egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis

Agatha Ribeiro da Silva, Felix Gunawan, Giulia Boezio, Emilie Faure, Alexis
Théron, Jean-François Avierinos, Soeun Lim, Shivam Govind Jha, Radhan
Ramadass, Stefan Guenther, et al.

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To cite this version:

Agatha Ribeiro da Silva, Felix Gunawan, Giulia Boezio, Emilie Faure, Alexis Théron, et al. egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis. Science Advances, 2024, 10 (20), 10.1126/sciadv.adl0633. hal-04582533

HAL Id: hal-04582533

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Submitted on 16 Apr 2025

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**Title**

* **Full title** Egr3 is a mechanosensitive transcription factor required for cardiac valve morphogenesis

* **Short title** Egr3 regulates cardiac valve morphogenesis and remodeling

**Authors**

Agatha Ribeiro da Silva¹,²,³, Felix Gunawan¹,²,³, †, Giulia L.M. Boezio¹,²,³, ‡, Emilie Faure⁴, Alexis Théron⁴,⁵, Jean-François Avierinos⁴,⁶, SoEun Lim¹, Shivam Govind Jha¹, Radhan Ramadass¹, Stefan Guenther²,³,⁷, Mario Looso²,³,⁸, Stéphane Zaffran⁴, Thomas Juan¹,²,³*, Didier Y.R. Stainier¹,²,³*

**Affiliations**

¹ Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany.

² German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany.

³ Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany.

⁴ Aix Marseille Université, INSERM, MMG, U1251, 13005 Marseille, France.

⁵ Service de Chirurgie Cardiaque, AP-HM, Hôpital de la Timone, 13005, Marseille, France.

⁶ Service de Cardiologie, AP-HM, Hôpital de la Timone, 13005, Marseille, France.

⁷ Bioinformatics and Deep Sequencing Platform, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

⁸ Bioinformatics Core Unit (BCU), Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

†Current address: Institute of Cell Biology, Center for Molecular Biology of Inflammation, University of Münster, Münster 48149, Germany.

‡ Current address: The Francis Crick Institute, Developmental Dynamics Laboratory, 1 Midland Road, London NW1 1AT, UK.

* Correspondence to Thomas Juan - thomas.juan@mpi-bn.mpg.de or to Didier Stainier - didier.stainier@mpi-bn.mpg.de
## Abstract

Biomechanical forces, and their molecular transducers, including key mechanosensitive transcription factors, such as KLF2, are required for cardiac valve morphogenesis.

However, *klf2* mutants fail to completely recapitulate the valveless phenotype observed under no-flow conditions. Here, we identify the transcription factor EGR3 as a conserved biomechanical force transducer critical for cardiac valve formation and disease. We show that zebrafish *egr3* null mutants display a complete and highly penetrant loss of valve leaflets leading to severe blood regurgitation and cardiac failure. Using tissue-specific loss- and gain-of-function tools, we find that during valve formation, Egr3 functions cell-autonomously in endocardial cells, and identify one of its effectors, the nuclear receptor Nr4a2b. We further find that mechanical forces upregulate *egr3/EGR3* expression in the developing zebrafish heart as well as during human aortic valve remodeling upon left ventricular assist device placement. Altogether, these findings reveal that EGR3 is necessary to transduce the biomechanical cues required for cardiac valve morphogenesis, and potentially for pathological aortic valve remodeling.

## Teaser

EGR3 is necessary to transduce the biomechanical forces required for valve morphogenesis and is a potential target to prevent pathological aortic valve remodeling.

# MAIN TEXT

## Introduction

Tissue morphogenesis is intrinsically linked to the mechanical forces that are required for the cellular and molecular responses defining cell fate (1-4). In this context, endothelial cells, which line the blood vessels throughout the body, are specialized according to the function and mechanical properties of the tissues they reside in (5-8). During heart development, the specialized endothelial cells lining the cardiac wall, known as endocardial cells (EdCs), invade the adjacent extracellular matrix (ECM) in the atrioventricular (AV) canal and outflow tract (OFT) in response to high shear forces.

These migrating EdCs acquire mesenchymal characteristics, differentiate into valve interstitial cells (VICs), and proliferate to give rise to the cardiac valves (9-17), which are essential for cardiac function as they guarantee unidirectional blood flow.

Extensive work in vertebrates has shown that perturbation of mechanical forces in the heart severely affects valve formation (18-21). In particular, the zebrafish model allows for the observation and manipulation of intracardiac mechanical forces while imaging
valve morphogenesis at single cell resolution. For example, stopping cardiac contraction, and consequently blood flow, results in a valveless phenotype (22). Molecularly, the classical endothelial flow-responsive transcription factor Klf2 has been described as the main mechanosensitive transcription factor required for cardiac valve formation (9, 14, 16, 19, 23-27). However, zebrafish *klf2a/b* double mutants only display a partially penetrant valveless phenotype (9, 25, 28), indicating that other transcription factors play critical roles in transducing the mechanical signals that promote valvulogenesis.

In this study, we show that the transcription factor Early Growth Response 3 (Egr3) is the master transducer of mechanical signaling during cardiac valve morphogenesis. We first show that loss of *egr3* causes a complete and highly penetrant lack of cardiac valves. Data from loss- and gain-of-function experiments indicate that mechanical forces activate the expression of *egr3* in endothelial cells, thereby inducing their migration towards the adjacent ECM. Using transcriptomic analyses followed by functional investigation, we also show that the nuclear receptor Nr4a2b is a target and effector of Egr3 during cardiac valve formation. Furthermore, we show that *EGR3* and its target/effector *NR4A2* may be involved in human cardiac valve remodeling upon ectopic biomechanical overload.

