Comparison of craniomaxillofacial morphology in children with unilateral cleft lip and palate treated with/without presurgical nasoalveolar molding - a retrospective study with a mean age of 5 years.

OBJECTIVES: To three-dimensionally assess differences in craniomaxillofacial skeletal development in patients with operated unilateral cleft lip and palate (UCLP) treated with/without presurgical nasoalveolar molding (PNAM) with a mean age of 5 years. MATERIALS AND METHODS: Cone-beam CT radiographs of 30 patients with UCLP who had undergone PNAM and 34 patients with UCLP who did not receive PNAM were analyzed. The data were stored in DICOM file format and were imported into the Dolphin Imaging program for 3D image reconstruction and landmark identification. 33 landmarks, 17 linear and three angular variables representing craniofacial morphology were analyzed and compared by using the Mann-Whitney U tests. RESULTS: The vast majority of linear variables and 3D coordinates of landmark points reflecting craniofacial skeletal symmetry were not significantly different between the two groups. In terms of craniofacial skeletal development, the PNAM group had a significantly smaller anterior nasal spine offset in the midsagittal plane and a greater maxillary length compared to the non-PNAM group. CONCLUSIONS: Evaluations performed in early childhood showed that treatment with/without PNAM in the neonatal period was not a major factor influencing craniomaxillofacial hard tissue development in patients with UCLP; moreover, PNAM treatment showed significant correction of skeletal deviation at the base of the nose. CLINICAL RELEVANCE: Follow-up in early childhood has shown that PNAM treatment administered during the neonatal stage does not impede maxillary development and has benefits in correcting nasal floor deviation. It is a viable option for improving nasal deformity in children with unilateral cleft lip and palate.

Emergence of a left-right symmetric body plan in vertebrate embryos.

External bilateral symmetry is a prevalent feature in vertebrates, which emerges during early embryonic development. To begin with, vertebrate embryos are largely radially symmetric before transitioning to bilaterally symmetry, after which, morphogenesis of various bilateral tissues (e.g somites, otic vesicle, limb bud), and structures (e.g palate, jaw) ensue. While a significant amount of work has probed the mechanisms behind symmetry breaking in the left-right axis leading to asymmetric positioning of internal organs, little is known about how bilateral tissues emerge at the same time with the same shape and size and at the same position on the two sides of the embryo. By discussing emergence of symmetry in many bilateral tissues and structures across vertebrate model systems, we highlight that understanding symmetry establishment is largely an open field, which will provide deep insights into fundamental problems in developmental biology for decades to come.

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

Orofacial clefts (OFCs) 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 0.50-0.83. Specifically, our model revealed that SNPs significantly associated with OFCs 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 OFCs. 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 OFCs, and the model can be extended to study other complex diseases or traits.

The ciliary protein C2cd3 is required for mandibular musculoskeletal tissue patterning.

The mandible is composed of several musculoskeletal tissues including bone, cartilage, and tendon that require precise patterning to ensure structural and functional integrity. Interestingly, most of these tissues are derived from one multipotent cell population called cranial neural crest cells (CNCCs). How CNCCs are properly instructed to differentiate into various tissue types remains nebulous. To better understand the mechanisms necessary for the patterning of mandibular musculoskeletal tissues we utilized the avian mutant talpid2 (ta2) which presents with several malformations of the facial skeleton including dysplastic tendons, mispatterned musculature, and bilateral ectopic cartilaginous processes extending off Meckel's cartilage. We found an ectopic epithelial BMP signaling domain in the ta2 mandibular prominence (MNP) that correlated with the subsequent expansion of SOX9+ cartilage precursors. These findings were validated with conditional murine models suggesting an evolutionarily conserved mechanism for CNCC-derived musculoskeletal patterning. Collectively, these data support a model in which cilia are required to define epithelial signal centers essential for proper musculoskeletal patterning of CNCC-derived mesenchyme.

Cross-sectional and retrospective study regarding the changes in the spatial position of the hyoid bone and upper airways before and after orthodontic therapy in developing patients with normocclusion.

