Interactions between the Bone Morphogenetic Protein and the Planar Cell Polarity Pathways lead to distinctive ethanol-induced facial defects

Interactions between the Bone Morphogenetic Protein and the Planar Cell Polarity Pathways lead to distinctive ethanol-induced facial defects | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Interactions between the Bone Morphogenetic Protein and the Planar Cell Polarity Pathways lead to distinctive ethanol-induced facial defects Raèden Gray , Anna Llyod , View ORCID Profile C. Ben Lovely doi: https://doi.org/10.1101/2025.04.23.650288 Raèden Gray 1 University of Louisville, School of Medicine, Department of Biochemistry and Molecular Genetics and Alcohol Research Center , Louisville, KY M.S Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anna Llyod 1 University of Louisville, School of Medicine, Department of Biochemistry and Molecular Genetics and Alcohol Research Center , Louisville, KY B.S Find this author on Google Scholar Find this author on PubMed Search for this author on this site C. Ben Lovely 1 University of Louisville, School of Medicine, Department of Biochemistry and Molecular Genetics and Alcohol Research Center , Louisville, KY Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for C. Ben Lovely For correspondence: ben.lovely{at}louisville.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Background Fetal Alcohol Spectrum Disorders (FASD) describes a spectrum of ethanol-induced neural and facial developmental defects. Ethanol susceptibility is modulated by genetics, but their underlying mechanisms remain poorly understood. In all vertebrates, a series of complex cellular events give rise to the body plan, including convergence & extension (C&E) and endoderm/ cranial neural crest (CNC-which gives rise to the facial skeleton) morphogenesis. These events are critical to establish complex signaling interactions, driving embryo development, including the facial skeleton. In zebrafish, C&E occurs between 6-10 hpf while endoderm/CNC morphogenesis occurs 10-24 hpf. Previous work shows that the PCP mutants are sensitive to ethanol from 6-24 hpf, covering both C&E and endoderm/CNC morphogenesis and exhibiting multiple defects to the forming head raising the question whether ethanol during both time windows drives PCP-ethanol defects. We hypothesize that PCP single and double mutants are ethanol sensitive 10-24 hpf, after C&E. We also hypothesize BMP signaling (sensitive 10-18 hpf) interacts with and sensitizes the PCP pathway to ethanol. Methods Here, we treated PCP/BMP mutants with ethanol from 6-10, 10-18, 10-24 or 24-30 hpf and combined morphometric and linear measurements to examine facial development. Results We show that PCP mutant larvae are ethanol-sensitive from 10-24 hpf, but not 6-10 or 24-30 hpf. We also show that BMP mutants sensitize PCP mutants to ethanol and lead to novel ethanol-independent midline craniofacial defects. Our results suggest that the ethanol-sensitive role of PCP pathway occurs after C&E, during endoderm/CNC morphogenesis and that the PCP and BMP pathways genetically interact during the morphogenesis events. Conclusions Ultimately, our work builds on a mechanistic paradigm of ethanol-induced birth defects we have been developing, connecting conceptual framework with concrete cellular events that could be ethanol-sensitive beyond facial development. Introduction Fetal Alcohol Spectrum Disorders (FASD) describes a wide range of birth defects due to prenatal alcohol exposure (PAE) ( Sokol et al. 2003 ; Hoyme et al. 2016 ). Although PAE is the most preventable cause of birth defects, FASD still impacts up to 5% of children in the US, with estimates even higher in other regions across the globe ( May et al., 2018 ; Popova et al., 2020 ). However, these are likely underestimates as up to 10% of women worldwide consume alcohol during pregnancy, though some estimates are higher such as 23.3% of women in the Caribbean, for example (Popova et al., 2018, 2017; Landgraf et al., 2013 ; Lange et al., 2017 ). In addition, up to 50% of pregnancies in the US are unplanned and many pediatricians fail to recognize and diagnose FASD (Rojmahamongkol et al., 2015; Finer and Zolna, 2016). Overall, PAE is one, if not the leading cause of birth defects. Fetal Alcohol Syndrome (FAS) is the most severe form of FASD and is characterized by neural, growth and craniofacial defects ( Landgraf et al., 2013 ; May et al., 2009 ). Not only can craniofacial defects negatively impact the ability for children to eat and drink ( Perkins et al., 1997 ), sociologists have shown that children who have craniofacial defects, such as cleft lip/palate, face a significant amount of stigma and social rejection ( Chung et al., 2019 ), increasing disadvantages the child will face in social environments. These factors together explain why the cost of correcting these and other congenital birth defects are over a billion dollars annually ( Swanson, 2023 ). While the developmental window of exposure and dosage of ethanol contribute to the etiology of FASD, human twin studies demonstrate that genetics also modulate the effects of prenatal ethanol exposure, where there is 100% concordance in FASD phenotypic outcomes in monozygotic twins over dizygotic twins (66%), full siblings (41%) and half siblings (22%) ( Hemingway et al., 2019 ). However, the ethanol-sensitive genetic loci that contribute to FAS susceptibility are still not well understood. The social disadvantages as well as the physical impact of the craniofacial defects caused by PAE highlights how imperative it is to understand how genetic and environmental factors intersect, driving craniofacial malformations observed in FASD. A key phenotype of FASD is jaw hypoplasia or reduction in jaw size. The jaw forms from the mandibular domain of the cranial neural crest (CNC) in the first pharyngeal arch ( Chai et al., 2000 ). CNC development into the jaw is dependent on the presence of the pharyngeal endoderm. Previous studies show that the pharyngeal endoderm acts as a signaling center through tissue-tissue interactions that provide extracellular cues to the CNC, oral ectoderm, and mesoderm ( Chai et al., 2006 ). In embryos that lack endoderm the CNC condense into the arches but undergo apoptosis and fail to form the facial skeleton ( Trumpp et al., 1999 ; David et al., 2002 ). Many genes involved in CNC morphogenesis of the jaw are not active until after the mandibular domain has already condensed, showing jaw development’s reliance on these cues ( Trumpp et al. 1999 ). Additional studies have shown that when the anterior pharyngeal endoderm is malformed, as seen in zebrafish mutants in Sphingosine-1-phosphate (S1P) signaling, it leads to jaw malformation ( Balczerski et al., 2012 ). Together, this emphasizes the importance of proper pharyngeal endoderm morphogenesis for development of the jaw. Multiple signaling pathways are critical for endoderm morphogenesis and jaw development, including S1P signaling (described above), Fibroblast Growth Factor (Fgf) signaling and Bone Morphogenetic Protein (BMP) Signaling ( Lovely et al., 2016 ). We and others have shown that the BMP pathway is critical for morphogenesis of the endoderm by regulating a series of downstream signaling targets regulating endodermal cell behaviors ( Lovely et al., 2016 ; Li et al., 2019 ). We have shown that when BMP signaling is blocked with the small chemical inhibitor Dorsomorphin (DM) from 10-18 hours post fertilization (hpf) in zebrafish embryos, overall endoderm morphogenesis is disrupted leading to a wide range of craniofacial malformations ( Lovely et al., 2016 ). We have recently shown that mutations in multiple genes of the BMP pathway, such as bmp4 , sensitize embryos to ethanol-induced defects to the anterior endoderm leading to defects in both the jaw and palate (Lovely, C.B., 2024; Klem et al., 2025 ). However, we show that ethanol does not disrupt BMP signaling directly, nor does it reduce expression of the downstream BMP target, nxk2.3 ( Klem et al., 2025 , Vo and Lovely, 2025 ). In addition, knockdown of nxk2.3 does not sensitive larvae to ethanol-induced facial defects ( Vo and Lovely, 2025 ). This suggests that ethanol is acting on additional loci, contributing to the facial defects in ethanol-treated BMP mutants. Previous work has shown that components of the Planar Cell Polarity (PCP) pathway are ethanol sensitive resulting in a range of phenotypes including craniofacial defects ( Sidik et al., 2021 ; Swartz et al., 2014 ). Mutants in vangl2 and gpc4 are ethanol sensitive leading to disrupted axonal projections, synophthalmia and a range of defects to the facial skeleton, which were exacerbated in the double mutant embryos ( Swartz et al., 2014 ; Sidik et al., 2021 ). Work from Sidik et al (2021) showed that vangl2 mutants are sensitive to ethanol leading from 6-24 hpf, covering both early convergence & extension and subsequent endoderm morphogenesis. Convergence & extension (C&E) describes a crucial part of embryogenesis in which germ layers elongate and narrow to develop the animal body plan ( Keller, 2002 ) and typically occurs in zebrafish between 6 and 10 hpf ( Warga & Kimmel, 1990 ; Topczewski et al., 2001 ; Jessen et al., 2002 ; Sepich et al., 2005 ;). The PCP pathway drives C&E by modulating the cell movement by regulating cell polarity and cell adhesion ( Muñoz-Soriano et al., 2012 ). Vangl2 is a transmembrane protein in the pathway whose phosphorylation is induced by Wnt ligands and is needed to promote PCP signaling and, in part, regulate E-cadherin levels and distribution ( Dush & Nascone-Yoder,2019 ; Yang et al., 2017 ). Gpc4 is a cell surface proteoglycan required for the transport of Wnt ligand and has been shown to be important in Wnt ligand transport from endoderm to other germ layers ( Hu et al., 2021 ). In addition, previous work shows that the PCP components vangl2 and gpc4 interact and play a key role during early C&E of the endoderm ( Miles et al., 2017 ). Noticeably, there were ethanol-induced jaw defects in both vangl2 and gpc4 mutants, suggesting that these ethanol-sensitive PCP mutants may play a role in craniofacial development ( Sidik et al., 2021 ; Swartz et al., 2014 ). However, the timing of ethanol sensitivity of these jaw defects was not further examined raising the question of the timing of the ethanol sensitive role vangl2 and gpc4 play in facial development. Here, we examine the role of vangl2 and gpc4, and their interaction with BMP signaling, in jaw development, post C&E, and what ethanol may be doing to disrupt that role. We show that vangl2 and gpc4 are sensitive to ethanol-induced jaw defects starting at 10 hpf, after C&E, with vangl2 ; gpc4 double mutants being hypersensitized. We go on to show that loss of bmp4 further sensitizes vangl2 or gpc4 mutants to ethanol-induced craniofacial defects, resulting in novel ethanol-induced midline facial defects not previously observed in PCP or BMP single mutants alone. Collectively, our data links perturbations in PCP and BMP signaling to ethanol susceptibility during jaw development, establishing a genetic pathway in gene-ethanol interactions for future studies in FASD. Materials and Methods Zebrafish (Danio rerio) care and use All zebrafish were raised and cared for using established IACUC protocols approved by the University of Louisville ( Westerfield, 2007 ). Adult fish were maintained at 28.5°C with a 14 / 10-hour light / dark cycle. The vangl2 m209 ( Solnica-Krezel et al., 1996 ; Stemple et al., 1996 ; Driever et al., 1996 ), gpc4 fr6 ( Topczewski et al., 2001 ) and bmp4 st72 ( Stickney et al., 2007 ) zebrafish lines used were previously described. Zebrafish staging and ethanol treatment Embryos were collected, morphologically staged as previously described ( Westerfield, 2007 ), sorted into sample groups and reared at 28.5°C in embryo media (EM) to desired developmental time points. At 6 hpf, 8 hpf, 10 hpf, and 24 hpf EM was changed to either fresh EM or EM containing 1% ethanol (v/v) depending on the time window being examined. At 10 hpf, 12 hpf, 18 hpf, 24 hpf and 30 hpf, EM containing ethanol was washed out with 3 fresh changes of EM. Alcian Blue cartilage staining Zebrafish larvae were fixed at 5 dpf (single mutants) and 4 dpf (double mutants due to increased die off at 5 dpf) and the facial cartilages were stained with alcian blue ( Walker & Kimmel, 2007 ). Whole mount, ventral view, brightfield images of the viscerocranium were taken on an Olympus BX53 compound microscope. Morphometric analyses Morphometric analysis of Alcian-stained larva was performed in TpsDig2 ( https://sbmorphomectrics.org ) and MorphoJ ( Klingenberg, 2011 ). Landmarks were placed on the following joints, Meckel’s cartilage midline joint, the joints between Meckel’s’ and the palatoquadrate, the palatoquadrate and ceratohyal and at the end of the hyomandibular cartilages. Linear measures were analyzed using TpsDig2. Principle component analysis (PCA), Procrustes ANOVA and wireframe graphs of facial variation were generated using MorphoJ. Statistical Analysis Cartilage angles and linear measures of Alcian-stained viscerocranium were analyzed with a two-way ANOVA (type III) and Sidak’s multiple comparison test in Graphpad Prism 10.4.1 (Graphpad Software Inc., La Jolla, CA). Results Ethanol sensitizes vangl2 and gpc4 to ethanol induced jaw defects from 10 – 18 hpf Previous work from Sidik et al., (2021) showed that when exposed to ethanol between 6-24 hpf, vangl2 and gpc4 zebrafish mutants exhibited a range of defects including those to the facial skeleton. This time window encompasses a host of developmental processes, including convergence & extension (C&E), somitogenesis, anterior pharyngeal endoderm (APE) morphogenesis, and cranial neural crest (CNC) migration and condensation. Because the role of CNC and APE in jaw development occur after C&E, we hypothesize that vangl2 & gpc4 are required for jaw development after 10 hpf. To test this, we exposed our wildtype and vangl2 or gpc4 mutant embryos to 1% ethanol from 10 hpf to 24 hpf. We selected 1% ethanol (v/v) as it is the highest dose that does not cause craniofacial defects in wild-type larvae, while higher doses have been shown to impact facial development ( Bilotta et al., 2004 ; Everson et al., 2022 ; McCarthy et al., 2013 ; Swartz et al., 2014 ; Zhang et al., 2014 ). 1% ethanol equilibrates to approximately 30% of the media within 5 minutes of exposure ( Flentke et al., 2014 ; Lovely et al., 2014 ; Reimers et al., 2004 ; Zhang et al., 2013 ), resulting in an embryonic ethanol concentration of 50 mM, roughly equivalent to a human Blood Alcohol Concentration of 0.23. While this is a binge dose, it is physiologically relevant to FASD with humans readily surpassing this amount ( Canfield et al., 2019 ; Ethen et al., 2009 ; Jones, 2008 ; Maier, 2001; Whaley et al., 2019 ). We stained the facial cartilage with Alcian blue and used morphometric analysis to observe facial shape changes between our untreated and ethanol-treated embryos. The benefit of utilizing morphometric analysis is that we can see the change in shape, independent of general size effects, which would not be identified using traditional linear measurements. We were able to generate a principal component analysis plot to visualize the variation between our groups. Our data show that untreated vangl2 mutants have mild facial shape changes compared to untreated and ethanol-treated wild-type siblings ( Figure 1A-C ). This results in a shift in PC1, which denotes a shortening and widening of the viscerocranium and a flattening of the ceratohyal (Ch) and Meckel’s (Mc) cartilages and represents 80% of all variation in the data ( Figure 1E ). When exposed to ethanol from 10-24 hpf, vangl2 mutants have increased variation in jaw shape relative to untreated mutants, shifting further along PC1 ( Figure 1D compared to C, E). PC2, which denotes subtle shape change in the position and angle of both the Ch and ( Figure 1A-C, E ). PC2. represents only 4% of variation in the data ( Figure 1E ). This suggests that while untreated vangl2 mutants have subtle facial shape changes not observed before ethanol exposure from 10-24 hpf, after C&E, leads to a significant change in facial shape. Download figure Open in new tab Figure 1 vangl2 mutants are sensitized to ethanol induced jaw hypoplasia. Whole-mount images of the viscerocranium of (A) wild type control embryos (B) wild type ethanol treated embryos, (C) vangl2 −/− control fish and (D) vangl2 −/− ethanol treated fish. (E) Principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n=25), cyan = wild type ethanol treated embryos (n=25), green n= vangl2 −/− control embryos (n=32), magenta = vangl2 −/− ethanol treated embryos (n=12), solid circles represent confidence ellipses in which 95% of all individual data points for each group lie, dashed circles represent 95% confidence ellipses for means. Wireframe graphs represent variation termed by each axis with black representing no variation and magenta representing variation relative to the black wireframe. For example, PC1 captures a shortening and widening in viscerocranial shape, while PC2 represents variation in midfacial width. Procrustes ANOVA showed significant change in the viscerocranial shape (F = 32.63, DF = 36, p = <.0001). Previous studies have shown that gpc4 mutants have craniofacial defects, including jaw hypoplasia, in control conditions ( Sisson et al., 2015 ; LeClair et al., 2009 ). We observed similar facial defects in our morphometric approach, with untreated gpc4 mutants having shorter and wider viscerocranium and flattened Ch and Mc cartilages ( Figure 2A-C, E ). Here, the shortening and widening of the viscerocranium in PC1 represents 86% of the variation in the data ( Figure 2E ). However, in contrast to ethanol-treated vangl2 mutants, ethanol-treated gpc4 mutants show little difference in facial shape compared to their untreated mutant siblings ( Figure 2D compared to C, E). We observed an interesting shift in facial shape between untreated and ethanol-treated gpc4 mutants. While untreated mutants were relatively consistent along PC1, they had greater spread over PC2 (representing 7% of the variation) which denotes a flattening of the Ch leading to greater distance between the midline joints of the Mc and the Ch ( Figure 2E ). On the other hand, ethanol-treated gpc4 