## Results

### Egr3 is a master regulator of cardiac valve formation

Seeking to identify additional transcription factors required for cardiac valve formation, we first determined and explored the transcriptional landscape of endocardial cells at the time when key morphogenetic events underlying valve development take place. Thus, we isolated wild-type zebrafish hearts at 50 hours post-fertilization (hpf) when valve identity has just been established, and at 80 hpf when forming valves are first observed (9-11, 29) (Fig. 1A). We carried out single cell RNA sequencing (scRNA-seq) of whole hearts and obtained 1310 and 2194 endocardial cells at 50 and 80 hpf, respectively (Fig. 1B). The valve population was clearly identified at both time points by the expression of valve endocardium specific genes such as *has2* (30) and *alcama* (29) (Fig. S1A). Differential expression analysis between valve and non-valve EdCs revealed *early growth response 3* (*egr3*) in the top 30 most enriched genes in the valve cluster and the only one encoding a gene with DNA-binding transcription factor activity (Fig. 1C). Notably, *egr3* expression appears more enriched in the valve endocardial cells when compared with other established valve transcription factor genes (14, 16, 20, 31), including *klf2a/b* (25) and *nfatc1* (11) (Fig. S1, B and C). We further analyzed *egr3* expression by *in situ*
hybridization, where we observed a similarly restricted mRNA localization in the AV canal (Fig. S1D). Two other Egr family members have been implicated in cardiac valve development. In the mouse, *Egr2* (*Krox20*) has been shown to influence aortic valve development (32, 33); while, in zebrafish and humans, *egr1/EGR1* has been shown to be expressed in cardiac valves (34-36) and its expression requires mechanical forces (34, 37). We observed *egr1* expression mostly at 80 hpf and in most endocardial cells (Fig. S1, B and C); neither *egr2a/b* nor *egr4* were present in our dataset (Fig. S1, B and C). Although the expression pattern and role of Egr3 in heart development have not been investigated yet, analyses of human and mouse heart scRNA-seq datasets (38, 39) reveal that *EGR3/Egr3* is expressed in cardiac valve endocardium and atrioventricular mesenchyme (Fig. S2, A and B). Furthermore, sequence alignment shows that the Egr3 DNA binding domain is highly conserved (Fig. S2, C and D).

Given the strong and early enrichment of *egr3* in the valve endocardium, we investigated its role in valvulogenesis by generating two mutant alleles using the CRISPR/Cas9 system, a 6.2 kb full locus deletion (*bns522*) (i.e., a null allele), and an 11 base pair deletion (Δ11) (*bns577*) resulting in a premature termination codon that is predicted to disrupt the DNA binding domain (Fig. 1D, Fig. S1, E and F). During early development, the valveless embryonic heart achieves unidirectional blood flow in part because of localized contraction waves and a simple vascular network (40) whose capacitance provides pressure storage and diminishes cardiac efforts (41). By 72 hpf, the AV endocardial cells have folded into a functional prevalvular structure capable of reducing retrograde blood flow and therefore supporting cardiac output (11, 12, 19). *egr3* mutants, of both alleles, are indistinguishable from their wild-type siblings until 72 hpf when they start to exhibit, at Mendelian ratios, noticeable pericardial edema (Fig. 1, E and F). In contrast to their wild-type siblings, *egr3* mutants fail to form AV valve leaflets as their endocardial cells remain as a monolayer in a fully-penetrant fashion in the null allele (19/19) and a nearly fully-penetrant fashion in the Δ11 allele (19/21) (Fig. 1, G and H, Fig. S1, G and H). This complete lack of AV valves results in severe retrograde blood flow (Fig. 1I, Movie S1) and leads to heart failure and impaired peripheral circulation (Fig. 1J, Movie S2); *egr3* mutants indeed no longer display cardiac output by 96 hpf (Movies S1, S2). Despite this impaired circulation, cardiac contraction and heart rate are not obviously affected in *egr3* mutants (Movie S1). In 96 hpf wild-type larvae, the AV valves are more mature and elongated and the forming outflow tract (OFT) valve leaflets are also present (11, 14, 42). At this stage, *egr3* mutant AV endocardial cells still remain as a monolayer
(Fig. 1, G-H, Fig. S1, G and H) and, similarly, the forming OFT valves also fail to develop (Fig. 1, K and L, Fig. S1I). Taken together, these data show that Egr3 is essential for cardiac valve formation.

## Endothelial cell-specific deletion of *egr3* results in the lack of atrioventricular valves

To precisely assess *egr3* expression in the developing heart, we generated a Gal4 knock-in reporter line (*Pt(egr3:gal4-VP16)*), hereafter referred to as *egr3:gal4*, by targeting exon 2 using the CRISPR/Cas9 system, thereby disrupting the zinc-finger DNA binding domain (Fig. 2A). Consequently, the *egr3:gal4* line has endogenous *egr3* reporter activity and is also an *egr3* mutant allele (Fig. S3B). *egr3* reporter<sup>+</sup> embryos display a strong expression in the olfactory bulb as well as in specific structures of the central and peripheral nervous systems (Fig. S3A). *egr3* reporter expression is also evident in the presumptive AV canal starting at 34 hpf (Fig. S3A), mostly in the AV endocardium, but also in the AV myocardium (Fig. 2B). Moreover, *egr3* reporter expression is observed in the OFT endocardium starting at 72 hpf (Fig. 2B), coinciding with the onset of OFT valve morphogenesis (14).

Considering that both endocardial and myocardial cells in the AV canal express *egr3* based on the *egr3:gal4* reporter expression, we sought to determine whether *egr3* deletion in one of these cell types was sufficient to recapitulate the *egr3* mutant phenotype. Thus, we used the CRISPR/Cas9 system to generate a sequential *loxP* site knock-in in the *egr3* locus, specifically 440 bp upstream of the TSS and 1.9 kb downstream of the stop codon (Fig. 2C). Injection of *cre* mRNA into embryos from *egr3<sup>flox/+</sup>* outcrossed with *egr3<sup>+/-</sup>* resulted in 72 hpf zebrafish larvae displaying pericardial edema and no valve leaflets, correlating with the recombination of the *egr3* floxed allele (Fig. S3, D to F). No phenotype was observed in the uninjected larvae, showing that the *egr3* floxed allele is functional. When crossing *egr3<sup>flox/+</sup>* zebrafish with *egr3<sup>+/-</sup>* zebrafish bearing an endothelial Cre driver (*kdrl:Cre*), we observed that deleting *egr3* in endothelial cells was sufficient to recapitulate the global *egr3* mutant phenotype, thereby resulting in the complete lack of AV valve leaflets (Fig. S3G, Fig. 2, D and E). Moreover, when crossing *egr3<sup>flox/+</sup>* zebrafish with *egr3<sup>+/-</sup>* zebrafish bearing a myocardial Cre driver (*myl7:Cre*), we observed that deleting *egr3* in myocardial cells led to no noticeable differences with their control siblings including in their valve leaflets (Fig. S3H, Fig. 2, D to F). Collectively, these data show that *egr3* functions in the endothelium to drive AV valve formation.
In order to gain better insight into the cellular and molecular causes of the cardiac valve phenotype in *egr3* mutants, we analyzed *egr3<sup>+/+</sup>* and *egr3<sup>-/-</sup>* sibling embryos at 48 hpf, a time when they cannot be visually distinguished from each other. At this stage, the AV canal is already morphologically and molecularly distinct from the atrial and ventricular chambers (29). Tissue convergence and a decrease in AV EdC size are important morphogenetic steps that precede AV valve formation (43, 44). Thus, in order to evaluate possible morphological differences in the endocardium leading to the *egr3* mutant phenotype, we analyzed EdC volume in each cardiac region (Fig. S4, A and B).

