Wnt signaling is a crucial regulator of embryogenesis through its regulation of body axis patterning, cell fate determination, cell migration, and cell proliferation (Logan and Nusse 2004, Steinhart and Angers 2018, Mehta, Hingole et al. 2021). Current mechanistic understanding of Wnt signaling during embryogenesis includes an extensive catalog of ligands, receptors, co-receptors, adaptors, and effector molecules (Clevers and Nusse 2012, Niehrs 2012, Loh, van Amerongen et al. 2016, Mehta, Hingole et al. 2021). The intricate spatiotemporal integration of Wnt signaling combinations is an important focus of developmental biology and tissue morphogenesis (Petersen and Reddien 2009, Clevers and Nusse 2012, Loh, van Amerongen et al. 2016, Wiese, Nusse et al. 2018). Disruptions of Wnt signaling-associated genes lead to a number of congenital malformations which often affect multiple organ systems given their pleotropic developmental functions (Hashimoto, Morita et al. 2014, Shi 2022). Craniofacial anomalies are among the most common structural congenital malformations and genes in the Wnt signaling pathway are frequently implicated (Ji, Hao et al. 2019, Reynolds, Kumari et al. 2019, Huybrechts, Mortier et al. 2020). The roles of certain Wnt signaling components may change in different temporal and spatial contexts, requiring detailed developmental and genetic analyses.

Genetic approaches in zebrafish have identified a number of key Wnt regulators of early development, with gastrulation and craniofacial phenotypes (Brand, Heisenberg et al. 1996, Hammerschmidt, Pelegri et al. 1996, Heisenberg, Brand et al. 1996, Piotrowski, Schilling et al. 1996, Solnica-Krezel, Stemple et al. 1996, Schilling and Le Pabic 2009). The silberblick (slb) mutant, later identified as a wnt11f2 allele, exhibited gastrulation and midline craniofacial phenotypes that encompassed aspects of multiple mutant classes (Heisenberg, Brand et al. 1996). During early segmentation in the somite stage, the wnt11f2 mutant developed a shortened anterior-posterior axis and partially fused eyes (Heisenberg, Brand et al. 1996). Subsequently as the cranial prominences converge and the anterior neurocranium (ANC) formed, instead of a fan-shaped structure observed in wildtype embryos, the wnt11f2 mutant ANC appeared rod-like, with a significant deficiency of the transverse dimension (Heisenberg, Brand et al. 1996, Heisenberg and Nusslein-Volhard 1997). Another mutant knypek, later identified as gpc4, an extracellular Wnt co-receptor, was identified as a gastrulation mutant also with a shortened body axis (Solnica-Krezel, Stemple et al. 1996). In contrast, the gpc4 mutant formed an ANC that is wider in the transverse dimension than the wildtype, in the opposite end of the ANC phenotypic spectrum compared to wnt11f2 (Topczewski, Sepich et al. 2001, Rochard, Monica et al. 2016). These observations beg the question how defects in early patterning and convergent extension of the embryo may be associated with later craniofacial morphogenesis. The observation that wnt11f2 and gpc4 mutant share similar gastrulation and axis extension phenotypes but contrasting ANC morphologies supports a hypothesis that convergent extension mechanisms regulated by these Wnt pathway genes are specific to the temporal and spatial context during embryogenesis. As such, there may be Wnt signaling modifiers that act in specific temporal windows during development.

Dact (aka frodo, dapper) are scaffolding proteins that were identified to regulate Dishevelled (Dvl)-mediated Wnt signaling, both positively and negatively (Gloy, Hikasa et al. 2002, Waxman, Hocking et al. 2004, Gao, Wen et al. 2008, Wen, Chiang et al. 2010, Ma, Liu et al. 2015, Lee, Cheong et al. 2018). Dact proteins bind directly to Dvl (Gloy, Hikasa et al. 2002, Brott and Sokol 2005, Lee, Cheong et al. 2018) and have been found to interact with and inhibit members of TGFβ and Nodal signaling pathways (Zhang, Zhou et al. 2004, Su, Zhang et al. 2007, Meng, Cheng et al. 2008, Kivimae, Yang et al. 2011). Previous experiments using morpholinos to disrupt dact1 and dact2 in zebrafish found dact1 morphants to be slightly smaller and developed a normal body axis (Waxman, Hocking et al. 2004). In contrast, dact2 morphants were found to phenocopy described zebrafish gastrulation mutants, with impaired convergent extension, shortened body axis, and medially displaced eyes (Waxman, Hocking et al. 2004). By disrupting dact1 and dact2 and various wnt genes concurrently, it was concluded that dact1 enhances wnt/β-catenin signaling, while dact2 interacts with the wnt/PCP pathway (Waxman, Hocking et al. 2004).

Here we investigated the genetic requirement of dact1 and dact2 during embryogenesis and craniofacial development, using germline mutant alleles generated by CRISPR-targeted mutagenesis. We examined the connection between convergent extension governing gastrulation, body axis segmentation, and craniofacial morphogenesis. Differential single-cell RNA sequencing between wildtype, dact1/2, and gpc4 mutants revealed distinct transcriptome profiles and the discovery of calpain 8 (capn8) calcium-dependent protease to be ectopically expressed in the dact1/2 mutants and functions to mediate Wnt signaling and craniofacial morphogenesis.


dact1 and dact2 have distinct expression patterns throughout embryogenesis

To determine the spatiotemporal gene expression of dact1 and dact2 during embryogenesis we performed wholemount RNA in situ hybridization (ISH) across key time points (Fig. 1) (Farrell, Wang et al. 2018, Lange, Granados et al. 2023). Given that the described craniofacial phenotypes of the dact2 morphant and the wnt11f2 mutant are similar (Heisenberg and Nusslein-Volhard 1997, Waxman, Hocking et al. 2004), we also performed wnt11f2 ISH to compare to dact1 and dact2 expression patterns. Until spatial transcriptomics is widely applied for zebrafish, the wholemount ISH provides anatomic registry not available through scRNAseq atlas, though the latter is increasingly helpful to provide details on cell types expressing the genes of interest.

