Cranial Neural Crest Development Research Articles

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway.

Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underlies facial shape variation, yet how those networks in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both TFAP2 family members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning and differentiation. Notably, Alx1, Alx3 and Alx4 (ALX) transcript levels are reduced, whereas ChIP-seq analyses suggest TFAP2 family members directly and positively regulate ALX gene expression. Tfap2a, Tfap2b and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish implies conservation of this regulatory axis across vertebrates. Consistent with this notion, tfap2a zebrafish mutants present with abnormal alx3 expression patterns, Tfap2a binds ALX loci and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development in part by activating expression of ALX transcription factor genes.

TFAP2 Paralogs Regulate Alx Genes During Midface Cranial Neural Crest Development

Craniofacial anomalies are the 2nd leading structural malformation present at birth. Those in the midface, such as orofacial clefts and hypertelorism, can arise from mutations in the Transcription Factor Activating Protein 2 genes TFAP2A and TFAP2B (abbreviated as TFAP2). These mutations do not reduce their gene expression, but instead generate mutant proteins that disrupt the activity of other wild‐type and functionally redundant paralogs. Previous studies showed that both paralogs are co‐expressed in the embryonic midface, hinting at their cooperative role during face development. Yet, how TFAP2 regulate midface development at the cellular and molecular level is poorly understood. To this end, we leveraged CRE‐based approaches in mice to delete both Tfap2 members in the neural crest (NC), a cell lineage that gives rise to craniofacial bone and cartilage. We show that NC‐specific knockout of Tfap2 (Tfap2NCKO) results in a fully penetrant midface cleft compared to controls and single paralog mutants. Lineage tracing analyses in Tfap2NCKO embryos suggest that cranial NC migration into the embryonic face is not severely impaired. Corroborating this result, we find that Tfap2 deletion in either pre‐migratory or late‐migratory NC cells still results in a midface cleft. This suggests this phenotype stems from disrupted TFAP2 activity in post‐migratory NC cells. Moreover, when examining Tfap2NCKO craniofacial skeletons, we observe severe bone malformations and ectopic structures in the frontonasal regions when compared to controls. Single‐cell RNA sequencing analysis reveals that loss of Tfap2 results in reduced cranial NC cell numbers and dysregulated Alx1/3/4 (Alx) gene expression. This is supported by the literature, as the Tfap2NCKO midface clefts phenocopy those with loss of various Alxgene combinations. Our ongoing efforts aim to test how TFAP2 regulates Alx genes, if loss of Tfap2 compromises midface NC survival or proliferation, and whether Tfap2 and Alx genetically interact. In sum, we propose the model that TFAP2 regulates Alx gene expression in NC cells during midface development.

KDM6B interacts with TFDP1 to activate P53 signaling in regulating mouse palatogenesis.

Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, the question of how epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains understudied. Using palatogenesis as a model, we reveal the functional significance of Kdm6b, an H3K27me3 demethylase, in regulating mouse embryonic development. Our study shows that Kdm6b plays an essential role in cranial neural crest development, and loss of Kdm6b disturbs P53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects in mice. Furthermore, activity of H3K27me3 on the promoter of Trp53 is antagonistically controlled by Kdm6b, and Ezh2 in cranial neural crest cells. More importantly, without Kdm6b, the transcription factor TFDP1, which normally binds to the promoter of Trp53, cannot activate Trp53 expression in palatal mesenchymal cells. Furthermore, the function of Kdm6b in activating Trp53 in these cells cannot be compensated for by the closely related histone demethylase Kdm6a. Collectively, our results highlight the important role of the epigenetic regulator KDM6B and how it specifically interacts with TFDP1 to achieve its functional specificity in regulating Trp53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.

The double-edged sword role of TGF-β signaling pathway between intrauterine inflammation and cranial neural crest development.

