 30 min incubation on ice, samples were sonicated, and lysates were cleared by centrifugation at 16,000 x g for 10 min at 4 °C. Lysates were analyzed by SDS-PAGE (4–12% NuPAGE Novex Bis-Tris gels) under reducing conditions and transferred to nitrocellulose membrane by wet transfer at 30 V and 300 mA for 1.5 h in 1x transfer buffer (25 mM Tris-HCl, pH 7.5, 192 mM glycine, 0.1% SDS, 10% methanol). The membrane was then blocked with 1x TBS, 0.05% Tween-20 and 10% non-fat milk and incubated with primary antibodies against HA and GFP (Rabbit anti-HA, Abcam, ab9110, 1:500; Mouse anti-GFP, Abcam, ab38689, 1:500). Enhanced chemiluminescence reagent (PerkinElmer, Waltham, MA) was used for protein detection. The cell supernatant was collected and centrifuged for 300 x g for 5 min at RT to remove any leftover cells and concentrated using a Vivaspin centrifugal concentrator (Sartorius).

FACS sorting and quantitative PCR
Larval hearts from Tg(myl7:GFP-CAAX); Tg(kdrl:nls-mCherry)) at 48 and 96 hpf were manually dissected in DMEM + 10% FBS, centrifuged for 3 min at 5000 rpm, washed in 1 ml of Hanks’ Balanced Salt Solution, and dissociated into single cells in 100 µl of Enzyme 1 and 5 µl of Enzyme 2 (Pierce Cardiomyocytes Dissociation Kit, ThermoFisher Scientific) for 30 min at 350 rpm shaking at 30 °C. The cells were centrifuged for 5 min at 5000 rpm, and the dissociation media was removed and replaced by DMEM + 10% FBS. The cells were then sorted using a BD FACSAria™ III—firstly by forward scatter amplitude and side scatter amplitude, and subsequently sorted by GFP and mCherry fluorescence into Trizol. More than 100 hearts were pooled together for each independent experiment. RNA extraction was then performed using Qiagen RNeasy Kit. At least 100 ng of total RNA was used for reverse transcription using Maxima First Strand cDNA synthesis kit (ThermoFisher Scientific). DyNAmo ColorFlash SYBR Green qPCR Mix (ThermoFisher Scientific) was used on a CFX connect Real-time System (Bio-Rad) with the following program: pre-amplification 95 °C for 7 min, amplification 95 °C for 10 s and 60 °C for 30 s (repeated for 39 cycles), melting curve 60-92 °C with increment of 1 °C every 5 s. Each point in the dot plots represents a biological replicate from three technical replicates. Gene expression values were normalized using the housekeeping gene rpl13a and fold changes were calculated using the 2-ΔΔCt method. The qPCR primers for gpr126 span Exon 3 to Exon 4.

Zebrafish transgenesis
Tol2 transgenesis was used to generate the Tg(myl7:gpr126NTF-p2a-tdTomato); Tg(fli1:gpr126-p2a-tdTomato) and Tg(fli1:tdTomato-p2a-gpr126CTF) lines. To generate stable lines, wild-type AB embryos (F0) were injected at the one-cell stage with an injection cocktail of transposase (tol2 mRNA, 12.5 pg per embryo) and plasmid DNA (25 pg per embryo). F0 embryos positive for tdTomato fluorescence were raised to adulthood and then screened for founder animals. The founders (three for each transgenic line) were further outcrossed to raise the F1 generation.

Morpholino injection
In order to stop contractility and blood flow in the embryos, 0.25 ng of tnnt2a morpholino (5′-CATGTTTGCTCTGATCTGACACGCA-3′) was injected into single-cell-stage embryos.

Chemical treatments
ErbB2 signaling was inhibited by treating the embryos with 10 µM of ErbB2 inhibitor PD168393 (Calbiochem). 10 µM working solution of PD168393 was prepared in 1x E3 media with 0.003% PTU. For the treatment, 10 embryos per well were placed in a 6-well plate containing the working solution of PD168393. As a control, 1% DMSO was used. The treatment was performed from 60 hpf to 120 hpf. At 120 hpf, the PD168393 solution was removed, and the embryos were washed three times with 1x E3 medium for 5 min and subsequently imaged.

WT and mutant embryos were treated with forskolin (2×6 h pulses, at 60-66 hpf and 84-90 hpf) at 25 µM or IBMX (3-isobutyl-1-methyl-xanthine) (continuous treatment, 60-96 hpf at 125 µM in 1x E3 medium with 0.003% PTU. Control embryos were treated with equivalent amounts of DMSO.

The ARPE-19 cells were treated with 3 µg/ml brefeldin A 24 h after transfection. Before the treatment, the cell medium was replaced with a serum-free medium, and the supernatant was collected 8 h after brefeldin A treatment.

Imaging
Image acquisition for immunostained cells as well as zebrafish hearts was performed using Carl Zeiss AG LSM800 confocal laser scanning microscope equipped with the ZEISS blue software. For live imaging of stopped zebrafish hearts, embryos and larvae were mounted in 1% low-melting agarose on glass-bottom dishes (MatTek). The larvae were oriented through a micromanipulator under the Carl Zeiss AG Axiozoom V16 stereoscope to enhance optical access to the heart. In order to stop the heart beating before imaging, 0.2% (w/v) tricaine was added to the low-melting agarose. A 40x (1.1 numerical aperture [NA]) water-immersion objective was used to acquire the images. The optical sections were 1 µm thick.

Image processing and Quantification
2D and 3D images were processed using ImageJ and the Fiji extension package. Figures were prepared with the help of Adobe Illustrator or Microsoft PowerPoint. The exon and protein structure in Figs. 1 and S7 were created by Illustrator for Biological Sequences 2.049. The criteria we have chosen for normal trabeculation were 3 or more trabeculae with 3 or more cardiomyocytes for 96 hpf hearts and 5 or more trabeculae with 3 or more cardiomyocytes for 120 hpf hearts. The quantification of intensity profiles of HA- and GFP-tag in ARPE-19 cells was performed using the “Measure” tool. The GFP intensity for each condition was measured relative to the HA intensity. For the quantification of myocardial Notch activity, intensity profiling of the YFP signal was performed. The total YFP signal relative to the YFP signal in the valve endocardium was plotted as the intensity of myocardial Notch. Three-dimensional surface rendering was performed using the Surfaces function of Imaris x64 (Bitplane). Bright-field images obtained from the stereoscope were processed using Zen blue (ZEISS) software.

Statistical analysis and reproducibility
Statistical analysis and p-value (p) calculation were performed using Graphpad Prism 8.3.0 and 10.2.0. Data are expressed as mean ± SEM or SD as indicated. Differences between the two groups were determined with a two-tailed Student’s t distribution test. To compare multiple conditions, one-way ANOVA was performed, and when data were compared to control groups, correction for multiple comparisons was performed by using Dunnett’s test. All tests were performed with a confidence level of 95%. All experiments were repeated at least three times unless otherwise specified.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Srivastava et al. explores the function of the Adhesion GPCR ADGRG6/Gpr126 in heart development. It follows up on previous studies of the Engel group (Patra et al. 2013) and shows that the receptor controls heart development and in particular, trabeculation in the zebrafish, potentially via two different mechanisms (adhesion and cell integrity) mediated separately by the receptor NTF and CTF. Finally, they propose an involvement of the receptor in the regulation of Notch activity.\n\nThe study by Srivastava et al. is very interesting and points towards exciting functions of the Adhesion GPCR ADGRG6/Gpr126, which also can give some indication on how Adhesion GPCRs in general function. However, many of the experiments are very preliminary and the interpretation sometimes is a little doubtful. I have several comments, especially regarding the use of the strains and constructs, which in my opinion need careful re-evaluation. In my opinion, the study in its current form is not complete and requires substantial more work to demonstrate many of the claims made, such as the CTF function and the regulation of Notch activity.\n\nMajor concerns:\n1. st49 mutant: I appreciate that the mutant has been characterized previously. However, both, st49 and st47 disrupt essential domains of the receptor, the CUB and the GAIN, respectively, which carries the risk of (partially) misfolded proteins. This probably does not matter for st47 as there is not much protein left and I agree that it can be considered a complete loss of function. However, it does matter for st49. The authors try to address this issue by expressing the construct in cells. However, as Fig. 1B shows, the construct accumulates in the cytoplasm. The authors state that this is due to the missing 7TM. I would assume that a missing 7TM leads to a liberated protein, which gets secreted rather than retained, especially as the signal peptide is still present. This is at least what we see for other GPCRs. The authors also postulate that this occurs in the st49 strain (p. 15, l. 326). I am highly worried what really happens to the protein. It might lead to dominant-negative effects in the fish and thus, to phenotypes\nI would like to see the experiments (or at least part of them) conducted in a fish strain carrying a “clean” version of the N terminus – liberated or even tethered to the membrane. I realize that this some effort, but it would generate much more solid data. With CRISPR-Cas this should be doable.\n2. Fig. 1B: It looks like there is less membrane localization of Gpr126 st49, but increased cytoplasmic localization. The authors state that this is due to the missing 7TM. As mention in comment 1, I would assume that the missing 7TM leads to a liberated protein that gets secreted rather than retained as the signal peptide is still present. I would assume other reasons for the cytoplasmic expression are more likely such as a partially misfolded protein.\n3. p. 9: Why does the st47 mutant not show a zygotic phenotype, but the st49 does? Maybe I am misunderstanding this, but if this is the case, it is a really interesting finding. It might either point towards a separate function of the NTF that is only visible when the CTF is absent, or it can be a dominant-negative effect of the st49 (see comment 1). The authors need to discriminate between the different options and explain this matter.\n4. p. 10: How does the CTF in Tg(fli1a:gpr126 CTF-p2a-tdTomato) look like? Does it have a signal peptide? Also, from the description in the Methods section, it seems that it does not have a tag or anything on its N-terminal side. A pure CTF might be rather instable and not be expressed too well at the membrane. This has been previously described in the context of Gpr126 (Liebscher et al. 2015). The authors do show expression in Fig. S3B, however, demonstrating proper expression of this construct at the cell membrane is absolutely necessary for the interpretation of the results. Thus, a tissue culture experiment would be good.\n5. p 10: The authors claim a CTF and an NTF function of Gpr126 in heart development. I am a bit worried about the postulated CTF function in this context. I agree that the CTF is required, but I am not sure that it is a sole CTF function. It has been shown for quite some aGPCR that NTF and CTF can reconstitute to form a (function) receptor (e.g. Silva et al. 2009, Prömel et al. 2012). As in st49, the N terminus is still present, providing the CTF in this context could lead to such a reconstitution.\nTo verify the results and to prove the interpretation the authors provide, the following control experiments would be necessary:\na) A rescue with a full-length Gpr126 construct specifically in this context.\nb) A rescue of the CTF in the st47 strain.\n6. Independently of whether the postulated CTF function is based solely on the CTF or on a combination of NTF/CTF, it remains elusive of which nature this function is. Obviously, the activation of a G protein cascade is the most likely CTF function. However, the authors show no such thing. Gpr126 signaling has been characterized quite well in zebrafish, such as receptor activation by a tethered agonist-derived peptide (Liebscher et al. 2014). It would be interesting to see whether a specific activation of the aGPCR by such a peptide rescues the trabeculation phenotype described on p. 8/9. The results of such an experiment will prove the involvement of the receptor CTF and shed light on the mechanism behind it.\n7. p. 13/14: How is the NTF in Tg(myI7:gpr126 NTF-p2a-tdTomato) designed? Is it the same as the construct in st49 or is it truly the NTF (stopping at the cleavage site within the GPS)?\n8. p. 14/15: The modulation of Notch signaling by Gpr126 is interesting. However, this data is very preliminary and highly speculative. At this point, it is definitely not solid enough to claim that Gpr126 regulates Notch activity.\nThe authors find that abrogating almost the entire receptor does not have a function on Notch activity. St49 leads to enhanced Notch activity, but overexpression of the NTF does not. From this, the authors conclude that the CTF inhibits Notch activity. However, several other scenarios are equally possible: Firstly, it can mean that the st49 construct conveys a dominant-negative effect (here, it is essential to know whether Tg(myI7:gpr126 NTF-p2a-tdTomato) carries the same construct as st49 – see my comment 7 above). Secondly, a purely compensatory mechanism is conceivable. The authors need to put some more effort into obtaining more and solid data on whether and how Gpr126 has an effect on the Notch pathway. The following controls might help:\na) A CTF expression in the st47 strain.\nb) An NTF expression in the st47 strain.\nc) A CTF expression in the st49 strain.\n8. p. 14/15: A highly puzzling issue is that if the CTF inhibits Notch activity as the authors postulate, one would expect that st47, in which the CTF is also missing, likewise leads to an increased Notch activity. This is, however, not the case and needs further clarification. I am wondering what the authors´ explanation for this Also, additional essential questions need to be addressed such as: Does the expression of the NTF in the st49 strain alter the expression of the Notch receptor/ligand? Altered expression levels of Notch pathway components could help explain changed Notch activity.\n10. p. 15: Again: How is the NTF in Tg(myI7:gpr126 NTF-p2a-tdTomato) designed? If it does not mimic st49, the data obtained with the construct cannot be compared to the ones on the st49 (Fig. 7A, B). If it does mimic st49, a potential dominant-negative effect of the construct cannot be excluded. Thus, in order to gain the most conclusive set of data, both constructs might need testing.\n11. p. 14/15: Simply showing an overexpression of the NTF in a wild-type background does not address the question which parts of the aGPCR are required for a potential function in the control of Notch activity. Further, it might help to also do the reciprocal experiment and e.g. express the CTF to see if a more pronounced downregulation effect occurs.\nMinor comments:\n1. Fig 1C: Information on statistics is missing: How many samples?\n2. There are some typos in the manuscript, e.g. p. 7 l. 147 “amino acid”. Please check spelling and grammar carefully.", "reviewer_2": "In this manuscript, Srivastava et al. studied the functions of adhesion G protein-coupled receptor 126 (Gpr126) in zebrafish heart trabeculation by analyzing two truncation mutants stl47 and st49. They found that maternal zygotic stl47 mutant exhibited hypotrabeculation phenotype with randomly distributed N-cadherin at membranes of the compact muscle layer. The st49 mutant lacking the C-terminal GPS motif exhibited a multilayered ventricular wall with normal N-cadherin distribution and elevated Notch signaling. They concluded that the N-terminal fragment (NTF) of Gpr126 maintains cell-cell adhesion and compact wall integrity in the myocardium, while the C-terminal fragment (CTF) is essential to establish the trabecular identity of cardiomyocytes. The finding here would provide novel insights into heart development and clarify the function of Gpr26 in myocardium trabeculation. However, as noted below, the conclusions might be a bit premature and require further analysis or clarification. The manuscript is well written and easy to follow.\n1. The authors used qPCR to show the relative expressions of gpr126 in CMs and endocardium, which is less optimal. Demonstrating the protein localization, either by an antibody staining or by tagging the gene, would be useful for mechanistic interpretations (e.g., a strategy used in a co-author’s previous paper: https://doi.org/10.1242/dev.171207 ). It is understandable that this request is likely beyond the scope of a revision. However, in situ hybridization of gpr126 showing the expression locations during trabeculation would be helpful.\n2. The authors only showed the myocardium in all figures. Since gpr126 is considered an endocardial-enriched gene in the heart, it seems necessary to assess any endocardial defects and endocardial cell-cardiomyocyte interactions in the mutants. Otherwise, the mechanism described in the current version seems to lack a significant component.\n3. Figure 1B. It is interesting that 36.67% of stl47 MZ mutant larvae and 26.67% of st49 MZ mutant larvae showed normal trabeculation. Could the authors speculate the reason? How do they define “normal trabeculation”?\n4. The authors showed that mosaic myocardial-specific NTF overexpression induced local cardiomyocyte multilayering and concluded that NTF has a function in maintaining cell-cell adhesion in the compact layer cardiomyocytes. However, gpr126 is considered an endocardial gene (Figure 3A). It is confusing how an endocardium secreted protein mediated cell adhesions within the myocardium. At least the outer layer of the multilayered myocardium seems to have no contact with the endocardium. Defining the expression domain of gpr126 (as noted above) seems necessary to clarify this. Also, an endocardial-specific overexpression strategy should be considered.\n5. Figure 7. The authors concluded that CTF signaling is required to inhibit myocardial Notch activity to allow cardiomyocytes to attain trabecular identity. However, the MZ stl47 mutant also lacks the CTF but showed normal myocardial Notch activity. Please explain.\n6. Figure S5. It is labeled as fli1a:gpr126 NTF-p2a-tdTomato in the figure but myl7:gpr126 NTF-p2a-tdTomato in the legend and main text. Please clarify. I assume it is myl7. The myocardium looks quite normal compared to the mosaic myocardial-specific NTF overexpression phenotype in Figure S4 (myl7:gpr126 NTF-p2a-tdTomato). Please clarify what makes the difference.\n7. Can the authors explain or speculate why zygotic stl47 mutant but not st49 mutant exhibited maternal effect?\n8. Control group is missing in the experiment shown in Figure S5\n9. Figure 4A and B lack quantification results.\n10. Page 8, lines 151-153. “The analysis of zygotic gpr126stl47/stl47 mutant hearts revealed no obvious gross morphological defects compared to WT siblings suggesting that Gpr126 is not required for heart development in zebrafish (Figure S2).” This statement is not accurate. The result only suggests that a zygotic mutant did not show a heart development defect. One cannot conclude that Gpr126 is not required for heart development.\n11. st49 and NTFdeltaGPS are used to represent the same mutant. It would increase the readability by using one label across the manuscript. Similarly, both gpr126stl47 and gpr126stl47/stl47 were used to denote the homozygous mutant in the figures (the same for st49). Please be consistent.