Unique and shared dact1 and dact2 gene expression domains during zebrafish development. (A-C) Whole-mount in situ hybridization showing dact1, dact2, and wnt11f2 gene expression patterns. Scale bar = 100 μm. A) At 8hpf, dact2 and wntllf2 are highly expressed in the dorsal margin and presumptive Nieuwkoop center of the gastrulating embryo, with dact1 being weakly detected (arrowhead). In contrast to wnt11f2, dact1 and dact2 are expressed in the presumptive dorsal mesoderm (asterisk). B) Lateral (anterior to the left of page) and anterior (dorsal side toward top of page) views of bud-stage embryos. dact2 and wnt11f2 transcripts are detected in the anterior neural plate (arrowhead) and tailbud (asterisk) while dact2 is additionally expressed in the axial mesoderm (arrow). dact1 gene expression is concentrated to the paraxial mesoderm (open arrowhead). C) Lateral and flat-mount views of 4 ss embryos. dact2 is expressed in the anterior neural plate and polster (P), notochord (N), paraxial and presomitic mesoderm (PM) and tailbud (TB). wnt11f2 is also expressed in these cells (Thisse 2001). In contrast, dact1 is expressed in the midbrain (MP) and the paraxial and presomitic mesoderm. D) RNAscope in situ hybridization analysis of dact1 (white) and dact2 (yellow) and irf6 (green) expression in transverse section of 72 hpf embryos. dact1 is expressed in the orofacial cartilage (pq), while dact2 is expressed in the oral epithelium (oe). The epithelial marker irf6 is expressed in the oe and surface epithelium (se). Dapi (blue). Scale bar = 100 μm. E) Daniocell single-cell RNAseq analysis with a display of dact1, dact2, wnt11f2 and gpc4 clusters from 3-120 hpf of development (Farrell, Wang et al. 2018).

During gastrulation at 8 hours post-fertilization (hpf; 75% epiboly), some regions of dact1 and dact2 gene expression were shared and some areas are distinct to each dact gene (Fig. 1A). Further, dact gene expression was distinct from wnt11f2. Transcripts of dact1, dact2 and wntllf2 were all detected in the blastoderm margin, as previously described (Gillhouse, Wagner Nyholm et al. 2004). Transcripts of dact2, and to a lesser extent dact1, were also detected in the prechordal plate and chordamesoderm (Fig. 1A). Additionally, dact2 gene expression was concentrated in the shield and presumptive organizer or Nieuwkoop center along with wnt11f2. This finding is consistent with previously described expression patterns in zebrafish and supports a role for dact1 and dact2 in mesoderm induction and dact2 in embryo dorsalization (Thisse 2001, Gillhouse, Wagner Nyholm et al. 2004, Muyskens and Kimmel 2007, Oteiza, Koppen et al. 2010). At the end of gastrulation and during somitogenesis the differences in the domains of dact1 and dact2 gene expressions became more distinct (Fig. 1B,C). At tailbud stage, dact1 transcripts were detected in the trunk and the posterior paraxial mesoderm, whereas dact2 transcripts were detected in the anterior neural plate, notochord, and tailbud, similar to wnt11f2 gene expression. However, dact2 was unique in that its expression demarcated the anterior border of the neural plate. As dact2 morphants exhibited a craniofacial defect with medially displaced eyes and midfacial hypoplasia (Waxman, Hocking et al. 2004), we examined dact1 and 2 expression in the developing orofacial tissues. We found that at 72 hpf dact2 and the epithelial gene irf6 were co-expressed in the surface and oral epithelium that surround the cartilaginous structures (Fig. 1D). This is in line with our prior finding of decreased dact2 expression in irf6 null embryos (Carroll, Macias Trevino et al. 2020). In contrast to dact2, dact1 was expressed in the developing cartilage of the ethmoid and palatoquadrate of the zebrafish larvae (Fig. 1D).

The differences in expression pattern between dact1 and dact2 using publicly available Daniocell single-cell sequencing data (Farrell, Wang et al. 2018) (Fig. 1E). During embryogenesis, dact2 was more highly expressed in anterior structures including cephalic mesoderm and neural ectoderm while dact1 was more highly expressed in mesenchyme and muscle (Fig. 1E). We also utilized this tool to compare dact1 and dact2 expression to wnt11f2 and gpc4 expression, known noncanonical Wnt pathway members with craniofacial phenotypes (Heisenberg, Brand et al. 1996, Solnica-Krezel, Stemple et al. 1996, Heisenberg and Nusslein-Volhard 1997). In general, we found dact1 spatiotemporal gene expression to be more similar to gpc4 while dact2 gene expression was more similar to wnt11f2. These results of shared but also distinct domains of spatiotemporal gene expression of dact1 and dact2 suggest that these paralogs may have some overlapping developmental functions while other roles are paralog specific.

dact1 and dact2 contribute to axis extension and dact1/2 compound mutants exhibit a convergent extension defect

dact1 and dact2 are known to interact with disheveled and regulate non-canonical Wnt signaling (Gloy, Hikasa et al. 2002, Waxman, Hocking et al. 2004, Gao, Wen et al. 2008, Wen, Chiang et al. 2010, Ma, Liu et al. 2015, Lee, Cheong et al. 2018). Previous work investigated the effect of dact1 and dact2 disruption during zebrafish embryogenesis using morpholinos and reported morphant phenotypes in embryonic axis extension and eye fusion (Waxman, Hocking et al. 2004). We were interested in how disruption of convergent extension in early embryogenesis may be related to anterior neurocranium (ANC) malformation, as has been described for slb (wnt11f2) and dsh mutants (Piotrowski, Schilling et al. 1996, Kimmel, Miller et al. 2001, Xing, Cheng et al. 2018). Further, we aimed to examine the genetic interactions between dact1/dact2 and key Wnt regulators such as wnt11f2, gpc4 and wls. These experiments required germline alleles in order to ensure the reproducibility of data and warranted generation of dact1 and dact2 alleles via CRISPR/Cas9 mediated targeted mutagenesis (Fig. S1). We generated a dact1 mutant allele (22 bp deletion, hereafter dact1-/-) and a dact2 allele (7 bp deletion, hereafter dact2-/-), both resulting in a premature stop codon and presumed protein truncation (Fig. S1). The specificity of the gene disruption was demonstrated by phenotypic rescue with injection of dact1 or dact2 mRNA (Fig. S1).

Analysis of compound dact1 and dact2 heterozygote and homozygote alleles during late gastrulation and early segmentation time points identified embryonic axis extension anomalies (Fig. 2A). dact1-/- or dact2-/- homozygotes developed normally. However, at 12 hpf, dact2-/- single mutants have a significantly shorter body axis relative to wildtype. In contrast, body length shortening phenotype was not observed in dact1-/- homozygotes. Compound heterozygotes of dact1+/-; dact2+/- also developed normally but exhibited shorter body axis relative to wildtype. The most significant axis shortening occurred in dact1-/-; dact2-/- double homozygotes with a less severe truncation phenotype in the compound heterozygote dact1+/-; dact2-/- (Fig. 2A). Interestingly, these changes in body axis extension do not preclude the compound heterozygous larvae from reaching adulthood, except in the dact1-/-; dact2-/- double homozygotes which did not survive from larval to juvenile stages.