OBJECTIVE: The objective was to study the effect of orthodontic therapy regarding the position of the hyoid bone and upper airways in nighty-one 6-21 year-old Caucasian patients with normoclussion. METHODS: Nineteen variables were analyzed by teleradiography. The differences were analyzed with the ANOVA test of repeated measures with an intra-group factor for two occasions. RESULTS: The statistical analysis revealed a greater influence on the variables Hy-MP (12.3 vs 14.2), Hps-C3 (25.2 vs 28.1), Hpi-C3 (28.8 vs 35.5), H°-C3 (14.4 vs 12.7), Hps-Rg (36.0 vs 42.7), Hpi-Rg (34.1 vs 39.7), USP (16.2 vs 20.2), MPP (12.9 vs 14.8), C3P (10.6 vs 12.8), PNS-Ba (43.5 vs 66.5), PtV-Ad (12.0 vs 17.1) and PtM-Ba (33.9 vs 35.9), and Ad2-SO (24.16 vs 20.87). DISCUSSION: Significant differences were observed between most of the airway and hyoid bone variables before and after orthodontic treatment in our Spanish pediatric population, especially in the nasopharynx.

Key Roles of Gli1 and Ihh Signaling in Craniofacial Development.

The Hedgehog (Hh) signaling pathway orchestrates its influence through a dynamic interplay of Hh proteins, the cell surface receptor Ptch1, Smo, and Gli transcription factors, contributing to a myriad of developmental events. Indian Hedgehog (Ihh) and Gli zinc finger transcription factor 1 (Gli1) play crucial roles in developmental regulation within the Hh signaling pathway. Ihh regulates chondrocyte proliferation, differentiation, and bone formation, impacting the development of cranial bones, cartilage, and the temporomandibular joint (TMJ). Losing Ihh results in cranial bone malformation and decreased ossification and affects the formation of cranial base cartilage unions, TMJ condyles, and joint discs. Gli1 is predominantly expressed during early craniofacial development, and Gli1+ cells are identified as the primary mesenchymal stem cells (MSCs) for craniofacial bones, crucial for cell differentiation and morphogenesis. In addition, a complex mutual regulatory mechanism exists between Gli1 and Ihh, ensuring the normal function of the Hh signaling pathway by directly or indirectly regulating each other's expression levels. And the interaction between Ihh and Gli1 significantly impacts the normal development of craniofacial tissues. This review summarizes the pivotal roles of Gli1 and Ihh in the intricate landscape of mammalian craniofacial development and outlines the molecular regulatory mechanisms and intricate interactions governing the growth of bone and cartilage exhibited by Gli1 and Ihh, which provides new insights into potential therapeutic strategies for related diseases or researches of tissue regeneration.

The society for craniofacial genetics and developmental biology 46th annual meeting.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 46th Annual Meeting at Cincinnati Children's Hospital Medical Center in Cincinnati, Ohio on October 10th-12th, 2023. On the first day of the meeting, Drs. Sally Moody and Justin Cotney were each honored with the SCGDB Distinguished Scientist Awards for their exceptional contributions to the field of craniofacial biology. The following two days of the meeting featured five sessions that highlighted new discoveries in signaling and genomic mechanisms regulating craniofacial development, human genetics, translational and regenerative approaches, and clinical management of craniofacial differences. Interactive workshops on spatial transcriptomics and scientific communication, as well as a poster session facilitated meaningful interactions among the 122 attendees representing diverse career stages and research backgrounds in developmental biology and genetics, strengthened the SCGDB community.

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.

The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

A common cis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation of PKDCC during chondrogenesis.

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, we demonstrate that the rs6740960 SNP is sufficient to confer chondrocyte-specific differences in PKDCC expression. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in the maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

Transforming growth factor beta signaling and craniofacial development: modeling human diseases in zebrafish.