Consistent with published data (44), AV EdCs had a smaller volume when compared with ventricular and atrial EdCs (Fig. S4C). Notably, there were no differences in EdC cell number or volume in any cardiac region between *egr3* mutants and their *egr3<sup>+/+</sup>* siblings (Fig. S4, C and D), such that *egr3* mutant EdCs were observed in expected numbers and underwent the cell shape changes typically observed during early AV valve morphogenesis.

To identify the molecular causes of the *egr3* mutant phenotype, we performed bulk RNA sequencing analysis of 48 hpf *egr3<sup>+/+</sup>* and *egr3<sup>-/-</sup>* dissected hearts (Fig. 3A). 154 genes were differentially expressed between *egr3* wild-type and mutant sibling embryos.
Notably, important valve regulators such as *klf2a/b*, *notch1b*, *dll4*, *nfatc1* or *wnt9a/b* did not appear to be differentially expressed in *egr3* mutants (Fig. S5A). Using *in situ* hybridization and immunostaining for Alcama, we indeed observed that the general molecular patterning of the AV canal was not affected in *egr3* mutants (Fig. S5, B and C), altogether suggesting that lack of *egr3* is sufficient to abolish cardiac valve formation in spite of normal expression of established cardiac valve markers and regulators. We further screened our dataset for differentially expressed genes with cardiac valve expression, and found that *nr4a2b*, *spp1*, and *nrg1* were significantly downregulated in *egr3* mutants (Fig. 3A). Nr4a2/Nurr1 is a nuclear receptor (45) with transcription factor activity and *nr4a2b* exhibits cardiac valve expression according to our scRNA-seq data (Fig. 3B), while *Spp1*/Osteopontin and *Nrg1* are expressed by valve EdCs (46, 47) (Fig. 3, B and C). Our observations that *nr4a2b* and *spp1* are targets of Egr3 agree with published reports in mammals showing that Egr3 is upstream of Nr4a2 (48) and that Nr4a2 activates *Spp1* expression by binding to its promoter (49). Moreover, Egr3 (50, 51), Nr4a2 (52), and *Spp1* (53, 54) promote endothelial cell migration downstream of VEGF, supporting the
model that Egr3 mediates a signaling axis regulating EdC migration during
valvulogenesis.

To test whether deficient cellular migration could be the cause of the *egr3* mutant phenotype, we evaluated EdC migration in the initial steps of AV valve morphogenesis. From 48 hpf onwards, the AV EdCs start to extend cellular protrusions into the adjacent ECM and, within a few hours, two to four AV EdCs collectively migrate in a ventricle to atrium direction (10, 11) (Fig. 3D). *egr3* mutants exhibit a two-fold reduction in the number of AV EdC protrusions towards the ECM (Fig. 3E), although this reduction was not significant. Strikingly, the subsequent collective EdC migration is severely affected, as only 5% (n=1/17) of the mutant embryos engaged in EdC migration compared with 70% (n=13/18) of their heterozygous siblings at 55 hpf (Fig. 3, F and G). Between two to four migrating EdCs were observed in these heterozygous hearts, while zero to one migrating EdC was generally observed in these *egr3* mutant hearts (Fig. 3H).

To investigate whether Egr3 is sufficient to induce EdC migration into the ECM, we generated a stable *egr3* overexpression line, *Tg(5XUAS:egr3-p2a-dTomato)*, hereafter referred to as ‘egr3 OE’. To induce ectopic *egr3* overexpression in the endothelium, we crossed *egr3* OE zebrafish with the endothelial *fli1a:Gal4* driver, *Tg(fli1a:Gal4ff)*. *egr3* overexpressing cells were clearly distinguished by dTomato fluorescence (Fig. 3I). In control hearts, valve interstitial cells (VICs) were present in the ECM of the AV canal, but not in the ECM of the ventricle or atrium (Fig. 3, J and K). However, in *egr3* OE hearts, not only was there more than a 2.5 fold increase in VICs in the ECM of the AV canal (Fig. 3, J and K), but also dTomato⁺ EdCs started to appear in the ventricular and atrial ECM adjacent to the EdC monolayer (Fig. 3, I to K). To better characterize the ectopic EdC in the ECM of *egr3* OE hearts, we assessed the expression of the endocardial valve marker Alcama (Fig. 3L). Notably, *egr3* overexpression was sufficient to induce ectopic Alcama expression in the ventricular and atrial chambers (Fig. 3L), as well as a significant increase of Alcama-expressing cells in the AV canal (Fig. 3M). Next, we tested whether overexpressing the Egr3 target, Nr4a2b, would be sufficient to rescue AV EdC migration in *egr3* mutants. Thus, we generated a *nr4a2b* overexpression stable line, *Tg(5XUAS:nr4a2b-p2a-dTomato)*, which, in the background of *egr3 bns577* mutant allele, was crossed with *egr3:gal4⁺/−*. In addition to the resulting transheterozygous embryos not producing a functional Egr3 protein, *nr4a2b* was overexpressed in *egr3:gal4*-expressing cells. The transheterozygous larvae displayed the expected *egr3* mutant phenotype at 72
hpf (Fig. S3B, Fig. 3, N and O). *egr3* transheterozygous larvae overexpressing *nr4a2b* in their AV endocardium displayed a significant increase in VIC number in the AV ECM, indicating a partial rescue of EdC migration (Fig. 3, N and O). Not all VICs in *nr4a2b* OE hearts were *dtomato+*, indicating Nr4a2b may act in a non-cell autonomous fashion. Collectively, these data indicate that the *egr3* mutant phenotype is a result of failed EdC migration into the adjacent ECM and that Egr3 is sufficient to induce EdC Alcama expression and migration, and suggest that Egr3 promotes AV EdC migration through its target Nr4a2b.