Impaired convergent extension in dact1 and dact2 compound mutants. A) Inter-cross of compound heterozygotes yield embryos with different degrees of axis extension that correspond to the dact1 and dact2 genotypes. Representative lateral images of embryos at 12 hpf. The yellow line indicates body axis angle measured from the anterior point of the head, the center of the yolk, to the end of the tail. B) Quantification of body axis angle. Numbers represent the difference in angle relative to the average wildtype embryo. Asterisk indicates genotypes with angles significantly different from wildtype. ANOVA p<0.5 n= 3-21 embryos. C) Representative bud stage wildtype and dact/2-/- mutant embryos stained for gsc (prechordal plate), pax2a (midbrain/hindbrain boundary) and krox20 (rhombomere 3). Asterisk indicates lack of krox20 expression in dact1/2 mutant. Scale bar = 200 μm D) Representative flat mounts of 1-2 ss wildtype and dact1/2 mutant embryos stained for zic1 (telencephalon), pax2a and tbx6 (ventrolateral mesoderm). E) Representative flat mounts of 10 ss wildtype and dact1/2-/- mutant embryos stained for ctsl1b (hatching gland), zic1, pax2a, krox20, and myo1d (somites).

Body axis truncation has been attributed to impaired convergent extension (CE) during gastrulation (Tada and Heisenberg 2012). In order to delineate CE hallmarks in the dact1-/-;dact2-/- mutants, we performed whole-mount RNA ISH detecting genes that are expressed in key domains along the body axis (Fig. 2B). At bud stage, dact1-/-; dact2-/- embryos demonstrate bifurcated expression of pax2a and decreased anterior extension of gsc expression, suggesting impaired midline convergence and anterior extension of the mesoderm. At the 1-2 somite stage, zic1, pax2a and tbx6 are expressed in neural plate, prospective midbrain and the tailbud, respectively, in both the wildtype and dact1-/-; dact2-/- embryos. However, the spacing of these genes clearly revealed the shortening of the anteroposterior body axis in the dact1-/-; dact2-/- embryos. Midline convergence is decreased and the anterior border of the neural plate (marked by zic1 expression) was narrower in the dact1-/-; dact2-/- embryos (Fig. 2C). At the 10 somite stage (ss), dact1-/-; dact2-/- embryos showed decreased spacing between ctslb1 and pax2a gene expression, suggesting impaired lengthening of the anterior portion of the embryo. Detection of muscle marker myo1d in the dact1-/-; dact2- /- embryos delineated impaired posterior lengthening as well as reduced somitogenesis, evidenced by the decreased number of somites (Fig. 2D). These data point to impaired CE of the mesoderm in dact1-/-; dact2-/- double mutants, which resulted in a shorter body axis. These findings were observed in a prior study using morpholinos to disrupt dact1 and dact2 expression, where CE defects were observed in dact2 but not dact1 or dact1/2 double morphants (Waxman, Hocking et al. 2004). These dact1-/-;dact2-/- CE phenotypes were similar to findings in other Wnt mutants, such as slb and kyn (Heisenberg, Tada et al. 2000, Topczewski, Sepich et al. 2001).

dact1/dact2 compound mutants exhibit anterior neurocranium dysmorphology similar to slb/wnt11f2 mutants

No craniofacial phenotype was observed in dact1 or dact2 single mutants (data not shown). However, in-crossing to generate dact1-/-; dact2-/- compound homozygotes resulted in dramatic craniofacial deformity (Fig. 3). Specificity of this phenotype to dact1/2 was confirmed via rescue with dact1 or dact2 mRNA injection (Fig. S1). The dact1-/-;dact2-/- mutant embryos exhibited midfacial hypoplasia and the eye fields converged in the midline (Fig. 3A). The forebrain protruded dorsally and the mouth opening and ventral cartilage structures were displaced ventrally (Fig. 3A). Alcian blue staining of cartilage elements revealed severe narrowing of the anterior neurocranium (ANC) into a rod-like structure, while the ventral cartilage elements were largely unaffected (Fig. 3B). This dact1-/-;dact2-/- double mutant phenotype is highly similar to that described for wnt11f2 (slb) mutants, a key regulator of non-canonical Wnt signaling and CE (Kimmel, Miller et al. 2001).

Midface development requires dact1 and dact2. A) Representative brightfield images of wildtype and dact1/2-/- compound mutants at 4 dpf. Lateral and ventral views show dact1/2-/- compound mutants have a hypoplastic midface, medially displaced eyes, and a displaced lower jaw. B) Representative flat-mount images of Alcian blue stained ANC and VC elements from 4 dpf wildtype and dact1/2-/- compound mutants. Dact1/2-/- mutants have a rod-shaped ANC with no distinct lateral and medial elements. No obvious differences were found in dact1/2 mutant VC. C) Representative images of Alcian blue stained dact1/2-/-, wntllf2-/-, and wnt11f2-/-,dact1/2-/- compound mutants. Lateral and ventral views show similar craniofacial phenotypes in each mutant. D) Representative flat-mount images of Alcian blue stained ANC and VC elements show a similar phenotype between dact1/2-/-, wntllf2-/-, and wnt11f2-/-,dact1/2-/- compound mutants. Scale bar: 200 μm.

We hypothesized that the shared phenotypes between wnt11f2 and dact1/2 mutants point to these genes acting in the same signaling pathway. To test for genetic epistasis between wnt11f2, dact1, and dact2 genes we generated wnt11f2-/-; dact1-/-; dact2-/- triple homozygous mutants. If wnt11f2 and dact1/2 had independent developmental requirements, the wnt11f2-/-; dact1-/-; dact2-/- mutant may exhibit a phenotype distinct from wnt11f2-/- or dact1-/-; dact2-/- mutants. We found that the wnt11f2-/-; dact1-/-; dact2-/- triple homozygous mutant phenotype of the linear rod-like ANC was the same as the wnt11f2-/- mutant or dact1-/-;dact2-/- double mutant, without exhibiting additional or neo-phenotypes in the craniofacial cartilages or body axis (Fig. 3C,D). This result supports dact1 and dact2 acting downstream of wnt11f2 signaling during ANC morphogenesis, where loss of dact1/2 function recapitulates a loss of wnt11f2 signaling. Alternatively, both wnt11f2 and dact1/2 are key regulators of convergent extension during craniofacial morphogenesis, so that disruption of either the wnt11f2 and dact1/2 signaling results in this common morphologic endpoint.