Humans and other jawed vertebrates rely heavily on their craniofacial skeleton for eating, breathing, and communicating. As such, it is vital that the elements of the craniofacial skeleton develop properly during embryogenesis to ensure a high quality of life and evolutionary fitness. Indeed, craniofacial abnormalities, including cleft palate and craniosynostosis, represent some of the most common congenital abnormalities in newborns. Like many other organ systems, the development of the craniofacial skeleton is complex, relying on specification and migration of the neural crest, patterning of the pharyngeal arches, and morphogenesis of each skeletal element into its final form. These processes must be carefully coordinated and integrated. One way this is achieved is through the spatial and temporal deployment of cell signaling pathways. Recent studies conducted using the zebrafish model underscore the importance of the Transforming Growth Factor Beta (TGF-ß) and Bone Morphogenetic Protein (BMP) pathways in craniofacial development. Although both pathways contain similar components, each pathway results in unique outcomes on a cellular level. In this review, we will cover studies conducted using zebrafish that show the necessity of these pathways in each stage of craniofacial development, starting with the induction of the neural crest, and ending with the morphogenesis of craniofacial elements. We will also cover human skeletal and craniofacial diseases and malformations caused by mutations in the components of these pathways (e.g., cleft palate, craniosynostosis, etc.) and the potential utility of zebrafish in studying the etiology of these diseases. We will also briefly cover the utility of the zebrafish model in joint development and biology and discuss the role of TGF-ß/BMP signaling in these processes and the diseases that result from aberrancies in these pathways, including osteoarthritis and multiple synostoses syndrome. Overall, this review will demonstrate the critical roles of TGF-ß/BMP signaling in craniofacial development and show the utility of the zebrafish model in development and disease.

Grainyhead-like 2 interacts with noggin to regulate tissue fusion in mouse.

Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2-/-;Nog+/- embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2-/- mice.

Cis-regulatory control of mammalian Trps1 gene expression.

TRPS1 serves as the causative gene for tricho-rhino phalangeal syndrome, known for its craniofacial and skeletal abnormalities. The Trps1 gene encodes a protein that represses Wnt signaling through strong interactions with Wnt signaling inhibitors. The identification of genomic cis-acting regulatory sequences governing Trps1 expression is crucial for understanding its role in embryogenesis. Nevertheless, to date, no investigations have been conducted concerning these aspects of Trps1. To identify deeply conserved noncoding elements (CNEs) within the Trps1 locus, we employed a comparative genomics approach, utilizing slowly evolving fish such as coelacanth and spotted gar. These analyses resulted in the identification of eight CNEs in the intronic region of the Trps1 gene. Functional characterization of these CNEs in zebrafish revealed their regulatory potential in various tissues, including pectoral fins, heart, and pharyngeal arches. RNA in-situ hybridization experiments revealed concordance between the reporter expression pattern induced by the identified set of CNEs and the spatial expression pattern of the trps1 gene in zebrafish. Comparative in vivo data from zebrafish and mice for CNE7/hs919 revealed conserved functions of these enhancers. Each of these eight CNEs was further investigated in cell line-based reporter assays, revealing their repressive potential. Taken together, in vivo and in vitro assays suggest a context-dependent dual functionality for the identified set of Trps1-associated CNE enhancers. This functionally characterized set of CNE-enhancers will contribute to a more comprehensive understanding of the developmental roles of Trps1 and can aid in the identification of noncoding DNA variants associated with human diseases.

Influence of BMI percentile on craniofacial morphology and development in adolescents,Part II: elevated BMI is associated with larger final facial dimensions.

BACKGROUND: Prevalence of adolescent obesity has markedly increased from 5.2% in 1974 to 19.7% in 2021. Understanding the impacts of obesity is important to orthodontists, as growth acceleration and greater pre-pubertal facial dimensions are seen in children with elevated body mass index (BMI). METHODS: To identify whether adolescent obesity shifts the timing and rate of craniofacial growth resulting in larger post-treatment dimensions, we evaluated cephalometric outcomes in overweight/obese (BMI > 85%, n = 168) and normal weight (n = 158) adolescents (N = 326 total). Cephalometric measurements were obtained from pre- and post-treatment records to measure growth rates and final dimensions and were statistically evaluated with repeated measures analysis of variance and linear regression models. RESULTS: Overweight and obese adolescents began and finished treatment with significantly larger, bimaxillary prognathic craniofacial dimensions, with elevated mandibular length [articulare-gnathion (Ar-Gn)], maxillary length [condylion-anterior nasal spine (Co-ANS), posterior nasal spine-ANS (PNS-ANS)], and anterior lower face height (ANS-Me), suggesting overweight children grow more overall. However, there was no difference between weight cohorts in the amount of cephalometric change during treatment, and regression analyses demonstrated no correlation between change in growth during treatment and BMI. BMI percentile was a significant linear predictor (P < 0.05) for cephalometric post-treatment outcomes, including Ar-Gn, Co-ANS, ANS-Me, upper face height percentage (UFH:total FH, inverse relationship), lower face height percentage (LFH:total FH), sella-nasion-A-point (SNA), and SN-B-point (SNB). LIMITATIONS: The study is retrospective. CONCLUSIONS: Growth begins earlier in overweight and obese adolescents and continues at a rate similar to normal-weight children during orthodontic treatment, resulting in larger final skeletal dimensions. Orthodontics could begin earlier in overweight patients to time care with growth, and clinicians can anticipate that overweight/obese patients will finish treatment with proportionally larger, bimaxillary-prognathic craniofacial dimensions.

