Embryonic Craniofacial Development Research Articles

Genetic requirement of dact1/2 to regulate noncanonical Wnt signaling and calpain 8 during embryonic convergent extension and craniofacial morphogenesis.

Wnt signaling plays crucial roles in embryonic patterning including the regulation of convergent extension during gastrulation, the establishment of the dorsal axis, and later, craniofacial morphogenesis. Further, Wnt signaling is a crucial regulator of craniofacial morphogenesis. The adapter proteins Dact1 and Dact2 modulate the Wnt signaling pathway through binding to Disheveled. However, the distinct relative functions of Dact1 and Dact2 during embryogenesis remain unclear. We found that dact1 and dact2 genes have dynamic spatiotemporal expression domains that are reciprocal to one another suggesting distinct functions during zebrafish embryogenesis. Both dact1 and dact2 contribute to axis extension, with compound mutants exhibiting a similar convergent extension defect and craniofacial phenotype to the wnt11f2 mutant. Utilizing single-cell RNAseq and an established noncanonical Wnt pathway mutant with a shortened axis ( gpc4 ), we identified dact1/2 specific roles during early development. Comparative whole transcriptome analysis between wildtype and gpc4 and wildtype and dact1/2 compound mutants revealed a novel role for dact1/2 in regulating the mRNA expression of the classical calpain capn8 . Over-expression of capn8 phenocopies dact1/2 craniofacial dysmorphology. These results identify a previously unappreciated role of capn8 and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of dact1 and dact2 in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling.

DeepFace: Deep-learning-based framework to contextualize orofacial-cleft-related variants during human embryonic craniofacial development

Orofacial clefts (OFC) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the ‘casual’ variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson Correlation Coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50 to 0.83. Specifically, our model revealed that SNPs significantly associated with OFC tended to exhibit higher SAD scores across various variant categories compared to less-related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFC. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFC, and the model that can be extended to study other complex diseases or traits.

Skeletal Class III Malocclusion Is Associated with ADAMTS2 Variants and Reduced Expression in a Familial Case.

Skeletal Class III malocclusion with maxillary deficiency is a severe maxillofacial disease with unclear pathogenic mechanisms. We recruited a Han Chinese family who was clinically diagnosed with skeletal Class III malocclusion and maxillary deficiency. Using whole exome sequencing, a missense variant in ADAMTS2 (NM_014244: c.3506G>T: p.G1169V) was identified and predicted as deleterious by in silico tools. We also found ADAMTS2 variants associated with deficient maxillary development in a cohort. ADAMTS2 expression in HEK293 cells showed significant decrease due to the variant, which was also consistent in dental pulp stem cells from the proband and a healthy control. In the adamts2-knockdown zebrafish model, the length and width of the ethmoid plate, as well as the length of the palatoquadrate became significantly shorter than the control group (p < 0.001), while there was no significant difference in the length and width of the mandible. The expression of Sox3, which was required in early embryonic craniofacial development, was significantly downregulated in the adamts2-knockdown zebrafish embryos. Bioinformatic and cellular studies showed that the decreased expression of ADAMTS2 may inhibit downstream ErbB signaling pathway transduction and restrain subsequent osteogenesis in human adult mesenchymal stromal cells. Collectively, these data showed that ADAMTS2 (c.3506G>T: p.G1169V) may confer susceptibility to risk of skeletal Class III malocclusion with maxillary deficiency.

Nucleolin loss of function leads to aberrant Fibroblast Growth Factor signaling and craniofacial anomalies.

ABSTRACTRibosomal RNA (rRNA) transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies; however, the cellular and molecular reasons for these defects are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. ncl mutant (ncl−/−) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observed that ncl−/− mutants exhibited decreased rRNA synthesis and p53-dependent apoptosis, consistent with a role in ribosome biogenesis. However, we found that Nucleolin also performs functions not associated with ribosome biogenesis. We discovered that the half-life of fgf8a mRNA was reduced in ncl−/− mutants, which perturbed Fgf signaling, resulting in misregulated Sox9a-mediated chondrogenesis and Runx2-mediated osteogenesis. Consistent with this model, exogenous FGF8 treatment significantly rescued the cranioskeletal phenotype in ncl−/− zebrafish, suggesting that Nucleolin regulates osteochondroprogenitor differentiation. Our work has therefore uncovered tissue-specific functions for Nucleolin in rRNA transcription and post-transcriptional regulation of growth factor signaling during embryonic craniofacial development.