\n12. Page 6, line 103. Please include citations for these 2 published mutant alleles.", "reviewer_3": "This is an interesting study that aims to clarify a long-standing controversy in the literature concerning the function of Adgrg6/Gpr126 in the zebrafish heart. Adgrg6/Gpr126 is a clinically important adhesion class GPCR (aGPCR) that is associated with a number of different disease states in humans, and so a better understanding of genetic variants in the zebrafish model is important and likely to be of general interest.\nSeveral studies in mice have revealed a function for Gpr126 in cardiac trabeculation. However, the murine cardiac defects did not appear to be recapitulated in the various zebrafish mutant alleles available, raising the possibility that a function for Gpr126 in the heart is not conserved. Nevertheless, many of the available zebrafish alleles are hypomorphic and/or predict expression of truncated protein variants, which has been offered as an explanation for the lack of cardiac defects. Other alleles have not yet been examined for a cardiac phenotype. A morpholino study in zebrafish (Patra et al., ref. 13) supported a role for the N-terminal fragment (NTF) of Gpr126 in cardiac trabeculation defects; as discussed in the current manuscript, genetic compensation could provide an explanation for the discrepancy between the morphant and mutant phenotypes. The Engel group have also already published a study linking gpr126 regulation to cardiac trabeculation (Musa et al., 2019), which is not cited in the current manuscript. The role for Gpr126 in the zebrafish heart therefore remains unclear.\nThe current manuscript re-visits this controversy through analysis of the zebrafish heart phenotype in two of the most severe truncating alleles available, using maternal-zygotic (MZ) as well as zygotic-only (Z-only) mutants to examine the phenotype. The authors clearly demonstrate that the MZ stl47 and MZ and Z-only st49 homozygous mutant zebrafish have defects in trabeculation of the endocardium, each showing variable penetrance and expressivity, and a range of different phenotypes (multilayering, hypotrabeculation or absence of trabeculation). Other data are promising but more preliminary, indicating possible links to blood flow, Erbb2 and Notch signalling.\nThe manuscript contradicts some previous findings (some of which are from the same group, or in papers with authors in common with the current manuscript). As the authors state on line 376, the work fails to corroborate their previous hypothesis that the NTFdelGPS is sufficient for cardiac trabeculation (line 376), instead now suggesting that the CTF plays a critical role. They also suggest that their data contradict the recent published interpretation that the heart phenotype in mouse results from a placental defect (Torregrosa-Carrion et al., 2021, ref. 15). These contradictions are not easy to follow in the manuscript. There are a number of concerns with presentation and interpretation of the data, and of citation and discussion of the previous literature, that need to be addressed.\nMajor comments\n1. One clear but surprising new finding is that the st49 allele, which the authors demonstrate is likely to express the NTFdelGPS fragment, is the only gpr126 allele described so far for which there is a cardiac trabeculation phenotype in zygotic (Z-only) mutants. This is contrary to the authors’ previous published analysis of gpr126 st49 mutants (Patra et al., 2013: ‘gpr126st49 mutants exhibited no pericardial edema or other cardiac abnormalities’) and their demonstration that the NTFdelGPS alone was sufficient to rescue a trabeculation defect in gpr126 morphants (Patra et al., 2013) (ref. 13). However, the interesting result of the Z-only phenotype in st49 is hidden in the manuscript—it is only presented as supplementary data (Fig. S2), and the title of the figure (Zygotic gpr126stl47 hearts do not exhibit trabeculation defects) completely overlooks the result. This figure should be moved to form a top row in the main Fig. 2, allowing a direct comparison of the Z-only and MZ phenotypes for each allele. The abstract should also mention this result.\nThe phenotypes shown are clearly incompletely penetrant and variably expressive for both the MZ and Z-only mutants for st49, and for MZ stl47 mutants, but this is not explicitly stated anywhere. Moreover, the distribution of cardiac phenotypes in the Z-only st49 mutants appears identical to that in MZ st49 mutants; given the low numbers of fish sampled (n=15 Z-only (Fig. S2) and n=30 MZ (Fig. 2)), there is no evidence for any significant difference in strength or nature of the phenotype between the Z-only and MZ fish. This finding should be presented more clearly, as suggested above. If the authors wish to document a difference in the distribution of cardiac phenotypes between Z-only st49 and MZ st49 fish, they will need to examine more animals and perform an appropriate statistical test on the data to confirm this.\n2. One interpretation of the data is that the NTFdelGPS has a dominant effect to give rise to the unique cardiac phenotype (multilayering) seen in some embryos for the st49 allele. Do the heterozygotes for this allele show any cardiac phenotype?\n3. The sections linking Gpr126 to blood flow, and the Erbb2 and Notch signalling pathways, are less well worked up and need additional experimental confirmation.\na. Figure 4. The description of the Erbb2 inhibitor and blood flow experiments shown in this figure is confusing. The genetic backgrounds tested (over-expressed gpr126, or the MZ st49 mutant, Fig. 4) both already have blocked trabeculation, so how can the authors conclude that the Erbb2 inhibitor blocks trabeculation in these backgrounds? Instead, both the inhibitor and lack of blood flow appear to rescue the multilayering phenotype in each background. This needs further discussion and explanation. Quantification of the phenotypes should also be provided in the figure legend.\nb. The authors document an increase in Notch signalling in the MZ st49 mutants in Fig. 7, and show that the CTF can rescue the multilayering phenotype in this allele in Fig. 3. Importantly, can over-expression of CTF also restore normal levels of Notch signalling in MZ st49? This is an essential control for the interpretation of the mechanism here.\n4. The authors also speculate on lines 421–422 that rescue by the CTF is likely to be through canonical G-protein-coupled signalling. This would be simple to test by raising cAMP with forskolin, a treatment that should rescue the cardiac phenotype in the st49 allele. This experiment is essential to support the argument.\n5. Over-expression of the NTF in a wild-type background results in multilayering (Fig. S4). Does over-expression of the NTF in MZ stl47 mutants result in an st49-like multilayering phenotype? This would help to clarify the role of the NTF.\n6. This also needs some more discussion in the manuscript: why does the maternal transcript fail to compensate for the loss of zygotic function for the st49 allele specifically? And why does the stl47 allele (which also lacks the CTF) not show any Z-only phenotype? Can the MZ stl47 phenotype also be rescued with forskolin?\n7. The imaging in Figure 6 is not entirely clear. A counterstain for cell membranes is necessary to show the position of cells, especially as the epithelium is a curved surface. In addition, the chosen ‘representative’ example for MZ st49 does not have the multilayering phenotype. It is essential to show what happens to cadherin expression in those MZ st49 mutant hearts that have a multilayering phenotype.\nMinor comments\n8. The order of the alleles presented in Figure 7 (in both the image panels and the graph) should be swapped round to match that in the rest of the manuscript, i.e. stl47 first, and then st49.\n9. The authors have undertaken a qPCR analysis of FACS-sorted cardiomyocytes (Fig. 3A) to confirm that Gpr126 mRNA is enriched in the endocardium, which is helpful. However, the Introduction should state that the gene is known to be expressed in the zebrafish heart (since a lack of expression in this organ in the zebrafish could be a simple explanation for any species-specific differences in function), citing relevant studies. The authors could refer to Geng et al. (2013) for expression at 24–48 hpf, or their own study for expression at 48 hpf (Patra et al., 2013 – although confusingly, note that the overall expression pattern shown in this paper does not match that shown in Monk et al., 2009 or Geng et al., 2013).\n10. In all relevant main and supplementary figures (including both images and diagrams), the authors should replace red with magenta, to make the images accessible to those with dichromatic colour vision. For the triple label in Figure 1, choose a suitable colour combination (e.g. magenta, green, white)\n11. There are some omissions or inconsistencies in discussion of the previous literature, including papers authored by some of the authors of the current manuscript, which make it difficult to follow the arguments.\na. D’Amato et al. (2016) (ref. 46) is cited in the context of myocardial patterning, but the work in this paper to elucidate the relationship between Gpr126 and Notch signalling in the mouse and zebrafish is not mentioned or discussed anywhere. D’Amato et al. (2016) used several lines of evidence to demonstrate that cardiac expression of Gpr126 is a downstream target of Notch signalling, including documenting a loss of gpr126 expression in the hearts of zebrafish antimorphic mib mutants (loss-of-function for Notch signalling). How do these results fit with the model that the authors propose for the zebrafish, with the enhanced myocardial Notch signalling in MZ Gpr126-st49 mutants?\nb. The authors do not cite or discuss previous work from their own group (Musa et al., 2019), in which they specifically linked miR-27a/b regulation of gpr126 expression to trabeculation in the zebrafish heart, and compared phenotypes to that of the gpr126 morphant. This paper appears to provide important background information that supports the current study. Why is this study not cited?\n12. A schematic diagram summarising the authors’ current model and thinking for how the NTF is working with the CTF, linking to Erbb2 and Notch signalling and cell polarisation, would be very helpful.\n13. Introduction, Line 56 – do the authors mean to say ‘is expressed in’ rather than ‘is essential for’ here? It seems odd to say that the gene is essential for development of the heart but then to go on to say this is controversial and needs testing.\n14. Much of the information presented in Supplementary Fig. S1 (panels A–C, i.e. both the sequence and the ear phenotype for both alleles) has been published by other groups elsewhere. It is good to see that the authors have been able to confirm these findings in their own hands, but it must be stated that they are confirming previous published findings, citing Petersen et al. (ref 65) for the analysis of stl47, and Monk et al. (ref 35) for st49, rather than presenting these as new data.\n15. Please note that the amino acid at the position of the indel in the stl47 allele is not a glutamine (Q), as previously published by Petersen et al. (2015) – it is an asparagine (N) (as the supplementary figure in the current manuscript clearly shows) (see Table 1 in Baxendale et al., genesis, 2021). This should be corrected in the text on line 104.", "reviewer_author_response": "Point-by-point response to\nreviewer comments\nWe sincerely thank the reviewers for thoroughly examining our manuscript and providing\nhelpful comments/suggestions to improve our manuscript. In the revised manuscript we have\naddressed all of the raised issues including adding a large amount of new experimental in vitro\nand in vivo data which further substantiate our conclusions and led to a significantly improved\nmanuscript. Please find below a detailed response to the individual questions.\nReviewer #1:\nThe study by Srivastava et al. is very interesting and points towards exciting functions of the\nAdhesion GPCR ADGRG6/Gpr126, which also can give some indication on how Adhesion\nGPCRs in general function. However, many of the experiments are very preliminary and the\ninterpretation sometimes is a little doubtful. I have several comments, especially regarding the\nuse of the strains and constructs, which in my opinion need careful re-evaluation. In my\nopinion, the study in its current form is not complete and requires substantial more work to\ndemonstrate many of the claims made, such as the CTF function and the regulation of Notch\nactivity.\nWe thank the reviewer for the overall positive view on the importance of our study and the\ndetailed comments and suggestions that allowed us to address the raised concerns resulting\nin a significantly improved manuscript.\nMajor concerns:\n1. st49 mutant: I appreciate that the mutant has been characterized previously. However, both,\nst49 and st47 disrupt essential domains of the receptor, the CUB and the GAIN, respectively,\nwhich carries the risk of (partially) misfolded proteins. This probably does not matter for st47\nas there is not much protein left and I agree that it can be considered a complete loss of\nfunction. However, it does matter for st49. The authors try to address this issue by expressing\nthe construct in cells. However, as Fig. 1B shows, the construct accumulates in the cytoplasm.\nThe authors state that this is due to the missing 7TM. I would assume that a missing 7TM leads\nto a liberated protein, which gets secreted rather than retained, especially as the signal peptide\nis still present. This is at least what we see for other GPCRs. The authors also postulate that\nthis occurs in the st49 strain (p. 15, l. 326). I am highly worried what really happens to the\nprotein. It might lead to dominant-negative effects in the fish and thus, to phenotypes. I would\n1\nlike to see the experiments (or at least part of them) conducted in a fish strain carrying a “clean”\nversion of the N terminus – liberated or even tethered to the membrane. I realize that this some\neffort, but it would generate much more solid data. With CRISPR-Cas this should be doable.\nWe agree with the reviewer that it is important to ensure that the gpr126st49 mutant expresses\nan NTFGPS that is not misfolded but is functional and secreted. We furthermore thank the\nreviewer for indicating that we have not properly presented our data resulting in the wrong\nassumption that the NTFGPS is accumulated in the cytoplasm. The corresponding image\nrepresents a minority of cells and was used to prove that the mutant forms are expressed. In\nour opinion the best proof that NTFGPS represents a functional NTF is to rescue gpr126st49\nmutant phenotypes by expressing endocardial CTF. To address this issue, we have changed\nthe text and added a large number of experiments:\n1) We provide overview images from cell cultures post transfection to show that NTFGPS is not\naccumulated in the cytoplasm (Fig. S2a) and clarify on page 7, line 151: “In contrast, the mutant\nGpr126 forms failed to localize to the membrane (Fig. 1b) and in a minority of cells cytoplasmic\nexpression was detected (Fig. S2a, b). Notably, previously we have shown that ectopic\nexpression of NTFGPS in Hela as well as H9c2 cells results in a stable, secreted protein.”\n2) To validate that NTFGPS is secreted, we performed western blot analyses of cell culture\nmedium in the absence and presence of brefeldin A (Fig. S2c). The results are stated on page\n7, line 157: “To confirm that a stable, secreted NTFGPS is produced by gpr126st49 cDNA, the\nculture medium from ARPE-19 cells transfected with WT or st49 constructs were analyzed by\nwestern blot (Fig. S2c). These data reveal that the culture medium contains NTF/NTFGPS.\nMoreover, treating the cells with brefeldin A, which blocks the trans-Golgi secretory pathway,\nsignificantly decreased the amount of NTF/NTFGPS in the medium. These data indicate that\ngpr126st49 zebrafish mutants express and secrete NTFGPS, which previously has been\nreported to be sufficient for heart development.”\n3) We state on page 7, line 155: “Thus, the failure to localize to the membrane and the reduced\nnumber of HA-positive cells could be explained with the loss of the 7TM domain in both\ntruncated forms.”\n4) Importantly, if the gpr126st49 mutation would result in the expression of a “(partially)\nmisfolded protein” possibly exhibiting dominant effects, it should not be possible to rescue this\nmutant phenotype by overexpression of the CTF. Therefore, we had provided in the original\nversion data regarding these rescue experiments. To further strengthen these data, we have\nnow added additional evidence for gpr126 being expressed in the endocardium in zebrafish\nheart (RNAscope, Fig. S5, page 10, line 229) and that overexpressed CTF localizes in the\ntransgenic animals in endothelial and endocardial cells at the membrane (Fig. 3c, end of page\n10). Furthermore, we provide new data showing that overexpression of CTF in the MZ\ngpr126st49 mutants, which restores normal trabeculation, also restores myocardial Notch\nactivity (Fig. 6e, f, page 16, line 362).\n5) Furthermore, if there were dominant negative effects of the st49-encoded protein, we would\nsee them in the heterozygotes. We did not observe any multilayering phenotypes in the\nheterozygous gpr126st49 larvae consistent to the absence of myelin phenotypes in\nheterozygous gpr126st49 in Petersen et al. 2015 (PMID: 25695270) (page 10, line 219).\nCollectively, these data show that NTFGPS represents a functional NTF.\nWe would also like to clarify that the fact that gpr126st49 mutants, lacking CTF, show enhanced\nNotch activity but MZ gpr126stl47 mutants, lacking NTF and CTF, does not, is not due to a\n2\ndominant effect of NTFGPS but due to the fact that NTF is required for compact wall integrity\nbefore trabeculation is initiated. To clarify this point we added on page 15, line 345:” It has\nbeen demonstrated that Notch signaling is activated during trabeculation upon cardiomyocyte\ndelamination in a subset of compact wall cardiomyocytes to inhibit their delamination38-40. As\nMZ gpr126stl47 mutants are characterized by hypotrabeculation, no change in myocardial Notch\nactivity is expected.”\nComment on “With CRISPR-Cas this should be doable”: We would like to note that the\nsuggested experiments would require precise gene editing which requires that cells utilize\nhomologous DNA repair (HDR). While HDR efficiently works in several model organisms\nincluding mouse, non-homologous end joining (NHEJ) is the preferred mechanism of repair in\nzebrafish. This is the reason why it is extremely difficult to generate zebrafish with precise\nmutations (PMID: 27795562).\n2. Fig. 1B: It looks like there is less membrane localization of Gpr126 st49, but increased\ncytoplasmic localization. The authors state that this is due to the missing 7TM. As mention in\ncomment 1, I would assume that the missing 7TM leads to a liberated protein that gets secreted\nrather than retained as the signal peptide is still present. I would assume other reasons for the\ncytoplasmic expression are more likely such as a partially misfolded protein.\nThis issue has been addressed in detail above (see response to Point 1).\n3. p. 9: Why does the st47 mutant not show a zygotic phenotype, but the st49 does? Maybe I\nam misunderstanding this, but if this is the case, it is a really interesting finding. It might either\npoint towards a separate function of the NTF that is only visible when the CTF is absent, or it\ncan be a dominant-negative effect of the st49 (see comment 1). The authors need to\ndiscriminate between the different options and explain this matter.\nWe agree with the reviewer that this is an interesting finding.\nOption 1: “dominant-negative effect”\nThis has been excluded as explained under Point 1.