Lineage tracing of NCC movements in dact1/2 mutants reveals ANC composition

The stereotypic convergent migration of cranial neural crest cells and their derivatives presents an excellent model to examine CE movements and their effects on tissue morphogenesis. The zebrafish ANC is formed from the joining of a midline frontonasal prominence derived from the anteromost cranial neural crest (NCC) cell population that migrate over the eyes and turn caudally, to join paired lateral maxillary prominences derived from the second stream of cranial NCC population that migrate rostrally (Kimmel, Miller et al. 2001, Wada, Javidan et al. 2005, Schilling and Le Pabic 2009, Dougherty, Kamel et al. 2012, Mork and Crump 2015). The ANC that forms is a planar fan-shaped structure where we and others have shown that the morphology is governed by Wnt signaling (Kimmel, Miller et al. 2001, Rochard, Monica et al. 2016).

Given the rod-like ANC we observed in the dact1-/-; dact2-/- double mutants, we hypothesized that the dysmorphology could be due to aberrant migration of the anteromost midline frontonasal stream of cranial NCCs or abrogated contribution from the second paired stream of maxillary NCCs. To distinguish between these possibilities, we carried out lineage tracing of the cranial NCC populations in wildtype and dact1/2 mutants. The dact1/2 compound mutants were bred onto a sox10:kaede transgenic background, where we and others have shown that the sox10 reporter is a reliable driver of cranial neural crest labeling (Wada, Javidan et al. 2005, Dutton, Antonellis et al. 2008, Swartz, Sheehan-Rooney et al. 2011, Dougherty, Kamel et al. 2012, Kague, Gallagher et al. 2012, Mork and Crump 2015). Cranial NCC populations in wildtype and dact1/2 mutants were targeted at 19 hpf to photoconvert Kaede reporter protein in either the anterior cranial NCCs that contribute to the frontonasal prominence, or the second stream of NCCs that contribute to the maxillary prominence, where the labeled cells were followed longitudinally over 4.5 days of development (Fig. 4). We found that the anterior NCCs of wildtype embryos migrated antero-dorsally to the eye and populated the medial ANC. To our surprise, the anterior cranial NCC also migrated to contribute to the median element of the rod-like ANC, suggesting the complex anterior then caudal migration of the anterior NCC is not disrupted by dact1/2 mutation. This finding is in contrast to lineage tracing in another midline mutant with a similarly shaped rod-like ANC, the syu (shh null) mutant, where the anterior NCCs failed to populate the ANC (Wada, Javidan et al. 2005).

Anterior neural crest cells of the dact1/t2-/- mutant migrate to the midline and populate the ANC. Lineage tracing of wildtype and dact1/2-/- double mutant zebrafish embryos using Tg(sox10:kaede) line. sox10:kaede fluorescence is shown in green and photo-converted kaede is shown in magenta and highlighted with an arrow. Asterisks indicate that the cell population is absent. 19 hpf embryo sagittal views showing photoconversion of anterior-most neural crest population. At 36 hpf frontal images show the migration of photoconverted neural crest cells to the frontonasal prominences in wildtype and dact1/2-/- double mutants. At 55 hpf, frontal images show photoconverted neural crest cells populating the region of the developing ANC in wildtype and dact1/2-/- mutants. At 4.5 dpf ventral images show photoconverted neural crest cells populating the medial ANC in wildtype. Similarly, neural crest cells in dact1/dact2-/- mutants populate the rod-shaped ANC. Scale bar:100 μm.

Next, the second stream of NCC population that contribute to the maxillary prominence was labeled, where they migrate and contribute to the lateral element of the ANC as expected in the wildtype (Fig 4). When the second stream of cranial CNN were labeled and followed in the dact1/2 mutants, the cells were found to migrate normally up to 36 hpf, but did not ultimately populate the ANC in the mutant, suggesting that the morphological movements after NCC migration were disrupted by loss of dact1/2 function. These results suggest that NCC migration itself is not regulated by dact1/2 but that loss of dact1/2 hinders the second stream of NCCs’ ability to populate the ANC. These results show that the rod-like ANC can be formed from 2 different NCC origins, where in the dact1/2 mutants the ANC is contributed by the anteromost frontonasal NCCs, in contrast to the similar rod-shaped ANC of the syu mutants that is formed from the more posterior stream of maxillary NCCs (Wada, Javidan et al. 2005).

Genetic interaction of dact1/2 with Wnt regulators gpc4 and wls to generate novel facial forms

Given the role of Dact/dapper as modifiers of Wnt signaling, we hypothesized that genetic interaction of dact1/2 with wls and gpc4 will modify facial morphology. Gpc4 is a glycoprotein that binds Wnt ligands and modulates Wnt signaling. gpc4 zebrafish mutants (kny) have impaired convergent extension which leads to a shortened body axis (Topczewski, Sepich et al. 2001). Wls is a posttranslational modifier of Wnt ligands which promotes their secretion (Banziger, Soldini et al. 2006, Bartscherer, Pelte et al. 2006). We previously described that these components of the Wnt/PCP pathway (gpc4 receptor, wls intracellular ligand chaperon, and Wnt ligands wnt9a and wnt5b) are required for craniofacial morphogenesis, where each gene affects particular morphologic aspects of chondrocytes arrangement in the cardinal axis of the ANC and Meckel’s cartilage (Rochard, Monica et al. 2016, Ling, Rochard et al. 2017). Using the ANC as a morphologic readout, we examined the genetic interaction of dact1 and dact2 with wls and gpc4. Compound mutants of dact1, dact2, gpc4 or wls were generated by breeding the single alleles. Compared to wildtype ANC morphology, abrogation of gpc4 led to increased width in the transverse axis, but shorter in the antero-posterior axis (Rochard, Monica et al. 2016). Disruption of wls leads to ANC morphology that is also wider in the transverse dimension, but to a lesser degree than observed in gpc4. Additionally, in the wls mutant, chondrocytes stack in greater layers in the sagittal axis (Rochard, Monica et al. 2016).

Disruption of gpc4 or wls in addition to dact1/2 generated ANC morphology that contained phenotypic attributes from each single mutant, so that the resultant ANC morphology represented a novel ANC form. The ANC of a triple homozygous dact1-/-; dact2-/-; gpc4-/- mutant was triangular, wider in the transverse axis and shorter in the antero-posterior axis compared to the rod-like ANC observed in the dact1-/-;dact2-/- double mutant (Fig. 5A). Similarly, the ANC of a triple homozygous dact1-/-; dact2-/-; wls-/- mutant was in the shape of a rod, shorter in the antero-posterior axis and thicker in the sagittal axis compared to the dact1-/-;dact2-/- double mutant, reflecting attributes of the wls mutant (Fig. 5B). In addition to the ANC phenotypes, the triple homozygous dact1-/-; dact2-/-; gpc4-/- mutant also had a short body axis and truncated tail similar but more severe than the gpc4 mutant (Fig. 5A). Since compound disruption of dact1, dact2, and gpc4 or wls resulted in a new phenotype we conclude that these genes function in different aspects of Wnt signaling during craniofacial development.