Retinoic Acid Deficiency Underlies the Etiology of Midfacial Defects

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that Rdh10 loss of function leads to a substantial reduction in retinoic acid (RA) signaling in the developing frontonasal process during early embryogenesis, which results in a variety of craniofacial anomalies, including midfacial cleft and ectopic chondrogenic nodules. Elevated apoptosis and perturbed cell proliferation in postmigratory cranial neural crest cells and a substantial reduction in Alx1 and Alx3 transcription in the developing frontonasal process were associated with midfacial cleft in Rdh10-deficient mice. More important, expanded Shh signaling in the ventral forebrain, as well as partial abrogation of midfacial defects in Rdh10 mutants via inhibition of Hh signaling, indicates that misregulation of Shh signaling underlies the pathogenesis of reduced RA signaling-associated midfacial defects. Taken together, these data illustrate the precise spatiotemporal function of Rdh10 and RA signaling during early embryogenesis and their importance in orchestrating molecular and cellular events essential for normal midfacial development.

Synergistic role of retinoic acid signaling and Gata3 during primitive choanae formation.

Developmental defects of primitive choanae, an anatomical path to connect the embryonic nasal and oral cavity, result in disorders called choanal atresia (CA), which are associated with many congenital diseases and require immediate clinical intervention after birth. Previous studies revealed that reduced retinoid signaling underlies the etiology of CA. In the present study, by using multiple mouse models which conditionally deleted Rdh10 and Gata3 during embryogenesis, we showed that Gata3 expression is regulated by retinoid signaling during embryonic craniofacial development and plays crucial roles for development of the primitive choanae. Interestingly, Gata3 loss of function is known to cause hypoparathyroidism, sensorineural deafness and renal disease (HDR) syndrome, which exhibits CA as one of the phenotypes in humans. Our model partially phenocopies HDR syndrome with CA, and is thus a useful tool for investigating the molecular and cellular mechanisms of HDR syndrome. We further uncovered critical synergy of Gata3 and retinoid signaling during embryonic development, which will shed light on novel molecular and cellular etiology of congenital defects in primitive choanae formation.

The Chromatin Regulator Ankrd11 Controls Palate and Cranial Bone Development.

Epigenetic and chromatin regulation of craniofacial development remains poorly understood. Ankyrin Repeat Domain 11 (ANKRD11) is a chromatin regulator that has previously been shown to control neural stem cell fates via modulation of histone acetylation. ANKRD11 gene variants, or microdeletions of the 16q24.3 chromosomal region encompassing the ANKRD11 gene, cause KBG syndrome, a rare autosomal dominant congenital disorder with variable neurodevelopmental and craniofacial involvement. Craniofacial abnormalities include a distinct facial gestalt, delayed bone age, tooth abnormalities, delayed fontanelle closure, and frequently cleft or submucosal palate. Despite this, the dramatic phenotype and precise role of ANKRD11 in embryonic craniofacial development remain unexplored. Quantitative analysis of 3D images of KBG syndromic subjects shows an overall reduction in the size of the middle and lower face. Here, we report that mice with heterozygous deletion of Ankrd11 in neural crest cells (Ankrd11nchet) display a mild midfacial hypoplasia including reduced midfacial width and a persistent open fontanelle, both of which mirror KBG syndrome patient facial phenotypes. Mice with a homozygous Ankrd11 deletion in neural crest cells (Ankrd11ncko) die at birth. They show increased severity of several clinical manifestations described for KBG syndrome, such as cleft palate, retrognathia, midfacial hypoplasia, and reduced calvarial growth. At E14.5, Ankrd11 expression in the craniofacial complex is closely associated with developing bony structures, while expression at birth is markedly decreased. Conditional deletion of Ankrd11 leads to a reduction in ossification of midfacial bones, with several ossification centers failing to expand and/or fuse. Intramembranous bones show features of delayed maturation, with bone remodeling severely curtailed at birth. Palatal shelves remain hypoplastic at all developmental stages, with a local reduction in proliferation at E13.5. Our study identifies Ankrd11 as a critical regulator of intramembranous ossification and palate development and suggests that Ankrd11nchet and Ankrd11ncko mice may serve as pre-clinical models for KBG syndrome in humans.