\nOption 2: “separate function of the NTF”\nOur data show that the NTF is required earlier in development than CTF. NTF is required early\nin development for compact wall formation by regulating N-cadherin localization, a pre-\nrequisite for proper trabeculation. In contrast, the CTF is required later for the subsequent\ncontrol of Notch activity and attainment of trabecular identity. As maternal mRNA is only\ntransiently present until early embryonic stages, these findings provide a likely explanation for\nwhy the Z gpr126stl47 mutant shows no phenotype, but the Z gpr126sl49 does.\nNotably, our data are in agreement with a previous report in the peripheral nervous\nsystem, where it has been shown that MZ gpr126stl47 mutants exhibited a more severe\nphenotype than Z gpr126stl47 mutants. In contrast, the severity of Z and MZ gpr126sl49 mutant\nphenotype were similar (PMID: 25695270).\nTo address this issue, we have modified the Discussion section accordingly (page 18, line\n404).\n4. p. 10: How does the CTF in Tg(fli1a:gpr126 CTF-p2a-tdTomato) look like? Does it have a\nsignal peptide? Also, from the description in the Methods section, it seems that it does not\nhave a tag or anything on its N-terminal side. A pure CTF might be rather instable and not be\n3\nexpressed too well at the membrane. This has been previously described in the context of\nGpr126 (Liebscher et al. 2015). The authors do show expression in Fig. S3B, however,\ndemonstrating proper expression of this construct at the cell membrane is absolutely\nnecessary for the interpretation of the results. Thus, a tissue culture experiment would be good.\nWe thank the reviewer for pointing out that we did not describe the construct in enough detail.\nPlease note, that the cytoplasmic red staining in Fig. S3d represents tdTomato which is\nseparately expressed from CTF and thus cannot localize to the membrane. The utilized\nconstruct to generate the Tg(fli1a:gpr126CTF-p2a-tdTomato) line is similar to the construct\nused by Liebscher et al. 2015 and encodes the CTF with a signal peptide as well as an HA-\ntag at the N-terminus and FLAG-tag at the C terminus. This CTF construct was shown to be\nfunctional and displayed increased basal activities in cAMP assays (PMID: 30849857). In\naddition, P2Y N-terminus peptide was fused to the Gpr126 CTF to facilitate the plasma\n12\nmembrane localization of the receptor.\nTo address this issue, we have modified the Methods section accordingly (page 25, line\n587) and added a scheme of the construct to Fig. 3. In addition, we provide anti-FLAG staining\nof 48 hpf Tg(fli1a:gpr126CTF-p2a-tdTomato) embryos that show membrane localization of the\nCTF in endothelial and endocardial cells in vivo (Fig. 3c). Notably, this construct was sufficient\nto rescue trabeculation- (Fig. 3d,e) and Notch-related (Fig. 6e,f) phenotypes in gpr126sl49\nmutants.\n5. p 10: The authors claim a CTF and an NTF function of Gpr126 in heart development. I am\na bit worried about the postulated CTF function in this context. I agree that the CTF is required,\nbut I am not sure that it is a sole CTF function. It has been shown for quite some aGPCR that\nNTF and CTF can reconstitute to form a (function) receptor (e.g. Silva et al. 2009, Prömel et\nal. 2012). As in st49, the N terminus is still present, providing the CTF in this context could lead\nto such a reconstitution.\nTo verify the results and to prove the interpretation the authors provide, the following control\nexperiments would be necessary:\na) A rescue with a full-length Gpr126 construct specifically in this context.\nb) A rescue of the CTF in the st47 strain.\nWe thank the reviewer for pointing out the issue and agree that our data do not prove that CTF\nhas an NTF-independent function but that it is required for heart development. Accordingly, we\nhave removed all statements that indicate that CTF has an NTF-independent function.\nIn addition, we tested whether the CTF can rescue phenotypes in MZ gpr126stl47 mutants.\nWe overexpressed CTF in the endothelium of MZ gpr126stl47 embryos and observed that CTF\noverexpression failed to rescue the adherens junction defects in MZ gpr126stl47 mutant (Fig.\nS9b). Moreover, overexpression of CTF in MZ gpr126stl47 mutants also failed to alter Notch\nactivity (Fig. S10c, d). These data substantiate the conclusion that CTF does not have an NTF-\nindependent effect.\n6. Independently of whether the postulated CTF function is based solely on the CTF or on a\ncombination of NTF/CTF, it remains elusive of which nature this function is. Obviously, the\nactivation of a G protein cascade is the most likely CTF function. However, the authors show\nno such thing. Gpr126 signaling has been characterized quite well in zebrafish, such as\nreceptor activation by a tethered agonist-derived peptide (Liebscher et al. 2014). It would be\ninteresting to see whether a specific activation of the aGPCR by such a peptide rescues the\ntrabeculation phenotype described on p. 8/9. The results of such an experiment will prove the\ninvolvement of the receptor CTF and shed light on the mechanism behind it.\ncascade”. Previously, it has been shown that ear and PNS phenotypes can be rescued in the\nabsence of the CTF by forskolin or IBMX treatment, which are both ubiquitously inducing cAMP\nsignaling. As stated on page 11, line 258, “Therefore, we tested whether forskolin/IBMX\ntreatment can also rescue the heart phenotype in gpr126st49 mutants. We confirmed that these\ntreatments rescued the puffy ear phenotype (Fig. S7a). Both treatments blocked trabeculation\nin WT hearts as well as multilayering in gpr126st49 hearts (Fig. S7b). As forskolin and IBMX\ninhibit also in WT hearts trabeculation, it remains unclear whether Gpr126 acts through cAMP\nactivation to promote trabeculation.”\nIt has to be noted that trabeculation is a dynamic process and general elevation of cAMP\nsignaling have a deleterious effect on trabeculation. We have also added new text in the\ndiscussion on page 19 line 443; “It is likely that CTF signals in the endocardium through\ncanonical G protein-coupled signaling, as observed in myelination during peripheral nervous\nsystem development. However, as the treatment with cAMP elevating drugs affected also\ntrabeculation in WT hearts, it could not be determined whether CTF acts through canonical G-\nprotein signaling. The effect on WT hearts is in agreement with recent reports that adrenergic\nsignaling strengthens cardiomyocyte cohesion, altered cAMP levels are associated with\nimpaired trabeculation, and that compartmentalized cAMP signaling is essential for proper\nheart development.”\nRegarding the use of a tethered agonist-derived peptide: This approach cannot be executed\nas such peptides only function in the presence of CTF, yet, neither gpr126stl47 or gpr126st49\nmutants express the CTF.\n7. p. 13/14: How is the NTF in Tg(myI7:gpr126 NTF-p2a-tdTomato) designed? Is it the same\nas the construct in st49 or is it truly the NTF (stopping at the cleavage site within the GPS)?\nThe NTF encoded in Tg(myI7:gpr126 NTF-p2a-tdTomato) is NTFGPS; the same as in the\ngpr126st49 mutants. To address this issue, we have modified the Methods section providing this\ninformation (page 25, line 587) and ensured that we defined NTFGPS in the beginning of the\nmanuscript (Abstract as well as Introduction) and consistently used NTFGPS.\n8. p. 14/15: The modulation of Notch signaling by Gpr126 is interesting. However, this data is\nvery preliminary and highly speculative. At this point, it is definitely not solid enough to claim\nthat Gpr126 regulates Notch activity.\nThe authors find that abrogating almost the entire receptor does not have a function on Notch\nactivity. St49 leads to enhanced Notch activity, but overexpression of the NTF does not. From\nthis, the authors conclude that the CTF inhibits Notch activity. However, several other\nscenarios are equally possible: Firstly, it can mean that the st49 construct conveys a dominant-\nnegative effect (here, it is essential to know whether Tg(myI7:gpr126 NTF-p2a-tdTomato)\ncarries the same construct as st49 – see my comment 7 above). Secondly, a purely\ncompensatory mechanism is conceivable. The authors need to put some more effort into\nobtaining more and solid data on whether and how Gpr126 has an effect on the Notch\npathway. The following controls might help:\na) A CTF expression in the st47 strain.\nb) An NTF expression in the st47 strain.\nc) A CTF expression in the st49 strain.\nWe agree with the reviewer that we do not show that Gpr126 or CTF directly regulate\nmyocardial Notch activity but that Gpr126 is required.\nall the suggested experiments which are described on page 16, line 360: “Notably, NTFGPS\noverexpression in MZ gpr126stl47 mutants [reviewer suggestion b], which leads to myocardial\nmultilayering (Fig. 4b,c), also results in a substantial increase of Notch activity at 120 hpf (Fig.\n6c, d). Overexpression of CTF in the MZ gpr126st49 [reviewer suggestion c] mutants, which\nrestores normal trabeculation, also restores the myocardial Notch activity (Fig. 6e, f).”\nFurthermore, we tested overexpression of NTF and CTF in the WT background (page 16,\nline 364): “Overexpression of NTFGPS and CTF in the WT background did not lead to any\nsubstantial changes in the myocardial Notch activity (Fig. S10a, b). Additionally,\noverexpression of CTF in MZ gpr126stl47 mutants also failed to alter Notch activity (Fig. S10c,\nd).” [reviewer suggestion a].\nWe conclude: “This suggests that CTF signaling is required to inhibit myocardial Notch\nactivity to allow cardiomyocytes to attain trabecular identity.” (page 16, line 366).\nIn addition, we provide an explanation why no alteration of Notch signaling was observed\nin the MZ gpr126stl47 mutants (page 20, line 460):” It has been demonstrated that Notch signaling\nis activated during trabeculation upon cardiomyocyte delamination in a subset of compact wall\ncardiomyocytes to inhibit their delamination. As MZ gpr126stl47 mutants are characterized by\nhypotrabeculation, no change in myocardial Notch activity is expected.”\nFinally, we removed in the text all notions that Gpr126 or CTF regulate myocardial Notch\nbut that the Gpr126/CTF is required for modulating myocardial Notch activity or that impaired\nsignaling of CTF causes alterations in myocardial Notch activity.\nRegarding the possible interpretations:\na) The possibility that NTFGPS conveys a dominant effect has been excluded (see\nresponse to Point 1).\nb) Compensatory mechanism: It has been shown that Notch signaling in cardiomyocytes\nhas an inhibitory effect on trabeculae formation. Thus, a compensatory mechanism\ndue to lack of trabeculation would not be the activation of Notch, which we observed\nhere, but its inhibition.\n9. p. 14/15: A highly puzzling issue is that if the CTF inhibits Notch activity as the authors\npostulate, one would expect that st47, in which the CTF is also missing, likewise leads to an\nincreased Notch activity. This is, however, not the case and needs further clarification. I am\nwondering what the authors´ explanation for this Also, additional essential questions need to\nbe addressed such as: Does the expression of the NTF in the st49 strain alter the expression\nof the Notch receptor/ligand? Altered expression levels of Notch pathway components could\nhelp explain changed Notch activity.\nPlease see our response to Point 1 and 8. In addition, please note that our data indicating that\nNTF and CTF are required at different timepoints during development is in agreement with\nobservations during myelination where NTF is required for radial sorting which is a prerequisite\nfor axon wrapping and myelin sheath formation which depends on CTF signaling (PMID:\n25695270) (page 18, line 413).\nWe also reasoned that due to the various methods of Notch signaling regulation, we\nexamined the most time-sensitive reagent available in zebrafish (Tg(TP1:Venus-PEST)). We\nwould also like to note that Notch signaling occurs not only in cardiomyocytes but also in\nendocardial cells explained now on page 20, line 451. Moreover, Notch signaling is very strong\nand constantly active in AVC and OFT (as shown in Fig. 6), which makes it very difficult to do\n6\nin-situ hybridization for Notch pathway components. This is the reason that in previous\npublications with myocardial Notch, the only readout has been the Venus-PEST (PMID:\n27926871, 33208950, 34363825). Additionally, myocardial Notch signaling is poorly\nunderstood and the identification of compact wall cardiomyocyte-specific Notch targets is\nbeyond the scope of this manuscript.\n10. p. 15: Again: How is the NTF in Tg(myI7:gpr126 NTF-p2a-tdTomato) designed? If it does\nnot mimic st49, the data obtained with the construct cannot be compared to the ones on the\nst49 (Fig. 7A, B). If it does mimic st49, a potential dominant-negative effect of the construct\ncannot be excluded. Thus, in order to gain the most conclusive set of data, both constructs\nmight need testing.\nThe NTF encoded in Tg(myI7:gpr126 NTF-p2a-tdTomato) is NTFGPS, same as the protein\nencoded in gpr126st49 mutants and based on our rescue experiments, we conclude that it\ndoes not have a dominant negative effect (see response to Point 1 and Point 7).\n11. p. 14/15: Simply showing an overexpression of the NTF in a wild-type background does\nnot address the question which parts of the aGPCR are required for a potential function in the\ncontrol of Notch activity. Further, it might help to also do the reciprocal experiment and e.g.\nexpress the CTF to see if a more pronounced downregulation effect occurs.\nPlease see our response to Point 8.\nMinor comments:\n1. Fig 1C: Information on statistics is missing: How many samples?\nWe have added the sample number in the figure legends.\n2. There are some typos in the manuscript, e.g. p. 7 l. 147 “amino acid”. Please check spelling\nand grammar carefully.\nWe thank the reviewer for pointing out the typos and errors in the manuscript. We have\ncarefully reviewed the manuscript and corrected spelling and grammar.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, Srivastava et al. studied the functions of adhesion G protein-coupled\nreceptor 126 (Gpr126) in zebrafish heart trabeculation by analyzing two truncation mutants\nstl47 and st49. They found that maternal zygotic stl47 mutant exhibited hypotrabeculation\nphenotype with randomly distributed N-cadherin at membranes of the compact muscle layer.\nThe st49 mutant lacking the C-terminal GPS motif exhibited a multilayered ventricular wall with\nnormal N-cadherin distribution and elevated Notch signaling. They concluded that the N-\nterminal fragment (NTF) of Gpr126 maintains cell-cell adhesion and compact wall integrity in\nthe myocardium, while the C-terminal fragment (CTF) is essential to establish the trabecular\nidentity of cardiomyocytes. The finding here would provide novel insights into heart\ndevelopment and clarify the function of Gpr26 in myocardium trabeculation. However, as noted\nbelow, the conclusions might be a bit premature and require further analysis or clarification.\nThe manuscript is well written and easy to follow.\nWe thank the reviewer for his/her constructive comments about our work and appreciate the\nsuggestions.\n7\n1. The authors used qPCR to show the relative expressions of gpr126 in CMs and\nendocardium, which is less optimal. Demonstrating the protein localization, either by an\nantibody staining or by tagging the gene, would be useful for mechanistic interpretations (e.g.,\na strategy used in a co-author’s previous paper: https://doi.org/10.1242/dev.171207 ). It is\nunderstandable that this request is likely beyond the scope of a revision. However, in situ\nhybridization of gpr126 showing the expression locations during trabeculation would be helpful.\nAfter testing all commercially available anti-Gpr126 antibodies, we could find no reliable\nworking antibody in mice, rat, or zebrafish. This is a common problem which is being faced by\nall labs working on Gpr126. Although precise tagging of Gpr126 is an ideal method, due to the\npreference of zebrafish in using non-homologous end joining instead of homologous DNA\nrepair, we believe that the probability of successfully generating a knock in is very low and\nbeyond the scope of this manuscript.\nTo address this issue, we decided for direct visualization of gpr126 transcripts by\ncombining RNAscope in whole mount zebrafish embryos with phalloidin immunostaining at 48\nhpf (before the onset of trabeculation) and 96 hpf (after the onset of trabeculation). The\nobtained data shown in Fig. S5 confirm our qPCR data that gpr126 is expressed in the\nendocardium in zebrafish (see page 10, line 229).\n2. The authors only showed the myocardium in all figures. Since gpr126 is considered an\nendocardial-enriched gene in the heart, it seems necessary to assess any endocardial defects\nand endocardial cell-cardiomyocyte interactions in the mutants. Otherwise, the mechanism\ndescribed in the current version seems to lack a significant component.\nPreviously, we have shown that the NTF binds to the myocardium (PMID: 24082093).\nConsequently, there is a myocardial and an endocardial component.\nTo address this issue, we have provided data showing that there are no obvious defects\nin the endocardium or the cardiac jelly between the endocardium and the myocardium at 72\nand 96 hpf. We have added this information to the revised manuscript and state on page 9,\nline 197: “Notably, MZ gpr126stl47 and MZ gpr126st49 mutants in the background of\nTg(myl7:mKATE-CAAX); Tg(krt4:GFP)sqet33 which label the myocardium and the endocardium,\nrespectively, did not exhibit obvious defects in the endocardium or the cardiac jelly between\nthe endocardium and the myocardium at 72 and 96 hpf (Fig. S3).“\nPlease note, that the interaction between endocardium and myocardium is still poorly\nunderstood (see page 20, line 466) and while a deeper analysis of the endocardium is of great\ninterest to us, it is beyond the scope of this manuscript.\n3. Figure 1B. It is interesting that 36.67% of stl47 MZ mutant larvae and 26.67% of st49 MZ\nmutant larvae showed normal trabeculation. Could the authors speculate the reason? How do\nthey define “normal trabeculation”?\nIt is true that the mutants exhibit variable penetrance and expressivity. It is fairly common in\nz\nin zebrafish mutants that even progenies of the same mutant fish exhibit different penetrance\n(PMID: 36533556).\nThe criteria we have chosen for normal trabeculation was 3 or more trabeculae with 3 or\nmore cardiomyocytes for 96 hpf hearts and 5 or more trabeculae with 3 or more\ncardiomyocytes for 120 hpf hearts. This information has been added to the Methods section\n(see page 30, line 694).\n4. The authors showed that mosaic myocardial-specific NTF overexpression induced local\ncardiomyocyte multilayering and concluded that NTF has a function in maintaining cell-cell\n8\nadhesion in the compact layer cardiomyocytes. However, gpr126 is considered an endocardial\ngene (Figure 3A). It is confusing how an endocardium secreted protein mediated cell\nadhesions within the myocardium. At least the outer layer of the multilayered myocardium\nseems to have no contact with the endocardium. Defining the expression domain of gpr126\n(as noted above) seems necessary to clarify this. Also, an endocardial-specific overexpression\nstrategy should be considered.\nAs requested, we have defined the expression domain of gpr126 (see response to Point 1),\nwhich is exclusively expressed in the endocardium in zebrafish larvae.