A nonoverlapping functional role for dact1, dact2, and gpc4 and wls. A) Representative Alcian blue stained whole-mount images of wildtype, dact1/2-/- double mutant, gpc4-/- mutant, and gpc4-/-,dact1/2-/- triple mutants at 4 dpf. Low magnification lateral images of embryos showing tail truncation in dact1/2-/- mutants, shortened and kinked tail in gpc4-/- mutants, and a combinatorial effect in gpc4-/-,dact1/2-/- triple mutants. Higher magnification lateral images show a shortened midface and displaced lower jaw in dact1/2-/- mutants, a shortened midface in gpc4-/- mutant, and a combinatorial effect in gpc4-/-,dact1/2-/- triple mutants. B) Representative flat-mount images of dissected Alcian blue-stained cartilage elements. dact1/2-/- mutants have a narrow rod-shaped ANC while gpc4-/- mutants have a broad and shortened ANC. dact1/2/gpc4 triple mutants have a combinatorial effect with a short, broad rod-shaped ANC. In ventral cartilages, dact1/2-/- mutants have a relatively normal morphology while Meckel’s cartilage in gpc4-/- mutants and gpc4-/-,dact1/2-/- triple mutants is truncated. C,D) Same as above except wls-/- mutant and wls-/-,dact1/2-/- triple mutant, with similar findings. E) Combinatorial genotypes of dact1, dact2, and gpc4. dact2-/- contributed the dact/gpc4 compound phenotype while dact1-/- did not. Scale: 200 μm.

As we analyzed the subsequent genotypes of our dact1/dact2/gpc4 triple heterozygote in-cross we gleaned more functional information about dact1 and dact2. We found that dact1 haploinsufficiency in the context of dact2-/-; gpc4-/- was sufficient to replicate the triple dact1/dact2/gpc4 homozygous phenotype (Fig. 5C). In contrast, dact2 haploinsufficiency in the context of dact1-/-; gpc4-/- double mutant produced ANC in the opposite phenotypic spectrum of ANC morphology, appearing similar to the gpc4- /- mutant phenotype (Fig. 5C). These results show that dact1 and dact2 do not have redundant function during craniofacial morphogenesis, and that dact2 function is more indispensable than dact1. These results also suggest that dact1 and gpc4 may have overlapping roles in craniofacial development.

dact1/2 and gpc4 regulate axis extension via overlapping and distinct cellular pathways

dact1 or dact2 are required for CE and anterior-posterior axis lengthening during gastrulation (Fig. 3). We aimed to investigate the relationship of this defect to the midfacial dysmorphism of the dact1/2 mutant. Phenotypic analyses of the interaction between dact1/2 and gpc4 (Fig. 5) indicate that dact1/2 function modifies the Wnt pathway, but that the development processes leading to the narrowing of the ANC in the dact1/2 double mutants cannot be explained by the same axis convergent extension defect observed the gpc4 mutant (Topczewski, Sepich et al. 2001). As dact1/2 mutants and gpc4 mutants have similar CE defects during late gastrulation but disparate ANC morphologies later during craniofacial morphogenesis, we performed single-cell transcriptional analysis to compare dact1/2 mutants, gpc4 mutants, and wildtype embryos during the segmentation stage. Single-cell encapsulation and barcoded cDNA libraries were prepared from dissociated 3 ss wildtype, dact1-/-;dact2-/- compound mutant and gpc4-/- mutant embryos using the 10X Genomics Chromium platform and Illumina next-generation sequencing. Twenty clusters were identified using Louvain clustering and identity was assigned by reviewing cluster-specific markers in light of published expression data (Farrell, Wang et al. 2018, Farnsworth, Saunders et al. 2020, Bradford, Van Slyke et al. 2022) (Fig. 6A,B). Qualitatively, we did not observe any significant difference in cluster abundance between genotype groups. We found that dact1, dact2, and gpc4 were detected at various levels across clusters, though dact1 expression was lower than dact2 (Fig. 6C), consistent with what we observed in RNA whole-mount ISH analysis (Fig. 1).

Single-cell RNAseq of 4 ss wildtype, dact1/2-/- mutant and gpc4-/- mutants. A) UMAP showing cluster identification. B) Dot plot showing the most differentially expressed genes between clusters. C) UMAP showing dact1, dact2, and gpc4 expression in wildtype embryos.

To assess the relative differences in gene expression between genotype groups, we merged clusters into broader cell lineages: ectoderm, axial mesoderm, and paraxial mesoderm (Fig. 7A). We focused on these cell types because they contribute significantly to convergent extension processes and axis establishment. For each of these cell lineages, we performed independent pseudobulk differential expression analyses (DEA) of wildtype vs. dact1-/-;dact2-/- mutant and wildtype vs. gpc4-/- mutant (Fig. 7B). In all 3 cases, we found differentially expressed genes (DEGs) that were commonly in dact1-/-;dact2-/- and gpc4-/- mutant relative to wildtype. To address the hypothesis that dact1 and dact2 regulate molecular pathways distinct from those regulated by gpc4 we also identified genes that were differentially expressed only in dact1-/-;dact2-/- mutants or only in gpc4-/- mutants (Fig. 7B). Functional analysis of these DEGs found unique enrichment of intermediate filament genes in gpc4-/- whereas dact1/2-/- mutants had enrichment for pathways associated with proteolysis (Fig. S2). Enrichment for pathways associated with calcium-binding were found in both gpc4-/- and dact1/2-/-, although the specific DEGs were distinct (Fig. S2). We performed functional analyses specifically for genes that were differentially expressed in dact1-/- ;dact2-/- mutants, but not in gpc4-/- mutants, and found enrichment in pathways associated with proteolysis (Fig. 7B) suggesting a novel role for Dact in embryogenesis.