Intrauterine infection would harm a developing embryo/fetus, thereby increasing the risk of developmental malformation. But, whether or not the infection-induced inflammation affects neural crest development still remains obscure. In this study, we employed meta-analysis to demonstrate the potential correlation between infection-induced inflammation and craniofacial anomalies, which was usually derived from the problems in neural crest cell development. The correlation was further verified by inflammatory cytokine release and the activation of nuclear factor kappa-light-chain enhancer of activated B cells signaling in lipopolysaccharide-treated HH10 chicken embryos. In such an inflammatory condition, AP-2α- and Pax7-labeled pre-migratory and migratory neural crest cells in HH10 chicken embryos were significantly less than the ones in control. The bioinformatics analysis of RNA-seq data demonstrated that the principal differential gene expression occurred in transforming growth factor-beta (TGF-β) signaling pathway, which was confirmed by the subsequent experimental results of quantitative PCR and immunofluorescent staining. Under this inflammatory circumstance, whole-mount in situ hybridization, immunofluorescence, and quantitative PCR showed the gene expression changes of key EMT-related transcription factors including upregulated Msx1, downregulated Slug, and FoxD3, as well as adhesion molecules and extracellular matrix protein including upregulated Cadherrin6B, E-cadherin, N-cadherin, and Laminin at the dorsal portion of neural tube of HH10 chicken embryos. Meanwhile, the bioinformatics analysis of RNA-seq data also manifested the differential gene expressions relevant to cell proliferation, which was confirmed by proliferating cell nuclear antigen Western blot data and co-immunofluorescence staining of human natural killer-1 and phosphorylated histone H3. In brief, this study revealed for the first time that the double-edged sword role of TGF-β signaling pathway between intrauterine inflammation (protective role) and cranial neural crest development (harmful role).

Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development.

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3′UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.

Single-Cell Multiomic Approaches Reveal Diverse Labeling of the Nervous System by Common Cre-Drivers.

Neural crest development involves a series of dynamic, carefully coordinated events that result in human disease when not properly orchestrated. Cranial neural crest cells acquire unique multipotent developmental potential upon specification to generate a broad variety of cell types. Studies of early mammalian neural crest and nervous system development often use the Cre-loxP system to lineage trace and mark cells for further investigation. Here, we carefully profile the activity of two common neural crest Cre-drivers at the end of neurulation in mice. RNA sequencing of labeled cells at E9.5 reveals that Wnt1-Cre2 marks cells with neuronal characteristics consistent with neuroepithelial expression, whereas Sox10-Cre predominantly labels the migratory neural crest. We used single-cell mRNA and single-cell ATAC sequencing to profile the expression of Wnt1 and Sox10 and identify transcription factors that may regulate the expression of Wnt1-Cre2 in the neuroepithelium and Sox10-Cre in the migratory neural crest. Our data identify cellular heterogeneity during cranial neural crest development and identify specific populations labeled by two Cre-drivers in the developing nervous system.

Transcriptomic Identification of Draxin-Responsive Targets During Cranial Neural Crest EMT.

Canonical Wnt signaling plays an essential role in proper craniofacial morphogenesis, at least partially due to regulation of various aspects of cranial neural crest development. In an effort to gain insight into the etiology of craniofacial abnormalities resulting from Wnt signaling and/or cranial neural crest dysfunction, we sought to identify Wnt-responsive targets during chick cranial neural crest development. To this end, we leveraged overexpression of a canonical Wnt antagonist, Draxin, in conjunction with RNA-sequencing of cranial neural crest cells that have just activated their epithelial–mesenchymal transition (EMT) program. Through differential expression analysis, gene list functional annotation, hybridization chain reaction (HCR), and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we validated a novel downstream target of canonical Wnt signaling in cranial neural crest – RHOB – and identified possible signaling pathway crosstalk underlying cranial neural crest migration. The results reveal novel putative targets of canonical Wnt signaling during cranial neural crest EMT and highlight important intersections across signaling pathways involved in craniofacial development.

Using an aquatic model, Xenopus laevis, to uncover the role of chromodomain 1 in craniofacial disorders.

The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders.