Genetic determinism and hemispheric influence in hair whorl formation.

BACKGROUND: The mechanisms determining the laterality and the rotation direction of hair whorls are unknown. Here we report observations on twins investigating the genetic bases of whorl pattern formation. Knowing that vortex phenomena may depend on geographic effects, we also provide comparative data on whorls from children born in the Northern hemisphere (France) versus children born in the Southern hemisphere (Chile). MATERIAL AND METHODS: We retrospectively included children from three populations: (1) Northern hemisphere general population, (2) Southern hemisphere general population, and (3) same-sex Northern hemisphere twins. We recorded whorl rotation direction (clockwise, counterclockwise), whorl position (left, right, central) and twinning type. Univariate logistic models were used to screen for associations between rotation direction and whorl position. For twins, the variable of interest was binary, i.e. same rotation direction (reference class) or opposite directions for each twin pair. For controls, all single combinations were included as virtual twins, and compared to real twins. Odds ratios (OR) were compared for both hemispheres, for real twins and virtual (control) twins. RESULTS: Seventy-four (37 pairs) twins and 50 children from the general population of each hemisphere were included. The OR for opposite rotation directions between two twins was ≠1 (p = 0.017), meaning that whorls rotated preferentially in the same direction in twins. ORs were <1 for Northern and Southern hemispheres, meaning that whorls rotated preferentially in the same direction in simulated twins. OR for the Northern hemisphere (0.04 [0.03; 0.05]) was less than the OR for the Southern hemisphere (0.28 [0.24; 0.32]) with no confidence interval superimposition, indicating than counterclockwise whorls were more frequent in the Southern hemisphere (p < 0.001). CONCLUSIONS: We suggest that hair whorl formation is a genetically determined developmental process that can be influenced by extrinsic environmental factors. Our results furthermore underline the general importance of studies focused on limit phenomena that can provide insights on general developmental mechanisms. We plead for large-scale epidemiological assessments of hair whorls in several Northern and Southern hemisphere populations to confirm these surprising findings suggesting significant modulations of craniofacial development by geographic effects.

Three-dimensional changes in the cranial base associated with soft-diet feeding.

BACKGROUND: Masticatory activity affects the morphology of the maxillo-mandibular complex, however, its influence on the cranial base remains to be elucidated. The recent integration of quantitative morphometric analysis with 3D imaging enabled a comprehensive and high-resolution morphological characterization of the craniofacial complex. We aimed to investigate the influence of masticatory activity on the morphology of the growing cranial base by three-dimensional (3D) geometric morphometric approach using micro-CT. METHODS: The micro-CT data was reanalyzed to illustrate the 3D shape of the cranial base, and wireframe models were generated by connecting landmarks on the images. In the original study, mice were fed a soft diet (SD) of powdered pellets or a conventional hard diet (HD) for 6 weeks from 3 to 9 weeks of age, immediately after weaning. A principal component (PC) analysis analyzed shape variations and assessed their significance, while canonical variate (CV) analysis facilitated the comparison and differentiation of groups based on shape, unveiling meaningful shape distinctions. RESULTS: Three PCs were extracted that significantly separated the SD and HD groups among those explaining variations in shape. These PCs were related to the length of the sphenoid bone, the width of the anterior part of the sphenoid bone, and the length of the cranial base. Furthermore, one CV effectively distinguished SD from HD, and CV analysis showed that the sphenoid was shortened in the length and narrowed at the border of the temporal bone in SD mice. CONCLUSIONS: Masticatory loading affects the skeletal development of the cranial base. The morphology of the sphenoid bone was affected in both the sagittal and transverse axes.

