Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

E-liquids and vanillin flavoring disrupts retinoic acid signaling and causes craniofacial defects in Xenopus embryos.

Environmental teratogens such as smoking are known risk factors for developmental disorders such as cleft palate. While smoking rates have declined, a new type of smoking, called vaping is on the rise. Vaping is the use of e-cigarettes to vaporize and inhale an e-liquid containing nicotine and food-like flavors. There is the potential that, like smoking, vaping could also pose a danger to the developing human. Rather than waiting for epidemiological and mammalian studies, we have turned to an aquatic developmental model, Xenopus laevis, to more quickly assess whether e-liquids contain teratogens that could lead to craniofacial malformations. Xenopus, like zebrafish, has the benefit of being a well-established developmental model and has also been effective in predicting whether a chemical could be a teratogen. We have determined that embryonic exposure to dessert flavored e-liquids can cause craniofacial abnormalities, including an orofacial cleft in Xenopus. To better understand the underlying mechanisms contributing to these defects, transcriptomic analysis of the facial tissues of embryos exposed to a representative dessert flavored e-liquid vapor extract was performed. Analysis of differentially expressed genes in these embryos revealed several genes associated with retinoic acid metabolism or the signaling pathway. Consistently, retinoic acid receptor inhibition phenocopied the craniofacial defects as those embryos exposed to the vapor extract of the e-liquid. Such malformations also correlated with a group of common differentially expressed genes, two of which are associated with midface birth defects in humans. Further, e-liquid exposure sensitized embryos to forming craniofacial malformations when they already had depressed retinoic acid signaling. Moreover, 13-cis-retinoic acid treatment could significantly reduce the e-liquid induced malformation in the midface. Such results suggest the possibility of an interaction between retinoic acid signaling and e-liquid exposure. One of the most popular and concentrated flavoring chemicals in dessert flavored e-liquids is vanillin. Xenopus embryos exposed to this chemical closely resembled embryos exposed to dessert-like e-liquids and a retinoic acid receptor antagonist. In summary, we determined that e-liquid chemicals, in particular vanillin, can cause craniofacial defects potentially by dysregulating retinoic acid signaling. This work warrants the evaluation of vanillin and other such flavoring additives in e-liquids on mammalian development.

Deep learning is widely applicable to phenotyping embryonic development and disease.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.

Ciliary Hedgehog signaling regulates cell survival to build the facial midline.

Craniofacial defects are among the most common phenotypes caused by ciliopathies, yet the developmental and molecular etiology of these defects is poorly understood. We investigated multiple mouse models of human ciliopathies (including Tctn2, Cc2d2a, and Tmem231 mutants) and discovered that each displays hypotelorism, a narrowing of the midface. As early in development as the end of gastrulation, Tctn2 mutants displayed reduced activation of the Hedgehog (HH) pathway in the prechordal plate, the head organizer. This prechordal plate defect preceded a reduction of HH pathway activation and Shh expression in the adjacent neurectoderm. Concomitant with the reduction of HH pathway activity, Tctn2 mutants exhibited increased cell death in the neurectoderm and facial ectoderm, culminating in a collapse of the facial midline. Enhancing HH signaling by decreasing the gene dosage of a negative regulator of the pathway, Ptch1, decreased cell death and rescued the midface defect in both Tctn2 and Cc2d2a mutants. These results reveal that ciliary HH signaling mediates communication between the prechordal plate and the neurectoderm to provide cellular survival cues essential for development of the facial midline.

GATA3 is essential for separating patterning domains during facial morphogenesis.

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

Prenatal diagnosis of recurrent mosaic ring chromosome 13 of maternal origin.

OBJECTIVE: We present prenatal diagnosis of recurrent mosaic ring chromosome 13 [r(13)] of maternal origin. CASE REPORT: A 27-year-old woman underwent amniocentesis at 17 weeks of gestation because of a past history of fetal abnormality caused by mosaic r(13) in the previous fetus associated with fetal intrauterine growth restriction (IUGR), a karyotype of 46,XY,r(13)[23]/45,XY,-13[10]/46,XY,idic r(13)[2] and a maternal origin of abnormal r(13). The parental karyotypes were normal. During this pregnancy, amniocentesis revealed a karyotype of 46,XX,r(13)[12]/45,XX,-13[8] and a 22.80-Mb deletion of chromosome 13q31.3-q34. The pregnancy was subsequently terminated, and a malformed fetus was delivered with craniofacial dysmorphism. Repeat amniocentesis revealed a karyotype of 46,XX,r(13)(p11.1q31)[18]/45,XX,-13[12]. The placenta had a karyotype of 46,XX,r(13)(p11.1q31)[27]/45,XY,-13[13]. Polymorphic DNA marker analysis using the DNA derived from the parental bloods and umbilical cord confirmed a maternal origin of the abnormal r(13). CONCLUSION: Prenatal diagnosis of mosaic r(13) in consecutive pregnancies should raise a suspicion of parental gonadal mosaicism, and polymorphic DNA marker analysis is useful for determination of the parental origin of recurrent aneuploidy under such a circumstance.