\nSeveral secreted factors from endocardial cells during cardiac development\nsimultaneously modulate cardiomyocyte proliferation and maturation, which are key cellular\nprocesses critical for ventricular compaction. How these endocardial factors affect the\nmyocardium is poorly understood (see page 20, line 470). Please note, that it has also recently\nbeen reported in zebrafish that the endocardium has direct contact with the myocardium\n(PMID: 35225788). In addition, we have previously shown (PMID: 24082093) that the NTFGPS\nis secreted from the endocardium, binds to the myocardium, and exerts its function.\nFurthermore, we would like to point out that the compact layer in the zebrafish heart ventricle\nis single layered, at least during the early stages of development. The NTF is acting at a stage\nwhen there is a single layered myocardial wall with no trabeculae formed yet. Regarding the\nmultilayering phenotype in gpr126st49 mutants, the NTF acts at the single myocardial layer\nstage to maintain the cell-cell adhesion between the cardiomyocytes. Yet, at the onset of\ntrabeculation, lack of CTF results in increased Notch activity resulting in decreased\ndelamination and multilayering.\n5. Figure 7. The authors concluded that CTF signaling is required to inhibit myocardial Notch\nactivity to allow cardiomyocytes to attain trabecular identity. However, the MZ stl47 mutant also\nlacks the CTF but showed normal myocardial Notch activity. Please explain.\nWe would like to clarify that the fact that gpr126st49 mutants, lacking CTF, show enhanced\nNotch activity but MZ gpr126stl47 mutants, lacking NTF and CTF, does not, is not due to a\ndominant effect of NTFGPS but due to the fact that NTF is required for compact wall integrity\nbefore trabeculation is initiated. To clarify this point we added on page 15, line 345:” It has\nbeen demonstrated that Notch signaling is activated during trabeculation upon cardiomyocyte\ndelamination in a subset of compact wall cardiomyocytes to inhibit their delamination. As MZ\ngpr126stl47 mutants are characterized by hypotrabeculation, no change in myocardial Notch\nactivity is expected.” We have also added an explanation in the Discussion (page 20, line 460):\n“The MZ gpr126stl47 mutants, however, do not form a proper compact wall due to the absence\nof NTF and hence do not reach the developmental stage where myocardial Notch activity could\nbe enhanced.”\nPlease note that our data indicating that NTF and CTF are required at different stages\nduring development are in agreement with observations during myelination where NTF is\nrequired for radial sorting which is a prerequisite for axon wrapping and myelin sheath\nformation which depends on CTF signaling, and we have also added this in the Discussion.\n(page 18, line 413)\n6. Figure S5. It is labeled as fli1a:gpr126 NTF-p2a-tdTomato in the figure but myl7:gpr126\nNTF-p2a-tdTomato in the legend and main text. Please clarify. I assume it is myl7. The\nmyocardium looks quite normal compared to the mosaic myocardial-specific NTF\noverexpression phenotype in Figure S4 (myl7:gpr126 NTF-p2a-tdTomato). Please clarify what\nmakes the difference.\nfigure accordingly (Fig S10a).\nThe observed differences are most likely due to the differences in expression: a) different\ncopy number. b) uniform (transgenic) vs. mosaic (plasmid injection) NTF overexpression\n(results in adhesion heterogeneity between cardiomyocytes).\nAs the mosaic expression was performed in WT, these data are difficult to interpret. In\naddition, plasmid injections in single cell-stage embryos are difficult to control. Therefore, we\nhave decided to remove these data and instead added data obtained by stable overexpression\nof NTFGPS in the myocardium of MZ gpr126stl47 mutants. This resulted in multilayering (Fig.\nS4a,b)(page 10, line 215) and enhanced myocardial Notch activity (Fig S6c,d)(page 16, line\n360) as observed in gpr126st49 mutants. This validates the role of NTFGPS in maintaining\ncompact wall architecture.\n7. Can the authors explain or speculate why zygotic stl47 mutant but not st49 mutant exhibited\nmaternal effect?\nOur data show that the NTF is required earlier in development than CTF. NTF is required early\nin development for compact wall formation by regulating N-cadherin localization, a pre-\nrequisite for proper trabeculation. In contrast, the CTF is required later for the subsequent\ncontrol of Notch activity and attainment of trabecular identity. As maternal mRNA is only\ntransiently present until early embryonic stages, these findings provide a likely explanation for\nwhy the Z gpr126stl47 mutant exhibits a maternal effect, but the Z gpr126st49 does not.\nNotably, our data are in agreement with a previous report in the peripheral nervous\nsystem, where it has been shown that MZ gpr126stl47 mutants exhibited a more severe\nphenotype than Z gpr126stl47 mutants. In contrast, the severity of Z and MZ gpr126st49 mutant\nphenotype were similar (PMID: 25695270).\nTo address this issue, we have modified the Discussion section accordingly (page 18, line\n404).\n8. Control group is missing in the experiment shown in Figure S5\nIt is unclear to the authors what the reviewer means. In this figure (now Fig. S10a), we wanted\nto determine whether Notch activity is affected in NTFGPS overexpressing hearts\n(Tg(myl7:gpr126NTF-p2a-tdTomato)) compared to control WT hearts (Tg(TP1:Venus-Pest).\nPlease note that we have added now additional experiments to further support that Gpr126\nis required for myocardial Notch activity (page 16, line 360): “Notably, NTFGPS overexpression\nin MZ gpr126stl47 mutants, which leads to myocardial multilayering (Fig. 4b,c), also results in a\nsubstantial increase of Notch activity at 120 hpf (Fig. 6c, d). Overexpression of CTF in the MZ\ngpr126st49 mutants, which restores normal trabeculation, also restores the myocardial Notch\nactivity (Fig. 6e, f).” Furthermore, we tested overexpression of NTF and CTF in the WT\nbackground (page 16, line 364): “Overexpression of NTFGPS and CTF in the WT background\ndid not lead to any substantial changes in the myocardial Notch activity (Fig. S10a, b).\nAdditionally, overexpression of CTF in MZ gpr126stl47 mutants also failed to alter Notch activity\n(Fig. S10c, d).” We conclude: “This suggests that CTF signaling is required to inhibit myocardial\nNotch activity to allow cardiomyocytes to attain trabecular identity.” (page 16, line 366).\n9. Figure 4A and B lack quantification results.\nAs requested, we have added quantifications (now Fig. S8b,c).\n10\n10. Page 8, lines 151-153. “The analysis of zygotic gpr126stl47/stl47 mutant hearts revealed\nno obvious gross morphological defects compared to WT siblings suggesting that Gpr126 is\nnot required for heart development in zebrafish (Figure S2).” This statement is not accurate.\nThe result only suggests that a zygotic mutant did not show a heart development defect.\nOne cannot conclude that Gpr126 is not required for heart development.\nTo address this issue, we changed the text on page 8, line 176 to “These data suggested\ninitially that Gpr126 is not required for heart development in zebrafish and that NTFGPS exhibits\na dominant effect. However, the lack of a phenotype in Z gpr126stl47 mutant hearts could also\nbe explained by compensation through maternal deposition of WT mRNA or protein into the\negg.“\n11a. st49 and NTFdeltaGPS are used to represent the same mutant. It would increase the\nreadability by using one label across the manuscript.\nPlease note that “st49” represents the mutated allele, while St49 represents the expression\nconstruct. Both of these encode for the protein NTFGPS. We have changed the manuscript to\nclarify this point. In addition, please note that the Tg(myl7:gpr126NTF-p2a-tdTomato) encodes\nalso NTFGPS but is not the same as the “st49” allele (endogenous gpr126 coding sequence\ncontaining the respective mutation).\n11b. Similarly, both gpr126stl47 and gpr126stl47/stl47 were used to denote the homozygous\nmutant in the figures (the same for st49). Please be consistent.\nThank you for indicating this issue. The manuscript has been changed accordingly.\n12. Page 6, line 103. Please include citations for these 2 published mutant alleles.\nWe have included the citations (page 6, line 121).\nReviewer #3 (Remarks to the Author):\nThis is an interesting study that aims to clarify a long-standing controversy in the literature\nconcerning the function of Adgrg6/Gpr126 in the zebrafish heart. Adgrg6/Gpr126 is a clinically\nimportant adhesion class GPCR (aGPCR) that is associated with a number of different disease\nstates in humans, and so a better understanding of genetic variants in the zebrafish model is\nimportant and likely to be of general interest.\nSeveral studies in mice have revealed a function for Gpr126 in cardiac trabeculation.\nHowever, the murine cardiac defects did not appear to be recapitulated in the various zebrafish\nmutant alleles available, raising the possibility that a function for Gpr126 in the heart is not\nconserved. Nevertheless, many of the available zebrafish alleles are hypomorphic and/or\npredict expression of truncated protein variants, which has been offered as an explanation for\nthe lack of cardiac defects. Other alleles have not yet been examined for a cardiac phenotype.\nA morpholino study in zebrafish (Patra et al., ref. 13) supported a role for the N-terminal\nfragment (NTF) of Gpr126 in cardiac trabeculation defects; as discussed in the current\nmanuscript, genetic compensation could provide an explanation for the discrepancy between\nthe morphant and mutant phenotypes. The Engel group have also already published a study\nlinking gpr126 regulation to cardiac trabeculation (Musa et al., 2019), which is not cited in the\ncurrent manuscript. The role for Gpr126 in the zebrafish heart therefore remains unclear.\nThe current manuscript re-visits this controversy through analysis of the zebrafish heart\nphenotype in two of the most severe truncating alleles available, using maternal-zygotic (MZ)\nas well as zygotic-only (Z-only) mutants to examine the phenotype. The authors clearly\n11\ndemonstrate that the MZ stl47 and MZ and Z-only st49 homozygous mutant zebrafish have\ndefects in trabeculation of the endocardium, each showing variable penetrance and\nexpressivity, and a range of different phenotypes (multilayering, hypotrabeculation or absence\nof trabeculation). Other data are promising but more preliminary, indicating possible links to\nblood flow, Erbb2 and Notch signalling.\nThe manuscript contradicts some previous findings (some of which are from the same\ngroup, or in papers with authors in common with the current manuscript). As the authors state\non line 376, the work fails to corroborate their previous hypothesis that the NTFdelGPS is\nsufficient for cardiac trabeculation (line 376), instead now suggesting that the CTF plays a\ncritical role. They also suggest that their data contradict the recent published interpretation that\nthe heart phenotype in mouse results from a placental defect (Torregrosa-Carrion et al., 2021,\nref. 15). These contradictions are not easy to follow in the manuscript. There are a number of\nconcerns with presentation and interpretation of the data, and of citation and discussion of the\nprevious literature,that need to be addressed.\nWe thank the reviewer for carefully reading the manuscript and the constructive comments,\nwhich we have all addressed by modifying the text as well as adding a significant amount of\nnew experimental data.\nMajor comments\n1. One clear but surprising new finding is that the st49 allele, which the authors demonstrate\nis likely to express the NTFdelGPS fragment, is the only gpr126 allele described so far for\nwhich there is a cardiac trabeculation phenotype in zygotic (Z-only) mutants. This is contrary\nto the authors’ previous published analysis of gpr126 st49 mutants (Patra et al., 2013:\n‘gpr126st49 mutants exhibited no pericardial edema or other cardiac abnormalities’) and their\ndemonstration that the NTFdelGPS alone was sufficient to rescue a trabeculation defect in\ngpr126 morphants (Patra et al., 2013) (ref. 13). However, the interesting result of the Z-only\nphenotype in st49 is hidden in the manuscript—it is only presented as supplementary data\n(Fig. S2), and the title of the figure (Zygotic gpr126stl47 hearts do not exhibit trabeculation\ndefects) completely overlooks the result. This figure should be moved to form a top row in the\nmain Fig. 2, allowing a direct comparison of the Z-only and MZ phenotypes for each allele. The\nabstract should also mention this result.\nAs requested, we have added the data for the zygotic mutants for both alleles in Fig. 2. In\naddition, we have added this information in the abstract (page 2, line 35) and discussed why\nonly Z gpr126st49, but not Z gpr126stl47, mutants exhibit a phenotype (page 18, line 404).\nAs discussed already in the previous manuscript (now page 18, line 417), in Patra et al.\n(PMID: 24082093) the mutants and morphants were only observed until 80 hpf, as morpholinos\nlose their effect at later stages. Here, we observed multilayering in the gpr126st49 mutants at\n96 hpf.\nThe phenotypes shown are clearly incompletely penetrant and variably expressive for both the\nMZ and Z-only mutants for st49, and for MZ stl47 mutants, but this is not explicitly stated\nanywhere. Moreover, the distribution of cardiac phenotypes in the Z-only st49 mutants appears\nidentical to that in MZ st49 mutants; given the low numbers of fish sampled (n=15 Z-only (Fig.\nS2) and n=30 MZ (Fig. 2)), there is no evidence for any significant difference in strength or\nnature of the phenotype between the Z-only and MZ fish. This finding should be presented\nmore clearly, as suggested above. If the authors wish to document a difference in the\ndistribution of cardiac phenotypes between Z-only st49 and MZ st49 fish, they will need to\nexamine more animals and perform an appropriate statistical test on the data to confirm this.\nvariable expressivity (page 9, line 210).\nIn regards to differences in strength or nature of the phenotype between Z and MZ\ngpr126st49, we do not expect any difference considering the late requirement of CTF (see\nDiscussion regarding maternal effect on page 18, line 404. Our current data is in agreement\nwith this conclusion (Fig. 2). This data is also in concordance with the study from Petersen et\nal (PMID: 25695270), where both Z and MZ gpr126st49 mutants exhibit similar phenotypic\nseverity.\n2. One interpretation of the data is that the NTFdelGPS has a dominant effect to give rise to\nthe unique cardiac phenotype (multilayering) seen in some embryos for the st49 allele. Do the\nheterozygotes for this allele show any cardiac phenotype?\nWe did not observe any multilayering phenotypes in the heterozygous gpr126st49 larvae\nconsistent to the absence of myelin phenotypes in heterozygous gpr126st49 in Petersen et al.\n2015 (PMID: 25695270) (page 10, line 219). This indicates that the observed phenotypes are\nnot due to the dominant effect of NTFGPS.\nTo further exclude the possibility that the gpr126st49 mutant phenotypes are due to a\ndominant effect of NTFGPS, we added a large number of additional experiments:\n1) We provide evidence that NTFGPS encoded by the st49 allele is a stable, secreted protein\n(Fig. 1, Fig. S2). Notably, previously we have already shown that ectopic expression of NTFGPS\nin Hela as well as H9c2 cells results in a stable, secreted protein.”\n2) Importantly, if the gpr126st49 mutation would result in the expression of a misfolded protein\nexhibiting dominant effects, it should not be possible to rescue this mutant phenotype by\noverexpression of the CTF. Therefore, we had provided in the original version data regarding\nthese rescue experiments. To further strengthen these data, we have now added additional\nevidence for gpr126 being expressed in the endocardium in zebrafish heart (RNAscope, Fig.\nS5, page 10, line 229) and that overexpressed CTF localizes in the transgenic animals in\nendothelial and endocardial cells at the membrane (Fig. 3c, end of page 10). Furthermore, we\nprovide new data showing that overexpression of CTF in the MZ gpr126st49 mutants, which\nrestores normal trabeculation, also restores myocardial Notch activity (Fig. 6e, f, page 16, line\n362).\nCollectively, these data show that NTFGPS represents a functional NTF.\n3. The sections linking Gpr126 to blood flow, and the Erbb2 and Notch signalling pathways,\nare less well worked up and need additional experimental confirmation.\na. Figure 4. The description of the Erbb2 inhibitor and blood flow experiments shown in this\nfigure is confusing. The genetic backgrounds tested (over-expressed gpr126, or the MZ st49\nmutant, Fig. 4) both already have blocked trabeculation, so how can the authors conclude that\nthe Erbb2 inhibitor blocks trabeculation in these backgrounds? Instead, both the inhibitor and\nlack of blood flow appear to rescue the multilayering phenotype in each background. This\nneeds further discussion and explanation. Quantification of the phenotypes should also be\nprovided in the figure legend.\nWe thank the reviewer for pointing out that this point needs more clarification.\nErbB2 signaling and blood flow are crucial regulators of trabeculation. In the absence of\neither of these factors in a WT heart, there is complete absence of trabeculation and the heart\nwall remains monolayered. The multilayering phenotype of gpr126st49 mutants and aneurysm-\nlike phenotype of the gpr126 overexpressing line raised the question whether Gpr126 has a\ntrabeculation promoting effect that might act in parallel to ErbB2 signaling overriding the\n13\nrequirement of ErbB2 signaling. Therefore, we tested whether inhibition of ErbB2 signaling has\nan effect on the phenotypes observed in gpr126st49 mutants and the gpr126 overexpressing\nline.\nTo address this issue, we have included this argument on page 12, line 277. In addition,\nwe quantified the data as requested (Fig. S8b,c).\nb. The authors document an increase in Notch signalling in the MZ st49 mutants in Fig. 7, and\nshow that the CTF can rescue the multilayering phenotype in this allele in Fig. 3. Importantly,\ncan over-expression of CTF also restore normal levels of Notch signalling in MZ st49? This is\nan essential control for the interpretation of the mechanism here.\nWe thank the reviewer for suggesting this important control experiment. We performed the\nsuggested experiments and observed that the overexpression of CTF restores normal levels\nof Notch signaling in MZ gpr126st49 mutants, and have included it in the manuscript. (page 16,\nline 362) (Fig. 6, f).\n4. The authors also speculate on lines 421–422 that rescue by the CTF is likely to be through\ncanonical G-protein-coupled signalling. This would be simple to test by raising cAMP with\nforskolin, a treatment that should rescue the cardiac phenotype in the st49 allele. This\nexperiment is essential to support the argument.\nWe agree with the reviewer that it likely that CTF signals via a activation of a G protein cascade.\nPreviously, it has been shown that ear and PNS phenotypes can be rescued in the\nabsence of the CTF by forskolin or IBMX treatment, which are both ubiquitously inducing cAMP\nsignaling. As stated on page 11, line 258, “Therefore, we tested whether forskolin/IBMX\ntreatment can also rescue the heart phenotype in gpr126st49 mutants. We confirmed that these\ntreatments rescued the puffy ear phenotype (Fig. S7a). Both treatments blocked trabeculation\nin WT hearts as well as multilayering in gpr126st49 hearts (Fig. S7b). As forskolin and IBMX\ninhibit also in WT hearts trabeculation, it remains unclear whether Gpr126 acts through cAMP\nactivation to promote trabeculation.”