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

BackgroundCraniofacial 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.ResultsZebrafish 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.ConclusionsOur 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.

Zinc oxide nanoparticles exposure-induced oxidative stress restricts cranial neural crest development during chicken embryogenesis.

Zinc oxide Nanoparticles (ZnO NPs) are widely used as emerging materials in agricultural and food-related fields, which exists potential safety hazards to public health and environment while bringing an added level of convenience to our original life. It has been proved that ZnO NPs could be taken up by pregnant women and passed through human placental barrier. However, the toxic potential for embryo development remains largely unanswered. In this study, we discovered that ZnO NPs caused the cytotoxicity in vitro. Inhibition of free Zn2+ ions in solution by EDTA or inhibition of Zn2+ ions absorption by CaCl2 could partially eliminate ZnO NPs-mediated cell toxicity, though not redeem completely. This indicated that both nanoparticles and the release of Zn2+ ions were involved in ZnO NPs-mediated cytotoxicity. In addition, we also found that both nanoparticles and Zn2+ ion release triggered reactive oxygen species (ROS) production, which further induced cell toxicity, inflammation and apoptosis, which are mediated by NF-κB signaling cascades and the mitochondria dysfunction, respectively. Eventually, these events lead to the suppressed production and migration of cranial neural crest cells (CNCCs), which subsequently prompts the craniofacial defects in chicken embryos. The application of the antioxidant N-Acetyl-L-cysteine (NAC) rescued the ZnO NPs-induced cell toxicity and malformation of the CNCCs, which further verified our hypothesis. Our results revealed the relevant mechanism of ZnO NPs exposure-inhibited the development of CNCCs, which absolutely contribute to assess the risk of nanoparticles application.

874: Overexpression of YAP rescue maternal diabetes induced neural tube defects

Maternal Diabetes is associated with higher risk of neural tube defects. The Hippo/YAP pathway play central role in cranial neural crest development. In present study, we aim to determine whether YAP is inactivated (phosphorylated at Ser127) in maternal diabetes induced neural tube defects and whether Hippo/YAP pathway plays a causal role in maternal diabetes induced neural tube defects. A transgenic mouse carry YAP wild-type under nestin promoter as well as Yap constitutive active form (Ser127Ala) was generated to study how these transgenic mice response to maternal diabetes induced neural tube defects. Neural epithelium conditional knockout (cKO) of the Hippo pathway effectors, YAP/TAZ, were also generated by crossing the YAP/TAZ floxed mice to mice with Cre driven under the nestin promoter. Neural tube defects were evaluated at the embryo day 9.5 or 10.5. We found that YAP is inactivated in maternal diabetes induced neural tube defects compare to normal littermates, while the constitutive active YAP transgenic mice also induce neural tube defects (51.3% vs Yap wild type) under normal condition. In the meantime, neural epithelium specific deletion of either YAP or YAP/TAZ results in 100% neural tube defects under normal condition. However, neural specific overexpression of YAP wild type significantly blocks maternal diabetes induced neural tube defects (from 35.0% for non transgenic mice to 12.1% for transgenic mice). Our further experiments suggest that this achieved probably through the restoring of the planar cell polarity pathway. Our findings indicate that the YAP is crucial for neural tube development and its activity is under fine-tune by different signaling pathways.

Zika virus induces abnormal cranial osteogenesis by negatively affecting cranial neural crest development