Pseudobulk differential expression analysis of single-cell RNAseq data. A) Heatmaps showing the 50 most differentially expressed genes in 3 major cell types; ectoderm (clusters 4,5,6,7), paraxial mesoderm (clusters 10,11,12), and lateral plate mesoderm (clusters 15, 16, 17,18) between dact1/2-/- mutants and wildtype and gpc4-/- mutants and wildtype. B) Venn diagrams showing unique and overlapping DEGs in dact1/2-/- and gpc4-/- mutants. C) GO analysis of dact1/2-/- mutant-specific DEGs in lateral plate mesoderm showing enrichment for proteolytic processes.

Interrogation of dact1-/-;dact2-/- mutant-specific DEGs found that the calcium-dependent cysteine protease calpain 8 (capn8) was significantly overexpressed in dact1-/-;dact2-/- mutants in paraxial mesoderm (103 fold), axial mesoderm (33 fold), and in ectoderm (3 fold; Fig. 7A). We also found that loss of dact1/2 causes significant changes to capn8 expression pattern (Fig. 8A). Whereas capn8 gene expression is principally restricted to the epidermis of wildtype embryos, loss of dact1/2 leads to significant expansion of ectopic capn8 gene expression in broader cell types such as in mesodermal tissues (Fig. 8A). We corroborated this finding with whole mount ISH for capn8 expression in wildtype versus dact1/2-/- 12 hpf embryos (Fig. 8B).

Expression of capn8 is significantly dysregulated in dact1/2-/- mutants. A) Single-cell RNAseq gene expression analysis of capn8 in wildtype and dact1/2-/- mutants. In wildtype embryos, capn8 expression is restricted predominantly to the epidermis whereas capn8 is widely expressed throughout the embryo in dact1/2-/- mutants, especially in the mesoderm. B) Whole-mount in situ hybridization of capn8 expression in wildtype and dact1/2-/- mutant embryos at 2 ss. Staining corroborates the single-cell RNAseq data, with expanded ectopic expression of capn8 throughout the embryo. C) Brightfield images and alcian blue staining of the ANC show ectopic expression of capn8 mRNA (200 pg) at the 1-cell stage in wildtype embryos recapitulates the dact1/2-/- compound mutant craniofacial phenotype at a low frequency (1/142 injected embryos). The mutant craniofacial phenotype did not manifest in gfp mRNA (200 pg) injected 1-cell stage embryos (0/192 injected embryos).

Capn8 is considered a “classical” calpain, with domain homology similar to Capn1 and 2 (Macqueen and Wilcox 2014). In adult human and mouse tissue, Capn8 expression is largely restricted to the gastrointestinal tract (Sorimachi, Ishiura et al. 1993, Macqueen and Wilcox 2014), however embryonic expression in mammals has not been characterized. Proteolytic targets of Capn8 have not been identified, however, other classical calpains have been implicated in Wnt and cell-cell/ECM signaling (Konze, van Diepen et al. 2014), including in Wnt/Ca+2 regulation of convergent extension in Xenopus (Zanardelli, Christodoulou et al. 2013). To determine whether the dact1-/-;dact2-/- mutant craniofacial phenotype could be attributed to capn8 overexpression, we performed injection of capn8 mRNA into 1 cell-stage zebrafish embryos. In wildtype zebrafish, exogenous capn8 mRNA caused the distinct dact1/2-/- craniofacial phenotype including a rod-like ANC at a very low frequency (1 in 142 injected embryos). This phenotype was never observed in wildtype larvae, or when wildtype embryos were injected with an equal concentration of gfp mRNA (0 in 192 injected embryos) (Fig. 8C). These findings suggest that dact-dependent suppression of capn8 expression is required for normal embryogenesis and craniofacial morphogenesis.


In this study, we examined how dact1 and dact2 interact with Wnt signaling during early embryogenesis and craniofacial morphogenesis. Wnt signaling is central to the orchestration of embryogenesis and numerous proteins have been identified as modulators of Wnt signaling, including Dact1 and Dact2. Several studies across Xenopus, zebrafish and mouse have ascribed roles to dact1 and dact2, including both promoting and antagonizing Wnt signaling, depending on the developmental context. Here, we show that dact1 and dact2 are required for axis extension during gastrulation and show a new example of CE defects during gastrulation associated with craniofacial defects. Our data supports the hypothesis that CE gastrulation defects are not causal to the craniofacial defect of medially displaced eyes and midfacial hypoplasia and that an additional morphological process is disrupted. We show that disruption of both dact1 and dact2 are required to generate the dysmorphic craniofacial phenotype, as single mutants develop normally. However, based on gene expression and epistasis experiments, it is clear that these paralogs are not redundant and have unique functions. We observed that dact1 and dact2 have distinct spatiotemporal expression patterns throughout embryogenesis, suggesting unique roles for each paralog in developmental processes. We found that dact1 and dact2 contribute to axis extension, and their compound mutants exhibit a CE defect. This finding aligns with previous studies that have implicated dact1 and dact2 in non-canonical wnt signaling and regulation of embryonic axis extension. Based on the data, we posit that dact1 expression in the mesoderm is required for dorsal CE during gastrulation through its role in noncanonical Wnt/PCP signaling, similar to the defect observed upon gpc4 disruption. Conversely, we propose dact2 functions in the prechordal mesoderm, anterior neural plate and pollster to promote anterior migration during gastrulation, a function which has also been ascribed to wntllf2. It is only upon loss of both functions of dact1 and dact2 that the craniofacial defect manifests.

Our results underscore the crucial roles of dact1 and dact2 in embryonic development, specifically in the connection between CE during gastrulation and ultimate craniofacial development. Our analysis of CNCC migration and contribution to the anterior neurocranium in dact1-/-;dact2-/- compound mutant suggests that embryonic fields determined during gastrulation effect the CNCC ability to contribute to the craniofacial skeleton. It will be important to test the generality of this phenomenon utilizing other gastrulation mutants and other model systems.

By comparing the transcriptome of a CE mutant, i.e. gpc4-/- with that of the dact1-/-;dact2-/- compound mutant, we identified a novel role for dact1/2 in the regulation of proteolysis, with significant misexpression of capn8 in the mesoderm of dact1-/-;dact2-/- mutants. Although at a very low frequency, ectopic expression of capn8 mRNA recapitulated the dact1-/-;dact2-/- mutant craniofacial phenotype, suggesting that inhibition of capn8 expression in the mesoderm by dact is required for normal morphogenesis. Genes involved in calcium ion binding (i.e. Purkinje cell protein 4 (pcp4a)) were also differentially expressed in the dact1-/-;dact2-/- mutants and we predict that altering intracellular calcium handling in conjunction with capn8 overexpression would increase the frequency of the recapitulated dact1-/-;dact2-/- mutant phenotype.