\nIt has to be noted that trabeculation is a dynamic process and general elevation of cAMP\nsignaling have a deleterious effect on trabeculation. We have also added new text in the\ndiscussion on page 19 line 443; “It is likely that CTF signals in the endocardium through\ncanonical G protein-coupled signaling, as observed in myelination during peripheral nervous\nsystem development. However, as the treatment with cAMP elevating drugs affected also\ntrabeculation in WT hearts, it could not be determined whether CTF acts through canonical G-\nprotein signaling. The effect on WT hearts is in agreement with recent reports that adrenergic\nsignaling strengthens cardiomyocyte cohesion, altered cAMP levels are associated with\nimpaired trabeculation, and that compartmentalized cAMP signaling is essential for proper\nheart development.”\n5. Over-expression of the NTF in a wild-type background results in multilayering (Fig. S4).\nDoes over-expression of the NTF in MZ stl47 mutants result in an st49-like multilayering\nphenotype? This would help to clarify the role of the NTF.\nWe thank the reviewer for this suggestion. We performed the suggested experiment and\nobserved that a stable overexpression of NTF in the myocardium of MZ gpr126stl47 mutants\nleads to multilayering (Fig. S4a,b)(page 10, line 215) and enhanced myocardial Notch activity\n(Fig S6c,d)(page 16, line 360) as observed in gpr126st49 mutants. This validates the role of\nNTFGPS in maintaining compact wall architecture.\naddition, plasmid injections in single cell-stage embryos are difficult to control. Therefore, we\nhave decided to remove these data and instead added data obtained by stable overexpression\nof NTFGPS in the myocardium of MZ gpr126stl47 mutants.\n6. This also needs some more discussion in the manuscript: why does the maternal transcript\nfail to compensate for the loss of zygotic function for the st49 allele specifically? And why does\nthe stl47 allele (which also lacks the CTF) not show any Z-only phenotype? Can the MZ stl47\nphenotype also be rescued with forskolin?\nOur data show that the NTF is required earlier in development than CTF. NTF is required early\nin development for compact wall formation by regulating N-cadherin localization, a pre-\nrequisite for proper trabeculation. In contrast, the CTF is required later for the subsequent\ncontrol of Notch activity and attainment of trabecular identity. As maternal mRNA is only\ntransiently present until early embryonic stages, these findings provide a likely explanation for\nwhy the Z gpr126stl47 mutant exhibits a maternal effect, but the Z gpr126st49 does not.\nNotably, our data are in agreement with a previous report in the peripheral nervous\nsystem, where it has been shown that MZ gpr126stl47 mutants exhibited a more severe\nphenotype than Z gpr126stl47 mutants. In contrast, the severity of Z and MZ gpr126st49 mutant\nphenotype were similar (PMID: 25695270).\nTo address this issue, we have modified the Discussion section accordingly (page 18, line\n404).\n7. The imaging in Figure 6 is not entirely clear. A counterstain for cell membranes is necessary\nto show the position of cells, especially as the epithelium is a curved surface. In addition, the\nchosen ‘representative’ example for MZ st49 does not have the multilayering phenotype. It is\nessential to show what happens to cadherin expression in those MZ st49 mutant hearts that\nhave a multilayering phenotype.\nAs requested, we have added the membrane counterstaining using Tg(myl7:mKATE-CAAX)\nwhich further substantiates our conclusion (Fig. S9a, c).\nRegarding the multilayering in gpr126st49 mutants: Please note that the multilayering\nphenotype cannot be observed at 72 hpf but only from 96 hpf on. Therefore, in Fig. 4 WT and\nmutant hearts have a single layered compact myocardial wall, where lateral localization of N-\ncadherin is only affected in MZ gpr126slt47 mutants, suggesting defects in compact wall\nformation. In Fig. 5, we show the N-cadherin phenotype for gpr126st49 mutants exhibiting\nmultilayering where the N-cadherin remodeling is affected.\nMinor comments\n8. The order of the alleles presented in Figure 7 (in both the image panels and the graph)\nshould be swapped round to match that in the rest of the manuscript, i.e. stl47 first, and then\nst49.\nWe have swapped the order as suggested in both, the image panel as well as the graph.\n9. The authors have undertaken a qPCR analysis of FACS-sorted cardiomyocytes (Fig. 3A)\nto confirm that Gpr126 mRNA is enriched in the endocardium, which is helpful. However,\nthe Introduction should state that the gene is known to be expressed in the zebrafish heart (since\na lack of expression in this organ in the zebrafish could be a simple explanation for any species-\nspecific differences in function), citing relevant studies. The authors could refer to Geng et al.\n(2013) for expression at 24–48 hpf, or their own study for expression at 48 hpf (Patra et al.,\n15\n2013 – although confusingly, note that the overall expression pattern shown in this paper does\nnot match that shown in Monk et al., 2009 or Geng et al., 2013).\nWe would like to thank the reviewer for pointing this out. We have edited the text\naccordingly (page 4, line 72). In addition, we decided for direct visualization of gpr126\ntranscripts by combining RNAscope in whole mount zebrafish embryos with phalloidin\nimmunostaining at 48 hpf (before the onset of trabeculation) and 96 hpf (after the onset of\ntrabeculation). The obtained data shown in Fig. S5 confirm our qPCR data that gpr126 is\nexpressed in the endocardium in zebrafish (see page 10, line 229).\n10. In all relevant main and supplementary figures (including both images and diagrams),\nthe authors should replace red with magenta, to make the images accessible to those with\ndichromatic colour vision. For the triple label in Figure 1, choose a suitable colour combination\n(e.g. magenta, green, white)\nWe have changed all figures to make them accessible to those with dichromatic color vision.\n11. There are some omissions or inconsistencies in discussion of the previous literature,\nincluding papers authored by some of the authors of the current manuscript, which make it\ndifficult to follow the arguments.\na. D’Amato et al. (2016) (ref. 46) is cited in the context of myocardial patterning, but the work\nin this paper to elucidate the relationship between Gpr126 and Notch signalling in the mouse\nand zebrafish is not mentioned or discussed anywhere. D’Amato et al. (2016) used several\nlines of evidence to demonstrate that cardiac expression of Gpr126 is a downstream target of\nNotch signalling, including documenting a loss of gpr126 expression in the hearts of zebrafish\nantimorphic mib mutants (loss-of-function for Notch signalling). How do these results fit with\nthe model that the authors propose for the zebrafish, with the enhanced myocardial Notch\nsignalling in MZ Gpr126-st49 mutants?\nIt is known that endocardial Notch lies upstream of most of the genetic pathways required for\ntrabeculation like EphrinB2, Bmp, as well as Nrg-ErbB2 signaling. In the manuscript of D’Amato\net al. (2016), it has been emphasized through various lines of evidence that endocardial\ndeletion of Notch signaling abrogates the cardiac expression of Gpr126 in zebrafish and mice,\nwhich suggested that Gpr126 is an important Notch effector throughout ventricular chamber\ndevelopment in the endocardium. The relationship of myocardial Notch activity and Gpr126,\nhowever, was not studied. The role of myocardial Notch activity during trabeculation has just\nrecently gained attention, especially in zebrafish studies. In Priya et al. 2020 (PMID: 33208950)\nand Han et al. 2016 (PMID: 27357797), it was shown that there is a myocardial Notch-mediated\nfate specification at play during trabeculation where the compact layer cardiomyocytes activate\na Notch reporter, while the trabecular layer cardiomyocytes do not.\nOur study indicates that Gpr126 has a role to play in this fate determination of compact\nvs. trabecular layer cardiomyocytes. In the gpr126st49 mutant, the compact wall is properly\nformed due to the presence of NTFGPS and the initial delamination events occur triggering the\nmyocardial Notch activity in the compact wall cardiomyocytes. The absence of CTF however\nleads to increased myocardial Notch activity.\nTo address this issue, we have clarified on page 20, line 451 that D’Amato et al. (2016)\nstudied the relation of Gpr126 and Notch in endocardial cells, while we discovered a new\nrelationship between Gpr126 and myocardial Notch.\nb. The authors do not cite or discuss previous work from their own group (Musa et al., 2019),\nin which they specifically linked miR-27a/b regulation of gpr126 expression to trabeculation in\n16\nthe zebrafish heart, and compared phenotypes to that of the gpr126 morphant. This paper\nappears to provide important background information that supports the current study. Why is\nthis study not cited?\nWe thank the reviewer for pointing this out. We have cited the study and added the text\naccordingly on page 4, line 84.\n12. A schematic diagram summarising the authors’ current model and thinking for how the NTF\nis working with the CTF, linking to Erbb2 and Notch signalling and cell polarisation, would be\nvery helpful.\nWe have added a schematic diagram of our current model (Fig. 7).\n13. Introduction, Line 56 – do the authors mean to say ‘is expressed in’ rather than ‘is essential\nfor’ here? It seems odd to say that the gene is essential for development of the heart but then\nto go on to say this is controversial and needs testing.\nWe have changed the text to “Adgrg6/Gpr126 has been reported to be required for the proper\ndevelopment of several tissues/organs including the peripheral nervous system, intervertebral\ndisc, ear, and placenta”(page 3, line 64) and added text about the cardiac expression of gpr126\n(page 4, line 72).\n14. Much of the information presented in Supplementary Fig. S1 (panels A–C, i.e. both the\nsequence and the ear phenotype for both alleles) has been published by other groups\nelsewhere. It is good to see that the authors have been able to confirm these findings in their\nown hands, but it must be stated that they are confirming previous published findings,\nciting Petersen et al. (ref 65) for the analysis of stl47, and Monk et al. (ref 35) for st49,\nrather than presenting these as new data.\nTo address this issue, we have cited the papers which first described the alleles and the\nphenotypes.\n15. Please note that the amino acid at the position of the indel in the stl47 allele is not a\nglutamine (Q), as previously published by Petersen et al. (2015) – it is an asparagine (N) (as\nthe supplementary figure in the current manuscript clearly shows) (see Table 1 in Baxendale\net al., genesis, 2021). This should be corrected in the text on line 104.\nWe would like to thank the reviewer for pointing out this. We have corrected the text\naccordingly.\nWe thank the reviewers for their in-depth review providing additional constructive comments to\nfurther improve our manuscript. In the revised manuscript we have addressed all the raised points\nby changing the text and/or providing a significant amount of new data.\nReviewer #1:\nThe revised version of the manuscript by Srivastava et al. has improved remarkably compared to\nthe initial one. I am very content with the additional data and arguments the authors present. They\nare convincing and I believe that the manuscript is a thoroughly studied piece now that is\ninteresting for a broad readership. However, I have one minor thing I am still a bit concerned\nabout. The authors show an interesting link between GPR126 and Notch activity, which is clearly\none of the most interesting aspects of the manuscript. However, there are still many open\nquestions regarding the interplay of the receptor and Notch. Thus, I suggest using more careful\nand less prominent phrasing e.g. in title and abstract and on the other side a few more details on\nthe open questions and the impact in the discussion.\nWe thank the reviewer for the overall positive comment. We have changed the title to\n“Adgrg6/Gpr126 is required for compact wall integrity and establishing trabecular identity during\ncardiac trabeculation” and used less prominent phrasing in the abstract. In addition, we have\nmodified the Discussion on page 24 (line 550 to 555) clarifying that there are open questions\nregarding the interplay of Gpr126 receptor and Notch.\nReviewer #2:\nThe authors have added significant new data and addressed most of my concerns in the revised\nmanuscript. However, a few concerns need further clarification or experimental validation. The\nauthors aimed to clarify a controversy in the function of gpr126 in the zebrafish heart. It is crucial\nto provide high-quality, mechanistic evidence, notably when the authors claimed some\nconclusions that contradict previous findings, including their own.\nWe thank the reviewer for acknowledging the importance of our new data. We agree that high-\nquality, mechanistic evidence is needed to “contradict previous findings”. Please note that neither\nthe involvement of Notch nor N-cadherin was the matter of controversy (both have previously not\nbeen analyzed in any Gpr126-modified animal model) but the overall involvement of Gpr126 in\nheart development. In this regard, we provide high-quality data (going in much more detail than\nthe previous study, providing transgenic rescue experiments) and on top provide evidence for\ndifferent roles of NTF and CTF and a link between GPR126 and Notch activity.\nContradictory findings, our own work: We have previously concluded that the NTF of Gpr126 is\nsufficient for proper heart development. This conclusion was drawn on morphological features,\nmeaning we simply looked at zebrafish embryos in which cardiomyocytes are labeled with GFP.\nA major limitation or difference to our current study was that morpholino-based studies did not\nallow to characterize morphants beyond 80 hpf. In addition, similar to the study by the de la Pompa\ngroup (Torregrosa-Carrion et al., Sci Adv 2021), we did not look at any other markers. Our detailed\nstudy (initiated and requested by our co-author Didier Stainier, also one of the authors in the\nheart development and that the lack of the CTF causes multilayering. Therefore, we revisited our\nprevious data. Notably, the published data (Fig. 4B, Patra et al., Proc Natl Acad Sci U S A. 2013)\nclearly show a multilayered heart. We had stated “MO2 morphants were characterized by\npericardial edema, lack of trabeculation, and reduced expression of mbp and krox20/egr2 at the\nPLLn. Injection of 100 pg of capped drgpr126-NTFΔGPS mRNA encoding amino acids 1–783\n(variant 1) rescued the pericardial edema and ventricular trabeculation phenotype in a subset of\nthe MO2 morphants (Fig. 4 A–C).” Apparently, we focused only on whether there is no\ntrabeculation (single layer of cardiomyocytes) or trabeculae (wall with luminal projections) but not\nmultilayering. This underlines the importance of the analysis of Notch and N-cadherin in our\ncurrent study.\nTo clarify this point, we have changed the text on page 11, line 255 to 259: “As our new data\nappeared to be in contrast to our previous findings, we revisited our previously published data\nregarding overexpressing NTFGPS in gpr126 morphants22. While previously the focus was only\non the “re-appearance of trabeculae”, a closer look revealed that the data could also be\ninterpreted as hypertrabeculation or multilayering (note, the analysis was limited to 80 hpf due to\nthe morpholino-based approach).”\nContradictory findings, work by de la Pompa’s group: We have provided a detailed explanation\non page 19, line 459, to page 20, line 473, why the group of de la Pompa might not have seen\nthe phenotype. Importantly, we have expanded the analysis beyond the characterization of hearts\nin which cardiomyocytes are labeled, identifying defects in N-cadherin localization and Notch\nactivation (unique phenotype). We determined that maternal RNA can compensate the depletion\nof gpr126. In addition, our data suggest that the CTF is required to balance NTF-mediated\nsignaling preventing overactivation of myocardial Notch signaling. This conclusion is based on a\nnumber of assays including in vivo rescue experiments.\nTo clarify the point of the need of balanced signaling, we have added a new paragraph to the\nDiscussion on page 20, line 473 to 485, which reads: “Another possible explanation is that a\nbalance of NTF- and CTF-mediated signaling is required for proper trabeculation. This is\nsupported by the observation of a substantial increase of Notch activity in MZ gpr126st49 mutants\nwhich could be normalized by the overexpression of CTF. Overall, all lines with an increased\nexpression of NTF compared to CTF exhibit the multilayering phenotype with the exception of the\nNTFGPS overexpression line (Fig. S13a). Notably, in this line cells express zygotic and maternal\nCTF and NTF and thus the ectopic expression of NTF might not be high enough to significantly\nimpair the balance between NTF and CTF or enough CTF is present to prevent increased Notch\nactivity. In contrast, mosaic expression, usually mediating a markedly higher expression was\nsufficient to induce local multilayering. In addition, the mosaic expression might result in adhesion\nheterogeneity between cardiomyocytes. Overall, our data suggest that the CTF is required to\nbalance NTF-mediated signaling preventing overactivation of myocardial Notch signaling.”\n2\n1. I'm afraid I have to disagree with the authors' claim that the probability of successfully\ngenerating a knock-in is very low. Numerous zebrafish knock-in alleles, including precise tagging\nalleles, have been published (e.g., PMIDs: 34495314, 32412410, 33681181, 35378361,\n36476416, 32064511, 36878640). It is doable to generate precise tagging of Gpr126. It would be\nthe authors' call whether it is necessary for the current study.\nWe agree that various studies have managed to successfully generate a knock-in in zebrafish\nand it is doable to generate precise tagging of Gpr126. However, the efficiency of a knock-in in\nzebrafish is very variable depending upon the gRNA used and the genomic location of the target\ngene. So far, our approaches haven’t been very efficient and also other Gpr126 researchers have\nnot been successful with this approach. In this study, we performed RNAScope analysis, which\nwas the most time-convenient way to identify the localization of Gpr126 transcript in the\nendocardium. In our opinion the available data are clearly showing the expression of Gpr126 in\nthe endocardium. Therefore, we have decided not to generate a zebrafish knock-in.\n2. The authors claimed that NTF is required for the junctional localization of N-cadherin in the\nworking model. However, N-cadherin is still localized to junctions in the stl47 mutant (Fig. 4, S9a).\nThe provided evidence only supports a correlation, and the impaired junctional localization might\nresult from other defects in the compact wall integrity. Notably, images in Figures 4 and S9a are\nsnapshots of representative samples. The phenotype is not clear to me. The WT images in Fig.\nS9a look very similar to the stl47 images in Fig. 4, depending on which confocal sections were\nchosen. It is not apparent how the schematic for the stl47 group was generated, which does not\nrecapitulate patterns in the image. The stl47 heart looks smaller than WT in the confocal section\nimage but not in the maximum projection image. More sophisticated presentation and\nquantification of the z-stack images would be necessary to support the claims.