Zika virus (ZIKV) infection during gestation is deemed to be coupled to birth defects through direct impairment of the nervous system during neurogenesis. However, in this study, our data showed that ZIKV infection dramatically suppressed cranial osteogenesis, shown by Safranin O/Fast Green and alizarin red staining, in chick embryos, which provides another possibility that craniofacial bone malformation caused by ZIKV may be a major cause of ZIKV-mediated birth defects. By immunofluorescent staining and electron microcopy, we confirmed ZIKV infection in chick embryo neural tubes and sites of neural crest. Next, in vivo (chick embryos) and in vitro [primary culture of neural crest cells (NCC)] ZIKV and HNK-1 double immunofluorescent staining demonstrated that ZIKV infection inhibited the production of migratory NCC. The reduction of both AP-2α- and Pax7-positive NCC in HH10 chick embryos infected by ZIKV confirmed that abnormal development of cranial NCC also occurred in the migratory process. Whole mount in situ hybridization demonstrated that cadherin 6B expression was elevated and Slug, FoxD3, and BMP4/Msx1 expressions decreased in ZIKV-infected HH10 chick embryos, implying that epithelial–mesenchymal transition (EMT) of neural crest production was blocked by ZIKV infection. Moreover, in vivo and in vitro pHIS3 and Pax7 double immunofluorescent staining showed that NCC proliferation was repressed by ZIKV infection. C-caspase-3 and AP-2α double immunofluorescent staining in HH10 chick embryos and western blotting showed that NCC apoptosis increased following ZIKV infection. Finally, electron microscopy showed multiple autophagosomes in ZIKV-infected embryos, and western blot and LC3B immunofluorescent staining demonstrated that autophagy-related genes were activated by ZIKV infection. Taken together, our data first showed that ZIKV infection during embryogenesis could interfere with cranial neural crest development, which in turn causes aberrant cranial osteogenesis. Our results provided new insights into brain malformations induced by ZIKV infection.

ZnO NPs exposure impairs cranial neural crest development by inducing excessive oxidant stress level during chick embryogenesis

Introduction: Zinc oxide Nanoparticles (ZnO NPs) could lead to defects in chick craniofacial skeleton during embryogenesis, but the further mechanism is still not clear. In this study, we found ZnO NPs exposure cause craniofacial development defect, including shorten coracoids, curved coracoids and impaired parietal bone, which is derived from the cranial neural crest cells. we observed that ZnO NPs could inhibit the production and migration of cranial neural crest cells, which mainly caused by altering expression of epithelial-mesenchymal transitions-related adhesion molecules. Moreover, we observed that ZnO NPs significantly induced the extent apoptosis in cranial neural crest cells but not affect the cell proliferation. Then, the level of oxidant stress of cranial neural crest cells is elevated by ZnO NPs treatment.

A new transgenic Xenopus reporter line reveals dynamic expression of snail2 during cranial neural crest development

During vertebrate embryogenesis, the cranial neural crest (CNC) is induced at the neural plate border (NPB) and subsequently migrate and differentiate into various cells and tissues. In Xenopus, the transcription factor Snail2 is induced at the NPB by canonical Wnt signaling and is required for CNC induction and migration, but the roles of Snail2 at later stages remain unclear. To monitor CNC development in live embryos, we generated a transgenic X. tropicalis line expressing Green Fluorescent Protein(GFP) driven by the snail2 promoter. Using whole‐genome sequencing, we mapped the transgene insertion to a noncoding region. Live imaging of the transgenic embryos reveals that snail2 is expressed in pre‐migratory and migrating CNC but is lost in CNC cells that have arrived at their destinations. However, snail2 is re‐expressed during CNC differentiation in all CNC derivatives that were examined. Defects in CNC differentiation, in addition to induction and migration, can be readily detected using this transgenic snail2 reporter line. Finally, comparison with a transgenic Wnt reporter line shows that Wnt signaling is consistently activated prior to snail2 expression, suggesting that the Wnt‐Snail2 axis may play important roles in multiple stages of CNC development. We are also developing a new transgenic line with a photoconvertible protein instead of GFP that can be used to monitor cell fate of various CNC branches for lineage tracing of CNC derivatives.Support or Funding InformationFunded by: NIH DE022813 and GM114105This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alcohol exposure induces chick craniofacial bone defects by negatively affecting cranial neural crest development