Egr3 is a mechanosensitive transcription factor in cardiac valve development and
disease

Considering that Egr3 is involved in mediating VEGF-induced endothelial migration (50) and that it is a VEGF target via ERK activation (55), we decided to test whether egr3 AV canal expression was dependent on VEGF signaling. We found that egr3 AV canal expression was unaltered after treatment with VEGF receptor inhibitors or a MEK inhibitor (Fig. S6A), suggesting that it is independent of VEGF/ERK signaling. Given the fundamental role of mechanical signaling in cardiac valve formation (19, 20, 22), we then decided to test whether egr3 expression was downstream of mechanical forces. We simulated a no-flow/no-contraction condition in vivo, by taking advantage of the zebrafish embryo's ability to withstand severe cardiac dysfunction (56). We used silent heart zebrafish mutants which lack the sarcomeric protein Tnnt2a and, therefore, do not display cardiac contraction (56). Notably, egr3 AV canal expression was completely abrogated in tnnt2a mutant hearts (Fig. 4A), and we also observed the loss of egr3 expression when treating zebrafish embryos with the myosin inhibitor BDM (Fig. S6A). To test whether egr3 expression responds to ectopic mechanical stimulation, we performed microsurgical insertion of a bead inside the embryonic heart through the inflow tract (18, 57) at 48 hpf (Fig. 4B). The bead remained inside the heart for 24 hours and moved rhythmically across the ventricular chamber following the heartbeats and without stopping the blood flow. Upon this ectopic mechanical stimulation, egr3 expression was expanded in the hearts of the bead-inserted larvae when compared with sham controls, as shown by the expression of the egr3:gal4 reporter (Fig. 4, C and D). As mechanical forces are the most upstream regulator of valve morphogenesis, we tested whether overexpressing egr3 in silent hearts could rescue their valve identity. For that, we injected tnnt2a morpholino (MO) into one-
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cell stage embryos of *egr3* OE zebrafish crossed with the endothelial *fli1a:Gal4* driver (Fig. 4E). Valve identity is absent in silent hearts as the morphology of valve cells is not distinguishable from the EdC chambers and the expression of valve markers is lost (22) (Fig. 4E). Notably, *egr3* OE in silent hearts induced the expression of Alcama and ectopic EdCs in the ECM of the cardiac chambers, aside of rescuing the cuboidal valve cell shape (Fig. 4E). Alcama-expressing cells were present in the chambers and the AV canal of silent hearts overexpressing *egr3* (Fig. 4F), suggesting no spatial valve specification in silent hearts.

In addition to playing an important role in valvulogenesis, mechanical forces are also pivotal during pathological valve remodeling. For instance, the use of a left ventricular assist device (LVAD) has become an established treatment option as well as a bridge for end-stage heart failure patients awaiting a heart transplant (58). Despite improving survival and heart function, LVAD use is correlated with aortic insufficiency, and 15 to 52% of patients may present worsening of previous aortic valve pathology or develop *de novo* aortic valve insufficiency (59). The LVAD increases cardiac output by diverting the blood from the left ventricle straight into the aorta, thereby decreasing the strain in the mitral valve and causing biomechanical overload in the aortic root and valves (60). This increased biomechanical overload in the aortic valves induces valve remodeling (i.e., aortic valve fusion) (61, 62). However, how the aortic valves transduce the biomechanical overload into a molecular response remains elusive. As zebrafish *egr3* expression responds to lack of, as well as increase in, mechanical forces during valve development (Fig. 4, A to D), we sought to investigate whether *EGR3* expression was altered during human aortic valve remodeling following LVAD placement. We collected samples of aortic valves from control donors and LVAD patients and performed RT-qPCR (Fig. 4H). *EGR3* expression was increased by almost six-fold in LVAD patient valves compared with donor control valves (Fig. 4I). Expression of the classical mechanosensitive transcription factor gene *KLF2* was increased by nearly four-fold (Fig. 4I). We also tested the EGR3 targets and observed that in LVAD patient valves, *NR4A2* expression was increased by more than 13-fold and *SPP1* expression by more than 20-fold, although the latter was not significant (Fig. 4I). Altogether, these data show that Egr3 is a mechanosensitive transcription factor during cardiac valve formation and that *EGR3* expression levels are upregulated in LVAD patient valves.

## Discussion
Here we identify the transcription factor Egr3 as a master transducer of the mechanical forces that guide valvulogenesis, and show that Egr3 is required for EdC migration into the adjacent ECM by activating the expression of the migration gene *nr4a2b* (52) (Fig. 3, A to C and L). *Klf2* has long been considered to be the main mechanosensitive transcription factor in cardiac valve formation. However, the broad endocardial expression of *klf2a/b* compared with the restricted expression of *egr3* in valve EdCs (Fig. S1, B and C) suggests that Egr3 has a more specific role in transducing mechanical forces during valvulogenesis. In fact, *klf2* mutations result in a 47-56% penetrance of the cardiac valveless phenotype observed in no-flow conditions (25, 28). Moreover, *klf2* mutants also display cardiomyocyte extrusion (28) and it is not clear how this phenotype affects cardiac valve formation. In contrast, the *egr3* mutant phenotype displays 90-100% penetrance among F2 to F4 embryos and it is specific to the valves (Fig. 1, G to L, Fig. 2, D and E), with no obvious defects in other cardiac regions (Fig. S4, A to D, Fig. S5, A to C).

*EGR3* is expressed in AV EdCs and fibroblasts in mammalian hearts (38, 39) (Fig. S2, A to D), consistent with a conserved function in valvulogenesis. *Egr3* mutant mice display a partially penetrant lethality (63), and underlying cardiac defects should be investigated. In humans, *EGR3* single nucleotide variants (SNVs) resulting in loss-of-function alleles are highly underrepresented with a loss-of-function observed/expected upper bound fraction (LOEUF) of 0.396 in the population (gnomAD v4.0.0), indicating that *EGR3* is haploinsufficient. Haploinsufficiency is typically observed in genes associated with bicuspic aortic valve (BAV) disease, such as GATA6(64) and NOTCH(65, 66). Of note, case reports have shown that chromosomal duplications of the region where *EGR3* is located (8p21.3) are associated with congenital heart disease (i.e., valve and septal defects) (67, 68), while a case-control study has reported an association between an *EGR3* polymorphism and coronary artery disease (CAD) (69); though with no mention of possible valve defects.

In parallel to *egr3* upregulation following mechanical stimulation in the developing zebrafish heart (Fig. 4, C and D), *EGR3* is upregulated in LVAD aortic valves upon biomechanical overload (Fig. 4F). EGR3 is necessary and sufficient to partially mediate the TGF-β fibrotic response in fibroblasts (70). Thus, we speculate that the biomechanical stimulus that promotes *EGR3* expression during cardiac development also promotes it in LVAD aortic valves, thereby contributing to pathological aortic valve remodeling and insufficiency (60-62). Further work should address whether the expression of *EGR3*, its
targets, and *KLF2* plays a role in the mechanotransduction of LVAD aortic valve pathogenesis.