\nWe thank the reviewer for pointing out that we did not explain well enough the N-cadherin\nphenotype and that our claim “NTF is required for the junctional localization of N-cadherin” is\nmisleading. In the Introduction, we provide a detailed description on N-cadherin localization during\nheart development and cite the relevant literature (page 5, line 110 to 114): “Second,\ncardiomyocyte crowding triggers their delamination during which adherens junctions between\ncardiomyocytes are remodeled39. N-cadherin, the major component of cardiac adherens\njunctions, re-localizes from the lateral to the basal domain in compact wall cardiomyocytes. In\ncontrast, trabecular cardiomyocytes exhibit a punctate N-cadherin distribution on their entire\nsurface39”\nIn addition, we have now better specified the phenotype. Regarding the N-cadherin\nlocalization in Fig. 4 and Fig. S9a (now S11a), we clarify that in the WTs, the N-cadherin\nlocalization is limited to the junctions (typical short straight lines between the neighboring cells or\nY-shaped patterns; see also original N-cadherin publication by Didier Stainier’s group). However,\nin the stl47 mutants, the N-cadherin localization is random, present still at the junctions but also\nat the apical and/or basal membrane (therefore, we have added green lines in the schematic at\nthe basal, apical, and junctional sites). To clarify this, we wrote on page 15, line 366, to page 16,\nline 382: “Single-plane confocal images of WT hearts showed that N-cadherin specifically\nlocalizes laterally in the membrane of compact layer cardiomyocytes with some expansion to the\nbasal site (Y-shape pattern) (Fig. 4, Fig. S11a) as previously published39. Maximum intensity\nprojection images confirmed the lateral localization of N-cadherin in WT hearts (Fig. 4, Fig. S11a).\nthe junctions but also at the apical and/or basal membrane of the compact layer cardiomyocytes,\nwhich was also obvious in maximum intensity projections (Fig. 4). The maximum intensity\nprojection images exhibit an organized distribution of N-cadherin in WT hearts, however in the\nMZ gpr126stl47 mutant hearts, the N-cadherin distribution is haphazard and mis-organized (Fig.\nS12). Notably, in the MZ gpr126st49 mutants, which express NTFGPS, N-cadherin was localized in\na WT-like pattern at the junctions (laterally) of compact layer cardiomyocytes (Fig.4, Fig. 11a).\nOverexpression of CTF alone in MZ gpr126stl47 mutants did not rescue the compact wall integrity\ndefects (Fig. S11b), further indicating the requirements of NTF to mediate cardiomyocyte\nadhesion. These data indicate that the NTFGPS is sufficient for the proper localization of adherens\njunctions in cardiomyocytes of the compact wall prior to trabeculation.”\nThe N-cadherin phenotype is clear and cannot be due to “where confocal sections were chosen”\n(see figures 4, S11, S12 and the original N-cadherin publication by Didier Stainier’s group). It is\nan on/off effect and thus is not quantified. To further clarify this point, we have provided Z-\nprojections of 7 or 8 different hearts from WT as well as stl47 larvae, respectively. These Z-stack\nimages show again clear differences between the WT and Stl47 hearts (Figure S12). The WT\nhearts exhibit N-cadherin localization at the junctions suggesting organized adherens junctions,\nwhile in the stl47 hearts, the haphazard/random distribution suggests the disorganization of\nadherens junctions.\nFinally, we agree with the reviewer that the N-cadherin localization defects could be secondary to\nother compact wall integrity defects. Yet, our data clearly show that Gpr126 is required for the\nproper localization of N-cadherin. Consequently, we have changed the title of the manuscript to\n“Adgrg6/Gpr126 is required for compact wall integrity and establishing trabecular identity during\ncardiac trabeculation” and toned down our conclusions in the manuscript and model and stated\nthat NTF is required for compact wall integrity. Here, we would like to emphasize that the aim of\nour manuscript is to point out that NTF and CTF have different functions during trabeculation,\nwhere NTF has a role in the correct formation of the compact wall which is essential for the CTF\nto play its role in providing trabecular identity to cardiomyocytes. Currently, the most feasible way\nto look for compact integrity defects is N-cadherin mislocalization.\n3. The authors claimed that CTF is required for N-cadherin relocalization. Again, the provided\nevidence only supports a correlation. There is no direct evidence supporting that CTF regulates\nN-cadherin localization. It is also unclear whether the impaired N-cad localization is an outcome\nor cause of the trabecular defect.\nWe agree that the impaired N-cadherin localization as well as the apico-basal polarity defects\ncould be an outcome or the cause of trabecular defects. However, please note that they are the\nonly cellular mechanisms known so far in zebrafish to identify trabecular cardiomyocytes from\nnon-trabecular cardiomyocytes, and we investigated them to emphasize that the trabecular\nidentity is compromised in st49 mutants.\nTo address this point, we have changed the title of the manuscript to “Adgrg6/Gpr126 is\nrequired for compact wall integrity and establishing trabecular identity during cardiac\ntrabeculation” and modified the model and now state that the CTF is required for trabecular\nidentity. Note, the overall aim of our manuscript is to point out that NTF and CTF have different\nfunctions during trabeculation, where NTF has a role in correct formation of the compact wall\nwhich is essential for the CTF to play its role in providing trabecular identity to cardiomyocytes.\nReviewer #3:\nThe authors have added a number of revisions to the original manuscript, including extra\nexperiments to address some of the reviewers’ comments. Some of the new findings have\nconfirmed previous findings, whereas others have changed their interpretations. In general, the\ndata are important and will be of interest to the scientific community, especially those working on\nGpr126 in different contexts and model systems. However, unanswered questions remain, and\nthe text and some of the interpretations and conclusions are sometimes unclear. Citation of other\nwork is sometimes erratic. There are a number of places where changes could be made to help\nthe reader follow the paper more easily.\nWe thank the reviewer for pointing out the importance of our work and the additional suggestions\nhow to further improve our manuscript.\nAnalysis of the transcripts and proteins produced\nLine 131 and Fig. S1D - It is surprising that neither mutant transcript is subject to nonsense-mediated mRNA decay, and that\ntranscript levels appear normal. Have the authors sequenced variants here? Could there be alternative splicing\n(possibly heart-specific) or transcription start sites that could compensate for the predicted non-functional transcripts? This might\nexplain the significant proportion of embryos in which trabeculation is normal in both Z and MZ mutants for both alleles.\nEven small amounts of alternatively spliced zygotic mRNA could result in functional protein.\nWe thank the reviewer for this very insightful comment.\n1) Note, that it has previously shown that gpr126 mRNA was detected at the same level in WT\nand gpr126st49 mutants by in situ hybridization utilizing riboprobes spanning exons 12 to 18 (PMID:\n19745155, Figure S5), confirming that at least in gpr126st49 mutants no nonsense-mediated\nmRNA decay occurs.\nTo clarify this point, we have added the following text to page 6, line 146: “These data suggest\nthat the mutant gpr126 mRNAs are not subjected to nonsense-mediated RNA decay, confirming\nprevious data for gpr126st49 15.”\n2) Alternative splicing is a common feature of adhesion GPCR’s (PMID: 31363148) and\ncompensation by a heart-specific splice isoform could be a feasible explanation for the variable\npenetrance of the trabeculation phenotypes in stl47 mutants as well as the reason why stl47\nzygotic mutants do not show a similar phenotype as st49 mutants. Therefore, as suggested by\nthe reviewer, we performed RNA sequencing of 3 dpf whole larvae as well as 4 dpf larval hearts\nfor WT, stl47, and st49 to assess the presence of splice isoforms. We looked at the differential\nexon usage (new Figure S7). Our data suggest that alternative splicing in the heart is not\ncompensating for the predicted non-functional transcripts. In addition, our data show that the\npredominant gpr126 isoform in the heart lacks exon counting bin (B) 008, corresponding to exon\n6, of the canonical transcript isoform. Notably, skipping of exon 6 results in deletion of 23 aa in\n5\nzebrafish affecting gpr126 protein conformation and signaling. Consistently, the construct used in\nthis study for rescuing NTF function lacks exon 6. For details see pages 12 and 13.\nAnalysis of the tagged proteins in Fig. 1 shows that there is quantifiable GFP fluorescence in cells expressing both mutant\nversions, although the Western blot is clean. Is the GFP fluorescence above untransfected background levels? If so, are\nsmall amounts of protein produced from these transfected constructs?\nThe GFP fluorescence in the transfected cells were at the same level as the untransfected cells.\nThe quantification never showed the fluorescence value as null, even though there was no\ndetectable fluorescence. The western blot analysis with anti-GFP antibody confirmed that there\nwere no detectable levels of GFP being produced from the two mutant constructs.\nIs maternal transcript detectable in WT and MZ stl47 and st49 embryos? As the authors are proposing a role for maternal\ntranscript or protein, an in situ should be shown from early (pre-ZGA) embryos to show whether there are detectable levels of\nmaternal mRNA.\nIt has been described before that 96% of aGPCR have at least some level of maternal expression\n(PMID: 25715737), including detectable gpr126 expression based on qPCR at 3 hpf, suggesting\nthe presence of maternal mRNA. In addition, we have performed in situ hybridization analyses for\npre ZGA embryos to visualize the maternal transcript and observed that WT as well as the mutants\nexhibit gpr126 expression at 2 hpf as shown in Fig. S3b. The data are described on page 9,\nline 209 to 215: “It has been described that 96% of aGPCR have at least some levels of maternal\nexpression46. The qPCR data for Gpr126 show detectable expression at 3 hpf, suggesting the\npresence of maternal mRNA46. To confirm the maternal deposition of gpr126 RNA, we performed\nin situ hybridization in embryos prior to zygotic genome activation to visualize the maternal\ntranscript and observed that WT as well as the MZ gpr126stl47 and MZ gpr126st49 mutants have\ngpr126 expression at 2 hpf (Fig. S3a).”\nTo further clarify the issue of maternal compensation and that this has been described before,\nwe have added the following text to the Discussion (page 19, line 459): “The analysis of the gpr126\nmutant zebrafish lines revealed that zygotic stl47 mutants exhibit no trabeculation phenotype.\nThis suggested that maternal gpr126 mRNA can compensate the loss of zygotic gpr126.\nConsequently, MZ gpr126stl47 mutants exhibit defective trabeculation. However, this raised the\nquestion how a maternal mRNA can compensate for such an extended time period the lack of\nzygotic mRNA. Notably, it had already previously been shown that MZ stl47 mutants exhibited a\nmore prominent phenotype regarding Schwann cell development. Importantly, at 96 hpf 42% of\nthe Z stl47 mutants showed normal myelin basic protein expression while none of MZ stl47\nmutants expressed myelin basic protein43. Collectively, our data indicate that the maternal gpr126\nmRNA and/or the encoded protein stability is high enough to allow normal trabeculation.”\nIt needs to be pointed out more explicitly that 36.67% of MZ stl47 mutants have WT trabeculation, and over 60% have some\ndegree of trabeculation. This indicates that under some circumstances (possibly for some genetic backgrounds), Gpr126 is\nnot required for heart development. This result is shown in Fig. 1 but is not described explicitly in the text (lines 189–192).\nTo address this point, we have modified the text and described explicitly the variable penetrance\nof the phenotypes observed (page 9, line 222): “Thus, in total 63.4% of the MZ gpr126stl47 mutants\nexhibited trabeculation defects, whereas 36.6% exhibited normal trabeculation. We did not\nobserve any other gross morphological defects in the MZ gpr126stl47 mutant larvae or\ndevelopmental delays compared with WT larvae (Fig. S3b).”\nWe agree that different genetic backgrounds are responsible for variable phenotype, as\nknown for many genes also in humans. Yet, trabeculation is a dynamic process, and previous\nanalyses of other mutant lines have revealed that it is common to observe different levels of\nseverity regarding trabeculation phenotypes. We suspect that compact wall integrity defects lead\nto reduced trabeculation in general, and in severe cases leads to a complete absence of\ntrabeculation in MZ stl47 mutants.\nCould the range of phenotypes seen in MZstl47 be explained by an overall general delay? What is the gross morphology of\nthese embryos? – this has not been shown anywhere.\nWe did not observe any overall general delay and provide now in Fig. S3b more data. The text\nhas been changed as follows (page 9, line 223): “We did not observe any other gross\nmorphological defects in the MZ gpr126stl47 mutant larvae or developmental delays compared with\nWT larvae (Fig. S3b).”\nSecretion of the NTFdelGPS\nThe authors report that the NTFdelGPS is detected in the cytoplasm, supported by immunostaining and Western blots of cell\nlysates (Fig. 1D). They say cytoplasmic expression was detected ‘in a minority of cells’. Does this mean only a minority of\ncells were transfected with the construct, or, of those transfected, only a minority show cytoplasmic expression? The authors\nnow also report secretion of the NTFdelGPS. This is very important given its apparent non-autonomous function on the\nmyocardium, and also because the anti-HA fluorescence and cell lysate blot shown in Fig. 1 imply that it is stuck in the\ncytoplasm. However, the main figure still shows the apparent cytoplasmic expression, while analysis of the supernatant to\nshow potential protein secretion, which the argument hinges on, is hidden in the supplementary material (Fig. S2C). What is\nthe relative amount of cytoplasmic vs secreted protein? – the Western blot should show the lysate and supernatant bands\nfrom the same samples side by side in the main figure.\nWe thank the reviewer for this important suggestion. We meant “of those transfected, only a\nminority show cytoplasmic expression”. To further clarify this point, we generated stable ARPE-19\nlines overexpressing the WT and the mutant forms and observed that the cytoplasmic localization\nwas significantly reduced in the stable lines (Fig. 1e,f), suggesting that the truncated proteins are\nnot getting stuck in the cytoplasm. The western blot analysis of the lysates and supernatants of\nWT and St49 overexpressing cells showed that the NTFGPS is secreted and treatment with\nbrefeldin A markedly blocked the secretion. We have also provided the lysate and supernatant\nbands for the transient overexpression in Figure S2c.\nPage 7, line 168: “In contrast, the mutant Gpr126 forms failed to localize to the membrane (Fig.\n1b) and in a minority of transfected cells cytoplasmic expression was detected (Fig. S2a, b).”\nPage 8, line 179: “Given that transient overexpression of proteins can lead to protein artifacts, we\ngenerated stable ARPE-19 lines expressing WT as well as St49 and St47 mutants of Gpr126 for\na more reliable analysis (Fig. 1e). We observed membrane localization of WT Gpr126, while the\nmutant forms failed to localize to the membrane. In addition, there was no detectable cytoplasmic\naccumulation of the mutant forms, indicating that the truncated forms are not retained in the\ncytoplasm. WB analyses of the lysates and culture medium from the stable lines, with and without\ntreatment with brefeldin A, confirmed that NTFGPS is secreted in the St49 overexpressing cells\n(Fig. 1f).”.\nPossible dominant effect of the NTF\nWe do not believe that the authors have sufficiently ruled out a possible dominant effect for the N terminal fragment produced\nby the st49 allele, a point that was queried by us and another reviewer for the original version.\nThe authors state on Line 221 – ‘no heart phenotypes were observed in heterozygous gpr126st49 mutants.’ It is essential to\nshow the data and quantification of the phenotypes here, as the authors have done for WTs, Z homozygotes and MZ\nhomozygotes. Given the incomplete penetrance of the phenotype in the homozygotes (26.6% of Z and MZ st49 mutants\nshow normal trabeculation), this also needs to be quantified for the heterozygotes. This may require further work and\ngenotyping, but is essential to rule out any dominant effect of the NTF.\nWe thank the reviewer for indicating that it is not well described in the manuscript why a dominant\neffect of NTFGPS can be excluded. In the revised manuscript, we have now clearly described why\nwe conclude that the NTFGPS has no dominant effect:\n1) Z and MZ heterozygous animals of both mutants, stl47 and st49 mutants, exhibit no\nphenotypes (see Fig. S5 and page 11, line 250)\n2) CTF overexpression can rescue the gpr126st49 mutant phenotype (see Fig. 3 and page 14,\nline 311).\n3) Overexpression of NTFGPS in WT animals does not cause a heart phenotype or Notch\nactivation (Figure S13a).\nNotably, the experiment presented in the previous manuscript (v1, Fig. S4), in which mosaic\noverexpression of the N terminus in the WT myocardium appeared to induce local multilayering—\nby definition a dominant effect—has now been removed from the current version of the\nmanuscript.\nWe agree that the first conclusion after observing a phenotype upon gene overexpression might\nbe that there is a dominant effect. Yet, there are also other explanations. We thank the reviewer\nfor forcing us to think more about the data to come up with an explanation, as based on our data\na dominant effect can be excluded (see above). If one considers the results of all mutant and\nrescue analyses, the fact that st49 mutants lacking the CTF exhibit enhanced Notch activity but\nNTFGPS overexpression in WT does not, as well as the role of the maternal RNA, one possible\nexplanation is that the CTF is required to balance NTF-mediated signaling preventing\noveractivation of myocardial Notch signaling.\nwhich reads: “Collectively, our data indicate that the maternal gpr126 mRNA and/or the encoded\nprotein stability is high enough to allow normal trabeculation. However, this raises the question,\nwhy maternal gpr126 mRNA/protein is not sufficient for normal trabeculation in gpr126st49 mutants.\nThe only difference is the presence of additional NTFGPS. Thus, one possible explanation is that\nNTFGPS acts in a dominant manner. However, our data argue against a dominant effect as for\nexample heterozygous St49 mutants show no phenotypes. Another possible explanation is that\na balance of NTF- and CTF-mediated signaling is required for proper trabeculation. This is\nsupported by the observation of a substantial increase of Notch activity in MZ gpr126st49 mutants\nwhich could be normalized by the overexpression of CTF. Overall, all lines with an increased\nexpression of NTF compared to CTF exhibit the multilayering phenotype with the exception of the\nNTFGPS overexpression line (Fig. S13a). Notably, in this line cells express zygotic and maternal\nCTF and NTF and thus the ectopic expression of NTF might not be high enough to significantly\nimpair the balance between NTF and CTF or enough CTF is present to prevent increased Notch\nactivity. In contrast, mosaic expression, usually mediating a markedly higher expression was\nsufficient to induce local multilayering. In addition, the mosaic expression might result in adhesion\nheterogeneity between cardiomyocytes. Overall, our data suggest that the CTF is required to\nbalance NTF-mediated signaling preventing overactivation of myocardial Notch signaling.”