mutants showed greater spread along PC1, which includes an inversion of the Ch with the shortening and widening of the viscerocranium ( Figure 2E ). Therefore, it appears that while more subtle in their ethanol-induced facial defects compared to vangl2 mutants, gpc4 mutants still display ethanol-induced defects in viscerocranial shape when treated from 10-24 hpf, after C&E. Download figure Open in new tab Figure 2 Ethanol drives greater variation in viscerocranial shape in gpc4 mutant embryos. Whole-mount i ages of the viscerocranium of (A) wild type control embryos (B) wild type ethanol treated embryos, (C) gpc4 −/− control fish and (D) gpc4 −/− ethanol treated fish. (E) Principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n=13), cyan = wild type ethanol treated embryos (n=12), green = gpc4 −/− control embryos (n=9), magenta = gpc4 −/− ethanol treated embryos (n=8), solid circles represent confidence ellipses in which 95% of all individual data points for ach group lie, dashed circles represent 95% confidence ellipses for means. Wireframe graphs represent variation termed by each axis with black representing no variation and magenta representing variation relative to the black wireframe. For example, PC1 captures a shortening and widening in viscerocranial shape, while C2 represents variation in midfacial width. Procrustes ANOVA showed significant change in the viscerocranial shape (F = 51.25, DF = 36, p = <.0001). In addition to facial defects ethanol-treated vangl2 mutants also showed eye field defects, where the majority of ethanol-treated vangl2 mutants displayed synophthalmia (eye field fusions) with a smaller minority showing full cyclopia (complete lens fusion; Swartz et al., 2014 ; Sidik et al., 2021 ). However, these studies both exposed vangl2 mutants to ethanol from 6-24 hpf, overlapping with C&E and Sidik et al, (2021) showed that failure of eye field separation was driven in part by disrupted C&E. In our exposure paradigm, we did observe synophthalmia in 50% of ethanol-treated vangl2 mutants but not in our gpc4 mutants ( Figures 1D , 2D , Table 1 ). While phenotypically consistent with previous work ( Swartz et al., 2014 ; Sidik et al., 2021 ), our exposure paradigm begins at 10 hpf, after C&E, suggesting that ethanol disrupts eye field separation independent of C&E, arguing other signaling mechanism may be disrupted. View this table: View inline View popup Download powerpoint Table 1. Mutations in vangl2 and gpc4 sensitize embryos to eye-field defects. A composite summary of the percentage of embryos that have specific eye-field phenotypes. The x-axis represents the genotypes (Wild-Type, vangl2 mutant, gpc4 mutant, vangl2 mut gpc4 het, gpc4 mut vangl2 het and double mutant) and treatment conditions (Control or Ethanol) evaluated. The y-axis represents the eye phenotypes: normal eye-field separation synophthalmia (eye fusion), synophthalmia (eye & lens fusion) and cyclopia. To further confirm that both vangl2 and gpc4 are ethanol sensitive after C&E and during APE and CNC morphogenesis, we explored the following multiple ethanol exposure time windows: 1. during C&E (6-10 hpf); 2. when we have previously shown that BMP signaling is required for APE morphogenesis (10-18 hpf); 3. after CNC condensation into the pharyngeal arches (24-30 hpf). When exposed to ethanol from 10-18 hpf, vangl2 and gpc4 mutants had very similar variation in viscerocranial shape and size compared to mutant embryos treated from 10-24 hpf ( Figure 3A-B ). Interestingly, we did observe subtle differences in viscerocranial morphology between our gpc4 mutant treatment groups. Unlike in Figure 2 , where PC2 drove differences in viscerocranial shape between untreated and ethanol-treated gpc4 mutants, exposure timing drove variation along PC1 (representing 87% of variation in data; Figure 3B ). The mean shape shifted further left (negative for PC1) as the duration ethanol exposure increased. To test that ethanol interacts with vangl2 and gpc4 during C&E or in the transition between C&E and APE/CNC morphogenesis (prior to APE/CNC morphogenesis), we treated our fish with ethanol from either 6-10 hpf or 8-12 hpf and analyzed viscerocranial shape ( Supplemental Figures 1 and 2 , respectively). Neither mutant displayed any significant difference viscerocranial morphology between untreated and ethanol-treated mutants, when exposed to ethanol from 6-10 hpf or 8-12 hpf. To examine if ethanol interacts with vangl2 and gpc4 after APE/CNC morphogenesis, when the CNC have condensed into the pharyngeal arches, we treated mutant larvae with ethanol from 24-30 hpf. We again did not observe any significant ethanol-induced changes in viscerocranial shape ( Supplemental Figure 3 ). Overall, this suggests that the ethanol sensitive time window for both vangl2 and gpc4 is between 10-18 hpf, after C&E and during APE/CNC morphogenesis. Download figure Open in new tab Supplemental Figure 1 vangl2 and gpc4 mutant embryos are not sensitive to ethanol induced jaw defects when treated between 6-10 hpf. (A) principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n=8), blue = wild type 6-10 hpf ethanol treated embryos (n=12), orange = wild type 10-24 hpf ethanol treated embryos (n=11), green = vangl2 −/− control embryos (n=18), cyan = vangl2 −/− 6-10 hpf ethanol treated embryos (n=14), magenta = vangl2 −/− 10-24 hpf ethanol treated embryos (n=13). Solid circles represent 95% confidence ellipses for means. Procrustes ANOVA showed significant change in the viscerocranial shape (F = 11.34, DF = 60, p = <.0001). (B) principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n= 9), blue = wild type 6-10 hpf ethanol treated embryos (n= 28), orange = wild type 10-24 hpf ethanol treated embryos (n=18), green = gpc4 −/− control embryos (n= 11), cyan = gpc4 −/− 6-10 hpf ethanol treated embryos (n= 26), magenta = gpc4 −/− 10-24 hpf ethanol treated embryos (n= 14). Solid circles represent 95% confidence ellipses for means. Procrustes ANOVA showed significant change in the viscerocranial shape (F =102.67, DF = 60, p =<0.0001). Download figure Open in new tab Supplemental Figure 2 vangl2 and gpc4 mutant embryos are not sensitive to ethanol induced jaw defects when treated between 8-12 hpf. (A) principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n=21), blue = wild type 8-12 hpf ethanol treated embryos (n=23), orange = wild type 10-24 hpf ethanol treated embryos (n=10), green = vangl2 −/− control embryos (n=25), cyan = vangl2 −/− 8-12 hpf ethanol treated embryos (n=21), magenta = vangl2 −/− 10-24 hpf ethanol treated embryos (n=30). Solid circles represent 95% confidence ellipses for means. Procrustes ANOVA showed significant change in the viscerocranial shape (F = 22.28, DF = 60, p = <.0001). (B) principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n= 11), blue = wild type 8-12 hpf ethanol treated embryos (n= 10), orange = wild type 10-24 hpf ethanol treated embryos (n=10), green = gpc4 −/− control embryos (n= 11), cyan = gpc4 −/− 8-12 hpf ethanol treated embryos (n= 18), magenta = gpc4 −/− 10-24 hpf ethanol treated embryos (n= 14). Solid circles represent 95% confidence ellipses for means. Procrustes ANOVA showed significant change in the viscerocranial shape (F =73.99, DF = 60, p =<0.0001). Download figure Open in new tab Supplemental Figure 3 vangl2 and gpc4 mutant embryos are not sensitive to ethanol induced jaw defects when treated between 24-30 hpf. (A) principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n=19), blue = wild type 24-30 hpf ethanol treated embryos (n=15), orange = wild type 10-24 hpf ethanol treated embryos (n=19), green = vangl2 −/− control embryos (n=22), cyan = vangl2 −/− 24-30 hpf ethanol treated embryos (n=18), magenta = vangl2 −/− 10-24 hpf ethanol treated embryos (n=19). Solid circles represent 95% confidence ellipses for means. Procrustes ANOVA showed significant change in the viscerocranial shape (F = 30.51, DF = 60, p = <.0001). (B) principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n= 9), blue = wild type 24-30 hpf ethanol treated embryos (n= 13), orange = wild type 10-24 hpf ethanol treated embryos (n=13), green = gpc4 −/− control embryos (n= 16), cyan = gpc4 −/− 24-30 hpf ethanol treated embryos (n= 12), magenta = gpc4 −/− 10-24 hpf ethanol treated embryos (n= 14). Solid circles represent 95% confidence ellipses for means. Procrustes ANOVA showed significant change in the viscerocranial shape (F =37.92, DF = 60, p =<0.0001). Download figure Open in new tab Figure 3 vangl2 and gpc4 mutant embryos are most sensitive to ethanol induced jaw defects between 10-18 hpf. (A) Principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n=21), blue = wild type 10-18 hpf ethanol treated embryos (n=14), orange = wild type 10-24 hpf ethanol treated embryos (n=22), green = vangl2 −/− control embryos (n=30), cyan = vangl2 −/− 10-18 hpf ethanol treated embryos (n=22), magenta = vangl2 −/− 10-24 hpf ethanol treated embryos (n=22). Procrustes ANOVA showed significant change in the viscerocranial shape (F = 16.75, DF = 60, p = <.0001). (B) principal component analysis plot and wireframes from morphometric analysis data. Landmarks were placed on the joints between the cartilage