Transduction of mechanical forces, which is partially mediated by VEGF signaling (71), also plays a role in lymphatic endothelial cell (LEC) physiology and development. VEGF is a known upstream regulator of *EGR3* and *NR4A2* expression in endothelial cells (50, 55, 72). In fact, human LECs upregulate *EGR3* and *NR4A2* expression following VEGF stimulation (73, 74), and a recent report on the transcriptional coactivator *Zmiz1* suggests that *Egr3* is involved in lymphatic valve formation (75).

In summary, our study reveals a novel signaling axis in which *EGR3* is necessary to transduce the mechanical signals required for cardiac valve formation and potentially also for LVAD valve remodeling. We anticipate that perturbations in the function or expression of *EGR3*, or its targets, also lead to cardiac valve defects in humans.

## Materials and Methods

### Zebrafish handling and lines

All zebrafish husbandry was performed under standard conditions in accordance with institutional (Max Planck Gesellschaft) and national (German) ethical and animal welfare regulations. All procedures performed on animals conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the Animal Protection Committee (Tierschutzkommission) of the Regierungspräsidium Darmstadt (reference: B2/1218).

The following mutant, knockin reporter, and transgenic lines were generated for this study: *egr3<sup>bns522</sup>, egr3<sup>bns577</sup>, Pt(egr3:Gal4-VP16)<sup>bns576</sup> abbreviated as egr3:Gal4, Pt(egr3:loxP-egr3-loxP)<sup>bns661</sup>, Tg(5xUAS:egr3-p2a-dTomato)<sup>bns607</sup> abbreviated as egr3:OE. The following transgenic and mutant lines were used in this study:

* tnnt2a<sup>mn0031Gt</sup> or Gt(GBT-R14)(76), Tg(kdrl:EGFP)<sup>s843 (29)</sup>, Tg(kdrl:Hsa.HRAS-mCherry)<sup>s896 (77)</sup>, Tg(kdrl:NLS-mCherry)<sup>is4 (78)</sup>, Tg(fli1a:Gal4FF)<sup>ubs4 (79)</sup>, Tg(myl7:BFP-CAAX)<sup>bns193 (80)</sup>, Tg(myl7:mCherry-CAAX)<sup>bns7 (81)</sup>, Tg(myl7:EGFP)<sup>twu26 (82)</sup>, Tg(5xUAS:EGFP)<sup>nkuasgfp1a (83)</sup>, Tg(UAS:Kaede)<sup>rk8 (84)</sup>, Tg(myl7:Cre)<sup>sd55 (85)</sup>, Tg(kdrl:Cre)<sup>s898 (86)</sup>.

### Generation of zebrafish lines

For Crispr/Cas9 mutagenesis, *in vitro* synthesis of Cas9 mRNA and design of gRNAs were performed as previously described (87, 88). gRNA efficiency was evaluated in single
AB wild-type injected embryos by High Resolution Melting Analysis (HRMA) or T7
endonuclease assay. Generation of egr3 full locus deletion (bns522) was achieved by
using one gRNA in the immediate 5' intergenic region and another in the 3' UTR, and F1
adults were screened by PCR followed by sequencing of PCR products. The egr3 Δ11
allele (bns577) was generated with commercially available egr3 gRNA (IDT), and F1
adults were screened by HRMA followed by sequencing of PCR products. The egr3:Gal4
reporter line (bns576) was generated according to the GeneWeld method(89) with 48 bp
homology arms flanking the p2a-Gal4-VP16-beta-actin sequence, and using the egr3
gRNA (IDT) as for the egr3 Δ11 allele (bns577). We used the previously published
sequential loxP knockin method(90) to generate the egr3 floxed allele (bns661) with
single-stranded donors containing loxp sites flanked by asymmetric 21 and 49 bp
homology arms. For the 5' loxP knockin we used the same 5' gRNA as for the full locus
deletion allele (bns522). Adult F1 zebrafish were screened for the 5' loxP integration
using a loxP-specific PCR primer and sequenced after performing a loxP-flanking PCR.
The 5' loxP founder fish was then crossed with AB wild type and the resulting one-cell
stage embryos were injected with a 3' gRNA in the neighboring intergenic region. Adult
F1 egr3 floxed allele zebrafish were screened by loxP-specific and flanking PCRs
followed by sequencing of PCR products. Despite observing a complete and adequate
integration of the 5' loxP site, we have not been able to sequence the full extent of the 3'
loxP site. Genetic polymorphisms and/or integration of multiple copies after mutagenesis
in the 3' region of the zebrafish used for 3' loxP knockin might explain why we could not
sequence the loxP integration. However, floxing of egr3 allele was confirmed by the
loxP-specific and flanking PCRs, as well as by functional analysis after Cre mRNA
injection, in which the egr3 phenotypes were observed at expected Mendelian ratios.
Conditional knockouts were obtained by crossing egr3<sup>flox/+</sup> zebrafish with egr3<sup>bns577 +/-</sup>
zebrafish carrying either Tg(kdrl:Cre) or Tg(myl7:Cre), for endothelial or myocardial cell-
specific knockout, respectively. We used tol2 transgenesis to generate the egr3
overexpression (egr3:OE) line (bns607), and adult F1 zebrafish were screened by crossing
with Tg(fli1a:Gal4FF) zebrafish and using the dTomato expression in the progeny as a
proxy. dTomato<sup>+</sup> embryos were used to confirm transgenesis by PCR amplification and
sequencing of PCR products. Of note, the data presented in this work were collected from
F2 to F4 animals and a reduction in the penetrance of the phenotype was observed in the
next generations. Sequences of all genotyping primers, CRISPR sites, and donor
oligonucleotides used are available in the Supplementary materials.
## Plasmid construction

The ORFs for *egr3*, *nr4a2b*, *spp1*, *nrg1*, *hey2*, and *has2* were amplified from cDNA generated from a pool of 78 hpf larvae, and cloned into a pCS2+ vector. To generate the *egr3:gal4* line, the pGTag-gal4-VP16-beta-actin plasmid was a gift from Jeffrey Essner (Addgene # 117817) and homology arms were added by vector ligation(91). To generate the *egr3:OE* line (*bns607*) and *nr4a2b:OE* construct, *egr3* and *nr4a2b* were amplified from pCS2+ and cloned into a *5XUAS:p2a-dTomato* vector. Vector ligation was performed by PCR amplification followed by *in vivo* cloning(92). All primers are listed in the Supplementary materials.