The effect of hypoxia on facial shape variation and disease phenotypes in chicken embryos

SUMMARYCraniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However, the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus) embryo model. Eggs were incubated in either hypoxic (7, 9, 11, 13, 15, 17 or 19% O2) or normoxic (21% O2) conditions. Embryos were photographed for morphological analysis at days 3–6. For analysis of skeletal development, 13-day embryos were cleared and stained with alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early-stage embryos (day 2) were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostaining for cleaved caspase-3, whereas later-stage embryos (days 4–6) were sectioned in paraffin for analysis of cell proliferation (BrdU), apoptosis (TUNEL) and metabolic stress (phospho-AMPK). Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation and, in early-stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed an increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development might induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are crucial to normal craniofacial morphogenesis.

Different expression patterns of Gli1–3 in mouse embryonic maxillofacial development

The Sonic hedgehog (SHH) signaling pathway plays many key roles in the development of Drosophila and vertebrate embryos, including regulating craniofacial development. The GLI family of transcription factors (GLI1, 2, and 3) mediates the SHH morphogenetic signal by regulating the expression of downstream target genes. Signaling aberrations seriously affect vertebrate development. To better understand the regulation of GLI transcription factors, we investigated the expression patterns of Gli1–3 during murine embryonic craniofacial development using in situ hybridization with whole-mounts and sections and quantitative real-time PCR. We found that Gli1 expression was mostly detected in the mesenchyme in embryonic dermis, and strongly detected in embryonic oral mucosa at late stages of face development. Gli2 was strong in mesenchyme, both in dermis and oral mucosa. Gli3 expression was detected fainter in the epithelium than the mesenchyme at 11 days post-coitum (dpc), but at 14.5dpc, expression was stronger in the epithelium. Of the three genes, Gli1 expression was highest in early stages of face development, Gli3 expression was highest in the second half of facial development (and remained stable thereafter), whereas Gli2 expression remained low throughout facial development. The present findings suggest that Gli1–3 may play different roles during facial development.

Expression of smoothened in mouse embryonic maxillofacial development

Hedgehog (Hh) signaling plays many key roles in the development of Drosophila and vertebrate embryos including regulation of craniofacial development. The seven-transmembrane protein, smoothened (Smo) transduces the Hh signal across the plasma membrane as an essential receptor of PTCHED1/2. There are few studies that evaluate the detailed expression of Smo in mouse embryonic craniofacial development. We investigated the expression patterns of Smo during murine embryonic craniofacial development using in situ hybridization (ISH), studies of whole-mounts and sections, immunohistochemistry, quantitative real time PCR, and Western blot analysis. We found that Smo mRNA was expressed in the face of mouse embryos at 11 and 12.5 days post coitum (dpc). After 13.5 dpc, the expression decreased to a low level and was faintly detected after birth. Smo protein could be detected also in embryos at 11, 12.5, and 14.5 dpc. After 15.5 dpc, the expression was very faint and paralleled the gene expression studies. No expression was detected in whisker follicle during facial development and faint signal was detected in Meckel's cartilage. These findings concerning Smo expression should guide further investigation of sonic Hh signaling pathway gene function during maxillofacial development.

Expression of Dpp6 in mouse embryonic craniofacial development

Dipeptidyl-peptidase-like protein 6 ( DPP6), a member of the dipeptidyl aminopeptidase family, plays distinct roles in brain development, but its expression in embryonic craniofacial development is unknown. The expression pattern of Dpp6 in the maxillofacial region during mouse embryonic craniofacial development was analyzed by whole-mount in situ hybridization on sections and by real-time PCR analysis. Dpp6 expression was detected during mouse embryonic craniofacial development in embryos 11–13.5 days post-coitum (dpc). Real-time PCR showed high Dpp6 expression present in 11.5–13.5 dpc, and this then decreased as development of maxillofacial region progressed. The expression pattern of Dpp6 suggests that Dpp6 may be involved in embryonic craniofacial development.

Visualizing canonical Wnt signaling during mouse craniofacial development.

Wnt signaling is critical for proper development of the head and face in the mouse embryo, playing important roles in various aspects of craniofacial development ranging from axis formation to survival of cranial neural crest cells to patterning of the brain. The signaling requirements for the development of different cell lineages in the head and face are active areas of investigation. In this study, we use a recently developed TCF/Lef-LacZ transgenic reporter mouse to characterize the expression of canonical Wnt signaling activity during craniofacial development. We present an atlas of representative sections to show embryonic craniofacial development. Our results demonstrate a pattern of sustained Wnt signaling reporter activity in most tissues which suggests sequential roles in craniofacial development.