structures of the viscerocranium. Each genotype is color-coded: black = wild type control embryos (n= 20), blue = wild type 10-18 hpf ethanol treated embryos (n= 29), orange = wild type 10-24 hpf ethanol treated embryos (n=13), green = gpc4 −/− control embryos (n= 28), cyan = gpc4 −/− 10-18 hpf ethanol treated embryos (n= 17), magenta = gpc4 −/− 10-24 hpf ethanol treated embryos (n= 22). solid circles represent confidence ellipses in which 95% of all individual data points for each group lie, dashed circles represent 95% confidence ellipses for means. Wireframe graphs represent variation termed by each axis with black representing no variation and magenta representing variation relative to the black wireframe. For example, PC1 captures a shortening and widening in viscerocranial shape, while PC2 represents variation in midfacial width. Procrustes ANOVA showed significant change in the viscerocranial shape (F =107.01, DF = 60, p =<0.0001). PCP mutants genetically interact during facial development Previous work from Mile et al. (2017) showed that vangl2 and gpc4 genetically interact during C&E leading to exacerbated endodermal defects over the single mutants ( Miles et al., 2017 ). We sought out to establish whether vangl2 & gpc4 also genetically interact and are further sensitized to ethanol-induced facial defects. To test this, we generated vangl2 +/− ; gpc4 +/− double heterozygous carriers and crossed them to produce vangl2 ; gpc4 compound heterozygous (heterozygous for one gene, homozygous mutant of the second gene) and double homozygous mutant larvae. Again, all larvae were treated with 1% Ethanol (v/v) from 10-24 hpf as described above. We observed severe facial and eye field defects in ethanol-treated compound heterozygous and homozygous double mutant larvae ( Figure 4A-J , Table 1 ). However, due to the severe morphological changes in our mutant larvae, we were not able to perform our morphometric analyses of shape on compound heterozygotes and homozygous double mutants. To simplify our measures, we quantified the length of the Mc and Ch cartilages, as well as the internal angle of the Ch cartilages ( Figure 4N-P, S-T ). We also measure eye field separation from lens to lens ( Figure 4M, R ). To account for variation in size due to ethanol exposure, we used overall head length in our linear measures ( Figure 4Q ) and report our Mc and Ch length measurements as Meckel’s-to-Head length ( Figure 4N ) and Ceratohyal-to-Head length ( Figure 4O ) ratios. Looking at head length measures alone ethanol did not impact head length in wild-type and gpc4 mutant larvae ( Supplemental Figure 4 ). We observed ethanol-induced decreases in head length in vangl2 mutants. Homozygous double mutant larvae were unaffected ( Supplemental Figure 4 ). Download figure Open in new tab Supplemental Figure 4 Comparison of Head length across all genotypes. Wild-type control embryos (n=11), Wild-Type ethanol treated embryos (n=9), vangl2 −/− control fish (n=9), vangl2 −/− ethanol treated fish (n=10), gpc4 −/− control fish (n=7), gpc4 −/− ethanol treated fish (n=13), vangl2 −/− ; gpc4 +/− control fish (n=16), vangl2 −/− ; gpc4 +/− ethanol treated fish (n=27), vangl2 +/− ; gpc4 −/− control fish (n=25), vangl2 +/− ; gpc4 −/− ethanol treated fish (n=23), vangl2 −/− ; gpc4 −/− control fish (n=5) and vangl2 −/− ; gpc4 −/− ethanol treated fish (n=11) were the genotypes used. (A) Comparison of head lengths of ethanol treated and untreated embryos within genotypes groups. Two-Way Anova was used to further analyze data. Download figure Open in new tab Figure 4 vangl2 and gpc4 genetically interact during jaw development. Whole-mount images of the viscerocranium of (A) wild type control embryos (n=11), (B) wild type ethanol treated embryos (n=9), (C) vangl2 −/− control fish (n=9), (D) vangl2 −/− ethanol treated fish (n=10), (E) gpc4 −/− control fish (n=7), (F) gpc4 −/− ethanol treated fish (n=13), (G) vangl2 −/− ; gpc4 +/− control fish (n=16), (H) vangl2 −/− ; gpc4 +/− ethanol treated fish (n=27), (I) vangl2 +/− ; gpc4 −/− control fish (n=25), (J) vangl2 +/− ; gpc4 −/− ethanol treated fish (n=23), (K) vangl2 −/− ; gpc4 −/− control fish (n=5) and (L) vangl2 −/− ; gpc4 −/− ethanol treated fish (n=11). (M) Eye-Field length. (N) Meckel length and (O) Ceratohyal length analyzed as ratios to overall head length. (P) Measures for the angle of the joints of the ceratohyals. Two-Way Anova was used to further analyze data. (Q-U) Whole-mount images of the viscerocranium in 4 dpf larvae showing linear measures and cartilage angles (cartilage is blue, ventral views, anterior to the left). The ethanol-induced reduction in head length in larvae either heterozygous or homozygous mutant for vangl2 was expected as the facial skeleton is significantly shorter and wider resulting in a shorter head length. Even after accounting for head length, we still observed significant ethanol-induced reductions in Mc length in vangl2 single mutant larvae ( Figure 4N ). In our homozygous double mutants, we did not observe ethanol-induced significant changes in Mc cartilage lengths. This was not surprising given that untreated double mutants already displayed severe craniofacial defects ( Figure 4K ). Ethanol did not result in significant reductions in Mc length in gpc4 mutants ( Figure 4N ). While, Ch length was unaffected in all groups ( Figure 4N, O ), ethanol did increase the size of the angle of Ch angle in our vangl2 single mutants and homozygous double mutants ( Figure 4P ) compared to their untreated siblings. Unlike our vangl2 single mutants and homozygous double mutants, our compound heterozygous mutants mainly resemble their single mutant siblings when untreated ( Figure 4G & I ). However, when exposed to ethanol, many of our compound heterozygous larvae had exacerbated Mc and Ch cartilage defects and eye field defects, comparable to the double mutant larvae ( Figure 4H , J compared to K, L). We observed that Mc length was significantly shorter in ethanol treated vangl2 heterozygous compound mutant larvae ( Figure 4N ). There was also a significant ethanol-induced increase in Ch angle in vangl2 heterozygous compound mutant larvae ( Figure 4P ). As with gpc4 single mutants, there is no significant difference in Mc length gpc4 compound mutant larvae although we do see a downward trend in that data ( Figure 4N ). We did observe a significant increase in Ch angle in ethanol-treated gpc4 compound mutants ( Figure 4P ), demonstrating that a flattening, or even an inversion, of the Ch occurs. While we are not able to perform morphometric analysis on our compound heterozygous and homozygous double mutants, similar shape changes did occur, and they were reminiscent of our single mutants ( Figures 1 & 2 ). In addition to facial phenotypes, we observed increasing rates of ethanol-induced synophthalmia (with or without partial lens fusion) and cyclopia (full lens fusion) in our compound heterozygous and homozygous double mutants ( Figure 4A-M , Table 1 ). As described above, we observed synophthalmia (without lens fusion) in ethanol-treated vangl2 single mutants but not in gpc4 single mutants ( Figure 1 , Table 1 ). Untreated homozygous double mutant larvae 80% showed synophthalmia (without lens fusion) and 20% cyclopia ( Figure 4K , Table 1 ). Ethanol-treatment increased synophthalmia with partial lens fusions to 64% (from 0% untreated, Table 1 ), though we did observe a decrease in full cyclopia (20% to 9%, Table 1 ). This demonstrates that gpc4 loss potentiates facial shape defects in vangl2 mutants, independent of ethanol, but ethanol exacerbates these defects. In both untreated gpc4 compound mutant larvae and vangl2 compound mutant larvae, 88% had complete eye field separation while only 12% had synophthalmia without lens fusions ( Table 1 ). However, ethanol treatment increased synophthalmia without lens fusions in gpc4 compound mutant larvae and vangl2 compound mutant larvae to 83% and 41%, respectively ( Table 1 ). Strikingly, we observed 4% cyclopia in gpc4 compound mutant larvae but 0% in vangl2 compound mutant larvae. To quantify these eye defects, we measured the lens-to-lens width untreated and ethanol-treated single, compound heterozygous and homozygous double mutant larvae. Homozygous double mutant larvae did not display ethanol-induced reductions in lens-to-lens width, though we did see a downward trend ( Figure 4M ). This is largely due to severity already observed in untreated double mutants with downward trend representing increased lens fusions ( Table 1 ). We observe significant ethanol-induced reductions in lens-to-lens width in vangl2 single and compound heterozygous mutant larvae ( Figure 4M ). We also observed significant ethanol-induced reductions in lens-to-lens width in gpc4 compound heterozygous mutants but not gpc4 single mutants. This suggests that loss of one copy of vangl2 sensitizes gpc4 mutants to ethanol-induced synophthalmia. Taken together, our data shows that like our single mutants, our compound heterozygous and homozygous double mutants are ethanol sensitive after C&E and interact increasing the penetrance and expressivity of ethanol-induced facial defects. Loss of BMP signaling leads to