Partial congenital arrhinia: never seen before adult presentation.

BACKGROUND: Arrhinia is defined as the partial or complete absence of the nasal structures. It is a defect of embryonal origin and can be seen in association with other craniofacial anomalies, central nervous system anomalies, absence of paranasal sinuses, and other palatal and ocular abnormalities. Very few patients with arrhinia have been reported so far in the history of modern medicine. CASE REPORT: This study reports an adult patient with congenital partial arrhinia and reviews the literature along with the embryological basis of such a rare disease. CONCLUSION: Arrhinia is a medical condition with scarce documentation in the literature. This article presents the clinical as well as radiological features of this rare entity.

The society for craniofacial genetics and developmental biology 43rd annual meeting.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 43rd annual meeting in a virtual format on October 19-20, 2020. The SCGDB meeting included the presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Marilyn Jones and Kerstin Ludwig and four scientific sessions on the molecular regulation of craniofacial development, craniofacial morphogenesis, translational craniofacial biology, and signaling during craniofacial development. The meeting also included workshops on career development, NIH/NIDCR funding, and the utility of the FaceBase database, as well as two poster sessions. Over 190 attendees from 21 states, representing over 50 different scientific institutions, participated. This diverse group of scientists included cell biologists, developmental biologists, and clinical geneticists. While in-person interactions were missed due to the virtual meeting format imposed by the COVID-19 pandemic, the meeting platform provided ample opportunities for participant interactions and discussions, thus strengthening the community.

Maternal Folic Acid Deficiency Is Associated to Developing Nasal and Palate Malformations in Mice.

Craniofacial development requires extremely fine-tuned developmental coordination of multiple specialized tissues. It has been evidenced that a folate deficiency (vitamin B9), or its synthetic form, folic acid (FA), in maternal diet could trigger multiple craniofacial malformations as oral clefts, tongue, or mandible abnormalities. In this study, a folic acid-deficient (FAD) diet was administered to eight-week-old C57/BL/6J female mouse for 2-16 weeks. The head symmetry, palate and nasal region were studied in 24 control and 260 experimental fetuses. Our results showed a significant reduction in the mean number of fetuses per litter according to maternal weeks on FAD diet (p < 0.01). Fetuses were affected by cleft palate (3.8%) as well as other severe congenital abnormalities, for the first time related to maternal FAD diet, as head asymmetries (4.6%), high arched palate (3.5%), nasal septum malformed (7.3%), nasopharynx duct shape (15%), and cilia and epithelium abnormalities (11.2% and 5.8%). Dysmorphologies of the nasal region were the most frequent, appearing at just four weeks following a maternal FAD diet. This is the first time that nasal region development is experimentally related to this vitamin deficiency. In conclusion, our report offers novel discoveries about the importance of maternal folate intake on midface craniofacial development of the embryos. Moreover, the longer the deficit lasts, the more serious the consequent effects appear to be.

Cilia, ciliopathies and hedgehog-related forebrain developmental disorders.

Development of the forebrain critically depends on the Sonic Hedgehog (Shh) signaling pathway, as illustrated in humans by the frequent perturbation of this pathway in holoprosencephaly, a condition defined as a defect in the formation of midline structures of the forebrain and face. The Shh pathway requires functional primary cilia, microtubule-based organelles present on virtually every cell and acting as cellular antennae to receive and transduce diverse chemical, mechanical or light signals. The dysfunction of cilia in humans leads to inherited diseases called ciliopathies, which often affect many organs and show diverse manifestations including forebrain malformations for the most severe forms. The purpose of this review is to provide the reader with a framework to understand the developmental origin of the forebrain defects observed in severe ciliopathies with respect to perturbations of the Shh pathway. We propose that many of these defects can be interpreted as an imbalance in the ratio of activator to repressor forms of the Gli transcription factors, which are effectors of the Shh pathway. We also discuss the complexity of ciliopathies and their relationships with forebrain disorders such as holoprosencephaly or malformations of cortical development, and emphasize the need for a closer examination of forebrain defects in ciliopathies, not only through the lens of animal models but also taking advantage of the increasing potential of the research on human tissues and organoids.

