Frontonasal malformations are caused by a failure in the growth of the frontonasal prominence during development. Although genetic studies have identified genes that are crucial for frontonasal development, it remains largely unknown how these genes are regulated during this process. Here, we show that microRNAs, which are short non-coding RNAs capable of targeting their target mRNAs for degradation or silencing their expression, play a crucial role in the regulation of genes related to frontonasal development in mice. Using the Mouse Genome Informatics (MGI) database, we curated a total of 25 mouse genes related to frontonasal malformations, including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism. MicroRNAs regulating the expression of these genes were predicted through bioinformatic analysis. We then experimentally evaluated the top three candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) for their effect on cell proliferation and target gene regulation in O9-1 cells, a neural crest cell line. Overexpression of these miRNAs significantly inhibited cell proliferation, and the genes related to frontonasal malformations (Alx1, Lrp2, and Sirt1 for miR-338-5p; Alx1, Cdc42, Sirt1, and Zic2 for miR-374c-5p; and Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 for miR-653-5p) were directly regulated by these miRNAs in a dose-dependent manner. Taken together, our results highlight miR-338-5p, miR-653-5p, and miR-374c-5p as pathogenic miRNAs related to the development of frontonasal malformations.

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.

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.

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.

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.

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-β) 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-β/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-β/BMP signaling in craniofacial development and show the utility of the zebrafish model in development and disease.

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.

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.

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.

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.