\nLink to Notch signalling\n20% of MZ stl47 mutants show a multilayering phenotype (Fig. 2D). Does this population also show an increase in Notch\nsignalling?\nWe did not observe any stl47 mutants with enhanced Notch signaling. We visualized active Notch\nsignaling in stl47 mutants with a membrane reporter line and did not observe increased\nmyocardial Notch signaling in the stl47 mutants exhibiting multilayering, as shown in the figure\nbelow.\nThe authors state in their rebuttal that there is an early requirement for the NTF in N-cad localisation (a prerequisite for\ntrabeculation), and a late requirement for the CTF in trabeculation. They say this difference in timing explains the difference\nin phenotype between the two alleles. It is stated several times in the MS and in the rebuttal that the maternal protein is\nunlikely to perform the later function. However, in the Z stl47 mutants, both early and late roles would have to be performed\nby the maternal transcript or protein, resulting in normal trabeculation. Action of the maternal transcript for such a late\nfuncton(96h+) is really puzzling. Thus, the proposed early/late functions in the model do not fit with the data shown.\nIf the maternal protein can perform both early and late functions (as demonstrated by the Z-only stl47 normal phenotype),\nthen why is it unable to do this in the presence of zygotically-produced NTFdelGPS in the Z st49 mutants? This is still\nunexplained, unless the NTF blocks the effects of the maternal protein in a dominant manner, or there is some heart-specific\ncompensation mechanism in the stl47 allele.\nThe authors do acknowledge that there are difficulties in their interpretation and that further work is needed at Line 413,\nbut this should be stated up-front more explicitly, and the model should either be revised or presented with much more caution.\nWe thank the reviewer for stressing this point which made us re-evaluating all data and re-thinking\nour conclusions. As detailed in our response to the points above we conclude that our data\nsuggest that a balance of NTF and CTF is required (see above and Discussion on page 20, line\n467).\nPharmacological rescue experiments\nParagraph starting at Line 255. It is really important to cite Petersen et al. (2015) here for pharmacological rescue of the stl47\nand st49 alleles. These authors only used forskolin, and importantly, showed that this could not rescue radial sorting or\nmyelination in stl47 mutants.\nFor use of IBMX, Geng et al. (2013) is cited, which is appropriate. However, it should be clarified here that the published use\nof IBMX was with other alleles, not stl47 or st49. In addition, the concentration used in this paper was 100 µM, not 125 µM.\nFinally, IBMX is not an AC activator - it is a phosphodiesterase inhibitor, raising cAMP levels independently of AC activity\nand thus an important way to confirm that the CTF works through cAMP.\nIt is important to get these details correct, as they are required to interpret the allele-specific and domain-specific effects for\nthese phenotypes, which are not straightforward.\nThank you for spotting this. We have changed the text accordingly and added the required details\nregarding IBMX and forskolin (see page 15, line 346).\nModel – autonomy and non-autonomy\nThe authors have still not explained how expression and activity of the CTF in the endocardium can lead to effects on the\nmyocardium, i.e. how the receptor acts in a non-autonomous manner. In other tissues (ear and Schwann cells), the\nphenotypes affect the cells in which the receptor is expressed. In the heart this appears to be different, but no suitable\nexplanation is provided.\nIn this context, it is really important to point out the cell types in which the experiments were done. For example, the over-\nexpression of the NTFdelGPS (lines 215–217) is done in the myocardium, whereas production of the NTF from the st49 allele is\nexpected to be in the endocardium.\nNote, we have previously shown that the NTF binds to the myocardium in mouse hearts and thus,\nwe have utilized a cardiac promoter to express NTF. In the revised manuscript, we have provided\nthis information and made sure that it is clearly stated in which cell type which experiments were\n10\nperformed (page 11, line 260): “Therefore, we analyzed mosaic cardiac expression of NTFGPS in\nWT larvae as well as stable overexpression of NTFGPS in cardiomyocytes (Tg(myl7:gpr126NTF-\np2a-tdTomato) in MZ gpr126stl47 mutants, as our previous data indicate that NTF acts as a\nsecreted molecule on cardiomyocytes22.\nRegarding the mechanism how the endocardial CTF affects the fate of cardiomyocytes, we\nhave provided possible explanations in the Discussion (see page 23, line 556). Importantly, “how\nendocardial factors affect cardiomyocyte behavior remains largely unknown and is currently under\ninvestigation. Recently it has been suggested that endocardial protrusions regulate\ncommunication between endocardium and myocardium by modulating Nrg/ErbB2 signaling58,59”\nOther comments and essential corrections\nThe sentence at Lines 52–53 is an exact copy of a sentence in the Abstract from Zaidman et al., PNAS USA 2020, and\nshould be reworded.\nWe have reworded the sentence.\nThe citations of references 18–20 supporting the information on Lines 66–70 are three primary papers covering pulmonary\nfunction, COPD and periodontitis. These references do not cover the other diseases that the authors refer to (scoliosis,\nintellectual disability, cancers). The authors should either cite all relevant primary papers to support their statements, or they\nshould cite a suitable review that covers all this information with comprehensive referencing.\nWe have cited the review from Baxendale et al (PMID: 33735533) which cover all the diseases\nwhich were referred.\nLines 88–89 ‘newly created zebrafish mutants’ (reference 17, Torregrosa-Carrion et al., 2021). It is essential to give full\ndetails of these alleles; one (bns342) is a promoter-less allele with undetectable transcript levels and normal cardiac\ntrabeculation, and so it is important to point this out. In addition, this study did not rely on ‘gross morphological analyses’ - it\npresents confocal images (ref. 17, Fig. 3) at the same level of detail as the current manuscript. This information should be\nacknowledged, described and cited correctly.\nWe have changed the text to add more details about the alleles (see page 4, line 101).\nLine 74 – depletion, not deletion\nWe have changed the text, as suggested.\nLines 121–127 Introduction to the alleles used. The authors now cite the original papers at the start of this section, and have\ncorrected the amino acid codon affected by the lesion, but still go on to present the data in the supplementary figures\nidentifying the molecular lesions as their own. It would be best to repeat the citations throughout the paragraph to make it\nclear that this figure is confirming previously published data, or to explain up front that these alleles have previously been\ncharacterised and that the figure is confirming these published data.\nWe have repeated the citations and added text to state that we are confirming previously\npublished data (see page 6, line 135).\ndata suggest that gpr126st49 mutants express and secrete the NTFdelGPS.’ (Not ‘indicate’.)\nWe have changed the text, as suggested (see page 8, line 187).\nLine 170–183 – The title of this section is about stl47. However, the important result of the multilayering in st49 is also\nintroduced here. It would help to have one section about stl47 and then another about st49.\nAs recommended, we have introduced in the revised manuscript two sub-sections to describe the\nphenotypes of the two mutant lines separately.\nLine 200 – it is stated that there are no obvious defects in the endocardium. However, it is not folded as normal, and this\ncould be interpreted as a defect.\nWe agree with the reviewer that there is a difference in the folding. However, it should be noted\nthat the endocardium and myocardium are in very close contact to each other during the analyzed\nstages and thus the missing folding appears to be due to the lack of trabeculation.\nTo address this point, we have added the following text to page 10, line 243: “In contrast, no\nobvious defect in the endocardium or the cardiac jelly between the endocardium and the\nmyocardium was detected at 72 and 96 hpf, when analyzing MZ gpr126stl47 and gpr126st49 mutants\nin the background of Tg(myl7:mKATE-CAAX); Tg(krt4:GFP)sqet33 46, which label the myocardium\nand the endocardium, respectively (Fig. S4). The only apparent difference is that the endocardium\nis not folded as normal, which appears to be due to the lack or a reduced number of trabeculae.”\nLine 209 – ‘experiments previously suggested that the NTFdelGPS is sufficient for trabeculation and heart development (ref.\n21). It would help to say here something like ‘However, we are now revising that view’.\nWe thank the reviewer for spotting that we did not clarify well that we revised our previous\nconclusion that the NTF is sufficient for proper heart development. In the revised manuscript we\nhave clarified/discussed this point on page 11 (line 255), page 11 (line 272), and page 21 (line\n508).\nLine 212 – this should be Z, not MZ\nWe have corrected the text accordingly.\nFigure 4 - N-Cadherin data\nThese images are still difficult to interpret. It would be good to show individual channels in inverted greyscale to show as\nmuch detail as possible.\nIn our opinion, the main problem is that we have not properly described the N-cadherin phenotype\nand do not think that inverted greyscale images will help.\nTo address this point, we provide in the Introduction a detailed description on N-cadherin\nlocalization during heart development and cite the relevant literature (page 5, line 110): “Second,\ncardiomyocyte crowding triggers their delamination during which adherens junctions between\ncardiomyocytes are remodeled39. N-cadherin, the major component of cardiac adherens\n12\njunctions, re-localizes from the lateral to the basal domain in compact wall cardiomyocytes. In\ncontrast, trabecular cardiomyocytes exhibit a punctate N-cadherin distribution on their entire\nsurface39.”\nIn addition, we have now better specified the phenotype. Regarding the N-cadherin\nlocalization in Fig. 4 and Fig. S9a (now S11a), we clarify that in the WTs, the N-cadherin\nlocalization is limited to the junctions (typical short straight lines between the neighboring cells or\nY-shaped patterns; see also original N-cadherin publication by Didier Stainier’s group). However,\nin the stl47 mutants, the N-cadherin localization is random, present still at the junctions but also\nat the apical and/or basal membrane (therefore, we have added green lines in the schematic at\nthe basal, apical, and junctional sites). To clarify this, we wrote on page 15, line 366, to page 16,\nline 382: “Single-plane confocal images of WT hearts showed that N-cadherin specifically\nlocalizes laterally in the membrane of compact layer cardiomyocytes with some expansion to the\nbasal site (Y-shape pattern) (Fig. 4, Fig. S11a) as previously published39. Maximum intensity\nprojection images confirmed the lateral localization of N-cadherin in WT hearts (Fig. 4, Fig. S11a).\nIn contrast, in MZ gpr126stl47 mutant hearts, N-cadherin was distributed randomly, still laterally at\nthe junctions but also at the apical and/or basal membrane of the compact layer cardiomyocytes,\nwhich was also obvious in maximum intensity projections (Fig. 4). The maximum intensity\nprojection images exhibit an organized distribution of N-cadherin in WT hearts, however in the\nMZ gpr126stl47 mutant hearts, the N-cadherin distribution is haphazard and mis-organized (Fig.\nS12). Notably, in the MZ gpr126st49 mutants, which express NTFGPS, N-cadherin was localized in\na WT-like pattern at the junctions (laterally) of compact layer cardiomyocytes (Fig.4, Fig. 11a).\nOverexpression of CTF alone in MZ gpr126stl47 mutants did not rescue the compact wall integrity\ndefects (Fig. S11b), further indicating the requirements of NTF to mediate cardiomyocyte\nadhesion. These data indicate that the NTFGPS is sufficient for the proper localization of adherens\njunctions in cardiomyocytes of the compact wall prior to trabeculation.”\nTo further clarify this point, we have provided Z-projections of 7 or 8 different hearts from WT\nas well as stl47, respectively. These Z-stack images show again clear differences between the\nWT and Stl47 hearts (Figure S12). The WT hearts exhibit N-cadherin localization at the junctions\nsuggesting organized adherens junctions, while in the stl47 hearts, the haphazard/random\ndistribution suggests the disorganization of adherens junctions.\nErbb2 section (Lines 267–290)\nAll data for this section are presented in the supplementary material. This is still too preliminary and it is recommended that\nthis section is removed from the manuscript.\nWe have removed this data from the manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed the concerns raised in the previous review. I appreciate that they\nhave toned down the language in several conclusions, adopting a more cautious and balanced\napproach in interpreting the phenotypes and addressing the discrepancy with previous work.\nThese revisions have improved the clarity and rigor of the manuscript.\nWe thank the reviewer for the many constructive comments and suggestions that greatly helped\nto improve the manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have added several new supplementary figures to the MS, including new data, and\nhave addressed many of the comments satisfactorily. These include some interesting new\nfindings relating to the prevalence of the exon-6-skipped transcript isoform in the heart. As stated\npreviously, the study is important and will be of value to the research community.\nThank you very much. We would like to thank the reviewer for acknowledging the importance of\nour work and for the constructive suggestions to improve and refine our manuscript.\nHowever, there are still a number of instances where the data and/or interpretations appear\ninconsistent or incomplete, and these may affect the way that the overall mechanistic model is\npresented. These issues are detailed below.\nThank you for spotting the inconsistencies of our manuscript. We have addressed all the\nremaining points as detailed below.\n1. Other alleles\na) It is good that the authors have added details of other mutant alleles when discussing previous\nwork. However, the Torregrosa-Carrión et al., 2021 study is still misquoted, as it was not 'based\non gross morphological analyses' in the added text at lines 99–100 - this statement needs to be\nremoved. The Torregrosa-Carrión paper presented detailed confocal imaging of trabeculae in\ntransgenic lines at 96 hpf, together with quantitative analysis of transcript levels at 48 hpf\n(Torregrosa-Carrión et al., 2021, Fig. 3).\nWe agree that “gross morphological analyses” is misleading. What we meant was that\nTorregrosa-Carrión et al. analyzed the mutants only based on the labeling of endocardial and\nmyocardial membranes and, in contrast to our study, did not utilize additional markers such as N-\ncadherin or markers to visualize depolarization.\nTo address this point, we have removed the statement “based on gross morphological\nanalysis” (page 4, line 100).\nb) The discrepancy with the current study can be explained possibly because this study only\nexamined Z-only mutants, although even this is problematical, because the authors demonstrated\na complete loss of transcript in zygotic bns342/bns342 mutants at 48 hpf (T-C et al., 2021, Fig.\n3B), and concluded that gpr126 is not required for trabeculation in the zebrafish heart. (It will\nobviously be interesting to test whether there is a heart phenotype in MZ bns342/bns342\nmutants.)\nWe agree with the reviewer that the discrepancy in both studies might be explained by the fact\nthat Torregrosa-Carrión et al. studied only zygotic mutants. That the authors did not observe a\nphenotype can have several reasons (e.g., also compensation). Notably, gpr126bns342/342 embryos\nhave an inflated swim bladder in their Fig 3E, in contrast to the gpr126bns342/342 embryo shown in\ntheir Fig. S10, whose appearance resembles our mutants (Fig. S1c). Clarifying why Torregrosa-\nthis manuscript.\nTo address this point, we have added the following sentence to the Discussion (page 22, line\n514): “It will be interesting to test whether there is a heart phenotype in MZ gpr126bns341 and MZ\ngpr126bns342 mutants.”\n2. Figure S3b\nAs requested, the authors have added Fig. S3b to show the whole-embryo appearance for stl47\nand st49 alleles. However, their description in the text and legend is at odds with the images\nshown. Gross abnormalities are evident for the stl47 allele in the figure: shorter body length, small\nhead and eye, underdeveloped jaw, and pericardial oedema. If these phenotypes are\nrepresentative, they need to be described. If not representative, another embryo should be shown.\nNote that incompletely penetrant pericardial oedema was previously reported for zygotic\nstl47/stl47 mutants (Fig. S1c, Petersen et al., 2015), matching the phenotype of the stl47 embryo\nin Fig. S3b. This published observation should be acknowledged and cited somewhere. An\nadditional issue with Fig. S3b is that the embryos look completely different to those shown in Figs.\nS1c and S10a, also labelled as 96 hpf. The puffy ears are not nearly as evident for either\ngenotype, and thus it is not convincing that these fish are actually mutant for gpr126. We suggest\nthat they are likely to be 72 hpf or younger – please check.\na) We sincerely thank the reviewer for spotting these points. The embryos shown in former Fig.\nS3 were actually 72 hpf embryos.\nTo address this point, we have now added images of 96 hpf embryos to Fig. S3. The majority\nof mutant embryos showed neither an obvious shorter body length, smaller head and/or eye, or\nunderdeveloped jaw.\nb) It is correct that we observed sporadically cardiac oedema in the MZ stl47 mutants (not\nappearing in all clutches), as described in Petersen et al. (2015, Fig. S1c). Embryos with strong\ncardiac oedema were excluded from this study.\nTo address this point, we have added the following two sentences to the Results (page 10,\nline 229): “Note, a pericardial oedema was sporadically observed in MZ gpr126stl47 mutants, as\npreviously reported43. Embryos with strong pericardial oedema were excluded from this study.”\n3. Figure S6\nThe authors state that they have previously shown that the NTF binds to the myocardium in mice\nas a justification for expressing it in the myocardium in zebrafish. However, they also confirm that\nGpr126 is normally expressed in the endocardium and that the NTF is secreted. It would have\nbeen more relevant to express the NTF in endocardium, to mimic the situation in the mutants\nmore closely. Expressing it in the myocardium adds an additional level of complexity to the\ninterpretation. Do you see similar phenotypes with endocardial (krt4-driven) or endothelial (fli1-\ndriven) expression of the NTF?