Sulfatase modifying factors control the timing of zebrafish convergence and extension morphogenesis

Domain: biology
Published: 2026-03-31
Editorial outcome: minor_revision

Abstract
Convergence and extension (C&E) cell movements that elongate the primary embryonic axis are precisely timed during vertebrate gastrulation, but mechanisms controlling their onset remain unknown. Using zebrafish embryonic explants that recapitulate C&E and its timing, we identified sulfatase modifying factor 2 (sumf2) as a candidate trigger gene for C&E onset. sumf2 and its paralog sumf1 encode negative and positive sulfatase regulators, respectively, whose expression levels invert and increase heparan sulfate sulfation during gastrulation. Overexpressing sumf1 or sumf2 causes delayed or precocious C&E, respectively, whereas their loss shifts C&E timing in the opposite direction. We identified Sulf1, a modifier of heparan sulfate proteoglycans (HSPGs), as their key downstream effector and found that altering heparan sulfate sulfation levels shifts C&E onset and suppresses sumf1 and sumf2 mutant phenotypes. This work supports a model in which sumf2 expression reduces sulfatase activity, rewriting HSPG sulfation patterns to promote the onset of C&E morphogenesis.

Introduction
Morphogenetic cell movements must be coordinated not only in space, but also in time to properly shape the embryonic body plan. Indeed, changes in the timing of developmental events, termed heterochrony, can underlie malformations in individuals and fuel evolutionary change in populations (1, 2). One striking example of morphogenetic timing is the onset of gastrulation cell movements that form the primordial germ layers and shape them into the nascent embryonic axes. In many species, multiple gastrulation movements occur simultaneously and/or in rapid succession, such that the absolute timing of one process is necessary to preserve its timing relative to the others. For example, many teleost fish and amphibian embryos exhibit epiboly (which thins and spreads the epiblast), internalization (which brings mesoderm and endoderm germ layers inside the embryo), and convergence & extension (C&E) (which elongate the anteroposterior axis) movements simultaneously. The onset of epiboly in zebrafish is thought to result from a rapid fluidization of the epiblast, a consequence of cell rounding during meta-synchronous cell cleavages (3, 4). Initial internalization of mesoderm and endoderm cells may be triggered by a threshold level of Nodal signaling activity (5), which promotes cell protrusions that un-jam cells to enable them to ingress at the margin (6) or ectopically (7). Once leader cells ingress at the margin, followers in adjacent spatial domains subsequently internalize in a temporally ordered fashion according to their expression of hox genes (8), similar to a mechanism reported during chick gastrulation (9). However, mechanisms controlling the timing of C&E morphogenesis remain elusive.

C&E movements simultaneously narrow and elongate embryonic tissues via polarized cell rearrangements, providing the major driving force of anteroposterior (AP) axis extension and, in many species, neural tube closure (10-13). C&E are driven by polarized cell behaviors including mediolateral (ML) cell elongation and alignment, ML-biased cell protrusions, and polarized contraction of cell interfaces by which cells exchange neighbors to form a longer and narrower array (14, 15). Vertebrate C&E movements and/or their underlying ML intercalation behaviors often begin at mid-gastrulation (16-18). In zebrafish, this is marked by a switch in the trajectory of cell movements toward the embryo’s dorsal side at ~75% epiboly (~8 hours post fertilization (hpf)) (18, 19). Evidence suggests that these movements do not result from chemotaxis (20), but rather from spatially restricted cell-cell interactions whose domains are defined by morphogen signaling gradients (21-23).

The signature ML polarity of vertebrate C&E behaviors is under the control of planar cell polarity (PCP) signaling, which orients cell behaviors with respect to the embryonic axes (24, 25). This results from the polarized membrane localization of core PCP signaling components (26-29), which directly precedes the start of C&E (22, 30). The precision and coordination of these events imply the existence of a timing cue that determines the onset of C&E cell behaviors. Several morphogen signaling pathways are essential for C&E, with BMP preventing C&E of the ventral mesoderm (21, 31, 32) and Nodal and FGF signaling promoting C&E dorsally (22, 33-37). However, all these pathways are active hours before C&E begins at mid-gastrulation, suggesting that their activity might not be directly responsible for the timing of C&E onset. Indeed, although Activin (which signals through the Nodal pathway) was sufficient to induce axial mesoderm exhibiting C&E in Xenopus animal cap explants, the timing of C&E remained constant regardless of when Activin was applied (38). This suggests that the timing mechanism functions in parallel with known C&E regulators, but its molecular basis is unknown.

Mechanisms controlling the timing of other early embryonic events have been extensively studied, including early cell cycles and zygotic genome activation (ZGA). For example, early cell cleavages are controlled by cyclical expression and degradation of maternally expressed Cyclins (39). After a set number of cell divisions, ZGA is triggered by a threshold nuclear/cytoplasmic (N/C) ratio (40, 41) (41-43), which is thought to function through titration of histones and other nuclear factors (42-45). In amphibian embryos, the timing of gastrulation was linked not to fertilization or ZGA, but to the first embryonic cleavage (46, 47), and this timing is reportedly regulated predominantly by cytoplasmic factors (46, 48-50). However, the onset of gastrulation morphogenesis is unchanged in embryos with varying cell size and cell cycle length (50-53), making mechanisms that count cell cycles or measure N/C ratios unlikely regulators of C&E timing. Zebrafish gastrulation morphogenesis requires zygotic transcription (54), but its timing is uncoupled from that of ZGA, instead supporting a model by which new gene expression triggers C&E at a specific time.

Using a reductive zebrafish embryonic explant model in which C&E is isolated from the other gastrulation cell movements, we determined that new transcription is required at gastrulation onset for C&E to occur, and that sulfatase modifying factor 2 (sumf2) is initially expressed during this critical window in both explants and intact embryos. sumf1 and sumf2 encode Formylglycine Generating Enzyme (FGE) (55-58) and its antagonist and paralog pFGE (59-61), respectively, a pair whose balance determines the activity of every sulfatase enzyme in the body (62-65). sumf1 is maternally expressed, and its transcript abundance drops just as sumf2 is expressed at gastrulation onset, inverting sumf1/sumf2 levels and altering sulfation in the embryo. We show that overexpression of sumf1 and sumf2 causes delayed or precocious C&E onset, respectively, in both explants and embryos, whereas loss of sumf1 and sumf2 function shifts C&E timing in the opposite direction. We further identified Sulf1 as a key sulfatase by which FGE (sumf1) and pFGE (sumf2) modify C&E timing. Reduced or increased levels of sulfated heparan sulfate, the predominant substrate of Sulf1 (66-68), similarly shift C&E onset and suppress sumf1 and sumf2 mutant phenotypes, indicating that heparan sulfate proteoglycans (HSPGs) mediate C&E timing downstream of sulfatase modifiers. Together, these data support a model in which sumf2 expression at gastrulation onset reduces sulfatase activity, triggering a switch in HSPG sulfation patterns to promote and/or permit C&E morphogenesis.

Results
Ex vivo convergence & extension requires new transcription at gastrulation onset
Blocking transcription prior to ZGA prevents all gastrulation morphogenesis in zebrafish embryos (54). However, it was not determined when new transcription is required for gastrulation cell movements nor the specific genes that are required. We previously showed that otherwise naïve zebrafish embryonic explants expressing Nodal signaling components recapitulate both C&E behaviors and their precise timing in culture, while lacking other gastrulation movements (36), making them an ideal system to study C&E timing. To identify the temporal window of gene expression required for C&E, we treated explants expressing the constitutively active Nodal receptor Acvr1b* (also known as TARAM-A-D (69)) with a time-course of the irreversible transcription inhibitor Triptolide (70). We then assessed their ability to undergo C&E by measuring their extension when sibling embryos reached the 4-somite stage (12 hpf) (Fig. 1A). acvr1b* explants treated when intact siblings reached 50% epiboly (5.3 hpf) failed entirely to extend, while those treated shortly thereafter at shield stage (6 hpf, gastrulation onset) or later were able to extend, albeit incompletely (Fig. 1B, C).

To rule out the possibility that Triptolide interferes with C&E by disrupting Nodal signaling dynamics, we repeated the experiment by activating Nodal under two additional experimental conditions. First, we expressed the Nodal ligand Ndr2/Cyc in explants, which display delayed Nodal activation compared to acvr1b* explants (71). Second, we expressed acvr1b* in explants generated from Nodal signaling-deficient maternal-zygotic (MZ)oep-/- embryos (72), which exhibit continued signaling in the absence of feed-forward synthesis of new Nodal ligands. Both conditions responded identically to wildtype (WT) acvr1b* explants, indicating that the requirement for transcription is independent of Nodal signaling per se (Supplementary Fig. 1A-B, D-E). This is consistent with Activin-induced Xenopus animal cap explants extending on time regardless of when in the competence window Activin was added (38). Finally, WT acvr1b* explants treated with the reversible transcription inhibitor Flavopiridol (73) at 50% epiboly and washed out at 90% epiboly (5.3–9 hpf) similarly failed to extend (Supplementary Fig. 1C, F), demonstrating that the stage of inhibition, rather than its duration, was responsible for failed C&E. These results indicate that new gene expression starting at gastrulation onset is required for ex vivo C&E movements, independent of Nodal signaling dynamics.

sumf1 and sumf2 transcript levels invert at gastrulation onset
Having established that transcription at gastrulation onset is crucial for C&E, we next identified genes whose expression is upregulated during this time window. We previously profiled transcription in ndr2, acvr1b*, and uninjected control explants by bulk RNA sequencing at seven developmental stages spanning gastrulation (71) (Fig. 1D). We examined these data for candidate trigger genes whose expression remained unchanged between sphere and 50% epiboly stages but then increased significantly at shield stage and beyond, corresponding to our experimentally determined transcriptional window. Because the timing of C&E onset is independent of Nodal signaling dynamics, we only considered genes with this temporal expression profile in explants of all three conditions, including uninjected controls. This analysis yielded a list of 180 genes, 129 of which also increased significantly between 50% epiboly and shield stages in intact zebrafish gastrulae (74) (Supplementary Data 1).

To focus our efforts on genes with the highest likelihood of functional relevance, we further selected for those exhibiting substantial increases in expression and overall expression levels. To this end, we selected an arbitrary cut-off of a 50% increase (Δ > 0.5x) in transcript level from 50% epiboly to shield stage, and an expression level of at least 5 transcripts per million (TPM) at shield stage in intact embryos. When narrowed down this way, we were left with 28 candidate genes (Fig. 1E) (Supplementary Data 1) that we hypothesized may trigger C&E cell behaviors when expressed at gastrulation onset. They encode proteins with a wide variety of biological functions, including transcription and chromatin factors (atf6, cramp1, gtf3c3, six7, tcf7l1a), cytoskeletal regulation (tuba1c, wdr35), cell signaling (adgrl2a, efnb3a, gli2a, mdkb, smad6b), biosynthetic processes (azin1a, dgat1b, lcat, xylt1), RNA binding (ddx31, nova2, pspc1), and others (Supplementary Data 1).