## Microscopy imaging

Live imaging of zebrafish stopped hearts was performed by mounting PTU-treated embryos and larvae in 1% agarose containing 0.2% tricaine. We used the following confocal microscopes: LSM700 Axio Imager 2 and LSM880 Axio Examiner with a W Plan-Apochromat 40×/1.0 dipping lens for image acquisition. We used Fiji (ImageJ 2.1.0/1.53o) to adjust the brightness and contrast of representative images. For three-dimensional rendering and analysis of confocal images, we used Imaris x64 (Bitplane, 10.0.1). We used the Imaris cell identification function to automatically segment endocardial cells based on their nucleus, marked by *Tg(kdrl:NLS-mCherry)* expression, and their cytoplasm, marked by *Tg(kdrl:EGFP)* expression. The endocardial cells where further segregated into ventricular, atrioventricular and atrial with the help of the x/y position object filtering followed by manual curation.

## In situ hybridization and immunostaining

Whole-mount RNA *in situ* hybridization was performed as previously described(93). *in situ* probes for *egr3*, *spp1*, *nrg1*, *hey2*, and *has2* were synthesized from previously cloned ORFs in pCS2+ vectors using PCR products with an incorporated T7 promoter. *in situ* probe for *egr1* was kindly provided by Julien Vermot in a pBSK vector(35). *in situ* probes for *klf2a(12)*, *klf4*, *nr4a2b*, *piezo2a(12)* were synthesized directly from a PCR product of 78 hpf wild-type AB cDNA, using primers with an incorporated T7 promoter. To visualize the heart outline, we used the primary Mouse anti-MHC antibody (1:500, MF-20, DSHB AB_2147781), followed by the secondary Goat anti-Mouse IgG Alexa Fluor 488 antibody (1:500, Invitrogen A11029). To assess atrioventricular valve identity, we
performed immunostaining for Alcama using Mouse anti-Alcama antibody (1:500, ZN-8, DSHB AB_531904) followed by Goat anti-Mouse-Alexa 647 antibody (1:500, Invitrogen A21236). *In situ* hybridizations were imaged using an SMZ25 stereomicroscope (Nikon) with a 2x/0.3 objective. Description of primers used to synthesize the *in situ* probes can be found in the Supplementary materials.

## AV blood flow fraction

Functional analysis of blood regurgitation was performed in 72 hpf zebrafish larvae mounted in 1% agarose without tricaine. The zebrafish hearts were live imaged using an inverted Cell Observer Spinning Disk microscope with a 25x/0.8 water-immersion objective at 240 frames per second (fps). Quantification of AV blood flow fraction was performed using a custom-made ImageJ script as previously described(12). For each imaged larva, AV retrograde blood flow is shown as a fraction of three averaged cardiac cycles.

## Microsurgery insertion of a bead

To assess the influence of ectopic mechanical stimulation on *egr3* expression *in vivo*, a bead was kept inside of the beating zebrafish heart for 24h(18, 57). For that manipulation, 48 hpf zebrafish embryos were mounted ventrally in 1% agarose and a microsurgical incision was performed in the yolk-sac using thin forceps. A bead (PureCube Glutathione MagBeads, Cube Biotech) was inserted with the forceps into the yolk cavity and gently pushed into the inflow tract, eventually reaching the heart through suction. Embryos were released from the agarose and kept in egg water at 28.5°C for 24 h, when *egr3:Gal4* reporter expression was assessed by confocal microscopy. Only larvae that had the bead inside of the ventricle and still displayed blood flow were considered for the analysis. Sham embryos underwent the same procedure of yolk piercing, but without bead insertion.

## Chemical treatments

To evaluate the influence of cardiac contraction on *egr3* expression, we treated wild-type embryos with 15 mM of the myosin inhibitor BDM (Sigma B0753). Possible regulation of AV *egr3* expression by VEGF signaling was tested by treating embryos with 1 µM and 2.5 µM of SKLB1002 (Selleck Chemicals S7258)(94) and 0.5 µM of SU5416 (Sigma S8442). Role of ERK signaling on *egr3* expression was assessed by treating embryos with 1 µM of the MERK inhibitor PD0325901 (Sigma PZ0162)(2). Concentrations were
adjusted in order to diminish side effects. 0.1% DMSO was administered as vehicle and all treatments were performed from 36 to 48 hpf, when embryos were collected and fixed for *in situ* hybridization.

## Research ethics for donated aortic valve samples

The study population consisted of left ventricular assist device (LVAD; n=6) patients referred for heart transplantation at La Timone Hospital (PMID: 34374902). Patients were not included if they had connective tissue disease and previous aortic valve dysfunction. The patients provided their written informed consent to participate in this study (approved by the Marseille ethic committee n°13.061). Aortic regurgitation (AR) was graded through an integrative approach(95, 96). Mechanism of AR was separated into prolapse of the fused leaflet, cusp restriction or both. Aortic valves were recognized by their anatomical landmarks under the microscope, and leaflets were isolated with minimal aortic wall contamination. Immediately following surgical removal, a portion of the aortic valve leaflet was placed in RNAlater solution (Sigma) and stored at -80°C until processing. Control valve samples were obtained at autopsy of individuals without cardiac problems who suffered a traumatic death (n=8). Control group was composed by individuals with an average age of 59 years +/- 4 years, and a male to female ratio (M/F) of 85%. LVAD group was composed by individuals with an average age of 54 years +/- 12 years, and M/F ratio of 71%. This study was performed according to the principles of the Declaration of Helsinki and in accordance with institutional guidelines.

## qRT-PCR

Total RNA from human aortic valve samples was purified using Trizol (Life Technologies) and RNeasy mini Kit (Qiagen). RNA was reverse transcribed using the AffinityScript Multiple Temperature cDNA synthesis kit (Agilent). Quantitative PCR was performed using SYBR Green (Roche) based qPCR on a LightCycler 480 (Roche). Graphs show the mean +/- standard deviation for 6-8 biological replicates. Samples were normalized to TBP or GAPDH as endogenous housekeeping genes. Relative expression levels were calculated by the comparative cycle threshold (ΔΔCt) method. All Ct and ΔCt values are listed in the Supplementary materials.