\nThe aim of our experimental approach was actually to reduce the complexity. As we currently do\nnot know how NTF is secreted from the endocardium and transported to the myocardium and\nwhether the CTF is involved or required in these processes, we decided to express NTFΔGPS in\ncardiomyocytes, as we had previously identified these cells as a target of NTF. In addition, we\nargued that if we see an effect, this will further support our hypothesis that the NTF acts on\ncardiomyocytes.\nWhile the endocardial expression of the NTF could further verify the secretory role of NTF on\nthe myocardium, we believe that these experiments will not add substantial information to the\nmanuscript and thus are beyond the scope of this manuscript.\n\"Therefore, we analyzed myocardial-specific (myl7-driven) mosaic expression of NTFΔGPS in\nWT larvae as well as stable expression in MZ gpr126stl47 mutants,\"\nAs requested, we have changed the text, however, we changed the order as we first show data\nfor stable expression and then for mosaic expression (page 11, line 269).\nIf the data from the stable line are available for MZ mutants, it is important to show the effects of\nexpression of this line in sibling embryos (Z mutants and WTs), as these will be more relevant\ncontrols for this experiment than the mosaic overexpression experiment.\nTo address this request, we have added a representative image of WT embryos overexpressing\nNTFΔGPS (Tg(myl7:gpr126NTF-p2a-tdTomato)) at 96 hpf in Fig. S6a. Stable NTF overexpression\nin WT embryos did not lead to any multilayering (Fig. S6b). The text has been changed\naccordingly (page 12, line 273).\nFig. S6b. In Fig. 2d, it is stated that 20% (n=30) of MZstl47 hearts have a multilayered ventricular\nwall. In Fig. S6b, this proportion has now halved to 10% (n=8), and this value is used for\ncomparison with the over-expression result (40%, n=10). However, is not clear how the\npercentage values for this graph in Fig. S6b were generated. Why are the data not presented\nlike the stacked bars in Fig. 2? Given the low numbers for the Fig. S6b experiment (did the data\npass a normality test?), and the Fig. 2d estimate of 20% (more accurate as based on higher\nnumbers), obtaining 4/10 embryos with multilayering by chance is very possible. Thus, as they\nstand, the results shown in Fig. S6b are not convincing.\nWe thank the reviewer for pointing out this inconsistency. We have revisited the images,\nquantified them according to Fig. 2d, added one more batch of stl47 MZ embryos, and presented\nthe data as stacked bars in Fig. S6b, similar to Fig. 2d. These data demonstrate that NTF\noverexpression in stl47 MZ embryos enhances multilayering.\n4. Figures 4, S11, S12\nThe description of the N-cadherin phenotype in MZstl47 mutants is now clearer, although it is still\nquite hard to see the differences between wild-type and mutant junctional arrangements in the\nimages, and this was also pointed out by another reviewer. The additional images in Fig. S12 are\nhelpful, although we still think that enlargements of selected regions of the MIPs would be useful.\nHowever, if the original images are retained, it would be helpful to include some labelling on the\nMIP images to highlight the main important features.\nTo address this point, we have added to Fig. 4 zoom-ins of the maximum projections. In addition,\nwe labeled the outline of the cardiomyocytes and utilized arrows and arrowheads to clarify the\nphenotype. One can clearly see that WT and st49 hearts exhibit clear boundaries of the N-\ncadherin localization at the lateral junctions, while in stl47 mutants, the boundaries are blurred\nand they appear more haphazard.\nNo additional images of MZst49 were provided, and it is stated that the MZst49 hearts have a\nwild-type arrangement of N-cadherin (line 377). To us, the MIP image of MZst49 (Fig. 4, lower\nright panel) has features that look more similar to the MZstl47 phenotype, especially towards the\nbottom and right hand side of the ventricle. If a phenotype is present, this would change the\ninterpretation here (line 381), instead indicating that the NTF is not sufficient for the correct\nlocalisation of N-cadherin in the compact layer. Can the authors comment and add some\nadditional images of the MZst49 hearts (it is stated that 12 samples were imaged) to Fig. S12,\nas for MZstl47?\nWe hope that the zoom-ins in Fig. 4 help the reviewer to understand the phenotype. In addition,\nwe have added 7 additional examples of MZ st49 hearts in Fig. S12.\nthe N-cadherin mis-localization phenotype, which might be not as obvious as the phenotype\nshown in mid-sagittal sections. Both representations of the phenotype together clearly show that\nthe N-cadherin is mis-localized in the stl47 hearts. Note, there is some variability in the maximum\nprojection data as trabeculation is a dynamic process which starts at around 60 hpf. Thus, there\nmight be already some sparse trabecular cardiomyocytes at 72 hpf which affect the appearance of\nclear boundaries. Furthermore, multilayering defects in st49 mutants might also affect the\nmaximum projections explaining the slight differences in N-cadherin localization at the lateral\njunctions between WT and st49 hearts. Notably, mid-sagittal sections in WT and st49 embryos\nclearly show that their hearts do not exhibit any mis-localized N-cadherin in the compact wall\ncardiomyocytes.\nThe mis-localization of N-cadherin in the compact wall cardiomyocytes in stl47 mutants show\nthat NTF is required for the correct localization of N-cadherin in the compact wall, whereas in st49\nmutants, the N-cadherin is correctly localized at the lateral side in compact wall cardiomyocytes,\nbut fail to relocalize to the basal membrane in the inner layer cardiomyocytes, as observed in\ntrabecular cardiomyocytes, suggesting the CTF is required for trabecular identity.\n5. Model (Figure 7)\nThe authors have revised their interpretation of the data, but the summary model in Fig. 7 remains\nunchanged, so it is hard to understand exactly what is new. It must be stated in the figure legend\n(as well as the text) that this is just a model for the role of Gpr126, as it incorporates a number of\nassumptions. Some basic details are unknown at the moment, such as any polarised location of\nthe receptor on the endocardial cell plasma membrane. It seems unlikely that all NTF would be\nsecreted and degraded, when the CTF persists – again, these details have not been tested.\nAs requested, we have changed the Figure legend (including the title) of Fig. 7 as well as the\nDiscussion (page 25, line 591) to clarify that our conclusion represents a model that has the\nindicated limitations.\nWhat do the dotted black arrows represent? Is this a second unidentified signal? This relates to\nour earlier comment that the autonomy and/or non-autonomy of action should be clarified (terms\nthat the authors have avoided, but seem to be relevant for the interpretation of the data). The\nauthors propose a non-autonomous role for both the NTF and the CTF, expressed in endocardial\ncells, on the myocardium. For the CTF, such a non-autonomous role is unexpected and would be\nlikely to need a second signal that can reach the myocardium. The authors do mention possible\nways in which the endocardial cells could contact the myocardium across the cardiac jelly, but\nthis is not depicted in the diagram.\nThe dotted black arrows were indicating that the CTF is required to determine the fate of\ntrabecular vs non-trabecular cardiomyocytes but the signaling to the myocardium is unknown.\nTo address this point, we have removed the arrows and added to Fig. 7 the information that\n“it has been suggested that endocardial protrusions regulate communication between\nendocardium and myocardium by modulating Nrg/ErbB2 signaling”, which is also stated in the\nIntroduction (page 24, line 576). In addition, we added to the figure legend of Fig. 7: “The\nendocardial cells form protrusions to interact directly with the cardiomyocytes and facilitate CTF\nsignaling to act on the myocardium.”\nThe diagram also does not help to explain what the authors mean by a ‘balance’ between the\nNTF and CTF, which now seems to be a key feature of their model, but is not mentioned in the\nfigure legend.\nThis is described in detail on page 20, line 485. To address this point, we have added the following\ntext to the figure legend of Figure 7: “Our data suggest that the CTF is required to balance NTF-\nmediated signaling preventing overactivation of myocardial Notch signaling.” and to the\ninhibition, which is integral for the myocardial pattern formation (Fig. 7).”.\nIn addition, the Notch signalling in Fig. 7 is shown in the myocardium, whereas in the Introduction\nand Discussion, it is stated that endocardial Notch signalling is important for trabeculation (line\n108, 558–9). The authors do cite work on Notch expression in cardiomyocytes at line 417, but\nthis needs a more detailed description, including known timing of events.\nTo address this point, we have added more information to the Introduction and the Discussion:\n• Page 5, line 107: “. First, endocardial Notch signaling, a critical early regulator of\ntrabeculation29-31, which in zebrafish is activated in the endocardium at 24 hpf and becomes\nrestricted to atrioventricular valves and the outflow tract by 72 hpf32,…”\n• Page 5, line 120: “The first appearance of Notch activity in ventricular cardiomyocytes overlaps\nwith the onset of trabeculation (60 hpf).”\n• page 24, line 578: “and heterogeneous expression of erbb2 in the single layer myocardium\nleads to tension heterogeneity initiating trabeculation, which triggers Notch activity in adjacent\ncardiomyocytes, suppressing erbb2 preventing their delamination64.”\nIn brief, endocardial Notch signaling is an activator of trabeculation, whereas myocardial Notch\nactivity inhibits the delamination of cardiomyocytes.\nAccording to the imaging with Tg(TP1:VenusPest) in Fig. 6, levels of Notch signalling in the\nmyocardium in WTs are normally low – the data here do not match the expression patterns\nsketched in Fig. 7. Some more explanation here, together with timing/embryo stages on the\ndiagram, would be helpful.\nNote, the myocardial Notch activity is only activated in compact wall cardiomyocytes. In addition,\nthe here utilized standard Notch reporter line Tg(TP1:VenusPest) drives the expression of\ndestabilized VenusPEST protein, which has a very short half-life. Thus, only cells with active\nNotch signaling are labelled. This is the reason, why the levels of Notch signaling in the\nmyocardium are low.\nIn our model (Fig. 7), we show Notch activity in the two cardiomyocytes next to a delaminating\ncardiomyocyte. To clarify the timing, we have now added the time periods of compact wall\nformation (48-72 hpf) and trabeculation (72-120 hpf).\n6. Other minor (but important) comments/corrections:\n• Line 70 ‘the membrane’ should be ‘the plasma membrane’\nWe have changed the text accordingly (page 3, line 71).\n• Lines 103–119 – It would help to outline the normal timing of cardiac trabeculation events and\nNotch signalling in the different cell types in this paragraph, so that the reader can relate them to the\nstages used in the experimental work.\nAs requested, we have now expanded the paragraph about cardiac trabeculation in the\nIntroduction providing information on the timing of cardiac trabeculation as well as myocardial and\nendocardial Notch signaling (page 4 line 103).\n• Line 138 – predicting, not resulting\nWe have changed the text accordingly (page 6, line 142).\n• Line 193 – it says 98 amino acids here, but 96 earlier in the MS and on the figure\nThank you for spotting this. We have corrected the text to 96 (page 8, line 197).\n5\n• Lines 208–209 – a reference is needed to support this statement.\nWe have added the reference (page 9, line 214).\n• Line 219 et seq. – it would help to state the corresponding n values here in the text with the %\nvalues, as well as in the figure legend.\nWe have added the information to the main text (page 9 line 224). The n numbers were already\nprovided in the figure legend. In addition, we have provided all n values for all experiments in the\nSource Data.\n• Line 231–232 ‘Gpr126 is required for cardiac chamber development in zebrafish’ - As 1/3 of MZ\nmutants of both genotypes have normal trabeculation, this statement should be qualified here to\nindicate that the phenotype shows incomplete penetrance and variable expressivity.\nTo address this point, we have changed the text to: “These data indicate that maternal\ncontribution of Gpr126 rescues the zygotic loss in around two third of gpr126stl47 mutants,\nindicating incomplete penetrance, and that Gpr126 is required for cardiac chamber development\nin zebrafish.” (page 10, line 238).\n• Line 354-356: Suggest rewording here to highlight the rescue aspects of this experiment, as well\nas describing the data in the order that they appear in the figure, e.g. \"Analysis of the heart\nrevealed that both treatments could partially rescue the multilayering phenotype in MZ gpr126st49\nmutants (Fig. S10b, upper panels). However, both treatments also blocked trabeculation, both in\nMZ gpr126st49 mutant hearts and in WT hearts (Fig. S10b, lower panels).”\nWe thank the reviewer for the suggestion and have replaced the text accordingly (page 15, line\n364).\n• Line 368 – side, rather than site\nWe have changed the text accordingly (page 16, line 380).\n• Line 378 – Fig. S11a, not Fig. 11a\nWe have changed the text accordingly (page 16, line 390).\n• Line 553–4 – give authors and year for the bioRxiv paper, and list full details and link in the\nBibliography, as for other references.\nThis paper is now published in Developmental Cell and we have cited it accordingly (page 24,\nline 568).\n• Line 574 – births\nWe have changed the text accordingly (page 25, line 600).\n• Fig. 1a, Fig. S7a The schematic diagram in Fig. 1a is not drawn to scale, and the term 'partial\nGAIN' is not explained anywhere. The similar diagram of the protein structure in Fig. S7a appears\nto be much more accurate in terms of scale - why not use this as a template to generate the\ndiagram of the tagged protein in Fig. 1a? It would help the reader if both diagrams matched each\nother. Note, however, there are a couple of typos in Fig. S7a - the allele name is st49, not stl49,\nand only 6 TM domains are depicted. The st49 mutation is also shown at a different position\nrelative to the GPS motif than that in Fig. 1a. This should be positioned as accurately as possible\n(and consistently) in both diagrams.\nWe thank the reviewer for their suggestion. We have now modified the schematic diagram in Fig.\n1a using the Fig. S7a gpr126 scheme as a template and have corrected the typos in Fig. S7a.\n6\n• Fig. 1b, e – Position of scale bars does not match between figure and legend.\nWe have placed the scale bars in both Fig1b and 1e and updated the figure legends.\n• Expression of constructs in ARPE-19 cells and BFA treatment. Fig. 1f (stable expression) and\nFig. S2c (transient transfection) – It is tricky to compare these figures as the lanes are presented\nin different orders. The position of bands relative to the size markers is inconsistent (WT bands\nare at or above the 115kDa marker in Fig. 1f, but well below it in Fig. S2c). Also, loading controls\n(e.g. of a constitutively secreted protein) are needed to confirm the reduction seen with BFA (for\nboth figures).\nThank you for spotting this. We have now presented the lanes in both figures in the same order.\nIn addition, we have corrected the position of the size markers.\n• Fig. 3 – order of panels (a, d, b, c, e, f) is very difficult to follow.\nWe have put the panels in order.\n• Fig. 3b and Line 326 - Is the P2Y12 N-terminus peptide the domain labelled P2A in the figure?\nThere is no explanation of this in the figure legend.\nThank you for spotting this lack of information. No, the P2Y12 N-terminus peptide is not the\ndomain labelled P2A in Fig. 3b. The P2A peptide is the self-cleaving peptide present between the\nGpr126 CTF and the tdTomato coding sequences which allows for cleavage right after protein\ntranslation to separate the Gpr126 CTF from the tdTomato fluorescent protein. The P2Y12 N-\nterminus peptide is attached to the HA tag to enhance the localization of the CTF to the\nmembrane.\nTo address this point, we have changed the figure legend of Fig. 3b to: “Schematic of the\nconstruct used in Tg(fli1a:gpr126CTF-p2a-tdTomato). The p2a peptide allows for cleavage right\nafter protein translation separating Gpr126 CTF and tdTomato fluorescent protein. The P2Y12 N-\nterminus peptide (blue line between HA tag and GPS) is fused to the HA tag to facilitate\nmembrane localization of Gpr126 CTF.”\n• Fig. 4, 5a schematics – it would help to include the endocardial layer in these diagrams.\nAs requested, we have added the endocardial layer in the schematics in Fig. 4 and 5a.\n• Fig. 5A schematics, Fig. S6c – These figures will be difficult to interpret for anyone with a red-\ngreen colour vision deficiency.\nWe have changed the colors to be easily interpreted by people with color vision deficiencies.\n• Fig. S6 – the data are not presented in the order they are described in the text. It would be\neasiest to follow if the mosaic WT data were presented first in the figure, following the order of\nthe description in the text.\nActually, the data are presented in the figure as they are described in the text. We first describe\nthe stable overexpression of NTFΔGPS in WT and gpr126stl47 larvae (Fig S6a, b) and then describe\nthe mosaic WT data (Figure S6c).\n• Fig. S6b legend - should say ‘Quantification of a’ (not b).\nWe have changed the text according to the changes made to the figure (new quantification).\n• Fig. 6 – title and legend to figure: Notch should have an upper case N\nThank you. We have changed the text accordingly.\n• Fig. S10 – the title for this figure says that ‘Forskolin and IBMX treatment impairs trabeculation\nin … MZ gpr126st49 hearts’. But trabeculation in these mutants is impaired anyway (Fig. 2a), so\n7\nthe title here is not an accurate reflection of the findings of the experiment. The text (line 355)\ninstead says that the treatment rescues multilayering. Please clarify.\nThank you for spotting this. We have changed the title to “Forskolin and IBMX treatment rescues\nmultilayering in MZ gpr126st49 hearts but impairs trabeculation in WT hearts.”\n• Fig. S10a - there is some developmental delay in the treated embryos in this figure (head shape,\njaw development and remaining yolk volume). This could account for some of the differences\nseen in the ears and hearts as well.\nPlease note, WT and mutant embryos were treated with forskolin (2x 6 h pulses, at 60-66 hpf and\n84-90 hpf) at 25 µM or IBMX (3-isobutyl-1-methylxanthine) (continuous treatment, 60-96 hpf at\n125 µM in 1x E3 medium with 0.003% PTU. Thus, an effect on development should be limited.\n• Fig. S13a – as membranes are not imaged here, it is difficult to tell whether or not there is any\nmultilayering phenotype in the WT hearts. As mosaic expression does result in multilayering (Fig.\nS6c), it is important to show whether there is any multilayering present, in addition to the lack of\nNotch activation.\nThis point is addressed by the changes made to Fig. S6. We have added a representative\nconfocal image of WT hearts with stable overexpression of NTFΔGPS in the myocardium\n((Tg(myl7:gpr126NTF-p2a-tdTomato)) crossed with the cardiomyocyte membrane reporter line\nTg(myl7:EGFP-hsa.HRAS) and quantified trabeculation phenotypes. These data show that\nmyocardial NTFΔGPS overexpression of in WT hearts does not cause multilayering.\n8"}, "author_response": ""}]

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