Among our candidate genes was sulfatase modifying factor 2 (sumf2). sumf2 encodes pFGE, a paralog and antagonist of Formylglycine Generating Enzyme (FGE) (65, 75), encoded by sulfatase modifying factor 1 (sumf1). As its name implies, FGE converts cysteine residues to formylglycine, a rare but essential post-translational modification required within the active site of all sulfatase enzymes for their activity (75). Sulfatases catalyze the removal of sulfate groups from a variety of substrates, including lipids, steroids, and glycosaminoglycans, modifying their biological activity (76, 77). Both pFGE (encoded by sumf2) and FGE (encoded by sumf1) are ER-resident proteins that share high amino acid sequence similarity, with the main distinction that pFGE lacks enzymatic activity (55, 75). Evidence suggests that pFGE binds FGE alone or in complex with its sulfatase substrates (59, 65), but it is not clear precisely how pFGE (sumf2) antagonizes FGE (sumf1) function. Zebrafish FGE (sumf1) shares a high degree of amino acid sequence similarity with its human and mouse homologs, including conservation of two essential cysteine residues in the FGE active site (Supplementary Fig. 2A). pFGE (sumf2) homologs also showed strong sequence conservation and all lacked the FGE active site (Supplementary Fig. 2AB). Zebrafish FGE and pFGE proteins share 46% amino acid identity and 62% similarity (Supplementary Fig. 2C), comparable to their human homologs (48% identity and 62% similarity) (75).

Although other candidate genes exhibited more dramatic expression increases at gastrulation onset, we selected sumf2 for further study because its function during vertebrate development has never been explored, and its intriguing complementary temporal expression patterns with sumf1 during gastrulation. In zebrafish embryos and explants, sumf1 is maternally expressed but exhibits a sharp reduction in transcript levels just prior to gastrulation onset, coinciding with increased sumf2 (74) (Fig. 1F, Supplementary Fig. 2D). Because sumf2 and sumf1 encode a negative and positive regulator of sulfatase activity, respectively, we hypothesized that this peak in the sumf2/sumf1 ratio at gastrulation onset (6hpf) (Fig. 1G) reduces sulfatase activity and thus increases sulfation of their substrates to trigger C&E movements.

Excess or loss of sulfatase modifying factors causes C&E defects in zebrafish gastrulae
To investigate the role of sulfatase modifying factors in C&E morphogenesis, we employed both gain- and loss-of-function approaches and performed morphometric analysis at the end of gastrulation (tailbud stage, 10 hpf). We first overexpressed sumf1 or sumf2 by mRNA injection into single-cell WT embryos. sumf1 overexpressing (OE) gastrulae exhibited a significant reduction in anteroposterior (AP) axis length and wider notochords, characteristic of C&E defects, and sumf2 OE embryos showed similar but milder phenotypes (Fig. 2). Next, we examined gastrulae of the recently characterized sumf1la015919Tg mutant line (hereafter sumf1-/-) harboring a viral insertion in exon 1 that abolishes FGE activity (78). Homozygous mutant embryos survived to adulthood (78), enabling examination of MZsumf1-/- gastrulae, which also exhibited reduced AP axis length and wider notochords indicative of impaired C&E (Fig. 2).

Because no existing sumf2 mutant line was available, we used CRISPR to generate a 7 bp insertion in exon 2 of sumf2 (named bcm126), resulting in a premature stop codon (Supplementary Fig. 3AA). These mutants also survived to adulthood, enabling us to maintain the line as homozygotes and analyze MZsumf2bcm126/bcm126 embryos (hereafter MZsumf2-/-). MZsumf2-/- embryos showed a substantial reduction in sumf2 transcript levels by RT-qPCR (Supplementary Fig. 3C), confirming this as a strong loss-of-function allele. Expression of the closely related sumf1 was unchanged at early and mid-gastrulation in MZsumf2-/- compared to WT embryos (Supplementary Fig. 3D), suggesting that transcriptional adaptation (79) is not induced by this mutation. Approximately 10% of MZsumf2-/- larvae displayed a tail-curled-up phenotype at 48 hpf and a variable number of MZsumf2-/- adults were undersized and scoliotic (Supplementary Fig. 3E, F), but the remaining larvae and adults had no obvious phenotypes. Although MZsumf2-/- mutant gastrulae did not exhibit C&E defects, sumf1 OE in this background produced more severe C&E phenotypes than in WT embryos (Fig. 2), supporting an antagonistic role for pFGE (sumf2) on FGE (sumf1).

To assess whether other gastrulation movements were affected by loss of sumf1 or sumf2, we imaged live MZsumf1-/- and MZsumf2-/- embryos throughout gastrulation and found no significant epiboly defects or delays, with blastopore closure occurring at similar times as in WT embryos (Supplementary Fig. 4A). We also analyzed mesendoderm internalization by quantifying the spread of sox17-expressing endoderm cells, which is coupled to mesodermal migration towards the animal pole (80). MZsumf2-/- gastrulae exhibited normal endoderm spreading, and although MZsumf1-/- gastrulae exhibited reduced spreading at mid-gastrulation, they were not different from WT by late gastrulation (Supplementary Fig. 4B-C). Together, these results demonstrate that altering levels of sumf1 or/and sumf2 specifically disrupts C&E without affecting other gastrulation movements in vivo.

sumf1 and sum2 levels modify the timing of C&E ex vivo
Next, we examined the effects of altered sumf1 and sumf2 levels in zebrafish embryonic explants, in which C&E is isolated from other concurrent morphogenetic processes. Surprisingly, neither OE nor deficiency of sumf1 or sumf2 impaired the extension of acvr1b* explants at the equivalent of 4-somite stage (Supplementary Fig. 5A, B), indicating that they are dispensable for C&E per se. To assess whether sumf1 or sumf2 levels instead influenced the timing of C&E morphogenesis, we acquired time-lapse recordings of acvr1b* explants to precisely determine the onset of morphological extension. Extension onset was defined as the time point when a visible tip first emerged from an initially round explant (Fig. 3A, Supplementary Movie 1). Importantly, all explants were staged relative to blastopore closure in age-matched intact embryos of the same genetic background mounted in the same dish. As previously described (36), WT acvr1b⁎ explants (WT control) and controls co-injected with superfold-GFP mRNA (sf-GFP OE) initiated extension around 8hpf (Fig. 3A, B). Strikingly, extension began significantly later upon sumf1 OE and significantly earlier upon sumf2 OE (Fig. 3B). Co-overexpression of sumf1 and sumf2 restored the normal time of extension onset (Fig. 3B), demonstrating that the balance between these factors (rather than their absolute levels) determines the time of C&E onset.

In the converse loss-of-function experiments, we found that extension onset was precocious in MZsumf1-/- explants and delayed in MZsumf2-/- explants (Fig. 3A, B), consistent with - but opposite to - sumf1 and sumf2 OE phenotypes. MZsumf1-/-; MZsumf2-/- double mutant explants phenocopied the precocious extension of MZsumf1-/- single mutants (Fig. 3B), consistent with pFGE (sumf2) modulating C&E timing via its role as an antagonist of FGE (sumf1). Notably, explants injected with a morpholino oligonucleotide (MO) against the core PCP component vangl2, which does not exhibit a trigger-like temporal expression pattern, exhibited severely impaired C&E morphogenesis both in vivo and ex vivo (Supplementary Fig. 5A-D) (36), but showed no change in the time of extension onset (Supplementary Fig. 5E). Together, these results demonstrate that the ratio of sumf1/sumf2 governs the timing of explant extension, distinct from general C&E defects, with higher sumf1 delaying and higher sumf2 advancing C&E onset.

To further characterize the dynamics of extension in MZsumf1-/- and MZsumf2-/- explants, we performed automated segmentation-based analysis of time-lapse movies and quantified changes in explant roundness over time (see Methods; Fig. 3C). Because explant extension is highly variable and not every explant extends even in control conditions (Supplementary Fig. 5A, B), only explants that reached roundness values below 0.5 (roundness of 1 = perfect circle) by 12 hpf were included in this analysis. While uninjected explants maintained roundness values close to 1 throughout the gastrulation period, the roundness of acvr1b* explants from all analyzed genotypes decreased over time (Fig. 3D). Sigmoidal curves were fitted to the roundness changes of each group, and the V50 (the time at which explants reached 50% of the total roundness change) and slope (reflecting the rate of shape change) were compared using an extra sum-of-squares F test. Significant differences in V50 were observed between groups (p < 0.0001), along with smaller but significant differences in the slope (p < 0.05), indicating changes to both the timing and pace of explant extension upon loss of sumf2 or sumf1. Consistent with previous results, MZsumf1-/- explants began extension precociously, as evidenced by a left-shifted curve and lower V50 compared with WT Controls, but their pace of extension was slower, as indicated by a shallower slope. The opposite was true of MZsumf2-/- explants, whose extension was delayed (higher V50) but faster (steeper slope). This likely explains why explants with different C&E onset ultimately reach similar degrees of elongation (Supplementary Fig. 5A, B).

sumf1 and sum2 levels modify the timing of C&E in vivo
To determine whether sumf1 and sumf2 similarly modify the timing of C&E cell movements in vivo, we acquired confocal time-lapse movies of H2B-scarlet-labeled gastrulae from 6.5 – 11 hpf and performed automated nuclear tracking (see Methods; Fig. 3E, Supplementary Movie 2). To ensure accurate stage matching across experiments, all embryos were staged relative to formation of the second somite, which was readily visible at the end of each movie. Convergence movements were quantified as the mediolateral (ML) displacement of dorsal and lateral cells over time (time frame = 5 minutes) (Fig. 3F–K, Supplementary Fig. 6). Because convergence movements are reduced within the embryonic midline where extension is more prominent (81), cells within 100 m of the dorsal midline were excluded from our analysis. In WT control embryos, we observed an upward inflection of ML displacement beginning around 7.75 - 8 hpf, marking the onset of convergence movements (consistent with previous findings (18, 19)), which continued to increase gradually thereafter (Fig. 3F–K, Supplementary Fig. 6).

Upon sumf1 OE, baseline convergence speed was reduced and, although convergence movements began at the same time as controls, they remained substantially slower (Fig. 3F). sumf2 OE embryos, on the other hand, had similar baseline convergence speeds as controls but with an earlier inflection point (Fig. 3G). For much of gastrulation, these movements were also faster than those of controls, indicating a likely change in both onset and pace of convergence movements. As in explants, co-OE of both sumf1 and sumf2 restored convergence movements to control timing and speeds (Fig. 3H).