Multifactorial Genetic and Environmental Hedgehog Pathway Disruption Sensitizes Embryos to Alcohol-Induced Craniofacial Defects.

BACKGROUND: Prenatal alcohol exposure (PAE) is perhaps the most common environmental cause of human birth defects. These exposures cause a range of structural and neurological defects, including facial dysmorphologies, collectively known as fetal alcohol spectrum disorders (FASD). While PAE causes FASD, phenotypic outcomes vary widely. It is thought that multifactorial genetic and environmental interactions modify the effects of PAE. However, little is known of the nature of these modifiers. Disruption of the Hedgehog (Hh) signaling pathway has been suggested as a modifier of ethanol teratogenicity. In addition to regulating the morphogenesis of craniofacial tissues commonly disrupted in FASD, a core member of the Hh pathway, Smoothened, is susceptible to modulation by structurally diverse chemicals. These include environmentally prevalent teratogens like piperonyl butoxide (PBO), a synergist found in thousands of pesticide formulations. METHODS: Here, we characterize multifactorial genetic and environmental interactions using a zebrafish model of craniofacial development. RESULTS: We show that loss of a single allele of shha sensitized embryos to both alcohol- and PBO-induced facial defects. Co-exposure of PBO and alcohol synergized to cause more frequent and severe defects. The effects of this co-exposure were even more profound in the genetically susceptible shha heterozygotes. CONCLUSIONS: Together, these findings shed light on the multifactorial basis of alcohol-induced craniofacial defects. In addition to further implicating genetic disruption of the Hh pathway in alcohol teratogenicity, our findings suggest that co-exposure to environmental chemicals that perturb Hh signaling may be important variables in FASD and related craniofacial disorders.

Chemical-induced craniofacial anomalies caused by disruption of neural crest cell development in a zebrafish model.

BACKGROUND: Craniofacial anomalies are among the most frequent birth defects worldwide, and are thought to be caused by gene-environment interactions. Genetically manipulated zebrafish simulate human diseases and provide great advantages for investigating the etiology and pathology of craniofacial anomalies. Although substantial advances have been made in understanding genetic factors causing craniofacial disorders, limited information about the etiology by which environmental factors, such as teratogens, induce craniofacial anomalies is available in zebrafish. RESULTS: Zebrafish embryos displayed craniofacial malformations after teratogen treatments. Further observations revealed characteristic disruption of chondrocyte number, shape and stacking. These findings suggested aberrant development of cranial neural crest (CNC) cells, which was confirmed by gene expression analysis of the CNC. Notably, these observations suggested conserved etiological pathways between zebrafish and mammals including human. Furthermore, several of these chemicals caused malformations of the eyes, otic vesicle, and/or heart, representing a phenocopy of neurocristopathy, and these chemicals altered the expression levels of the responsible genes. CONCLUSIONS: Our results demonstrate that chemical-induced craniofacial malformation is caused by aberrant development of neural crest. This study indicates that zebrafish provide a platform for investigating contributions of environmental factors as causative agents of craniofacial anomalies and neurocristopathy.

Dose addition in chemical mixtures inducing craniofacial malformations in zebrafish (Danio rerio) embryos.

A challenge in cumulative risk assessment is to model hazard of mixtures. EFSA proposed to only combine chemicals linked to a defined endpoint, in so-called cumulative assessment groups, and use the dose-addition model as a default to predict combined effects. We investigated the effect of binary mixtures of compounds known to cause craniofacial malformations, by assessing the effect in the head skeleton (M-PQ angle) in 120hpf zebrafish embryos. We combined chemicals with similar mode of action (MOA), i.e. the triazoles cyproconazole, triadimefon and flusilazole; next, reference compounds cyproconazole or triadimefon were combined with dissimilar acting compounds, TCDD, thiram, VPA, prochloraz, fenpropimorph, PFOS, or endosulfan. These mixtures were designed as (near) equipotent combinations of the contributing compounds, in a range of cumulative concentrations. Dose-addition was assessed by evaluation of the overlap of responses of each of the 14 tested binary mixtures with those of the single compounds. All 10 test compounds induced an increase of the M-PQ angle, with varying potency and specificity. Mixture responses as predicted by dose-addition did not deviate from the observed responses, supporting dose-addition as a valid assumption for mixture risk assessment. Importantly, dose-addition was found irrespective of MOA of contributing chemicals.

Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2-/- embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5' upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.

Maternal choline supplementation mitigates alcohol-induced fetal cranio-facial abnormalities detected using an ultrasonographic examination in a sheep model.