Capn8 is described as a stomach-specific calpain and a role during embryogenesis has not been previously described. Calpains are typically calcium-activated proteases and it is feasible that Capn8 is active in response to Wnt/Ca2+ signaling. A close family member, Capn2 has been found to modulate Wnt signaling by degradation of beta-catenin (Zanardelli, Christodoulou et al. 2013, Konze, van Diepen et al. 2014). These findings suggest that dact-dependent suppression of capn8 expression is necessary for normal embryogenesis and craniofacial morphogenesis, further expanding the functional repertoire of dact1/2. Further research is required to examine the direct regulatory role of dacts on capn8 expression. Our findings also warrant investigation into the role of capn8 during embryogenesis and possible implications for known craniofacial or other disorders.

Another gene differentially expressed specifically in the dact1-/-,dact2-/- embryos is smad1. Smad1 acts in the TGF-β signaling pathway and dact2 has been described to inhibit TGF-β and Nodal signaling by promoting the degradation of Nodal receptors (Zhang, Zhou et al. 2004, Su, Zhang et al. 2007, Meng, Cheng et al. 2008, Kivimae, Yang et al. 2011). It is robustly feasible that dysregulation of TGF-β signaling in the dact1-/-;dact2-/- mutant contributes to the craniofacial phenotype. Future research will examine the role of dact1 and dact2 in the coordination of Wnt and TGF-β signaling and the importance of this coordination in the context of craniofacial development.

The zebrafish has proven to be an important tool in studying early developmental biology. Early days of forward genetic screens, careful descriptive analyses, and gene epistasis experiments were seminal to our understanding of the key regulators of development and embryogenesis, including the Wnt pathway. With the substantial technological advances that have become available, including gene regulation analyses, transcriptomics, and advanced real-time imaging, we can revisit those initial discoveries and expand the breadth and depth of scientific knowledge. Moving forward we are positioned to make progress in our understanding of the complexities of spatial and temporal regulation of the key developmental signaling pathways as well as discover how these different pathways interact with each other.


Animals and gene editing

All animal husbandry and experiments were performed in accordance with approval from Massachusetts General Hospital Institutional Animal Care and Usage Committee. Zebrafish (Danio rerio) embryos and adults were maintained in accordance with institutional protocols. Embryos were raised at 28.5 C in E3 medium (Carroll, Macias Trevino et al. 2020) and staged visually and according to standardized developmental timepoints (Westerfield 1993). All zebrafish lines used for experiments and gene editing were generated from the Tubingen or AB strain. The wnt11f2 mutant line and gpc4-/- mutant line were obtained from ZIRC (wnt11f2tx226/+ and gpc4hi1688Tg/+ respectively). The wls-/- mutant line was originally gifted to the lab and independently generated, as previously described (Rochard, Monica et al. 2016). The sox10:kaede transgenic line was previously generated and described by our lab (Dougherty, Kamel et al. 2012).

CRISPR sgRNA guides were designed using computational programs ZiFiT Targeter v4.2 ( (Sander, Zaback et al. 2007), ( (Ran, Hsu et al. 2013), and ChopChop ( (Montague, Cruz et al. 2014) with traditional sequence constraints. Guides were chosen that were predicted to give high efficiency and specificity and located near the 5’ end of the gene. Guides best meeting these parameters were selected in exon 2 of dact1 and exon 4 of dact2. No suitable gRNA with sufficient efficiency were identified for dact2 5’ of exon 4 and the resulting phenotype was reassuring compared to previous morpholino published results. Guides for dact1 and dact2 and Cas9 protein were prepared and microinjected into 1 cell-stage zebrafish embryos and founders were identified as previously described (Carroll, Macias Trevino et al. 2020). Primers flanking the sgRNA guide site were designed for genotyping and fragment analysis was performed on genomic DNA to detect base pair insertion/deletion. Sanger sequencing was performed to verify targeted gene mutation and confirm the inclusion of a premature stop codon.

Microinjection of mRNA

mRNA for injection was generated using an in vitro kit (Invitrogen mMessage mMachine) and cloned cDNA as a template. One-cell stage zebrafish embryos were injected with 2nL mRNA solution.

Whole-mount and RNAscope in situ hybridization

Whole-mount in situ hybridization (WISH) was performed as previously described (Carroll, Macias Trevino et al. 2020). Zebrafish embryonic cDNA was used as a template to generate riboprobes. Primers were designed to PCR amplify the specific riboprobe sequence with a T7 promoter sequence linked to the reverse primer. In vitro transcription was performed using a T7 polymerase and DIG-labeled nucleotides (Roche). RNAscope was performed on sectioned zebrafish larvae as previously described (Carroll, Macias Trevino et al. 2020). Probes were designed by and purchased from ACD Bio. Hybridization and detection was performed according to the manufacturer’s protocol. Sections were imaged using a confocal microscope (Leica SP8) and z-stack maximum projections were generated using Fiji software.

Alcian blue staining and imaging

Alcian blue staining and imaging were performed at 4 dpf as previously described (Carroll, Macias Trevino et al. 2020). Briefly, larvae were fixed in 4% formaldehyde overnight at 4C. Larvae were dehydrated in 50% ethanol and stained with Alcian blue as described (Walker and Kimmel 2007). Whole and dissected larvae were imaged in 3% methylcellulose using a Nikon Eclipse 80i compound microscope with a Nikon DS Ri1 camera. Z-stacked images were taken and extended depth of field was calculated using NIS Element BR 3.2 software. Images were processed using Fiji software.

Axis measurements

Compound dact1+/-; dact2+/- zebrafish were in-crossed and progeny were collected from 2 separate clutches and fixed in 4% formaldehyde at approximately 8ss. Embryos were individually imaged using a Zeiss Axiozoom stereoscope and processed for DNA extraction and genotyping (Westerfield 1993). Images were analyzed using Fiji. A circle was drawn to overlay the yolk and the geometric center was determined using the function on Fiji. Using the Fiji angle tool, lines were drawn from the center point to the anterior-most point of the embryo and from the center to the posterior-most point of the embryo. The resulting inner angle of these lines was determined. Each angle measurement was then calculated as a ratio to the average angle of the wildtype embryos. ANOVA was performed to determine statistical significance, p<0.05.