Also as in explants, in vivo cell tracking of MZsumf1-/- and MZsumf2-/- mutants revealed opposite effects to their overexpression. Convergence onset in MZsumf1-/- embryos trended earlier than controls and baseline speed trended higher, although these movements plateaued later in gastrulation (Figure 3I). Conversely, baseline convergence in MZsumf2-/- mutants was slower than controls with a later inflection point, indicating delayed convergence (Fig. 3J). MZsumf1-/-; MZsumf2-/- embryos displayed an earlier onset of convergence movements (Fig. 3K) as MZsumf1-/- single mutants did, as expected if pFGE (sumf2) functions upstream of FGE (sumf1). Additional analysis revealed small increases in the persistence of cell movements within MZsumf1-/- gastrulae, but not in other sumf1 and/or sumf2 gain- or loss-of-function conditions (Supplementary Fig. 7A), while overall cellular speed remained unchanged in all groups (Supplementary Fig. 7B). Together with our ex vivo results, these data support a model in which pFGE (sumf2) counters the activity of FGE (sumf1) to control the onset and pace of C&E cell movements during gastrulation.

Sulfatase modifiers govern C&E via the extracellular sulfatase Sulf1
Because FGE (sumf1) and pFGE (sumf2) together regulate sulfatase activity levels, we hypothesized that high sumf2/sumf1 ratios trigger C&E onset by reducing the activity of one or more key sulfatases. Indeed, increased sulfatase activity was shown to disrupt gastrulation morphogenesis in Xenopus and sea urchin (64, 82-85). The zebrafish genome encodes 17 sulfatases, 11 of which are expressed during peri-gastrulation stages (74) (Supplementary Fig. 8A). To identify which of these mediates the effect of sumf1/sumf2 on C&E timing, we overexpressed each of these 11 sulfatases by mRNA injection and performed morphometric analysis at tailbud stage and scored axis phenotypes at 24 hpf. Notably, three of them — the heparan sulfate endosulfatases sulf1 and sulf2a, and the chondroitin sulfatase arsb — produced severe AP axis extension defects and increased notochord width, consistent with C&E defects (Supplementary Fig. 8B-E). Unlike sumf1 and sumf2 OE, overexpression of these three sulfatases also prevented full extension of acvr1b⁎ explants at the equivalent of 4-somite stage (Supplementary Fig. 8F, G). However, only sulf1 OE significantly delayed acvr1b* explant extension, while the other two had no effect on C&E timing (Fig. 4A, Supplementary Movie 3). Thus, of the 17 zebrafish sulfatases, only Sulf1 was found to affect both C&E morphogenesis and its timing.

To further investigate the role of Sulf1 in zebrafish gastrulation morphogenesis, we examined C&E in MZsulf1sjr9/sjr9 full-locus deletion mutants (Gursimran Kaur Bajwa , Gülsüm Kayman Kürekçi, and Rubén Marín-Juez, manuscript under consideration) (hereafter MZsulf1-/-). MZsulf1-/- embryos exhibited reduced AP axis length at tailbud stage (Fig. 4B-D) and MZsulf1-/- acvr1b* explants showed impaired extension (Supplementary Fig. 8F, G), indicating that both gain and loss of Sulf1 reduces C&E. Strikingly, MZsulf1-/- acvr1b* explants exhibited precocious extension (Fig. 4A, Supplementary Movie 3), similar to MZsumf1-/- and sumf2 OE explants (but opposite to sulf1 OE). We next quantified the effect of Sulf1 levels on C&E onset in vivo (Supplementary Movie 4). Convergence speed was drastically reduced upon sulf1 OE (Fig. 4E, Supplementary Fig. 6) while MZsulf1-/- gastrulae exhibited earlier and enhanced convergence movements compared to WT controls (Fig. 4F, Supplementary Fig. 6). No changes in persistence or cellular speed were detected upon loss or overexpression of sulf1 (Supplementary Fig. 7). These results highlight Sulf1 as a strong candidate for the key sulfatase controlling C&E onset.

To examine the relationship between Sulf1 and FGE (sumf1), we generated acvr1b* explants co-injected with low doses of each mRNA alone or in combination. The combination of otherwise sub-phenotypic doses of sumf1 and sulf1 synergistically delayed and reduced explant extension (Fig. 4G, Supplementary Fig. 8F, G), consistent with FGE (sumf1) enhancing Sulf1 activity. Finally, we tested whether sumf1 OE could delay explant extension in the absence of sulf1. We found that although sumf1 OE significantly delayed extension onset in WT explants, it had no effect on the onset of extension in MZsulf1-/- explants, which remained precocious (Fig. 4G). Together, these results implicate Sulf1 as the primary sulfatase through which FGE (sumf1) and pFGE (sumf2) govern the timing of C&E morphogenesis.

Heparan sulfate sulfation patterns change with C&E onset and are regulated by sumf1/sumf2
Sulf1 is an extracellular sulfatase that removes 6-O sulfate groups from heparan sulfate (HS) polysaccharides, with preference towards highly sulfated domains containing IdoA2S-GlcNS6S (D2S6) and, to a lesser degree, GlcA-GlcNS6S (D0S6) disaccharides (67). We would therefore expect the inversion of FGE (sumf1) and pFGE (sumf2) levels at the beginning of gastrulation to decrease Sulf1 activity, leading to increased sulfated HS levels at late gastrulation. Indeed, Alcian Blue staining (at pH 1) (86) for overall glycosaminoglycan (GAG) sulfation increased dramatically from early (50% epiboly) to late (90% epiboly) gastrulation stages, indicating increased embryo-wide GAG sulfation. Treatment with sodium chlorate, which prevents the formation of the sulfate donor 3’-phosphoadenosine 5’-phosphosulfate (PAPS) (87, 88), drastically diminished Alcian Blue staining (Fig. 5A), confirming its specificity.

Because Alcian Blue non-specifically stains all negatively charged GAGs, we next isolated HS from WT zebrafish at early and late gastrulation stages by anion-exchange chromatography and quantified levels of HS disaccharides by hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry (HILIC-Q-TOF-MS), using published methods (89). From this analysis, we found that total HS levels increased significantly between 50% and 80% epiboly stages (Fig. 5B). Although we observed a slight decrease in the percentage of D0S6 disaccharides at late compared to early gastrulation stages (Fig. 5C), there was no significant change in the total abundance of these 6-O sulfated disaccharides (reflecting the observed increase in total HS levels) (Fig. 5D). In contrast, while the relative proportion of D2S6 disaccharides remained unchanged during gastrulation (Fig. 5C), their absolute abundance significantly increased (Fig. 5D). These results indicate that abundance of the preferred Sulf1 substrate D2S6 increases during gastrulation, consistent with our hypothesis that the peak sumf2/sumf1 ratio at gastrulation onset leads to reduced Sulf1 activity.

We next investigated how loss of sulfatase modifying factors affects HS disaccharide composition at late gastrulation (80% epiboly, 8.5hpf). As expected for loss of the key sulfatase activator FGE (sumf1), the proportion of unsulfated disaccharides (D0A0) was reduced while proportions of the sulfated disaccharides D0S6 and D2S6 were increased in MZsumf1-/- compared with WT (TUwt) gastrulae (Fig. 5E). We also observed changes in HS disaccharide composition upon loss of pFGE (sumf2). Strikingly, the proportion of D2S6 disaccharides was reduced in MZsumf2-/- compared with WT (ABwt) gastrulae (Fig. 5F), consistent with increased sulfatase activity in these mutants. The proportion of D0S6 disaccharides, on the other hand, was increased in MZsumf2 mutants, which may reflect compensatory activity of sulfotransferases or other HS modifiers. Importantly, loss of FGE (sumf1) and pFGE (sumf2) produced opposite effects on the predominant substrate of Sulf1 during gastrulation, supporting their roles as positive and negative regulators, respectively, of Sulf1 activity. The observation that D2S6 disaccharides also increased throughout gastrulation in WT embryos suggests an important role for HS sulfation in C&E morphogenesis.

HSPGs mediate the effects of sulfatase modifiers on C&E timing
Sulfatases modify a large number of biological substrates, including lipids, steroids, and multiple GAGs (90). To test whether sulfated heparan sulfate proteoglycans (HSPGs) influence C&E morphogenesis and its timing, we experimentally decreased and increased sulfated HS levels within embryos and explants. To inhibit overall sulfation, including of GAGs like HS, we treated dome stage embryos (4.3 hpf) with two different doses of sodium chlorate (200 mM and 50 mM), which we previously showed reduced Alcian Blue staining in zebrafish gastrulae (Fig. 5A). We observed severe AP axis shortening and cyclopia in 24 hpf embryos treated with the higher dose, and milder axis defects at the lower dose (Fig. 6A). A dose-dependent effect on C&E was also observed at tailbud stage (Fig. 6B-D), demonstrating that sulfation is required for proper C&E morphogenesis (as previously reported (91-93)). Although explants treated with the higher dose were not viable, treatment with the lower dose of sodium chlorate dramatically reduced extension of acvr1b⁎ explants (Supplementary Fig. 9A, B). Sodium chlorate treatment also significantly delayed extension onset in both WT control and, importantly, in MZsumf1-/- explants in which extension otherwise occurred early (Fig. 6E, Supplementary Fig. 9C). This finding strongly suggests that loss of sumf1 induces precocious C&E by enhancing sulfation.

In a complementary experiment, we increased levels of sulfated HS by injecting the extracellular space of 256-cell stage embryos with 10 ng/ml HS (or water as a control) (Fig. 6F). As previously reported (94), HS-injected embryos exhibited dorsalized phenotypes (Supplementary Fig. 9D), likely reflecting increased FGF signaling (95). Notably, while increased HS did not affect the extension of acvr1b⁎ explants at the equivalent of 4-somite stage (Supplementary Fig. 9A, B), their extension onset was significantly earlier than control explants (Fig. 6G, Supplementary Fig. 9EI). HS injection also advanced extension onset in MZsumf2-/- explants (Fig. 6G, Supplementary Fig. 9E), indicating that increased HS levels are sufficient to trigger precocious C&E ex vivo and can override the delay caused by sumf2 deficiency. Taken together, these results support a model in which sumf2 expression at gastrulation triggers the timely onset of C&E cell movements by reducing Sulf1 activity and consequently increasing sulfation of HSPGs.

Methods
All zebrafish experiments were performed in compliance with the Baylor College of Medicine Institutional Animal Care and Use Committee (IACUC), Animal Welfare Assurance number D16-00475. All use of recombinant nucleic acids was performed according to a protocol approved by the Baylor College of Medicine Institutional Biosafety Committee.