Expression of Dlx and Lhx family homeobox genes in fetal thymus and thymocytes

Homeobox genes comprise a nearly ubiquitous and highly conserved superfamily of developmental regulatory genes that encode transcription factors involved in the determination of axis and tissue identity. While homeobox gene expression has been well characterized in a variety of embryonic tissues, their expression has not been extensively studied in lymphoid progenitor cells or in sites of lymphogenesis. To examine homeobox gene expression in the developing thymus, we screened an embryonic day 13.5 thymus cDNA library by polymerase chain reaction (PCR) using degenerate oligonucleotides within the highly conserved homeodomain region of eight homeobox gene families. The resulting PCR products were then cloned and sequenced. Transcripts for multiple Dlx family members and Lhx2 were repeatedly detected in this screen. Screening of embryonic day 16.5 and adult murine thymus and Thy1 + thymocytes was performed for selected members of these homeobox gene families. Transcripts encoding Lhx2, Lhx3, and Lhx9, as well as Dlx1 and Dlx2 were detected in both thymus and purified thymocytes. Dlx1 is a member of the distal-less homeobox gene family that has been shown to regulate embryonic craniofacial development. Significantly, Dlx1 is expressed in the third branchial arch, which contributes to the thymus. Although Dlx1 knockout mice did not display any obvious developmental defects in thymus or thymocyte development, the expression of these homeobox genes in neural crest derivatives suggests a possible role in cell migration and development that may overlap with other homeobox genes.

Expression of DLX5 during human embryonic craniofacial development

Dlx (distal-less gene) homeogenes encode transcription factors that are involved in the patterning of orofacial skeleton derived from cephalic neural crest cells. In order to study the role of DLX genes during embryonic development in human, DLX5 expression pattern was investigated in 6- to 11-week-old human embryos. A DLX5 PCR fragment was amplified from a human dental cDNA library subcloned and used for in situ hybridization investigations. DLX5 gene expression was primarily detected in the mandible at 6 weeks and then, after in the maxilla. DLX5 gene expression became restricted to progenitor cells of developing tooth germs, bones and cartilages of mandible and maxilla. During odontogenesis from bud to late cap stages, DLX5 transcripts were present in both dental epithelium and mesenchyme tissues. DLX5 expression was restricted to few cells in the vestibular aspect of the dental epithelium, while DLX5 mRNA signal was more widely distributed in dental mesenchyme. The observed expression pattern of DLX5 homeogene extends the proposed site-specific combination of homeogene expression in neural crest derived cells to human specific dentition. Furthermore, during the bud and cap stages of tooth morphogenesis, the asymmetric expression of DLX5 in the dental epithelium and dental mesenchyme may contribute to the complex patterning of human tooth shape.

Epilogue Symposium on "Current Concepts in Embryonic Craniofacial Development"

Epilogue Symposium on "Current Concepts in Embryonic Craniofacial Development"

Current concepts in embryonic craniofacial development.

Embryology mirrors phylogeny. The phenotypic expression of the genome is the result of differential gene transcription, the critically timed turning on and off of specific genes by transcription factors to produce cyto-, histo-, and morpho-differentiation that fleetingly reflects evolutionary stages of development during ontogeny. Hox genes regulate transcription of other structural genes and are responsible for patterning of the facial primordia. Cephalic development involves extremely complex morphogenetic mechanisms built on conserved elements that have undergone enormous evolutionary changes. Transient expression of phylogenetic origins characterize ontogeny and are reflected in defective development that may be due to inappropriate expression of Hox genes or distorted or disrupted epignetic processes. The mechanisms by which genetic information is transformed into morphological patterning by the actions of growth factors, morphogenes, and receptors are currently being identified. Biochemical, immunological, and allometric analyses of embryos and fetuses in experimental and descriptive studies are elucidating details of units of craniofacial morphogenesis--faciogenesis, palatogenesis, gnathogenesis, odontogenesis. Three-dimensional model computer-assisted reconstruction of sectioned embryos and fetuses provides a further technique for understanding the complex configurations of tissue migratory patterns and growth sites that account for normal and abnormal craniofaciogenesis.