Single-cell RNA sequencing (scRNA-seq)

Single-cell transcriptomic analyses were performed on 10 zebrafish embryos, including 4 wildtype, 3 dact1-/-;dact2-/- compound mutant, and 3 gpc4-/- mutant embryos. Embryos were collected at 3 ss with dact1-/-,dact2-/- and gpc4-/- mutants being identified by their truncated body axis. Embryos were dechorionated with a short (approximately 10 min) incubation in 1 mg/ml Pronase and then washed 3x in embryo medium. Cell dissociation was performed with modifications as previously described (Farrell, Wang et al. 2018). Each embryo was transferred to 50 μl DMEM/F12 media on ice. To dissociate cells, media was replaced with 200 μl DPBS (without Ca2+ and Mg2+) with 0.1% BSA. Embryos were disrupted by pipetting 10x with a P200 pipette tip. 500 μl of DPBS + 0.1% BSA was added and cells were centrifuged at 300xg for 1 min. Cell pellets were resuspended in 200 μl DPBS + 0.1% BSA and kept on ice. Just prior to encapsulation, cells were passed through a 40 μm cell strainer, and cell counts and viability were measured. After droplet encapsulation, barcoding, and library preparation using the 10X Genomics Chromium Single Cell 3’ kit (version 3), data were sequenced on an Illumina NovaSeq 6000 sequencer.

FASTQ files were demultiplexed and aligned to the GRCz11 build of the zebrafish genome using Cellranger (version 6.1.0) (Zheng, Terry et al. 2017). Raw Cellranger count matrices were imported into R (version 4.1.2) using Seurat (version 4.1.0) (Hao, Hao et al. 2021). First, we reviewed data for quality and excluded any droplet that did not meet all of the following criteria: (i) have at least 1,500 unique molecular identifiers (UMIs), (ii) covering at least 750 distinct genes; (iii) have <5% of genes mapping to the mitochondrial genome; and (iv) have a log10 of detected genes per UMI >80%. After quality control, the dataset was also filtered to exclude genes with a detection rate below 1 in 3,000 cells, leaving a total of 20,078 distinct genes expressed across 19,457 cells for analysis.

The quality-controlled count data were normalized using Pearson’s residuals from the regularized negative binomial regression model, as implemented in Seurat::SCTransfrom (Hafemeister and Satija 2019). When computing the SCT model, the effect of the total number of UMIs and number of detected genes per cell were regressed out. After normalization, the top 3,000 most variably expressed genes were used to calculate principal components (PCs). Data were then integrated by source sample using Harmony (version 0.1.0) (Korsunsky, Millard et al. 2019). A two-dimensional uniform manifold approximation and projection (UMAP) (Becht, McInnes et al. 2018) was then derived from the first 40 Harmony embeddings for visualization. Using the integrated Harmony embeddings, clusters were defined with the Louvain clustering method, as implemented within Seurat. A resolution of 0.3 was used for cluster definition. Cluster identities were assigned by manually reviewing the results of Seurat::FindAllMarkers, searching for genes associated to known developmental lineages. Gene expression data for key markers that guided cluster identity assignment were visualized using Seurat::DotPlot.

Following this detailed annotation, some clusters were grouped to focus downstream analyses on 3 major lineages: ventral mesoderm (grouping cells from the pronephros, vasculogenic/myeloid precursors, hematopoietic cells, heart primordium, and cephalic mesoderm clusters), dorsal mesoderm (adaxial cells, segmental plate, and paraxial mesoderm), ectoderm (CNS, mid/hindbrain boundary, spinal cord, and neural crest). For those 3 lineages, single-cell level data were aggregated per sample and cluster to perform pseudobulk differential expression analyses (DEA) contrasting genotypes. Independent pairwise comparisons of dact1-/-;dact2-/- versus wildtype and gpc4-/- vs wildtype were performed using DESeq2 (v1.34.0) (Love, Huber et al. 2014). P-values were corrected for multiple testing using the default Benjamini-Hochberg method; log2 fold change values were corrected using the apeglm shrinkage estimator (Zhu, Ibrahim et al. 2019). Significance was defined as an adjusted p-value < 0.1 and log2 fold change > 0.58 in absolute value. Heatmaps of the top most significant differentially expressed genes were generated from the regularized log transformed data using pheatmap (version 1.0.12). Overlap in significant genes across pairwise comparisons were determined and visualized in Venn diagrams. Over-representation analyses against the Gene Ontology (GO) database were ran using clusterProfiler (version 4.2.2) (Wu, Hu et al. 2021), using as input the set of genes found to be differentially expressed in the comparison of dact1-/-;dact-/- versus wildtype but not gpc4-/- versus wildtype. Sequencing data have been deposited in GEO under accession code GSE240264.

Statistical analysis

Analyses were performed using Prism Software (GraphPad) unless otherwise specified. An unpaired Student’s t test or one-way ANOVA with multiple comparisons was used as indicated and a P-value <0.05 was considered significant. Graphs represent the mean +/- the SEM and n represents biological replicates.


We thank Christoph Seiler, Adele Donohue and team at Children’s Hospital of Philadelphia and Jessica Bethoney at Massachusetts General Hospital (MGH) for their management of our aquatics facilities. We thank the MGH Next Generation Sequencing Core for cell encapsulation, cDNA library preparation, and sequencing. Single-cell sequencing analysis was performed by the Harvard Chan Bioinformatics Core. Work by A.J. was funded in part by the Harvard Stem Cell Institute. This work was supported by the National Institutes of Health grant R01DE027983 to E.C.L.

Supplemental Figures

Characterization of CRISPR/Cas9 generated dact1-/- and dact2-/- mutants. A) Schematic representations of dact1 and dact2 exons, positions of guide RNA target site, introduced premature stop codon (arrow), and sequences of mutations. B) Expression levels of dact1 and dact2 mRNA by RT-qPCR in 12 hpf dact1-/- mutants, dact2-/- mutants, and dact1/2-/- compound mutants. 8 embryos were pooled for mRNA isolation per sample. C) Injection of dact1 mRNA, dact2 mRNA, or a combination of dact1 and dact2 mRNA rescues the rod-shaped ANC phenotype in dact1/2-/- compound mutants. Representative images of Alcian blue stained dact1/2-/- double mutant treated with 300 pg dact1 mRNA and 300 pg dact2 mRNA. Arrow highlights normal ANC. D) Quantification of the mutant craniofacial phenotype observed in a dact1-/-,dact2+/- breeding in-cross. Without mRNA injection, the mutant phenotype was observed at approximately the expected Mendelian ratio of 25%. Injection with dact1 mRNA, dact2 mRNA, or a combination of dact1 and dact2 mRNA decreased the frequency that the mutant craniofacial phenotype was observed.

Loss of gpc4 and loss of dact1/2 leads to distinct changes in gene expression profiles but with some overlapping functions. GO analysis of DEGs identified between gpc4-/- and wildtype embryos and dact1/2-/- and wildtype embryos identified changes in calcium ion binding and actin interaction in both mutants.