Zebrafish
Adult zebrafish were maintained following established protocols (129) in compliance with the Baylor College of Medicine IACUC, Animal Welfare Assurance number D16-00475. Embryos were obtained through natural mating, and staging was based on established morphology (130). Fish were chosen from their home tank to be crossed at random, and embryos were randomly chosen from the clutch for injection and inclusion in experiments. WT and mutant embryos were collected in parallel in a period not longer than 10–15 min to minimize developmental differences and raised in egg water at 28.5C under identical conditions. Experiments on WT embryos were conducted in either the AB or TU background, depending on the background of the mutant line used, which were obtained from the Zebrafish International Resource Center (ZIRC) via the Baylor College of Medicine zebrafish facility. The mutant lines used were sumf1la015919Tg (obtained from ZIRC, ZFIN ID: ZDBALT-120806-11568), sulf1sjr9 (obtained from the University of Montreal)(Gursimran Kaur Bajwa , Gülsüm Kayman Kürekçi, and Rubén Marín-Juez, manuscript under consideration), sumf2bcm126 (this study, described further below), and oeptz257 (103) (obtained from Washington University School of Medicine). oep-/- embryos were rescued to viability by injection of 50 pg of oep mRNA (131) and raised to adulthood, then intercrossed to generate MZoep-/- embryos for explantation. MZsumf1-/-; MZsumf2-/- double mutants were generated by crossing single homozygous mutants to obtain double heterozygotes, which were subsequently incrossed.

sumf2bcm126 generation and genotyping
sumf2 zebrafish mutants were generated on the AB background using the CRISPR–Cas9 system. A single guide (sg)RNA targeting exon 2 of sumf2 (5′-GGATGGAGAATCGCCAACAC-3′) was designed using CRISPRscan (132). sgRNAs were transcribed using T7 RNA polymerase (NEB, M0251S) from DNA templates generated with the forward primer (Fw): gaaattaatacgactcactataGGATGGAGAATCGCCAACACgttttagagctagaaatagc, and the reverse primer (Rv): aaaagcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaac. 1 μl of gRNA was pre-incubated for 10 min at 37 °C with Cas9 protein (NEB, M0646M) and 300mM KCl (as described by (133)). AB WT Embryos were injected at the single-cell stage with 1 nl of the gRNA–Cas9 complex and cutting efficiency was assessed using a T7 endonuclease I assay (134). A PCR fragment encompassing the target sequence was amplified from genomic DNA (gDNA) of individual embryos (using the following primers: Fw: AGATGGTGTTTATTCCTGGTGG, Rv: TCCTCTGATACAAATCCTGGAA) and incubated with T7 endonuclease I (NEB #M0302L) in NEB 10× Buffer 2. Injected F0 embryos were raised and outcrossed to AB WT fish. F1 progeny were genotyped by Sanger sequencing to identify transmitted mutations. A 7 bp insertion in exon 2 of sumf2 (sumf2bcm126), resulting in an early stop codon, was selected (Supplementary Fig. 2A). This mutation introduced an XcmI restriction site, enabling genotyping by digestion of the PCR-amplified target fragment with XcmI (NEB, R0533S) (Supplementary Fig. 2B). Heterozygous F1 carriers were incrossed to produce the F2 generation. Homozygotes F2 fish were raised and incrossed to obtained MZsumf2-/- embryos for experiments.

RT-qPCR
RNA was isolated from 50 pooled WT and MZsumf2-/- embryos at 30% epiboly (4.66 hpf), 75% epiboly (8 hpf), and 24 hpf from three independent clutches. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, 15596018), purified by sodium acetate precipitation, eluted in 10 mM Tris pH7.5 and treated with Turbo DNAase I (Invitrogen, AM2238) at 37C for 30 min. For each sample, 1g of the RNA was reverse-transcribed using iScript Reverse Transcription Supermix for RT-qPCR (Biorad, #1708840). sumf2 specific primers were designed to span exon-exon junctions [Exon 1-2 (pair 1) FW: CACAGTGTCTTGTGCAGCAG, RV: AGTTGGAGTTGGTGACAGGA; Exon 3-5 (pair 2) FW: GGCTGAAACATTTGGCTGGA, RV: GGCATCATTCCAGCTGACCT]. sumf1 specific primers were designed to span exon-exon junctions [Exon 4-5 (pair 1) FW: GGACCAGATTCAACCATACACA, RV: GCTTCTGTAGGAAGTCTGCGTT; Exon 7-9 (pair 2) FW: CTGTATGACATGGTGGGAAATG, RV: GCTGCACACCTGTATCTGTAGC]. rpl13a-1 was used as a housekeeping gene for normalization [FW: TCTGGAGGACTGTAAGAGGTATGC, RV: AGACGCACAATCTTGAGAGCAG]. qPCR was performed in technical triplicates using SsoAdvanced Universal SYBR Green Supermix (Biorad, #1725270). Relative expression levels were calculated using the 2−ΔΔCt method.

Amino acid sequence alignment
Amino acid sequence alignment and visualization of human, mouse and zebrafish FGE (SUMF1) and pFGE (SUMF2) orthologues were performed using Jalview2.11.5.1 (135). Multiple sequence alignment was generated with Clustal Omega, and sequence identity and similarity were calculated using the BLOSUM62 substitution matrix.

Preparation and microinjection of mRNA
All mRNAs were transcribed using the SP6 mMessage mMachine Kit (Fisher Scientific, AM1340) and purified using Bio-Rad Microbiospin columns (Bio-Rad, 7326250). Single-celled embryos were placed in agarose molds (Adaptive Science Tools, I-34) and injected with 0.5-1 nl volumes using pulled glass needles (Fisher Scientific, 50-821-984). Doses of mRNA per embryo were as follows: 0.5 pg acvr1b* (69), 10 pg ndr2 (136), 50-100 pg sumf1, 100 pg sumf2, 500 pg sfGFP (a gift from Dr. Jimann Shin), 500 pg sulf2b, 500 pg sulf2a, 500pg gnsb, 50-500 pg sulf1, 500 pg sgsh, 500 pg arsb, 500 pg gnsa, 500 pg ids, 500 pg galns, 500 pg arsa, 500 pg sts, 100 pg mem-GFP (11), and 100 pg H2B-scarlet (a gift from Dr. Lance Davidson). Templates for sumf1, sumf2, and all sulfatases were generated by Gibson cloning (137) each of their open reading frames (synthesized by Twist Biosciences) into a PJS2 vector linearized with EcoRI. Requests for these reagents can be addressed to the corresponding author. Injection of 2 ng MO4-tri/vangl2 (138) was carried out as for synthetic RNA.

Whole mount in situ hybridization (WISH)
tbxta (brachyury, t), dlx3b, egr2b (krox20), and sox17 antisense riboprobes were transcribed using NEB T7 RNA polymerase (NEB, M0251s) and labeled with digoxigenin (DIG) NTPs (Sigma/Millipore, 11277073910). WISH was performed according to (139). Embryos were fixed overnight in at 4°C 4% PFA in PBS, rinsed in PBS + 10% tween-20 (PBT), and dehydrated into methanol. Following rehydration into PBT, embryos were pre-incubated for at least two hours in hybridization buffer with 50% formamide (in 0.75 M sodium chloride, 75 mM sodium citrate, 0.1% tween 20, 50 mg/mL heparin (Sigma), and 200 mg/mL tRNA) at 70°C, and hybridized overnight at 70°C with antisense probes (1-5 ng/mL) in hybridization buffer. Samples were washed gradually into 2X SSC buffer (0.3 M sodium chloride, 30 mM sodium citrate), and then gradually from SSC to PBT. Samples were blocked at room temperature for several hours in PBT with 2% goat serum and 2 mg/mL bovine serum albumin (BSA), then incubated overnight at 4°C with anti-DIG antibody (Roche #11093274910) at 1:5000 in block. After extensive washes in PBT, embryos were rinsed in staining buffer (PBT +100 mM Tris pH 9.5, 50 mM MgCl2, and 100 mM NaCl) and developed in BM Purple AP substrate (Roche) until the desired staining intensity was achieved. Staining was stopped with 10 mM EDTA in PBT before imaging.

Blastoderm explants
Blastoderm explants were prepared as described by (140). Briefly, uninjected, acvr1b*-, or ndr2-injected embryos were dechorionated at the 256-cell stage using pronase (Roche; 1 ml of 20 mg/ml stock in 15 ml 3× Danieau’s solution). At the 512-cell stage, approximately 1/3 of the most animal blastoderm cells were excised using Dumont #55 forceps (Fisher Scientific, NC9791564) on an agarose-coated plate containing 3× Danieau’s solution. Explants were allowed to heal briefly before being transferred into agarose-coated 6-well plates containing explant medium [Dulbecco’s modified eagle medium with nutrient mixture F-12 (Gibco 11330032) containing 2.5 mM L-glutamine, 15 mM HEPES, 3% newborn calf serum (Invitrogen 26010–066), 50 units/mL penicillin, and 50 mg/mL streptomycin (10,000 U/mL pen-strep at 1:200, Gibco 15140163)]. Explants were incubated at 28.5 °C until sibling embryos from the same genetic background reached the desired developmental stage.

Alcian Blue staining
Embryos were fixed in PFA 4% overnight, extensively washed in PBS, and incubated in Alcian Blue staining solution pH 1 (0.2% Alcian Blue 8 GX (Sigma # A5268), 50% ethanol, 0.1N HCl) for 48 h at room temperature, protected from light. Samples were then gradually rehydrated into PBS, post-fixed in 4% PFA, washed with PBS, and rinsed in 2% KOH. Embryos were cleared through a graded series of glycerol in 2% KOH (20%, 40%, 60%) and stored in 80% glycerol in 2% KOH for imaging and long-term storage.

Transcription inhibitor treatment
1 M Triptolide (Sigma # T3652) or an equivalent volume of DMSO was added to the media of blastoderm explants in agarose-coated 6-well plates at 50% (4.7 hpf), shield (6 hpf), 70% (7.5 hpf) or 80% (8.5 hpf). 0.5 M Flavopiridol (Selleck, S1230) or an equivalent volume of DMSO was added to the media of blastoderm explants at 50% (4.7 hpf) and washed-out twice with 0.3x Danieau solution, before incubation with fresh explant medium.

Sodium Chlorate treatment
Dome stage (4.33 hpf) embryos or blastoderm explants were treated with 200 or 50 mM of Sodium Chlorate (VWR # 7775-09-9) in egg water or explant medium.

Heparan Sulfate injections
Uninjected or acvr1b*-injected embryos were dechorionated at the 64-cell stage using pronase (Roche; 1 mL of 20 mg/mL stock in 15 mL 3× Danieau’s solution). Embryos were transferred to agarose-coated plates containing cubical depressions (Adaptive Science Tools, PT-1) filled with 0.3× Danieau’s solution. At the 256-cell stage, embryos were injected with 2 nL of 5 ng/mL heparan sulfate (Sigma-Aldrich, H7640) or nuclease-free water into the extracellular space.