exacerbated craniofacial phenotypes in PCP mutants We have previously shown that multiple members of the BMP pathway, including the ligand bmp4 , are ethanol sensitive from 10-18 hpf, disrupting endoderm morphogenesis and resulting in defects to the viscerocranium ( Klem et al., 2025 ). Based on phenotypic and exposure paradigm similarities, we predicted that the BMP and PCP interact and sensitize embryos to ethanol-induced viscerocranial defects. To test this, we generated compound bmp4; vangl2 and bmp4; gpc4 double heterozygous crosses to analyze this potential interaction. Loss of one or both bmp4 alleles further sensitize vangl2 and gpc4 mutants to ethanol, increasing both the penetrance and expressivity of jaw defects and synophthalmia ( Figure 5A,B,D,G , Tables 2 & 3 ). We observed increases in severity of jaw hypoplasia, increases in synophthalmia in vangl2 mutant / bmp4 heterozygous larvae and homozygous double mutant larvae but not in bmp4 mutant / vangl2 heterozygous larvae ( Table 2 ). We observed similar increases in gpc4 mutant / bmp4 heterozygous larvae and homozygous double mutant larvae only, including synophthalmia which has not been observed in gpc4 single mutants ( Table 3 ). Strikingly, we also observed several new craniofacial defects not originally seen in ethanol-treated BMP mutants or PCP mutants alone. We observed ectopic cartilages ( Figure 5C ), midline defects (which includes asymmetry of the midline joint between the ceratohyals; Figure 5E & F ), basihyal defects (the basihyal is either split, crooked and/or rodded; Figure 5H, I & J ) or palate defects (rodded ethmoid palate or jagged trabecula; Figure 5K & L ). In our screen, these novel midline defects only marginally increased in ethanol suggesting they may be a novel ethanol-independent set of phenotypes in BMP/PCP interactions ( Tables 2 & 3 ; Supplemental Figure 5 ). Taken together, our data show that disruptions to BMP signaling sensitizes PCP mutants and generates novel midline defects not observed in BMP or PCP mutants. This suggests that BMP/PCP interactions may lie at the heart of ethanol-sensitive endoderm morphogenesis driving facial defects. Download figure Open in new tab Supplemental Figure 5 Bar graphs demonstrate the occurrence of midline defects in PCP/BMP mutant crosses. A) The occurrence of midline defects in bmp4 +/− ; vangl2 +/− cross. B) The occurrence of midline defects in bmp4 +/− ; gpc4 +/− cross. The percentages for the number of embryos that have no midline defects (Green) and those that have Midline defects (Magenta) are displayed for all genotype and treatment groups. Download figure Open in new tab Figure 5 Representative images of phenotypes found in PCP/BMP crossed embryos. Whole-mount images of the malformations found in the viscerocranium of PCP/BMP embryos (ventral views, anterior to the left). the phenotypes displayed are (A) jaw hypoplasia, (B) severe jaw hypoplasia, (C) ectopic cartilage, (D) flattened ceratohyals, (E) asymmetry of ceratohyals, (F) asymmetry of midline joint alignment, (G) synophthalmia, (H) split basihyal, (I) crooked basihyal, (J) rodded basihyal, (K) rodded ethmoid plate and (l) jagged trabecula. arrows indicate the malformation or phenotype described. View this table: View inline View popup Download powerpoint Table 2. Summarized data on the occurrence of craniofacial phenotypes under different treatment conditions in vangl2/bmp4 cross. Column A represents the genotype of the embryos (Wild-Type, bmp4 mut, vangl2 mut, bmp4 mut vangl2 het, vangl2 mut bmp4 het and double mut) while Column B defines the treatment condition (control or ethanol). Column C displays the total number of fish for each genotype under the specified conditions. Column D represents the number of fish in the group with jaw hypoplasia, and Column E represents the number of fish in the group with jaw loss. Jaw hypoplasia and jaw loss are mutually exclusive, meaning that if an embryo has one, it cannot have the other. Column F shows the number of fish in the group with ectopic cartilage, while Column G represents the number of fish with flattened ceratohyals. Column H provides the number of fish with craniofacial midline defects, Column I indicates the number with synophthalmia, and Column J details the number with basihyal defects. Column K represents the number of fish in the group with palate defects. The numerical values represent the number of fish that have the phenotype described within a given genotype and treatment group. Empty cells represent 0 phenotype observed in the group. The parentheses represent the percentage of embryos with the phenotype out of the total number of fish. View this table: View inline View popup Download powerpoint Table 3. Summarized data on the occurrence of craniofacial phenotypes under different treatment conditions in gpc4/bmp4 cross. Column A represents the genotype of the embryos (Wild-Type, bmp4 mut, gpc4 mut, bmp4 mut gpc4 het, gpc4 mut bmp4 het and double mut) while Column B defines the treatment condition (control or ethanol). Column C displays the total number of fish for each genotype under the specified conditions. Column D represents the number of fish in the group with jaw hypoplasia, and Column E represents the number of fish in the group with jaw loss. Jaw hypoplasia and jaw loss are mutually exclusive, meaning that if an embryo has one, it cannot have the other. Column F shows the number of fish in the group with ectopic cartilage, while Column G represents the number of fish with flattened ceratohyals. Column H provides the number of fish with craniofacial midline defects, Column I indicates the number with synophthalmia, and Column J details the number with basihyal defects. Column K represents the number of fish in the group with palate defects. The numerical values represent the number of fish that have the phenotype described within a given genotype and treatment group. Empty cells represent 0 phenotype observed in the group. The parentheses represent the percentage of embryos with the phenotype out of the total number of fish. Discussion Prenatal alcohol exposure (PAE) results in a wide range of structural malformations, including those to the facial skeleton ( Sokol et al. 2003 ; Hoyme et al. 2016 ). Described as Fetal Alcohol Spectrum Disorders (FASD), the malformations include, thin upper lip, microcephaly and, for purposes of this work, jaw hypoplasia ( Lovely, 2020 ). Formation of the facial skeleton is a highly complex process requiring reciprocal signaling events between multiple cell types, including the pharyngeal endoderm and the cranial neural crest (CNC). These signaling interactions rely greatly on separate morphogenesis events in each tissue ( Swartz et al., 2012 ; Lovely et al., 2016 ). We and others have shown that many of the genetic loci that regulate these events can be sensitive to ethanol, disrupting facial formation ( Swartz et al., 2014 ; Klem et al., 2025 ; Vo and Lovely, 2025 ). One such set of ethanol-sensitive genetic pathways is the highly conserved Planar Cell Polarity (PCP) pathway. PCP plays a major role in a host of developmental processes, including convergence and extension (C&E), endoderm morphogenesis and facial development ( Miles et al., 2017 ; Scar et al., 2021; Ulrich et al., 2003 ; Ulrich et al., 2005 , Ling et al., 2017 ). Loss of either vangl2 or gpc4 results in C&E defects in the endoderm leading to a shorter trunk and wider body axis, which is exacerbated in vangl2; gpc4 double mutants ( Miles et al., 2017 ). Moreover, gpc4 has been shown to play a role in cartilage polarity and cell stacking during neural crest development ( Sisson et al., 2015 ; Ling et al., 2017 ). PCP-ethanol interactions lead to facial defects Previous work showed that vangl2 and gpc4 mutants are sensitive to ethanol-induced facial defects and synophthalmia when treated from 6-24 hpf ( Sidik et al., 2021 ). However, this time window covers both C&E (6-10 hpf) and endoderm/CNC morphogenesis (10-24 hpf). This work examining ethanol-induced phenotypes in vangl2 and gpc4 mutants never separated these two developmental events through varying the time windows of ethanol exposure. To focus on the ethanol-sensitive role that vangl2 and gpc4 play in endoderm/CNC morphogenesis, we examined multiple ethanol-exposure time windows spanning C&E (6-10 hpf), endoderm/CNC morphogenesis (10 hpf-24 hpf) and after the CNC condense into the pharyngeal arches (24-30 hpf). We observed that both vangl2 and gpc4 are ethanol sensitive during endoderm/CNC morphogenesis (10-24 hpf), but not C&E (6-10 hpf). In addition, both mutants were insensitive to ethanol after 24 hpf demonstrating that vangl2 and gpc4 were ethanol sensitive during endoderm/CNC morphogenesis (10-24 hpf) leading to increased synophthalmia and facial defects. However, given that our focus was on facial development, we did not examine expression changes in C&E markers that Sidik et al., (2021) observed in their work. It is possible that when isolated, the significant reductions in expression domain size of these C&E markers may not manifest in the subsequent facial phenotypes. This suggests that the facial phenotypes we observed in our 10-24 hpf exposure