Early detection of prenatal alcohol exposure is critical for designing and testing effectiveness of interventional therapeutics. Choline supplementation during and after prenatal alcohol exposure has shown promising benefits in improving outcomes in rodent models and clinical studies. A sheep model of first trimester-equivalent binge alcohol exposure was used in this study to model the dose of maternal choline supplementation used in an ongoing prospective clinical trial involving pregnancies at risk for FASD. Pregnant sheep were randomly assigned to six groups: Saline + Placebo control, Saline + Choline, binge Alcohol + Placebo (light binging), binge Alcohol + Choline, Heavy binge Alcohol + Placebo (heavy binging), and Heavy binge Alcohol + Choline. Ewes received intravenous alcohol or saline on three consecutive days per week from gestation day (GD) 4-41 to mimic a first trimester-equivalent weekend binge-drinking paradigm. Choline (10 mg/kg in the daily food ration) was administered from GD 4 until term. On GD 76, 11 fetal ultrasonographic measurements were collected transabdominally. Heavy binge alcohol exposure reduced fetal Frontothalamic Distance (FTD), Mean Orbital Diameter (MOD), and Mean Lens Diameter (MLD), and increased Interorbital Distance (IOD) and Thalamic Width (TW). Maternal choline supplementation mitigated most of these alcohol-induced effects. Maternal choline supplementation also improved overall fetal femur and humerus bone lengths, compared to their respective placebo groups. Taken together, these results indicate a potential dose-dependent effect that could impact the sensitivity of these ultrasonographic measures in predicting prenatal alcohol exposure. This is the first study in the sheep model to identify biomarkers of prenatal alcohol exposure in utero with ultrasound and co-administration of maternal choline supplementation.

The location of the fetal ears: A hint for prenatal diagnosis of agnathia-otocephaly complex.

Otocephaly is an extremely rare lethal congenital anomaly characterized by the absence or underdevelopment of the mandible. The clinical presentation is variable. Some cases may present with severe micrognathia as the only anomaly seen prenatally. The key to early diagnosis is careful assessment of the location of the fetal ears on 2D ultrasound examination.

Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors.

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.

Relative potency ranking of azoles altering craniofacial morphogenesis in rats: An in vitro data modelling approach.

Facial malformations represent one of the most frequent abnormality in humans. The adverse outcome pathway involved in facial defects seems to be related to retinoic acid (RA) pathway imbalance. Environmental agents inducing craniofacial malformations in experimental models include pesticides (especially azole fungicides). By using the in vitro alternative method postimplantation rat whole embryo culture (WEC), we evaluated the intrinsic embryotoxic activity of some azole antifungals (cyproconazole, CYPRO; triadimefon, FON; flusilazole, FLUSI; and prochloraz, PCZ), in comparison to RA. All the tested molecules induced in a dose-related manner specific defects of the craniofacial structures (fused branchial arches), similar to those induced by RA. Collected data were modelled using PROAST 65.5 software to characterise the relative potency factors (RPFs) versus RA. In comparison to RA, all the evaluated azoles were less potent, showing among them a similar potency. Our data suggest a possible azole-related RA signalling perturbation to be further investigated. Moreover, the present results indicate the approach used in this work to be an interesting tool applicable to the hazard evaluation of novel compounds or the assessment of combined exposure to azoles or other dismorphogens.

Ectopic expression of Hoxb1 induces cardiac and craniofacial malformations.

Members of the large family of Hox transcription factors are encoded by genes whose tightly regulated expression in development and in space within different embryonic tissues confer positional identity from the neck to the tips of the limbs. Many structures of the face, head, and heart develop from cell populations expressing few or no Hox genes. Hoxb1 is the member of its chromosomal cluster expressed in the most rostral domain during vertebrate development, but never by the multipotent neural crest cell population anterior to the cerebellum. We have developed a novel floxed transgenic mouse line, CAG-Hoxb1,-EGFP (CAG-Hoxb1), which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP overexpression. When induced within the neural crest lineage, pups die at birth. A variable phenotype develops from E11.5 on, associating frontonasal hypoplasia/aplasia, micrognathia/agnathia, major ocular and forebrain anomalies, and cardiovascular malformations. Neural crest derivatives in the body appear unaffected. Transcription of effectors of developmental signaling pathways (Bmp, Shh, Vegfa) and transcription factors (Pax3, Sox9) is altered in mutants. These outcomes emphasize that repression of Hoxb1, along with other paralog group 1 and 2 Hox genes, is strictly necessary in anterior cephalic NC for craniofacial, visual, auditory, and cardiovascular development.