Microscopy
Live embryos injected with H2B-mScarlet and mem-GFP mRNA were manually dechorionated and mounted in 0.3-0.35% low-melt agarose (Thermo Fisher Scientific, 16520100) in glass-bottomed 35 mm Petri dishes (Fisher Scientific, FB0875711YZ) for imaging using a Nikon ECLIPSE Ti2 inverted confocal microscope equipped with a Yokogawa W1 spinning disk unit, PFS4 camera, and 405/488/561 nm lasers (emission filters: 455/50, 525/36, 605/52). Temperature was maintained at 28.5°C during imaging using a Tokai Hit STX stage top incubator. Images were acquired using Nikon NIS Elements software. For live time-lapse series, 100 μm z-stacks with a 2 μm step were collected every 5 minutes for 5 hours using a Plan Apo Lambda 20x dry objective lens. Live blastoderm explants were mounted in rounded chambers made in 1% low-melt agarose in glass-bottomed dishes containing explants medium and imaged in the same scope. For live time-lapses, 14 μm z-stacks were obtained with a 2 μm step every 10 minutes for 6 hours using a Plan Apo Lambda 10x dry objective lens. Images of WISH and alcian blue-stained embryos and live embryos, larvae and explants were taken with a Nikon Fi3 color camera on a Nikon SMZ745T stereoscope. To monitor gastrulation progression in bright-field, live embryos were placed in 1.5% methylcellulose in egg water from the sphere stage and imaged periodically from (4 hpf) to tailbud stage (10 hpf) using a Nikon color camera and stereoscope as above. Embryos were staged according to WT controls.

Image analysis
ImageJ/Fiji was used to visualize and measure all microscopy data sets.

Morphometric analyses
During analysis, researchers were kept unaware of the conditions of all image data using the blind_renamer Perl script (https://github.com/jimsalterjrs/blindanalysis) (blindanalysis: v.1.0.) prior to analysis. To measure the length/width ratios of explants, the length of a segmented line drawn along the midline of each explant (accounting for curvature) was divided by the length of a perpendicular line spanning the maximal width of the explant. Tailbud morphometries were performed in whole-mount embryos staged using egr2b expression at the future mid-hindbrain boundary. Notochord width was quantified in dorsal-view images as the mediolateral extent of the tbxta expression domain at the midline. Anteroposterior (AP) axis length was measured in lateral-view images from the anterior to posterior boundaries of the dlx3b expression domain. To quantify the extent of endoderm migration toward the animal pole, embryos were imaged laterally at shield, 75% epiboly, and 90% epiboly stages and the spread of sox17-positive cells was measured on the ventral side.

Explant onset of extension
The onset of explant extension was assessed in a blinded manner as the time point when a visible tip first emerged from an initially rounded explant. If an explant protrusion extended toward or away from the objective in a way that interfered with determining its onset of extension, it was excluded from the analysis. Explant were staged according to sibling intact embryos from the same genetic background cultured in the same plate.

Explants Shape analysis
Explant time-lapse movies were saved as image sequences (one image per time point) and segmented using Cellpose 2.0 (141) implemented in Python. Using FIJI, each individual explant ROI was overlaid onto its corresponding image, and explant roundness was quantified using the standard shape descriptor, where a value of 1 indicates a perfect circle and decreasing values reflect explant elongation. To distinguish changes in the timing or dynamics of extension from defects in C&E morphogenesis, only explants that reached roundness values below 0.5 by 12 hpf were included in the analysis

Cell tracking analysis
Automated nuclear tracking was performed using the ImageJ TrackMate7 plugin (142) in the dorsal hemisphere (encompassing dorsal and lateral cells) of zebrafish gastrulae injected with H2B-scarlet mRNA. TrackMate generated color-coded trajectories and measurements of mediolateral (ML) displacement. Embryos were staged relative to formation of the second somite. To minimize noise from reduced convergence movements near the dorsal midline, cells within 100 μm of the midline were excluded from the analysis. Average displacement in the X (ML) dimension was calculated per time frame, smoothed using a sliding window of four time points, and plotted over time using GraphPad Prism 10. For persistence and speed analyses, only cells that were tracked for at least 15 minutes (three consecutive time frames) were included. Persistence of motion was quantified as the ratio between the linear distance traveled by a cell and the total length of its migration path. Straight-line speed was calculated as the total linear displacement of a cell divided by the total tracking time. Instantaneous velocity was defined as the cell’s spatial displacement between two consecutive frames divided by the frame interval (5 min).

GAG isolation from zebrafish embryos
To compare GAG composition between stages, sibling AB WT embryos were collected and flash frozen in liquid nitrogen at 5.3 (8 biological replicates) and 8.5 hpf (7 biological replicates), with each replicate containing 100 embryos. To compare GAG composition between WT and mutants, MZsumf1-/- (on a Tuebingen background, 5 biological replicates), MZsumf2-/- (on an AB background, 4 biological replicates), and control WT embryos of Tuebingen and AB strains, respectively (3 biological replicates each) were collected and flash frozen at 8.5 hpf, with each replicate containing 100 embryos. Whole embryos were homogenized and lysed in 0.5% CHAPS lysis buffer (50 mM HEPES, 120 mM NaCl, 2 mM EDTA, pH 7.4) containing a protease inhibitor cocktail (Roche). 50 µL of cell lysate was set aside for protein quantification via BCA assay. Homogenates were diluted 1:10 in a wash buffer (50 mM sodium acetate, 200 mM NaCl, 0.1% Triton X-100, pH 6.0) and incubated with Pronase (0.4 mg/ml, Sigma) overnight at 37 °C with mild agitation. The product was centrifuged (4,000 xg, 20 minutes) then passed through a DEAE-Sephacel (Cytiva) column equilibrated in 50 mM sodium acetate buffer, pH 6.0, containing 200 mM NaCl, and desalted using a PD-10 desalting column (Cytiva), and lyophilized to dryness overnight.

HS depolymerization and HILIC-Q-TOF-MS disaccharide analysis
For HS disaccharide analysis, purified GAGs were digested with 2 mU each of heparin lyases I, II, and III (IBEX) for 16 hours at 37 °C in a buffer containing 40 mM ammonium acetate and 3.3 mM calcium acetate, pH 7. Samples were dried in a centrifugal evaporator then aniline-tagged, as previously described (89). Briefly, [12C6]aniline (17 μL, Sigma-Aldrich) and 17 μL of 1M NaCNBH3 (Sigma-Aldrich) freshly prepared in dimethyl sulfoxide:acetic acid (7:3, v/v) were added to each sample. Reactions were carried out at 37 °C for 16 hours and then dried in a centrifugal evaporator. Dried samples were resuspended in LC-MS grade water and spiked with an equimolar mixture of [13C6]aniline-tagged HS disaccharide standards (20 pmol, Iduron) prior to HILIC-Q-TOF-MS analysis. HILIC-UPLC was performed on a Waters Acquity UPLC system (Waters Corporation). Separation was achieved on a Waters Acquity UPLC Amide BEH column (2.1 mm × 150 mm, 1.7 μm) maintained at 40 °C. Mobile phases consisted of (A) acetonitrile and (B) 50 mM ammonium formate in water at pH 4.4 (Ammonium Formate Solution - Glycan Analysis, Waters). For HS disaccharide analyses, the following gradient program was used: 0–5 min, 90% A; 5–48 min, 90–67% A; 48–60 min, 67% A; 60–65 min, 67–90% A; and 65–70 min, 90% A. The flow rate was 0.5 mL/min in all runs. The injection volume was 2 μL. The UHPLC system was coupled to a Waters Synapt XS Q-TOF mass spectrometer equipped with an electrospray ionization source operated in negative ion mode. The following source parameters were used: source temperature 80 °C, desolvation temperature 250 °C, cone gas flow 50 L/h, desolvation gas flow 1000 L/h, capillary voltage 2.0 kV, sampling cone 35 V, and source offset 4.0 V. Data were acquired in resolution mode from 200 to 1000 m/z. MassLynx v4.2 software (Waters Corporation) was used for molecular feature extraction and data processing. Samples were quantified using isotopically labeled [13C6]aniline-tagged internal disaccharide standards and normalized to total protein, as measured by BCA.