window result from additional developmental events occurring after C&E, in particular endoderm/CNC morphogenesis. Future work varying exposure paradigms and broader mechanistic analyses will be needed to fully address this discrepancy. Further analysis with our morphometric approach showed subtle variation in facial shape that was not previously observed. Ethanol-treated wild-type larvae displayed altered facial morphology compared to untreated wild-type siblings, though the shape change was not as pronounced as observed in untreated vangl2 mutants. This suggests ethanol increases facial variation and loss of vangl2 potentiates these defects. While we did not observe similar differences between untreated and ethanol-treated gpc4 mutants when exposed from 10-24 hpf, we did see that when exposed from 10-18 hpf the facial shape changes were not as expressive as those exposed 10-24 hpf. Given the gpc4 plays roles in both endoderm morphogenesis and CNC development ( Sisson et al., 2015 ; Miles et al., 2017 ; Ling et al., 2017 ), the subtle differences in facial shape in the different exposure windows are not surprising. It is possible that gpc4 may be playing roles in two different yet mechanistically linked developmental processes driving facial development. While our exposure time windows suggest these distinct temporal roles of gpc4 , future will be needed to separate these dual roles for gpc4 in facial development. Overall, this suggests that while exposure and genotype drive facial shape changes in vangl2 mutants, timing of exposure drives these changes in gpc4 mutants and vangl2 mutation fully sensitizes larvae to ethanol-induced facial defects but gpc4 only mildly sensitizes larvae depending on the exposure window. While vangl2 - or gpc4 -ethanol interactions result in unique facial malformations, they both impact facial development suggesting they interact hyper-sensitizing larvae to ethanol. Here, we show that heterozygous loss of gpc4 sensitized vangl2 larvae to ethanol-induced facial malformations and increased synophthalmia/cyclopia, consistent with the work from Sidik et al., (2021) . However, our work narrows the vangl2 - and gpc4 -ethanol interactions to endoderm/CNC morphogenesis after C&E (10-24 hpf), suggesting that either CNC development or endoderm morphogenesis (or both) may be impacted by these interactions. Previous work has shown that gpc4 is necessary for CNC development in part by regulating CNC polarity ( Ling et al., 2017 ). However, loss of gpc4 does not appear to disrupt pharyngeal arch morphology at 36 hpf though it does act cell autonomously in the CNC suggesting it plays a role during CNC morphogenesis out of the pharyngeal arches to form the facial skeleton ( Sisson, et al., 2015 ). While it’s possible that ethanol may be interacting with gpc4 mutants to exacerbate polarity defects earlier during endoderm/CNC morphogenesis that then result in later CNC developmental defects, we can’t rule out that ethanol may be synergizing with gpc4 mutants through additional targets. It is possible that vangl2 is the target of interaction with ethanol as we see vangl2 mutation sensitizing larvae to ethanol-induced facial malformations. This suggests that CNC develop could be disrupted in ethanol-treated vangl2 mutants. While vangl2 mutants do impact CNC migration, this impact is mild and may not fully explain the impact of ethanol on facial development in vangl2 mutants ( Matthews et al., 2008 ). However, ethanol does exacerbate facial phenotypes in mutants of CNC migration ( McCarthy et al., 2013 ). This suggests that vangl2 mutation may sensitize CNC to ethanol-induced defects not observed in ethanol or vangl2 loss of function alone. For gpc4 single mutants, ethanol may be disrupting vangl2 function, impacting CNC development. It is also possible that vangl2 and gpc4 may be disrupting endoderm morphogenesis. Both play roles in endoderm C&E ( Miles et al., 2017 ) and cannot be ruled out of impacting endoderm morphogenesis after C&E and disrupting endoderm signaling to the CNC resulting in facial malformations. This would be consistent with our previous results showing that BMP-ethanol interactions disrupt endoderm morphogenesis driving CNC defects ( Klem et al., 2025 ; Vo and Lovely, 2025 ). While we have previously shown that BMP signaling is required in the endoderm for its morphogenesis ( Lovely et al., 2016 ), we would need to test cell autonomy of vangl2 and gpc4 to determine whether morphogenesis of the endoderm, CNC, both or neither are impacted in our ethanol-treated mutants. BMP-PCP interactions yield novel phenotypes We have previously shown that multiple BMP mutants are ethanol-sensitive from 10-18 hpf leading to disrupted endoderm morphology ( Klem et al., 2025 ). However, ethanol does not attenuate BMP signaling nor its downstream target nkx2.3 but does increase CNC apoptosis in BMP mutants through a yet unknown signaling mechanism ( Klem et al., 2025 ; Vo and Lovely, 2025 ). Throughout our study, we see overlapping similarities between the BMP and PCP pathways. BMP and PCP mutant larvae share ethanol sensitive time window of 10-18 hpf during endoderm/CNC morphogenesis ( Klem et al., 2025 ). Ethanol-treated PCP mutants phenocopy ethanol-induced shorter and wider facial shape observed in BMP mutants ( Klem et al., 2025 ). Both BMP and PCP regulate endoderm development ( Lovely et al., 2016 ; Miles et al., 2017 ). All this strongly supports our analysis of BMP-PCP interactions in ethanol-sensitive facial development where we observed that loss one or both bmp4 alleles further sensitizes PCP mutants to ethanol-induced facial defects and synophthalmia, phenocopying vangl2; gpc4 double mutants and showing increased penetrance of facial defects and synophthalmia. Surprisingly, we observed additional midline craniofacial defects in untreated larvae not seen in our PCP double mutants. Here, ethanol mildly increases the penetrance of these midline defects. This suggests that the PCP and BMP pathways interact in two ways: 1. An ethanol-dependent exacerbation facial defects and synophthalmia; 2. An ethanol-independent generation of novel midline defects. Given the complexity of facial development, several mechanisms may be driving these two distinct interactions. BMP may be upstream, regulating PCP component gene expression in the endoderm. This would lead to defects in endoderm morphogenesis resulting in facial defects. However, this does not explain how we only see synophthalmia in PCP and BMP-PCP mutants but not BMP mutants alone. It also leaves the open question of how defects in endoderm morphogenesis give rise to CNC apoptosis. It is possible that BMP drives ethanol-induced defects in endoderm morphogenesis while PCP drives ethanol-induced defects in the CNC. Here we would predict a synergy between BMP-endoderm and PCP-CNC defects driving the overall phenotypes. If this were true, we would predict that increased CNC apoptosis in ethanol-treated PCP mutants would result in similar facial malformations as seen in ethanol-treated BMP mutants ( Klem et al., 2025 ). However, our morphometric and genetic interaction analyses argue against this as PCP mutants, both single and double, do not fully recapitulate the ethanol-induced facial phenotypes of BMP mutants. While ethanol-treated PCP mutants do not fully phenocopy ethanol-treated BMP mutants, it is possible that ethanol-induced CNC apoptosis in PCP mutants leads to unique outcomes in CNC development compared to ethanol-treated BMP mutants. Building on this, the novel midline phenotypes in our PCP/BMP double mutants suggest a more complex relationship between BMP and PCP signaling. We previously showed that BMP signaling regulates formation of lateral endoderm ( Lovely et al., 2016 ) and PCP plays a major role in cell endoderm cell polarity ( Miles et al., 2017 ). The presence of midline defects in our PCP/BMP double mutants suggests that medial-lateral patterning may also be affected leading to exacerbation of existing phenotypes and generation of the novel midline defects. Beyond studies of cell autonomy described above, detail analyses of BMP and PCP signaling dynamics and cell behavior will be needed to dissect the complex ethanol-sensitive relationship between BMP and PCP signaling. Combined our results show that work in animal models like zebrafish are key to dissecting the complex relationship between genetics and ethanol exposure paradigms driving ethanol-induced development defects. Given the genetic and developmental homology between zebrafish and humans, our work provides a powerful model to examine complex genetic signaling pathways and generate a deeper mechanistic understanding of ethanol interactions with these pathways on craniofacial development. Here, we show that PCP mutants are ethanol sensitive after C&E, during endoderm/CNC morphogenesis, and interact with BMP signaling resulting in unique facial defects and synophthalmia. The expressivity and penetrance of these phenotypes suggest a novel and complex ethanol-sensitive signaling pathway driving endoderm/CNC morphogenesis during facial development. Future studies to identify the mechanism on a cellular level will be needed to dissect the role PCP and BMP during endoderm/CNC morphogenesis, and how ethanol fits into the story. Ultimately, this will continue to build on a conceptual framework and a mechanistic paradigm of ethanol-induced birth defects we have been developing, connecting ethanol exposure with concrete cellular events that could be sensitive beyond facial development. Funding National Institute on Alcohol Abuse and Alcoholism, https://ror.org/02jzrsm59 , R00AA023560 , R01AA031043 Footnotes Support: This work was funded by National Institutes of Health/National Institute on Alcohol Abuse (NIH/NIAAA) R00AA023560 and R01AA031043 to CBL. References Antinucci , P. , & Hindges , R . ( 2016 ). A crystal-clear zebrafish for in vivo imaging . Scientific reports , 6 ( 1 ), 1 – 10 . OpenUrl CrossRef PubMed ↵ Balczerski , B. , Matsutani , M. , Castillo , P. , Osborne , N. , Stainier , D. Y. , & Crump , J. G . ( 2012 ). Analysis of sphingosine-1-phosphate signaling mutants reveals endodermal requirements for the growth but not dorsoventral patterning of jaw skeletal precursors . Developmental biology , 362 ( 2 ), 230 – 241 . OpenUrl CrossRef PubMed ↵ Bilotta , J. , Barnett , J. A. , Hancock , L. , & Saszik , S . ( 2004 ). Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome . Neurotoxicology and teratology , 26 ( 6 ), 737 – 743 . OpenUrl CrossRef PubMed Web of Science ↵ Canfield , D.V. ; Forster , E.M. ; Cheong , Z.-I. ; Cowan , J.M . Breath/Blood Alcohol Concentration as an Indicator of Alcohol Use Problems . Aerosp. Med. Hum. Perform . 2019 , 90 , 488 – 491 . OpenUrl CrossRef PubMed ↵ Chai Y , & Maxson RE Jr. ( 2006 ). Developmental Dynamics , 235 , 2353 – 2375 . [PubMed: 16680722] OpenUrl CrossRef PubMed Web of Science ↵ Chai Y , Jiang X , Ito Y , Bringas P Jr ., Han J , Rowitch DH , et al. ( 2000 ). Development , 127 , 1671 – 1679 . [PubMed: 10725243] OpenUrl Abstract Chung WS , Stainier DYR . Intra-Endodermal Interactions Are Required for Pancreatic β Cell Induction . Developmental Cell . 2008 Apr; 14 ( 4 ): 582 – 93 . OpenUrl CrossRef PubMed Web of Science ↵ Chung , K. Y. , Sorouri , K. , Wang , L. , Suryavanshi , T. , & Fisher , D . ( 2019 ). The impact of social stigma for children with cleft lip and/or palate in low-resource areas: a systematic review . Plastic and Reconstructive Surgery–Global Open , 7 ( 10 ), e2487 . OpenUrl CrossRef ↵ David NB , Saint-Etienne L , Tsang M , Schilling TF , Rosa FM ( 2002 ) Requirement for endoderm and FGF3 in ventral head skeleton formation . Development 129 , 4457 – 4468 . OpenUrl Abstract / FREE Full Text ↵ Driever , W. , Solnica-Krezel , L. , Schier , A. F. , Neuhauss , S. C. F. , Malicki , J. , Stemple , D. L. , … & Boggs , C. ( 1996 ). A genetic screen for mutations affecting embryogenesis in zebrafish . Development , 123 ( 1 ), 37 – 46 . OpenUrl Abstract / FREE Full Text ↵ Dush , M. K. , & Nascone-Yoder , N. M. ( 2019 ). Vangl2 coordinates cell rearrangements during gut elongation . Developmental Dynamics , 248 ( 7 ), 569 – 582 . OpenUrl CrossRef PubMed ↵ Ethen , M. K. , Ramadhani , T. A. , Scheuerle , A. E. , Canfield , M. A. , Wyszynski , D. F. , Druschel , C. M. , … & National Birth Defects Prevention Study . ( 2009 ). Alcohol consumption by women before and during pregnancy . Maternal and child health journal , 13 , 274 – 285 . OpenUrl CrossRef PubMed Web of Science ↵ Everson , J.L. ; Tseng , Y. ; Eberhart , J.K . High-throughput Detection of Craniofacial Defects in Fluorescent Zebrafish . Birth Defects Res . 2022 , 115 , 371 – 389 . OpenUrl PubMed ↵ Flentke , G. R. , Klingler , R. H. , Tanguay , R. L. , Carvan III , M. J. , & Smith , S. M . ( 2014 ). An evolutionarily conserved mechanism of calcium-dependent neurotoxicity in a zebrafish model of fetal alcohol spectrum disorders . Alcoholism: Clinical and Experimental Research , 38 ( 5 ), 1255 – 1265 . OpenUrl CrossRef PubMed ↵ Hemingway , S. J. A. , Bledsoe , J. M. , Brooks , A. , Davies , J. K. , Jirikowic , T. , Olson , E. M. , & Thorne , J. C . ( 2019 ). Twin study confirms virtually identical prenatal alcohol exposures can lead to markedly different fetal alcohol spectrum disorder outcomes—fetal genetics influences fetal vulnerability . Advances in pediatric research , 5 ( 3 ), 23 . OpenUrl ↵ Hoyme , H. E. , Kalberg , W. O. , Elliott , A. J. , Blankenship , J. , Buckley , D. , Marais , A. S. , … & May , P. A. ( 2016 ). Updated clinical guidelines for diagnosing fetal alcohol spectrum disorders . Pediatrics , 138 ( 2 ). OpenUrl ↵ Hu , B. , Rodriguez , J. J. , Kakkerla Balaraju , A. , Gao , Y. , Nguyen , N. T. , Steen , H. , … & Lin , F. ( 2021 ). Glypican 4 mediates Wnt transport between germ layers via signaling filopodia . Journal of Cell Biology , 220 ( 12 ), e202009082 . OpenUrl CrossRef PubMed Ibarra-García-Padilla R , Howard AGA , Singleton EW , Uribe RA . A protocol for whole-mount immuno-coupled hybridization chain reaction (WICHCR) in zebrafish embryos and larvae . STAR Protocols . 2021 ; 2 ( 3 ): 100709 . 819 76. OpenUrl CrossRef PubMed ↵ Jessen , J. R. , Topczewski , J. , Bingham , S. , Sepich , D. S. , Marlow , F. , Chandrasekhar , A. , & Solnica-Krezel , L . ( 2002 ). Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements . Nature cell biology , 4 ( 8 ), 610 – 615 . OpenUrl CrossRef PubMed Web of Science ↵ Jones , A.W . Ultra-Rapid Rate of Ethanol Elimination from Blood in Drunken Drivers with Extremely High Blood-Alcohol Concentrations . Int. J. Legal Med . 2008 , 122 , 129 – 134 . OpenUrl CrossRef PubMed ↵ Keller , R . ( 2002 ). Shaping the vertebrate body plan by polarized embryonic cell movements . Science 298 , 1950 – 1954 . OpenUrl Abstract / FREE Full Text ↵ Klem , J. R. , Schwantes-An , T. H. , Abreu , M. , Suttie , M. , Gray , R. , Vo , H. , … & Lovely , C. B. ( 2025 ). Mutations in the Bone Morphogenetic Protein signaling pathway sensitize zebrafish and humans to ethanol-induced jaw malformations . Disease Models & Mechanisms , dmm-052223 . ↵ Klingenberg CP . MorphoJ: an integrated software package for geometric morphometrics: COMPUTER PROGRAM NOTE . Molecular Ecology Resources . 2011 Mar; 11 ( 2 ): 353 – 7 . OpenUrl CrossRef PubMed Kuehn E , Clausen DS , Null RW , Metzger BM , Willis AD , Özpolat BD . Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii . J Exp Zool Pt B . 2022 Jun; 338 ( 4 ): 225 – 40 . OpenUrl CrossRef ↵ Landgraf , M. N. , Nothacker , M. , & Heinen , F . ( 2013 ). Diagnosis of fetal alcohol syndrome (FAS): German guideline version 2013 . european journal of paediatric neurology , 17 ( 5 ), 437 – 446 . OpenUrl CrossRef PubMed ↵ Lange , S. , Probst , C. , Heer , N. , Roerecke , M. , Rehm , J. , Monteiro , M. G. , … & Popova , S. ( 2017 ). Actual and predicted prevalence of alcohol consumption during pregnancy in Latin America and the Caribbean: systematic literature review and meta-analysis . Revista Panamericana de Salud Pública , 41 , e89 . OpenUrl ↵ LeClair , E. E. , Mui , S. R. , Huang , A. , Topczewska , J. M. , & Topczewski , J . ( 2009 ). Craniofacial skeletal defects of adult zebrafish Glypican 4 (knypek) mutants . Developmental dynamics: an official publication of the American Association of Anatomists , 238 ( 10 ), 2550 – 2563 . OpenUrl PubMed ↵ Li , L. , Ning , G. , Yang , S. , Yan , Y. , Cao , Y. , & Wang , Q . ( 2019 ). BMP signaling is required for nkx2. 3-positive pharyngeal pouch progenitor specification in zebrafish . PLoS Genetics , 15 ( 2 ), e1007996 . OpenUrl CrossRef ↵ Ling , I. T. , Rochard , L. , & Liao , E. C . ( 2017 ). Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification . Developmental Biology , 421 ( 2 ), 219 – 232 . OpenUrl CrossRef PubMed ↵ Lovely , C. B. , Nobles , R. D. , & Eberhart , J. K . ( 2014 ). Developmental age strengthens barriers to ethanol accumulation in zebrafish . Alcohol , 48 ( 6 ), 595 – 602 . OpenUrl CrossRef PubMed ↵ Lovely , C. B. , Swartz , M. E. , McCarthy , N. , Norrie , J. L. , & Eberhart , J. K . ( 2016 ). Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish . Development , 143 ( 11 ), 2000 – 2011 . OpenUrl Abstract / FREE Full Text ↵ Lovely , C.B . Animal Models of Gene–Alcohol Interactions . Birth Defects Res . 2020 , 112 , 367 – 379 . OpenUrl CrossRef PubMed Maier , S. E. , & West , J. R . ( 2001 ). Drinking patterns and alcohol-related birth defects . Alcohol Research & Health , 25 ( 3 ), 168 . OpenUrl PubMed Web of Science Mani , R. , St. Onge , R. P. , Hartman IV , J. L. , Giaever , G. , & Roth , F. P . ( 2008 ). Defining genetic interaction . Proceedings of the National Academy of Sciences , 105 ( 9 ), 3461 – 3466 . OpenUrl Abstract / FREE Full Text ↵ Matthews , H. K. , Marchant , L. , Carmona-Fontaine , C. , Kuriyama , S. , Larraín , J. , Holt , M. R. , … & Mayor , R. ( 2008 ). Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA . ↵ May , P. A. , Chambers , C. D. , Kalberg , W. O. , Zellner , J. , Feldman , H. , Buckley , D. , … & Hoyme , H. E. ( 2018 ). Prevalence of fetal alcohol spectrum disorders in 4 US communities . Jama , 319 ( 5 ), 474 – 482 . OpenUrl CrossRef PubMed ↵ May , P.A. , Gossage , J.P. , Kalberg , W.O. , Robinson , L.K. , Buckley , D. , Manning , M. , Hoyme , H.E ., 2009 . Prevalence and Epidemiologic Characteristics of FASD from Various Research Methods with an Emphasis on Recent Inschool Studies Developmental Disabilities 15 , 176 – 192 . OpenUrl ↵ McCarthy , N. , Wetherill , L. , Lovely , C. B. , Swartz , M. E. , Foroud , T. M. , & Eberhart , J. K . ( 2013 ). Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD . Development , 140 ( 15 ), 3 OpenUrl Abstract / FREE Full Text ↵ Miles , L. B. , Mizoguchi , T. , Kikuchi , Y. , & Verkade , H . ( 2017 ). A role for planar cell polarity during early endoderm morphogenesis . Biology open , 6 ( 5 ), 531 – 539 . OpenUrl Abstract / FREE Full Text ↵ Muñoz-Soriano , V. , Belacortu , Y. , & Paricio , N . ( 2012 ). Planar cell polarity signaling in collective cell movements during morphogenesis and disease . Current genomics , 13 ( 8 ), 609 – 622 . OpenUrl CrossRef PubMed ↵ Perkins , Jonathan A. , et al. “ Airway management in children with craniofacial anomalies .” The Cleft palate-craniofacial journal 34 . 2 ( 1997 ): 135 – 140 . OpenUrl CrossRef ↵ Popova , S. , Dozet , D. , & Burd , L . ( 2020 ). Fetal alcohol spectrum disorder: can we change the future? . Alcoholism, clinical and experimental research , 44 ( 4 ), 815 . OpenUrl CrossRef PubMed ↵ Reimers , M. J. , Flockton , A. R. , & Tanguay , R. L . ( 2004 ). Ethanol-and acetaldehyde-mediated developmental toxicity in zebrafish . Neurotoxicology and teratology , 26 ( 6 ), 769 – 781 . OpenUrl CrossRef PubMed Web of Science Scar Hu , B. , Rodriguez , J. J. , Kakkerla Balaraju , A. , Gao , Y. , Nguyen , N. T. , Steen , H. , … & Lin , F. ( 2021 ). Glypican 4 mediates Wnt transport between germ layers via signaling filopodia . Journal of Cell Biology , 220 ( 12 ), e202009082 . OpenUrl CrossRef PubMed pa , E. , & Mayor , R. ( 2016 ). Collective cell migration in development . Journal of Cell Biology , 212 ( 2 ), 143 – 155 . OpenUrl Abstract / FREE Full Text ↵ Sepich , D. S. , Calmelet , C. , Kiskowski , M. , & Solnica-Krezel , L. ( 2005 ). Initiation of convergence and extension movements of lateral mesoderm during zebrafish gastrulation . Developmental dynamics: an official publication of the American Association of Anatomists , 234 ( 2 ), 279 – 292 . OpenUrl PubMed ↵ Sidik , A. , Dixon , G. , Buckley , D.M. , Kirby , H.G. , Sun , S. , Eberhart , J.K . ( 2021 ). Exposure to ethanol leads to midfacial hypoplasia in a zebrafish model of FASD via indirect interactions with the Shh pathway . BMC Biol . 19 ( 1 ): 134 . OpenUrl CrossRef PubMed ↵ Sisson , B. E. , Dale , R. M. , Mui , S. R. , Topczewska , J. M. , & Topczewski , J . ( 2015 ). A role of glypican4 and wnt5b in chondrocyte stacking underlying craniofacial cartilage morphogenesis . Mechanisms of development , 138 , 279 – 290 . OpenUrl CrossRef PubMed ↵ Sokol , R. J. , Delaney-Black , V. , & Nordstrom , B . ( 2003 ). Fetal alcohol spectrum disorder . Jama , 290 ( 22 ), 2996 – 2999 . OpenUrl CrossRef PubMed Web of Science ↵ Solnica-Krezel , L. , Stemple , D. L. , Mountcastle-Shah , E. , Rangini , Z. , Neuhauss , S. C. , Malicki , J. , … & Driever , W. ( 1996 ). Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish . Development , 123 ( 1 ), 67 – 80 . OpenUrl Abstract / FREE Full Text ↵ Stemple , D. L. , Solnica-Krezel , L. , Zwartkruis , F. , Neuhauss , S. C. , Schier , A. F. , Malicki , J. , … & Driever , W. ( 1996 ). Mutations affecting development of the notochord in zebrafish . Development , 123 ( 1 ), 117 – 128 . OpenUrl Abstract / FREE Full Text ↵ Stickney , H. L. , Imai , Y. , Draper , B. , Moens , C. , & Talbot , W. S . ( 2007 ). Zebrafish bmp4 functions during late gastrulation to specify ventroposterior cell fates . Developmental biology , 310 ( 1 ), 71 – 84 . OpenUrl CrossRef PubMed Web of Science Streissguth , A.P. , Dehaene , P ., 1993 . Fetal alcohol syndrome in twins of alcoholic mothers: concordance of diagnosis and IQ . Am J Med Genet 47 , 857 – 861 . OpenUrl CrossRef PubMed Web of Science ↵ Swanson , J . ( 2023 ). Inpatient Hospitalization Costs Associated with Birth Defects Among Persons Aged 65 Years—United States, 2019 . MMWR. Morbidity and Mortality Weekly Report , 72 . ↵ Swartz , M. E. , Nguyen , V. , McCarthy , N. Q. , & Eberhart , J. K . ( 2012 ). Hh signaling regulates patterning and morphogenesis of the pharyngeal arch-derived skeleton . Developmental biology , 369 ( 1 ), 65 – 75 . OpenUrl CrossRef PubMed ↵ Swartz , M. E. , Wells , M. B. , Griffin , M. , McCarthy , N. , Lovely , C. B. , McGurk , P. , … & Eberhart , J. K. ( 2014 ). A screen of zebrafish mutants identifies ethanol-sensitive genetic loci . Alcoholism: Clinical and Experimental Research , 38 ( 3 ), 694 – 703 . OpenUrl CrossRef PubMed Swartz , M. E. , Wells , M. B. , Griffin , M. , McCarthy , N. , Lovely , C. B. , McGurk , P. , … & Eberhart , J. K. ( 2014 ). A screen of zebrafish mutants identifies ethanol-sensitive genetic loci . Alcoholism: Clinical and Experimental Research , 38 ( 3 ), 694 – 703 . OpenUrl CrossRef PubMed ↵ Topczewski , J. , Sepich , D. S. , Myers , D. C. , Walker , C. , Amores , A. , Lele , Z. , … & Solnica-Krezel , L. ( 2001 ). The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension . Developmental cell , 1 ( 2 ), 251 – 264 . OpenUrl CrossRef PubMed Web of Science Topczewski , J. , Sepich , D. S. , Myers , D. C. , Walker , C. , Amores , A. , Lele , Z. , … & Solnica-Krezel , L. ( 2001 ). The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension . Developmental cell , 1 ( 2 ), 251 – 264 . OpenUrl CrossRef PubMed Web of Science ↵ Trumpp A , Depew MJ , Rubenstein JLR , Bishop JM , & Martin GR ( 1999 ). Genes & Development , 13 , 3136 – 3148 . [PubMed: 10601039] OpenUrl Abstract / FREE Full Text ↵ Ulrich , F. , Concha , M. L. , Heid , P. J. , Voss , E. , Witzel , S. , Roehl , H. , … & Heisenberg , C. P. ( 2003 ). Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation . ↵ Ulrich , F. , Krieg , M. , Schötz , E. M. , Link , V. , Castanon , I. , Schnabel , V. , … & Heisenberg , C. P. ( 2005 ). Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin . Developmental cell , 9 ( 4 ), 555 – 564 . OpenUrl CrossRef PubMed Web of Science ↵ Vo , H. D. , & Lovely , C. B . ( 2025 ). Ethanol induces craniofacial defects in Bmp mutants independent of nkx2. 3 by elevating cranial neural crest cell apoptosis . Biomedicines , 13 ( 3 ), 755 . OpenUrl CrossRef PubMed ↵ Walker M , Kimmel C . A two-color acid-free cartilage and bone stain for zebrafish larvae . Biotechnic & Histochemistry . 2007 Jan; 82 ( 1 ): 23 – 8 . OpenUrl CrossRef PubMed Web of Science ↵ Warga , R. M. , & Kimmel , C. B . ( 1990 ). Cell movements during epiboly and gastrulation in zebrafish . Development , 108 ( 4 ), 569 – 580 . OpenUrl Abstract / FREE Full Text ↵ Westerfield M. The zebrafish book . A guide for the laboratory use of zebrafish (Danio rerio) . 5th ed . 2007 . ↵ Whaley , C.C. ; Young , M.M. ; Gaynor , B.G . Very High Blood Alcohol Concentration and Fatal Hemorrhage in Acute Subdural Hematoma . World Neurosurg . 2019 , 130 , 454 – 458 . OpenUrl CrossRef PubMed Williams , M. L. K. , & Solnica-Krezel , L . ( 2020 ). Cellular and molecular mechanisms of convergence and extension in zebrafish . Current topics in developmental biology , 136 , 377 – 407 . OpenUrl CrossRef PubMed ↵ Yang , W. , Garrett , L. , Feng , D. , Elliott , G. , Liu , X. , Wang , N. , … & Gao , B. ( 2017 ). Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development . Cell research , 27 ( 12 ), 1466 – 1484 . OpenUrl CrossRef PubMed ↵ Zhang , C. , Frazier , J. M. , Chen , H. , Liu , Y. , Lee , J. A. , & Cole , G. J . ( 2014 ). Molecular and morphological changes in zebrafish following transient ethanol exposure during defined developmental stages . Neurotoxicology and teratology , 44 , 70-80 . 254 – 3265 . OpenUrl ↵ Zhang , C. , Ojiaku , P. , & Cole , G. J . ( 2013 ). Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin, Fgf, and sonic hedgehog function . Birth Defects Research Part A: Clinical and Molecular Teratology , 97 ( 1 ), 8 – 27 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted April 26, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Interactions between the Bone Morphogenetic Protein and the Planar Cell Polarity Pathways lead to distinctive ethanol-induced facial defects Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Interactions between the Bone Morphogenetic Protein and the Planar Cell Polarity Pathways lead to distinctive ethanol-induced facial defects Raèden Gray , Anna Llyod , C. Ben Lovely bioRxiv 2025.04.23.650288; doi: https://doi.org/10.1101/2025.04.23.650288 Share This Article: Copy Citation Tools Interactions between the Bone Morphogenetic Protein and the Planar Cell Polarity Pathways lead to distinctive ethanol-induced facial defects Raèden Gray , Anna Llyod , C. Ben Lovely bioRxiv 2025.04.23.650288; doi: https://doi.org/10.1101/2025.04.23.650288 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13895) Bioinformatics (41951) Biophysics (21456) Cancer Biology (18594) Cell Biology (25520) Clinical Trials (138) Developmental Biology (13381) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24323) Genetics (15612) Genomics (22510) Immunology (17738) Microbiology (40401) Molecular Biology (17184) Neuroscience (88622) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)