Excess alcohol consumption during pregnancy could lead to fetal alcohol syndrome (FAS). However, the molecular mechanism leading to craniofacial abnormality, a feature of FAS, is still poorly understood. The cranial neural crest cells (NCCs) contribute to the formation of the craniofacial bones. Therefore, NCCs exposed to ethanol was investigated − using chick embryos and in vitro explant culture as experimental models. We demonstrated that exposure to 2% ethanol induced craniofacial defects, which includes parietal defect, in the developing chick fetus. Immunofluorescent staining revealed that ethanol treatment downregulated Ap-2ɑ, Pax7 and HNK-1 expressions by cranial NCCs. Using double-immunofluorescent stainings for Ap-2ɑ/pHIS3 and Ap-2ɑ/c-Caspase3, we showed that ethanol treatment inhibited cranial NCC proliferation and increased NCC apoptosis, respectively. Moreover, ethanol treatment of the dorsal neuroepithelium increased Laminin, N-Cadherin and Cadherin 6B expressions while Cadherin 7 expression was repressed. In situ hybridization also revealed that ethanol treatment up-regulated Cadherin 6B expression but down-regulated slug, Msx1, FoxD3 and BMP4 expressions. In summary, our experimental results demonstrated that ethanol treatment interferes with the production of cranial NCCs by affecting the proliferation and apoptosis of these cells. In addition, ethanol affected the delamination, epithelial-mesenchymal transition (EMT) and cell migration of cranial NCCs, which may have contributed to the etiology of the craniofacial defects.

From the Cover: Usage of Dexamethasone Increases the Risk of Cranial Neural Crest Dysplasia in the Chick Embryo.

Dexamethasone (Dex) is commonly used in the treatment of a variety of benign and malignant conditions. Unfortunately, although it has a variety of teratogenic effects, it remains used in clinical practice for pregnant women mainly due to limited alternatives. However, there is limited knowledge of the mechanisms that lead to the observed teratogenic effects. In this study, the effects of Dex during embryogenesis on neural crest development were evaluated in the early chick embryos. First, we demonstrated that 100 µl 10-6 M Dex treatment leads to craniofacial developmental defects, and also retards embryo growth and plausibly can cause embryo demise. Second, we demonstrated that Dex represses the production of HNK-1, PAX7, and AP-2α labeled cranial neural crest cells, the progenitor cells of the craniofacial skeleton. Third, double immunofluorescent staining of pHIS3/PAX7 and AP-2α/c-Caspase3 revealed that Dex promotes cell apoptosis but does not change cell proliferation rates. Last, fibroblast growth factor signaling molecules were inhibited by Dex treatment. Dex also inhibited neural crest cells production by repressing Msx1 expression in the developing neural tube and by altering expression of epithelial-mesenchymal transition-related adhesion molecules and cell migration genes. Overall, we obtained experimental evidence that Dex treatment during embryogenesis disrupts cranial neural crest development which in turn causes defective cranial bone development.

Face off against ROS: Tcof1/Treacle safeguards neuroepithelial cells and progenitor neural crest cells from oxidative stress during craniofacial development.

One-third of all congenital birth defects affect the head and face, and most craniofacial anomalies are considered to arise through defects in the development of cranial neural crest cells. Cranial neural crest cells give rise to the majority of craniofacial bones, cartilages and connective tissues. Therefore, understanding the events that control normal cranial neural crest and subsequent craniofacial development is important for elucidating the pathogenetic mechanisms of craniofacial anomalies and for the exploring potential therapeutic avenues for their prevention. Treacher Collins syndrome (TCS) is a congenital disorder characterized by severe craniofacial anomalies. An animal model of TCS, generated through mutation of Tcof1, the mouse (Mus musculus) homologue of the gene primarily mutated in association with TCS in humans, has recently revealed significant insights into the pathogenesis of TCS. Apoptotic elimination of neuroepithelial cells including neural crest cells is the primary cause of craniofacial defects in Tcof1 mutant embryos. However, our understanding of the mechanisms that induce tissue-specific apoptosis remains incomplete. In this review, we describe recent advances in our understanding of the pathogenesis TCS. Furthermore, we discuss the role of Tcof1 in normal embryonic development, the correlation between genetic and environmental factors on the severity of craniofacial abnormalities, and the prospect for prenatal prevention of craniofacial anomalies.