Statistical analysis
Number of embryos or explants (n) and independent experimental replicates (N) for embryo or explant studies are stated in graphs. All experiments were performed at least in triplicate. GraphPad Prism 10 software was used to perform statistical analyses and generate graphs for all data analyzed. Datasets were tested for normality prior to analysis and statistical tests were chosen accordingly. The statistical tests used for each data set are noted in figure legends, and all tests used were two-sided. For explant roundness changes over time, curves were fitted using a Boltzmann sigmoidal model. Curve comparisons were performed using an extra sum-of-squares F test, comparing a model in which parameters were shared across datasets with a model in which parameters were allowed to vary between groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "NCOMMS-25-85681\nTitle: Sulfatase modifying factors control the timing of zebrafish convergence and extension morphogenesis\nConvergence and extension (C&E) movements are essential for proper embryogenesis and must occur at a precise time\nduring gastrulation. However, the mechanisms controlling this timing is unknown. The primary goal of this study was to\nelucidate the molecular factors involved in C&E timing using a zebrafish explant model previously developed by the lab.\nThrough bulk RNA-sequencing analysis, the authors identified two genes involved in sulfatase modification- sumf1 and\nsumf2- with reciprocal expression dynamics at the onset of C&E movements in gastrulation. The authors followed with\noverexpression and knock-out experiments in explants and in vivo to confirm it is the ratio of Sumf1 and Sumf2 that is\nrequired for C&E onset in gastrulation. Further analysis revealed Sumf1 and Sumf2 affect the activity of the sulfatase Sulf1.\nThe authors were able to establish a mechanism wherein low Sumf1/high Sumf2 prevents the activity of Sulf1 leading to\nmore sulfated heparan sulfate proteoglycans presence in the embryo at the onset of C&E movements during gastrulation.\nThis study is well-done; manuscript is well prepared with robust data. I have but a few minor comments to be addressed:\n1. More explanation about how the cut-offs were determined for identifying the 28 candidates from a list of 129\n(lines 140-142) would be useful.\n2. The manuscript did not address whether sumf2-/- embryos have a phenotype, although presumably since only MZsumf2-/-\nwas described, they did not. Is this true? It is curious that only the MZ have a phenotype considering Sumf2 is not maternally\ndeposited. Is there perhaps a paralog of Sumf2 that is compensating or another biological reason? Please clarify.\na. Related to this point, the manuscript describes sumf2-/- adults were scoliotic and undersized (line 195-196) however the\nreferenced supplementary figure labeled these fish as MZsumf2-/-. Please clarify or relabel.\n3. The manuscript does show the ratios of Sumf1/Sumf2 is important for onset of C&E in gastrulation. This argument may be\nstrengthened by the addition of an additional condition wherein both Sumf1 and Sumf2 are absent via double mutants\n(sumf1-/-; sumf2-/-), knockdown of sumf1 in MZsumf2-/- or vice versa to see if CE is still affected.\n4. On page 20, line 432, the authors refer to “craniofacial abnormalities” present in some MZsumf2-/- zebrafish. These\n“abnormalities” are not evident from the current images in Supplementary Fig. 2D-2E. Please either remove this phrase or\noffer additional images depicting the craniofacial phenotype in the zebrafish and/or Alcian blue staining to visualize the\ncraniofacial structures.\n5. What is the sumf1 expression level in the MZsumf2-/- mutant embryos? How does it change before vs. during\ngastrulation? Answering these questions via qPCR would offer additional background for the MZsumf2-/- + sumf1 OE\nexperiments.\nMinor critiques:\n1. Scale bar is missing in Figure 1B.\n2. Scale bar is missing in Figure 3A.\n3. To make Figure 3A more accessible to readers that cannot easily discern red from green, the authors are encouraged to\nuse different colors to depict arrows.", "reviewer_2": "This work presents novel findings relating to the role of sulfatases in regulating the timing of gastrulation onset in zebrafish\nembryos. While we know a relatively large amount about the molecular mechanisms governing the control of cell\nintercalation events that act to produce convergence and extension movements during gastrulation, relatively little is known\nabout how these events are coordinated in time during development. This work provides the first evidence of a novel\nmechanism governing this process that acts at the level of controlling the production of heparan-sulfate proteoglycans.\nThese results are novel and interesting, and will set the stage for future studies investigating the role of post-translational\nmodifiers in controlling the timing of developmental events. The discussion places the observations in a broader context of\nwhat is known about HPSGs in the regulation of known events critical to the progression of gastrulation: the regulation of\nmorphogen activity for multiple signalling pathways. I have some suggestions for further improving the manuscript before it is\nready for publication:\n1) The characterisation of 'trigger' genes associated with gastrulation onset, and are expressed within the critical window of\ngene expression required for C&E onset is interesting. The authors select two genes to follow up with, a provide a\nsupplementary table with the other gene expression changes. It would be informative to dig a little deeper into these\nadditional gene expression changes and summarise them within the main text of the paper.\n2) Many of the experiments involve the quantification of elongation onset in embryonic explants. This was performed in a\nblinded manner by visually determining when the first protrusion is generated from an explant. Is this a reliable method for\nquantification? There could be issues with instances where explants protrusion towards or away from the objective, rather\nthan laterally. It would be helpful to discuss these potential caveats or look for automated shape analysis that could be\nemployed to validated the findings.\n3) Fig. 3. It is not clear whether the traces shown for ML displacement are representative traces from single movies, or\ncombined statistics from multiple independent movies. If the former, can the authors include the additional data in\nsupplementary materials?\n4) Resumably a major impact of HFPG modification would be on components of the ECM, that might in turn explain the\ndefects in gastrulation morphogenesis. Could the authors discuss this alongside their speculation over the potential\ninteraction with known signalling pathways?", "reviewer_3": "In this manuscript, Cervino et al address the fundamental, yet poorly understood, question of how the timing of\nmorphogenetic movements are controlled during gastrulation. The authors identify a role for the ratio of sumf2 and its\nparalog sumf1 in controlling the onset and dynamics of CE movements in zebrafish. They further identify the Sulf1, an\nextracellular sulfatase that modifies heparan sulfate proteoglycans (HSPGs), as a key effector by which sumf1 and sumf2\nregulate tissue movements.\nThe manuscript addresses an exciting open question in biology and identifies an unexpected role for sulfatase enzymes in\nregulating morphogenesis in zebrafish. This is a very interesting avenue of research, however, in my view, the mechanism\nand interpretation of the phenotypes remain rather unclear, with important assumptions from the authors or knowledge gaps\nrequiring, in my view, further testing/work. Moreover, the in vivo data is, in my view, more consistent with an important\nregulatory role for sulfatase activity in regulating CE movements, rather than a timer mechanism (which I nonetheless find\nvery exciting!). See my detailed points below.\n1. How clear is the notion that sumf1 and sumf2 have opposite roles on sulfatase activity in zebrafish? Is this consistent with\nthe domain organization and homology of both enzymes to well-studied FGE and pFGE enzymes in other organisms?\n2. Do the levels of sulfated HSPG levels change systematically, and as expected, in the context of overexpression and\ndownregulation of either enzyme and w/the expected kinetics? This will also clarify how much the differential expression\npattern of these 2 enzymes would be expected to be sufficient to tip sulfatase activity in vivo…\n3. A key point in the manuscript is that the shift in the ratio between sumf1/sumf2 triggers CE movements – how is the\nexpression of sumf2 regulated?\nThese points are at the core of the mechanism and require additional work in my view.\nIt is unclear to me why the sumf1 and sumf2 manipulations would display CE defects in vivo, as shown by the authors.\nBased on the timer model, I would have only expected a delay/accelerated onset (more consistent w/the observations in the\nexplants, but not with the in vivo work). Instead, the defects ML movements and axis extension suggest rather a regulatory\nrole in CE movements. In line with this, upon sumf1 OE in vivo, the difference in CE onset is rather modest, with a much\nclearer delay in mediolateral displacement during CE (as also pointed out by the authors). This is also consistent with the\nphenotypes of Sul1 and the treatments with sodium chlorate.\n4. Additional characterization of the tracks for persistence, directionality is also needed to better understand\nthese cellular defects in CE.\n5. The authors should track ML displacements in explants, where no obvious macroscopic defect in CE is detected? Are\nthere only timing differences or also changes in cell movement in this context? Could the in vivo phenotype be a result of a\nshorter window for CE? (that would still not explain why the embryos that start CE earlier, also shows defects though)\n6. How do these phenotypes compare with well-established regulators of CE? Would these not produce both a change in\nonset and in the speed of ML movements during CE, just like the manipulation of sulfation?\n7. How does sulfation act to regulate CE movements? Are there differences in morphogen signaling for instance (e.g. BMP\nor FGF range, levels, dynamics) or rather in the distribution of PCP components? This is also important to understand\nwhether this is a timer mechanism or a regulator of CE.\n8. In vivo, are other morphogenetic movements affected by manipulations of sulfatase activity? This is another important\npoint to better characterize the phenotypes observed in vivo.\n9. A final important point in my view is to characterize cell type proportions upon OE/knockout of the sumf1/sumf2/sul1.\nOverall, based on the in vivo data, however, it is, in my view, hard to conclude that this is a timer mechanism for CE rather\nthan an additional and important regulator of cell behaviors driving CE. This is the center premise of the manuscript, so\nadditional work is, in my view, required in this regard to clarify the core of the author’s model."}, "author_response": "Response to Reviewers\nNCOMMS-25-85681\nCervino et al.\nSulfatase modifying factors control the timing of zebrafish convergence and extension morphogenesis\nReviewer #1 (Remarks to the Author)\nConvergence and extension (C&E) movements are essential for proper embryogenesis and must occur at a\nprecise time during gastrulation. However, the mechanisms controlling this timing is unknown. The primary\ngoal of this study was to elucidate the molecular factors involved in C&E timing using a zebrafish explant\nmodel previously developed by the lab. Through bulk RNA-sequencing analysis, the authors identified two\ngenes involved in sulfatase modification- sumf1 and sumf2- with reciprocal expression dynamics at the onset\nof C&E movements in gastrulation. The authors followed with overexpression and knock-out experiments in\nexplants and in vivo to confirm it is the ratio of Sumf1 and Sumf2 that is required for C&E onset in gastrulation.\nFurther analysis revealed Sumf1 and Sumf2 affect the activity of the sulfatase Sulf1. The authors were able to\nestablish a mechanism wherein low Sumf1/high Sumf2 prevents the activity of Sulf1 leading to more sulfated\nheparan sulfate proteoglycans presence in the embryo at the onset of C&E movements during gastrulation.\nThis study is well-done; manuscript is well prepared with robust data. I have but a few minor comments to be\naddressed:\nWe thank the Reviewer for their kind words and their appreciation of the significance and rigor of our study.\n1. More explanation about how the cut-offs were determined for identifying the 28 candidates from a list of 129\n(lines 140-142) would be useful.\nBased on the outcome of our time course of Triptolide treatment in explants, we hypothesized that genes\nrequired for timely C&E onset would exhibit a “trigger” like pattern, meaning that their expression would\nincrease substantially between 50% epiboly and shield stages. To identify such genes in explants, we sorted\nfor transcripts whose expression did not increase prior to shield stage but then increased significantly and\nremain elevated at shield stage and beyond. To focus in on genes with potential in vivo relevance, we further\nselected for genes with similar expression patterns in intact zebrafish embryos but whose 1) increase in\nexpression and 2) expression level were substantial enough to reasonably affect our biology of interest. To this\nend, we selected an arbitrary cut-off of 1) a 50% increase in transcript level from 50% epiboly to shield stage in\nintact embryos and 2) an expression level of at least 5 TPM at shield stage. When narrowed down this way, we\nwere left with 28 candidate genes to choose from. This expanded description has been added to the Results\nsection in lines 131-146.