## scRNA-seq sample preparation and data analysis
In order to obtain a longitudinal transcriptional signature of single endocardial cells, we isolated hearts from *Tg(myl7:EGFP)* wild-type zebrafish at 50 and 80 hpf. Up to 150 hearts were isolated per condition as previously described(97). Briefly, zebrafish embryos and larvae were fragmented using a needle and syringe in an Eppendorf tube with 1 ml of DMEM (ThermoFisher 88281) with 10% FBS (Sigma F2442). The homogenate was filtered through a 100 µm mesh filter. An additional wash with DMEM with 10% FBS was performed to remove the remaining hearts in the Eppendorf tube, followed by filtering. The isolated hearts were manually collected from the flow-through under a fluorescence microscope. The hearts were further dissociated using the Pierce Primary Cardiomyocyte Isolation Kit (ThermoFisher 88281) and incubated for 25 minutes at 30°C (shaker 300 rpm) with pipetting every 5-10 minutes. Dead cells were removed from the final cell isolate by FACs sorting in PBS without Calcium and Magnesium (Lonza, 17-516F) and with 0.04% BSA. The cell suspensions were counted with a Moxi cell counter and diluted according to the manufacturer's protocol to obtain 10.000 single cell data points per sample. Each sample was run separately on a lane in a Chromium controller with Chromium Next GEM Single Cell 3' Reagent Kits v3,1 (10xGenomics). scRNA-seq library preparation was done using a standard protocol, and sequencing was done on a Nextseq2000. scRNA-seq analysis was performed as previously described(98) and final data visualization was done using a CellxGene package (doi:10.5281/zenodo.3235020).
Endocardial cells were identified by the expression of endothelial markers (e.g., *cdh5*, *flilα*, *kdrl*, *tie1*) and represented 3504 from the whole 6476 sequenced heart cells. Valve endocardial cells were identified by the expression of valve markers (e.g., *alcama*, *has2*) and represented 1343 cells. Data analysis was performed using only the endocardial cells, and the other cell types are not displayed for visualization purposes.

RNA-seq sample preparation and data analysis

To evaluate the transcriptional differences underlying the *egr3* mutant phenotype, we conducted a bulk RNA-seq analysis in dissected zebrafish hearts at 48 hpf. As *egr3* mutants cannot be distinguished from their siblings at this stage, embryos were live genotyped at 36 hpf as previously described(99). We dissected 20 hearts for each biological duplicate of *egr3* mutant and wild-type sibling samples in cold DMEM with 10% FBS. Total RNA was isolated using the miRNeasy micro Kit (Qiagen 217084), followed by on-column DNase digestion (DNase-Free DNase Set, Qiagen 79254). Final elution was performed in 12 µl of RNase-free water, and subsequent RNA quality control,
cDNA preparation, sequencing, and analyses were performed as previously described(94).
Analysis of the obtained reads confirmed the 11 bp deletion in the mutant samples (data
not shown). A total of 154 genes were significantly dysregulated based on a padj-
value<0.05. Heatmaps were obtained using the Webbased Interactive Omics
visualizatioN – Applications (WIlSOn)(100).

Statistical analysis

GraphPad Prism (v.9) was used to perform all statistical analyses. We used two-tailed
Student's t-test for comparing two samples (Fig. 3, H and K, Fig. 4, D and F, Fig. S4D),
and one-way ANOVA followed by Tukey's post hoc test for multiple comparisons (Fig.
1I, Fig. S4C); data are presented as mean ± SEM and actual p-values are shown. Two-
sided fisher's exact test was used to compare categorical data between two groups (Fig.
1H and J and L, Fig. 2, E and F, Fig. 3E and G, Fig. S3C) and data were transformed into
percentages after statistical analysis to improve visualization. To allow for statistical
analysis, the classifications "Superior valve leaflet" and "Superior s-shape leaflet" were
collapsed into "Superior valve leaflet", and the "Monolayer / one VIC" and "Monolayer"
were collapsed into "Monolayer" (Fig. 1H); all classifications and proportions are in Fig.
S1, G to I and Fig. S3I. All zebrafish experiments were performed independently at least
twice, using different batches of embryos and in different days unless otherwise indicated.
Exact sample sizes and p-values are described in the figures and figure legends. p-
values<0.05 were considered significant.
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## Acknowledgements

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We thank Y. Xu for discussion and comments on the manuscript, A. Atzberger, K.

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Khrievono, N. Fukuda, S. Howard, S. Perathoner and H.-M. Maischein for their technical support, J. Vermot for providing the *egr1 in situ* probe, S. Abdelilah-Seyfried and V.

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Lecaudey for their comments and suggestions as thesis advisory committee members. We thank A. Gentile, C. Dooley, J. Martinez-Meyer, and P. Panza for discussions and important input during the development of this work.

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## Funding

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This work was supported by funds from the Max Planck Society to D.Y.R.S.

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## Author contributions

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Conceptualization: ARS, FG, SZ, TJ, DYRS

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Methodology: ARS, FG, EF, AT, JFA, RR, SG, ML, TJ

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Investigation: ARS, FG, GLMB, EF, AT, JFA, SL, SG, TJ

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Visualization: ARS, RR, TJ

863
Supervision: FG, SZ, TJ, DYRS

Writing – original draft: ARS

Writing – review & editing: FG, GLMB, SZ, TJ, DYRS

**Competing interests:** The authors declare no competing interests.

**Data and materials availability:**

scRNA-seq data of 50 and 80 hpf whole isolated hearts have been deposited in NCBI's

Gene Expression Omnibus under accession number GSE242483 and it can be interactively
accessed at:

https://bioinformatics-public.mpi-bn.mpg.de/mampok-cluster-bn/dst127-boezio/cellxgene-new/

Analyzed Bulk RNA-seq of 48 hpf dissected hearts is provided in the Supplementary
materials. Raw bulk RNA-seq data have been deposited in NCBI's Gene Expression
Omnibus under accession number GSE241935.

Previously published data are available at:

Human snRNA-seq data are available online at the Broad Single Cell Portal under study number: SCP1852 (38).

Mouse scRNA-seq data were available online at the Broad Single Cell Portal (39).

Uniprot was used to align zebrafish, human, and mouse Egr1-3: P26632, P18146, P08046, E7F266, Q05159, P11161, P08152, Q6DG27, Q06889, and P43300.