In vitro models of cranial neural crest development toward toxicity tests: frog, mouse, and human.

During craniofacial development, cranial neural crest (NC)-derived mesenchymal cells migrate to pharyngeal arches and contribute extensively to neurons, Schwann cells, smooth muscle cells, osteoblasts, chondrocytes, and odontoblasts, forming maxillofacial structures. In vitro models using model organism cells, such as African clawed frog (Xenopus Laevis) and mouse (Mus Musculus), were developed to understand cellular and molecular mechanisms of cranial NC development. Recent studies using human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have enabled the generation of human NC cells (NCCs) invitro to provide insight into human NC development. Understanding molecular mechanisms underlying craniofacial development will contribute to develop novel embryotoxicity tests and to decrease the incidence of drug-induced congenital anomalies in the craniofacial region, such as cleft lip or cleft palate. Here, we review culture methods to derive NCCs invitro from Xenopus presumptive ectoderm (animal caps), mouse embryonic stem cells (mESCs), and human pluripotent stem cells (hPSCs) and discuss how these invitro models can be used to help clarify the mechanisms underlying craniofacial development and for developing embryotoxicity tests predicting drug-induced congenital anomalies in the craniofacial region.

Retinoic Acid and Pitx2 Regulate Early Neural Crest Survival and Migration in Craniofacial and Ocular Development.

Congenital eye and craniofacial anomalies are associated with the dysregulation of retinoic acid (RA) levels during embryogenesis. In the present study, we observed that RA and pitx2a cooperatively regulate early cranial neural crest migration from the rhombencephalon to the pharyngeal arches and from the mesencephalon and prosencephalon to the periocular mesenchyme and frontonasal processes. The cranial neural crest tracked toward areas of high RA activity (i.e., developing eye) and circumvented areas of low RA activity (i.e., mesencephalon). Although previous studies have shown that RA increased pitx2a expression at later stages of cranial neural crest development, in these studies we found that RA inhibited pitx2a expression in the early migrating ventral cranial neural crest. Increased RA or decreased Pitx2a expression decreased cell survival and inhibited ventral neural crest migration. Decreased RA or increased pitx2a expression markedly disrupted both dorsal and ventral neural crest migration. The tight control of RA and subsequent regulation of pitx2 were required for precise cranial neural crest survival and migration. These alterations in the neural crest in the periocular mesenchyme and frontonasal processes may reflect the craniofacial dysmorphism and microphthalmia observed in cases of increased (i.e., as resulting from isoretinoin exposure) or decreased (i.e., as may occur in fetal alcohol syndrome) RA signaling during pregnancy

Meis2 is essential for cranial and cardiac neural crest development.

BackgroundTALE-class homeodomain transcription factors Meis and Pbx play important roles in formation of the embryonic brain, eye, heart, cartilage or hematopoiesis. Loss-of-function studies of Pbx1, 2 and 3 and Meis1 documented specific functions in embryogenesis, however, functional studies of Meis2 in mouse are still missing. We have generated a conditional allele of Meis2 in mice and shown that systemic inactivation of the Meis2 gene results in lethality by the embryonic day 14 that is accompanied with hemorrhaging.ResultsWe show that neural crest cells express Meis2 and Meis2-defficient embryos display defects in tissues that are derived from the neural crest, such as an abnormal heart outflow tract with the persistent truncus arteriosus and abnormal cranial nerves. The importance of Meis2 for neural crest cells is further confirmed by means of conditional inactivation of Meis2 using crest-specific AP2α-IRES-Cre mouse. Conditional mutants display perturbed development of the craniofacial skeleton with severe anomalies in cranial bones and cartilages, heart and cranial nerve abnormalities.ConclusionsMeis2-null mice are embryonic lethal. Our results reveal a critical role of Meis2 during cranial and cardiac neural crest cells development in mouse.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0093-6) contains supplementary material, which is available to authorized users.