\n2. The manuscript did not address whether sumf2-/- embryos have a phenotype, although presumably since\nonly MZsumf2-/- was described, they did not. Is this true? It is curious that only the MZ have a phenotype\nconsidering Sumf2 is not maternally deposited. Is there perhaps a paralog of Sumf2 that is compensating or\nanother biological reason? Please clarify.\na. Related to this point, the manuscript describes sumf2-/- adults were scoliotic and undersized (line 195-196)\nhowever the referenced supplementary figure labeled these fish as MZsumf2-/-. Please clarify or relabel.\nThe Reviewer raises a great point. Because sumf2 is not maternally expressed, generating MZsumf2-/-\nembryos is not strictly necessary. However, because these Zsumf2-/- mutants are viable and develop into\nfertile adults, we chose to maintain this line as homozygotes for practical reasons, namely that it eliminated the\nneed to genotype individual explants, which is infeasible. We did not examine the phenotypes of zygotic\nmutants during gastrulation or at the tailbud stage, but we did observe larvae with axis defects and adults with\nscoliosis among Zsumf2-/- progeny obtained from F1 incrosses (which were similar to the larval and adult\nphenotypes observed in MZsumf2-/- animals). sumf1 is the only known paralog of sumf2, but because these\ntwo genes exhibit antagonistic functions, we don’t expect genetic compensation by sumf1 in sumf2 mutants.\nIndeed, as described further in point 5 below, we observe no change in sumf1 expression levels within\nMZsumf2-/- embryos. Finally, we have corrected the text to reflect that the animals shown in the supplementary\nfigure are in fact MZsumf2-/-. We apologize for this oversight.\n3. The manuscript does show the ratios of Sumf1/Sumf2 is important for onset of C&E in gastrulation. This\nargument may be strengthened by the addition of an additional condition wherein both Sumf1 and Sumf2 are\nabsent via double mutants (sumf1-/-; sumf2-/-), knockdown of sumf1 in MZsumf2-/- or vice versa to see if CE is\nstill affected.\nWe agree entirely with the Reviewer’s comment and have added the analysis of in vivo convergence\nmovements in MZsumf1-/-; MZsumf2-/- embryos. These can be found in the new Figure 3K. We find that\nconvergence movements in double mutants resemble those of MZsumf1-/- single mutants, demonstrating that\nsumf1 is epistatic to sumf2, as would be expected if sumf2/pFGE functions by antagonizing sumf1/FGE. This\ndescription was added to the results section in lines 316-321.\n4. On page 20, line 432, the authors refer to “craniofacial abnormalities” present in some MZsumf2-/- zebrafish.\nThese “abnormalities” are not evident from the current images in Supplementary Fig. 2D-2E. Please either\nremove this phrase or offer additional images depicting the craniofacial phenotype in the zebrafish and/or\nAlcian blue staining to visualize the craniofacial structures.\nWe agree, and as characterization of skeletal abnormalities resulting from Sumf2 loss of function is beyond the\nscope of the present manuscript, we have removed the sentence referring to this phenotype.\n5. What is the sumf1 expression level in the MZsumf2-/- mutant embryos? How does it change before vs.\nduring gastrulation? Answering these questions via qPCR would offer additional background for the MZsumf2-\n/- + sumf1 OE experiments.\nWe thank the Reviewer for this suggestion and have now added a qPCR analysis of sumf1 expression at pre-\ngastrulation and late gastrulation stages in WT and MZsumf2-/- embryos. These data are now shown in Suppl.\nFig. 3D (formerly Suppl. Fig. 2) and demonstrate that sumf1 expression levels in MZsumf2-/- embryos are not\ndifferent from WT at either time point. A description of these results was added to the results section in lines\n201-203.\nMinor critiques:\n1. Scale bar is missing in Figure 1B.\n2. Scale bar is missing in Figure 3A.\n3. To make Figure 3A more accessible to readers that cannot easily discern red from green, the authors are\nencouraged to use different colors to depict arrows.\nWe appreciate the Reviewer’s comments and have made all the suggested corrections.\nReviewer #2 (Remarks to the Author):\nThis work presents novel findings relating to the role of sulfatases in regulating the timing of gastrulation onset\nin zebrafish embryos. While we know a relatively large amount about the molecular mechanisms governing the\ncontrol of cell intercalation events that act to produce convergence and extension movements during\ngastrulation, relatively little is known about how these events are coordinated in time during development. This\nwork provides the first evidence of a novel mechanism governing this process that acts at the level of\ncontrolling the production of heparan-sulfate proteoglycans. These results are novel and interesting, and will\nset the stage for future studies investigating the role of post-translational modifiers in controlling the timing of\ndevelopmental events. The discussion places the observations in a broader context of what is known about\nHPSGs in the regulation of known events critical to the progression of gastrulation: the regulation of\nmorphogen activity for multiple signalling pathways. I have some suggestions for further improving the\nmanuscript before it is ready for publication:\nWe thank the Reviewer for their positive evaluation of our study and their recommendations to improve it.\n1) The characterisation of 'trigger' genes associated with gastrulation onset, and are expressed within the\ncritical window of gene expression required for C&E onset is interesting. The authors select two genes to follow\nup with, a provide a supplementary table with the other gene expression changes. It would be informative to\ndig a little deeper into these additional gene expression changes and summarise them within the main text of\nthe paper.\nAlthough we did not provide much detail about these other candidates in the original submission, we agree that\nthese additional genes may be of interest to the readership. First, we provided gene annotation information in\nSupplementary table 1, including a new tab with GO, KEGG, and INTERPRO terms for all 28 candidate genes.\nWe have further added a discussion of these genes and their functions to the main text in the results section in\nlines 142-146.\n2) Many of the experiments involve the quantification of elongation onset in embryonic explants. This was\nperformed in a blinded manner by visually determining when the first protrusion is generated from an explant.\nIs this a reliable method for quantification? There could be issues with instances where explants protrusion\ntowards or away from the objective, rather than laterally. It would be helpful to discuss these potential caveats\nor look for automated shape analysis that could be employed to validated the findings.\nThe reviewer raises an important point. If an explant protrusion extended toward or away from the objective in\na way that interfered with determining its onset of extension, it was scored as “out of focus” and excluded from\nthe analysis. We have added a statement in the Methods section to clarify this. In addition, we performed\nautomated shape analysis of WT, MZsumf1, and MZsumf2 mutant explants and plotted their roundness over\ntime, which is now shown in our new Figure 3C. We found that this type of analysis was not suitable for reliably\ndetermining the precise onset of extension because the emergence of an extension tip did not substantially\nalter explant roundness until well after it first became visually apparent. However, this automated did enable\ncomparisons of the dynamics of explant extension across conditions. As expected, these curves were shifted\nleftward (earlier) and rightward (later) in MZsumf1-/- and MZsumf2-/- explants, respectively, but the rate of\nelongation (i.e. the slope of explant roundness over time) also differed between these conditions. This analysis\ncorroborates and strengthens our conclusion that the onset of C&E movements is altered by levels of sulfatase\nmodifiers and revealed additional effects on the pace of C&E. A description of these results was added to the\nresults section in lines 253-268.\n3) Fig. 3. It is not clear whether the traces shown for ML displacement are representative traces from single\nmovies, or combined statistics from multiple independent movies. If the former, can the authors include the\nadditional data in supplementary materials?\nThe plots in Figure 3 are the mediolateral displacement of cells combined from multiple embryos, with the\nnumber of analyzed embryos (n) from independent experiments (N) indicated in the graph. Statistical analyses\nwere also performed on these combined traces, comparing each experimental group with its corresponding\nWT control. Per the Reviewer’s suggestion, we have now also added single-embryo tracks in Figure Suppl. 6.\n4) Resumably a major impact of HFPG modification would be on components of the ECM, that might in turn\nexplain the defects in gastrulation morphogenesis. Could the authors discuss this alongside their speculation\nover the potential interaction with known signalling pathways?\nThe Reviewer raises an excellent point. We have now added a discussion of potential effects of altered HSPG\nsulfation on the ECM and how this may contribute to the observed C&E defects in lines 619-622.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Cervino et al address the fundamental, yet poorly understood, question of how the timing of\nmorphogenetic movements are controlled during gastrulation. The authors identify a role for the ratio of sumf2\nand its paralog sumf1 in controlling the onset and dynamics of CE movements in zebrafish. They further\nidentify the Sulf1, an extracellular sulfatase that modifies heparan sulfate proteoglycans (HSPGs), as a key\neffector by which sumf1 and sumf2 regulate tissue movements.\nThe manuscript addresses an exciting open question in biology and identifies an unexpected role for sulfatase\nenzymes in regulating morphogenesis in zebrafish. This is a very interesting avenue of research, however, in\nmy view, the mechanism and interpretation of the phenotypes remain rather unclear, with important\nassumptions from the authors or knowledge gaps requiring, in my view, further testing/work. Moreover, the in\nvivo data is, in my view, more consistent with an important regulatory role for sulfatase activity in regulating CE\nmovements, rather than a timer mechanism (which I nonetheless find very exciting!). See my detailed points\nbelow.\nWe are glad to hear this Reviewer found our study to be interesting and exciting. We are confident that our\nrevisions have improved the manuscript and hope they will satisfactorily address the Reviewer’s concerns.\n1. How clear is the notion that sumf1 and sumf2 have opposite roles on sulfatase activity in zebrafish? Is this\nconsistent with the domain organization and homology of both enzymes to well-studied FGE and pFGE\nenzymes in other organisms?\nWe thank the reviewer for raising this point. The opposing roles of SUMF1/FGE and SUMF2/pFGE have been\nestablished in human cell lines, and this study represents, to our knowledge, the first functional analysis of\nsumf2 in zebrafish. We have added to Figure Suppl. 2 an alignment of the amino acid sequences of zebrafish\nsumf1 and sumf2, showing high homology of each with their human and mouse homologs. Consistent with the\nmammalian homologs, zebrafish sumf2 lacks the two cysteine residues present in the active site of sumf1.\nDiscussion of these findings was added to the results section in lines 156-161. We also present strong\nfunctional data demonstrating opposing roles on sulfatase function. First, overexpression of sumf1 delays C&E\nonset which is ‘cancelled out’ by co-overexpression of sumf2. Second, double MZsumf1; MZsumf2 mutant\nexplants and now embryos (newly added to Figure 3J) resemble MZsumf1 single mutants, demonstrating that\nsumf1 is epistatic to sumf2, as we would expect if sumf2/pFGE functions by antagonizing sumf1/FGE. Finally,\nwe performed HILIC-Q-TOF-MS profiling of HS disaccharides in MZsumf1 and MZsumf2 mutant embryos as\nsuggested by the Reviewer in point 2 below. We found that these mutations had opposite effects on the\nproportion of sulfated D2S6 disaccharides, which are the predominant substrate of Sulf1. Together, the strong\nconservation with mammalian homologs and our functional experiments support an antagonistic role between\nsumf1 and sumf2 in zebrafish.\n2. Do the levels of sulfated HSPG levels change systematically, and as expected, in the context of\noverexpression and downregulation of either enzyme and w/the expected kinetics? This will also clarify how\nmuch the differential expression pattern of these 2 enzymes would be expected to be sufficient to tip sulfatase\nactivity in vivo…\nThe reviewer raises an important point. To address it, we have profiled HS sulfation by HILIC-Q-TOF-MS in\nMZsumf1 and MZsumf2 mutant embryos at the 80% epiboly time point when C&E is active. The resulting\nfindings have been added to Figure 5E and F, and show that the composition of sulfated disaccharides is\nindeed altered in both mutants compared with their respective WT controls. We found that MZsumf1-/-\nembryos had an increased proportion of sulfated D2S6 disaccharides, the predominant substrate of Sulf1,\nwhile MZsumf2-/- embryos had a decreased proportion of this same disaccharide. This demonstrates a\nsignificant change in sulfatase activity and downstream HSPG profiles upon altered levels of both sulfatase\nmodifiers. A description of these results was added to the results section in lines 400-412.\n3. A key point in the manuscript is that the shift in the ratio between sumf1/sumf2 triggers CE movements –\nhow is the expression of sumf2 regulated?\nWe agree entirely that this is an important and interesting question! However, addressing it would likely require\nan entire additional manuscript’s worth of work and is thus, beyond the scope of the current study. We hope to\nbe able to answer this question in the future.