A Feasible Role of Neuropilin Signaling in Pharyngeal Pouch Formation in Zebrafish.

Pharyngeal pouches are an important epithelial structure controlling facial skeletal development in vertebrates. A series of pouches arise sequentially in the pharyngeal endoderm through collective cell migration followed by rearrangement of pouch-forming cells. While crucial transcription factors and signaling molecules have been identified in pouch formation, a role for Neuropilins (Nrps) in pouch development has not yet been analyzed in any vertebrates. Nrps are cell surface receptors essential for angiogenesis and axon guidance. In all vertebrates, the two Nrp family members, Nrp1 and Nrp2, are conserved in the genome, with two paralogs for Nrp1 (Nrp1a and Nrp1b) and Nrp2 (Nrp2a and Nrp2b) being identified in zebrafish. Here, I report a potential requirement of Nrp signaling in pouch development in zebrafish. nrp1a and nrp2b were expressed in the developing pouches, with sema3d, a ligand for Nrps, being expressed in the pouches. Knocking down Nrps signaling in the pharyngeal endoderm led to severe defects in pouches and facial cartilages. In addition, blocking Mitogen-activated protein kinase (MAPK) activities, a downstream effector of Nrp signaling, in the pharyngeal endoderm caused similar defects in pouches and facial skeleton to those by knocking down Nrps signaling. My results suggest that Nrp signaling acts for pouch formation through MAPK.

Comparative analysis of Hmx expression and the distribution of neuronal somata in the trigeminal ganglion in lamprey and shark: insights into the homology of the trigeminal nerve branches and the evolutionary origin of the vertebrate jaw.

The evolutionary origin of the jaw remains one of the most enigmatic events in vertebrate evolution. The trigeminal nerve is a key component for understanding jaw evolution, as it plays a crucial role as a sensorimotor interface for the effective manipulation of the jaw. This nerve is also found in the lamprey, an extant jawless vertebrate. The trigeminal nerve has three major branches in both the lamprey and jawed vertebrates. Although each of these branches was classically thought to be homologous between these two taxa, this homology is now in doubt. In the present study, we compared expression patterns of Hmx, a candidate genetic marker of the mandibular nerve (rV3, the third branch of the trigeminal nerve in jawed vertebrates), and the distribution of neuronal somata of trigeminal nerve branches in the trigeminal ganglion in lamprey and shark. We first confirmed the conserved expression pattern of Hmx1 in the shark rV3 neuronal somata, which are distributed in the caudal part of the trigeminal ganglion. By contrast, lamprey Hmx genes showed peculiar expression patterns, with expression in the ventrocaudal part of the trigeminal ganglion similar to Hmx1 expression in jawed vertebrates, which labeled the neuronal somata of the second branch. Based on these results, we propose two alternative hypotheses regarding the homology of the trigeminal nerve branches, providing new insights into the evolutionary origin of the vertebrate jaw.

Pax3 lineage-specific deletion of Gpr161 is associated with spinal neural tube and craniofacial malformations during embryonic development.

Sonic hedgehog (Shh) signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality associated with craniofacial defects and neural tube defects. However, the structural defects of later embryonic stages and cell lineages underlying abnormalities have not been well characterized due to the limited lifespan of Gpr161 null mice. We found that embryos with Pax3 lineage-specific deletion of Gpr161 presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest), vertebral abnormalities (somite) and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida was partly due to reduced Pax3 and Cdx4 gene expression in the posterior dorsal neural tubes of Gpr161 mutant embryos with decreased Wnt signaling, whereas Shh signaling was increased. We describe a previously unreported role for Gpr161 in the development of posterior neural tubes and confirm its role in cranial neural crest- and somite-derived skeletogenesis and midbrain morphogenesis in mice.