All data are available in the main text or the supplementary materials. Materials and raw images are available upon request to the corresponding authors.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Egr3 is required for cardiac valve formation. (A), Schematic of hearts isolated for scRNA-seq; valve endocardial cells (magenta), non-valve endocardial cells (blue). (B), scRNA-seq of endocardial cells from 50 and 80 hpf dissected zebrafish hearts. (C), Dot plot of 30 most differentially expressed genes in valve compared with non-valve endocardial cells. (D), Schematic of generation of egr3 full locus deletion (bns522) and Δ11 (bns577) alleles. (E-F), Brightfield images of egr3<sup>+/+</sup> and egr3<sup>-/-</sup> sibling larvae at 80 hpf. (G), Confocal images of representative hearts from 72 and 96 hpf egr3<sup>+/+</sup> and egr3<sup>-/-</sup> sibling larvae. (H), Percentage of egr3<sup>+/+</sup> and egr3<sup>-/-</sup> sibling larvae with a superior valve leaflet and without a superior valve leaflet (i.e., endocardial monolayer) at 72 and 96 hpf;</figcaption></figure>

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7 and 4 independent experiments, respectively. (I), AV retrograde blood flow shown as a
fraction of three cardiac cycles; n = 6, 11, and 3; 1 experiment; mean ± SEM, one-way
ANOVA followed by Tukey's post hoc test. (J), Peripheral circulation in the caudal vein
plexus at 80 hpf, 1 experiment. (K), Confocal images of representative outflow tract
valves from 96 hpf egr3⁺/⁺ and egr3⁻/⁻ sibling larvae. (L), Percentage of larvae with
forming outflow tract leaflets and with no outflow tract leaflets at 96 hpf; 4 independent
experiments. (G, K), Endocardial cells are marked by Tg(kdrl:eGFP) expression (cyan),
myocardial cells by Tg(myl7:BFP-CAAX) expression (magenta). # indicates illustrative
vectorized cartoons of the valves. (H, j, L), Fisher's exact test. AV = atrioventricular, V
= ventricle, A = atrium, OFT = outflow tract, DBD = DNA binding domain, EdC =
endocardial cell. Scale bars (E, F) = 400 µm, (G, K) = 20 µm.
A

<figure><img src="image_4.png" /></figure>

C egr3 floxed

<figure><img src="image_5.png" /></figure>

B
Endocardium egr3
Myocardium egr3

<figure><img src="image_6.png" /></figure>

D

<figure><img src="image_7.png" /></figure>

E

<figure><img src="image_8.png" /></figure>

F

<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Endothelial-specific deletion of *egr3* recapitulates the global mutant phenotype. (A), Schematic of the knockin reporter *Pt(egr3:gal4-VP16)* generated by inserting *gal4* in exon 2 of *egr3*. (B), *Pt(egr3:gal4-VP16)*; *Tg(UAS:eGFP)* expression (grey) at 72 hpf; myocardial cells are marked by *Tg(myl7:BFP-CAAX)* expression</figcaption></figure>
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(magenta) and endocardial cells by Tg(kdrl:Hsa. HRAS-mCherry) expression (cyan);
yellow arrowheads point to AV egr3+ cardiomyocytes; (B') maximum projection of
Pt(egr3:gal4-VP16); Tg(UAS:eGFP) expression with image segmentation of egr3
reporter+ AV endocardial cells (cyan), egr3 reporter+ AV myocardial cells (magenta), and
egr3 reporter+ outflow tract (yellow). (C), Schematic of egr3 floxed allele Pt(egr3:loxP-
egr3-loxP). (D), Confocal images of representative hearts from 72 hpf egr3 +/,
endothelial cell-specific egr3 -/-, and myocardial cell-specific egr3 -/ larvae using the egr3
floxed allele line. (E-F), Percentage of 96 hpf egr3 +/?, endothelial cell-specific egr3 -/ (E),
and myocardial cell-specific egr3 -/ (F) larvae with a superior valve leaflet and without a
superior valve leaflet (i.e., endocardial monolayer); 1 and 2 independent experiments,
respectively; Fisher's exact test. AV = atrioventricular, V = ventricle, A = atrium. Scale
bars = 20 µm.
<figure><img src="image_10.png" /><figcaption>**Fig. 3.** *egr3* directs endocardial cell migration, which is required for cardiac valve formation. (A), RNA-seq heatmap analysis of differentially expressed genes; fold-change ± 1.5, padj < 0.05, mean > 5. (B), scRNA-seq endocardial expression pattern of Egr3 targets. (C), *in situ* hybridization for Egr3 targets *nr4a2b* and *spp1* in *egr3*<sup>+/+</sup> and *egr3*<sup>-/-</sup> siblings at 48 hpf. (D), Confocal images of representative hearts from 48 hpf *egr3*<sup>+/-</sup> and *egr3*<sup>-/-</sup> sibling embryos displaying, respectively, AV endocardial cell protrusions towards the ECM and no protrusions; endocardial cells are marked by Tg(kdrl:eGFP) expression</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 4.** *egr3* expression in the heart is regulated by biomechanical forces. (A), *in situ* hybridization for *egr3* expression in 48 hpf *tnnt2a*<sup>+/+</sup> and *tnnt2a*<sup>-/-</sup> sibling embryos. (B), Illustration of bead insertion into the heart (48 hpf) via an incision in the yolk and the use of fine forceps to guide the bead into the inflow tract. (C), *Pt(egr3:gal4-VP16)*; *Tg(UAS:eGFP)* expression (grey) at 72 hpf in bead-inserted larvae vs sham; myocardial cells are marked by *Tg(myl7:BFP-CAAX)* expression (magenta) and endocardial cells by *Tg(kdrl:Hsa. HRAS-mCherry)* expression (cyan). Red dotted circle highlights inserted bead; yellow arrowhead points to *egr3+* cells near the inserted bead. (D), Quantification of total volume of *egr3* reporter<sup>+</sup> cells in bead-inserted larvae and sham; n = 3 and 5; 1 experiment. (E), Representative control (*Tg(fli1a:gal4);Tg(UAS:Kaede)*), and endothelial-specific *egr3* overexpressing (*Tg(fli1a:gal4);Tg(UAS:Kaede);Tg(UAS:egr3-p2a-dTomato)*) hearts of *tnnt2a* MO injected embryos stained for Alcama expression; cropped regions show no alcama expression in the AV canal of *tnnt2a* MO injected wild-type hearts and AV Alcama expression in *tnnt2a* MO injected *Tg(UAS:egr3-p2a-dTomato)*<sup>+</sup> embryos. (F), Quantification of Alcama + endocardial cells present in the</figcaption></figure>
ECM per cardiac region of *tnnt2a* MO injected embryos. (G), High shear forces induce the expression of the mechanosensitive transcription factor Egr3 in valve EdCs where it orchestrates valvulogenesis by promoting their migration via Nr4a2b. (H), Schematic of aortic valve collection from left ventricular assist device (LVAD) patients and control donors. (I), Relative mRNA levels of *EGR3*, *KLF2*, *NR4A2*, and *SPP1*, from aortic valves of LVAD patients and control donors; n = 4 and 4-6; 1 experiment. (D, F, I), Student's t test, mean ± SEM. V = ventricle, A = atrium. Ct values can be found in the Source Data File. Scale bars = 20 µm.