\nThese points are at the core of the mechanism and require additional work in my view.\nIt is unclear to me why the sumf1 and sumf2 manipulations would display CE defects in vivo, as shown by the\nauthors. Based on the timer model, I would have only expected a delay/accelerated onset (more consistent\nw/the observations in the explants, but not with the in vivo work). Instead, the defects ML movements and axis\nextension suggest rather a regulatory role in CE movements. In line with this, upon sumf1 OE in vivo, the\ndifference in CE onset is rather modest, with a much clearer delay in mediolateral displacement during CE (as\nalso pointed out by the authors). This is also consistent with the phenotypes of Sulf1 and the treatments with\nsodium chlorate.\nThe Reviewer raises an important point. Indeed, our explant assay allows us to detect changes in the onset of\nC&E (seen in MZsumf1 and MZsumf2 mutants) as well as more general C&E defects (seen in MZsulf1-/- and\nchlorate treated explants). Changes in C&E onset can also be detected in intact embryos by nuclear tracking,\nas the Reviewer noted, even if C&E is not disrupted per se. We speculate that defects in axis length and width\nmeasured in fixed mutant embryos result not from a general C&E defect but rather from a miscoordination of\nC&E with other concurrent cell movements. For example, if epiboly finishes on time (which it does in both\nMZsumf1 and MZsumf2 mutants) but C&E is delayed or precocious, these two movements would be out of\nsync with one another and could therefore manifest as the observed C&E defects. Further speculation on this\npoint can be found in the discussion section in lines 572-574. The fact that these embryos eventually recover\nto form larvae and adults of normal length further suggests that this is a timing effect, since embryos with\ngeneral C&E defects (for example, trilobite and knypek mutants) develop into very short larvae that don’t\nsurvive beyond ~5 days.\n4. Additional characterization of the tracks for persistence, directionality is also needed to better understand\nthese cellular defects in CE.\nWe thank the reviewer for this suggestion, and have added these cell tracking statistics in the new Suppl. Fig.\n7. A description of these results was also added to the results section in lines 316-321.\n5. The authors should track ML displacements in explants, where no obvious macroscopic defect in CE is\ndetected? Are there only timing differences or also changes in cell movement in this context? Could the in vivo\nphenotype be a result of a shorter window for CE? (that would still not explain why the embryos that start CE\nearlier, also shows defects though)\nThis is a great idea. We are indeed able to track labeled nuclei within explants (as we showed in PMID\n32319426), but convergence movements are very challenging to detect and measure ex vivo. In intact\nembryos, cells converge from the lateral regions of the embryo across the surface of the yolk, resulting in\nsubstantial ML displacement that we can easily measure. By contrast, explants have no yolk and thus,\nalthough extension movements are readily apparent, convergence movements are not. However, we are able\nto address this question without nuclear tracking. We performed automated analysis of WT, MZsumf1-/-, and\nMZsumf2-/- explant shape and plotted explant roundness over time (shown in our new Figure 3C-D), providing\na readout of explant extension dynamics. This curve was shifted earlier and later for MZsumf1-/- and\nMZsumf1-/- explants, respectively, corroborating our conclusion that C&E onset changes with loss of sulfatase\nmodifiers. However, these curves also had modest changes in slope, revealing that precocious C&E\nmovements were slightly slower and delayed C&E movements were slightly faster than WT controls. The result\nis that the period of C&E is the same in all conditions. A description of these results was added to the\nresults section in lines 253-268.\n6. How do these phenotypes compare with well-established regulators of CE? Would these not produce both a\nchange in onset and in the speed of ML movements during CE, just like the manipulation of sulfation?\nThis is an excellent point. To address it, we analyzed the onset of extension in explants cut from vangl2 MO-\ninjected embryos, which exhibit severe C&E defects due to disruption of planar cell polarity (PCP) signaling.\nAs expected, the overall extension of vangl2 MO explants was significantly impaired, as we previously described\nin PMID 32319426. However, we found that their extension onset time was not affected (in the new Suppl. Fig.\n5D). This demonstrates that a general C&E defect need not manifest as a change in C&E onset and vice\nversa, and that regulation of each can be molecularly distinct. A description of these results was added to the\nresults section in lines 248-250.\n7. How does sulfation act to regulate CE movements? Are there differences in morphogen signaling for\ninstance (e.g. BMP or FGF range, levels, dynamics) or rather in the distribution of PCP components? This is\nalso important to understand whether this is a timer mechanism or a regulator of CE.\nWe completely agree that this is an exciting area of research, and we hope to pursue it in the future. However,\nto answer this question would require months or even years of further experiments that would likely comprise\nan entire additional manuscript. For this reason, we believe it is beyond the scope of the current study.\n8. In vivo, are other morphogenetic movements affected by manipulations of sulfatase activity? This is another\nimportant point to better characterize the phenotypes observed in vivo.\nIndeed, we feel this study is novel and exciting because manipulation of sulfatase modifier levels affects the\ntiming specifically of C&E without creating a general developmental delay or altering the other gastrulation cell\nmovements of epiboly and ingression. To further support this claim, we have provided a new Suppl. Fig. 4.\nIn panel A, we include representative images of WT, MZsumf1-/-, and MZsumf2-/- gastrulae showing no major\ndelays in epiboly movements, as all embryos achieve blastopore closure within similar time frames. We also\nadded analyses of endoderm spreading (which, because they are coupled, also indicates mesoderm\nspreading) in these genetic backgrounds (Suppl. Fig. 4B,C). Although reduced endodermal spreading was\nobserved in MZsumf1-/- embryos at mid-gastrulation, the embryos ‘caught up’ by late gastrulation, reaching\nlevels comparable to WT embryos. Overall, while we cannot rule out minor roles for sulfatase modifying factors\nin other morphogenetic processes, their loss of function does not severely impair these behaviors during\ngastrulation. A description of these results was added to the results section in lines 209-217.\n9. A final important point in my view is to characterize cell type proportions upon OE/knockout of the\nsumf1/sumf2/sul1.\nBased on whole mount in situ hybridization analyses of ectodermal (dlx3b, egr2b), mesodermal (tbxta), and\nendodermal (sox17) marker genes (shown in multiple figures throughout the manuscript), we did not observe\nobvious changes in germ layer specification or cell type proportions in either loss- or gain-of-function conditions\nfor sumf1 or sumf2. The spatial expression patterns and domain size of these markers were comparable to\nthose in WT embryos, indicating that overall tissue patterning is preserved. Further, these embryos survive to\nadulthood, demonstrating that all cell types must be present in approximately normal numbers. Although we\ncannot rule out small changes in cell type proportions, we have no evidence that sumf1 and sumf2 play a\nmajor role in early cell fate specification.\nOverall, based on the in vivo data, however, it is, in my view, hard to conclude that this is a timer mechanism\nfor CE rather than an additional and important regulator of cell behaviors driving CE. This is the center premise\nof the manuscript, so additional work is, in my view, required in this regard to clarify the core of the author’s\nmodel.\nWe are confident that the additional evidence described here strengthens the support for our model that sumf1\nand sumf2 regulate the timing of C&E during zebrafish gastrulation and satisfactorily address the Reviewers’\nconcerns.\nWe thank all three Reviewers for their careful assessment of the revised version of this manuscript. Our\nresponses to the remaining concerns raised are below:\nReviewer #3 (Remarks to the Author):\nI much appreciated the clarifications and additional experiments provided by Cervino, et al in the revised\nmanuscript, which has improved the clarity of their manuscript and strengthened their findings substantially in\nmy view.\nI would still request a few more clarifications and analysis from the authors before publication, namely:\n1. I am not convinced that the rate of mediolateral movements would be affected in Sumf1 and Sumf2 mutants\nand OE in vivo simply due to a change in onset alone – even if the total duration of these movements is\naffected as indicated by the authors in the rebuttal, again the expectation here would be that they proceed at\nregular speed. Thus, I think it is important to directly and explicitly discuss these different possibilities in the\nDiscussion section.\nWhen completing our first round of revisions, we found that the rate of extension does indeed differ between\nexplants with precocious and delayed C&E onset, such that the total period of C&E does not differ between\nconditions. Evidence from our nuclear tracking in vivo supports this ex vivo finding, and we accordingly state in\nthe Results and Discussion sections (see lines 452, 475, and 707) that both onset and speed of convergence\nmovements are affected by manipulating levels of sumf1 or sumf2.\nIn addition, please provide the quantification of the epiboly rates and larvae length discussed in the rebuttal for\nthe Sumf1 and Sumf2 in the manuscript.\nIn Supplemental Fig. 3E, we show that about 90% of MZsumf2-/- larvae are indistinguishable from WT,\nalthough we have not quantified their length. Supplemental Fig. 4A also shows 3 representative, age-matched\nembryos for each WT, MZsumf1-/-, and MZsumf2-/- embryos undergoing epiboly. Although these were not\nformally quantified, these images show clearly that the rate of epiboly is not affected in these mutants.\n2. Please move the data from S9 into the corresponding main figure.\nWe have moved these data into Figure 5.\n3. Please include, for comparison, the expression dynamics for well-known regulators of CE (e.g. vangl2) in the\ncorresponding plots (or as supplementary data).\nWe now show log2fold expression changes for vangl2 in explants of all 3 conditions and in intact embryos in\nSupplemental Fig. 5.\n4. A discussion on the specificity of the timing defects for CE vs other morphogenetic processes would be\nbeneficial, especially considering the slight defects in internalization rate in the MZsumf1-/ embryos.\nWe indeed found that the reported timing defects are specific to C&E and do not affect the other gastrulation\ncell movements of epiboly and internalization. This is discussed in lines 693-701.\n5. While I agree that gross defects in the cell type proportions are unlikely, an imbalance between axial and\nparaxial populations in the context of the mutant is still important to address in my view.\nI fear that we cannot fully address this concern without performing single-cell RNA-sequencing, which would\nrequire substantial time and resources. Because we cannot formally rule out the possibility that altered\nproportions of different cell types contribute to the observed phenotypes, we state this in the Discussion\nsection (see line 724). However, we also note that our data do not support this as the primary mechanism by\nwhich sulfatase modifiers affect C&E timing."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have fully addressed my comments and I have no additional comments.\nThank you for the opportunity to review this work.", "reviewer_2": "The authors have satisfactorily address all of my previous concerns.", "reviewer_3": "I much appreciated the clarifications and additional experiments provided by Cervino, et al in the revised manuscript, which\nhas improved the clarity of their manuscript and strengthened their findings substantially in my view.\nI would still request a few more clarifications and analysis from the authors before publication, namely:\n1. I am not convinced that the rate of mediolateral movements would be affected in Sumf1 and Sumf2 mutants and OE in\nvivo simply due to a change in onset alone – even if the total duration of these movements is affected as indicated by the\nauthors in the rebuttal, again the expectation here would be that they proceed at regular speed. Thus, I think it is important to\ndirectly and explicitly discuss these different possibilities in the Discussion section. In addition, please provide the\nquantification of the epiboly rates and larvae length discussed in the rebuttal for the Sumf1 and Sumf2 in the manuscript.\n2. Please move the data from S9 into the corresponding main figure.\n3. Please include, for comparison, the expression dynamics for well-known regulators of CE (e.g. vangl2) in the\ncorresponding plots (or as supplementary data).\n4. A discussion on the specificity of the timing defects for CE vs other morphogenetic processes would be beneficial,\nespecially considering the slight defects in internalization rate in the MZsumf1-/ embryos.\n5. While I agree that gross defects in the cell type proportions are unlikely, an imbalance between axial and paraxial\npopulations in the context of the mutant is still important to address in my view."}, "author_response": ""}]

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