Jaw Cartilage Research Articles

Exposure effects of synthetic glucocorticoid drugs on skeletal developmental and immune cell function in zebrafish

Synthetic glucocorticoids (GCs) are used to treat a wide range of human health conditions and as such are frequently detected in the aquatic environment. This, together with the highly conserved nature of the glucocorticoid system across vertebrates means that the potential for biological effects of GCs in fish is relatively high. Here, we found that exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of 4 of the most widely used synthetic GCs (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone), from 0 to 4 days post fertilisation (dpf), resulted in no effects on embryo-larval development or bone and cartilage formation. However, after exposure to equivalents of human therapeutic plasma levels, developmental abnormalities were observed that included pericardial oedema, blood pooling and alterations in jaw cartilage. Furthermore, using a double transgenic zebrafish osteoblast and chondrocyte reporter line, exposure up to 10 dpf resulted in alterations to lower jaw cartilage and bone development for all compounds at, and above, human therapeutic plasma concentrations. In the case of beclomethasone dipropionate, a reduction in lower jaw intercranial distance was observed at the environmentally relevant concentration of 0.1 μg/L. Using further transgenic reporter lines with fluorescently tagged neutrophils and macrophages, we also show exposure of embryo-larvae (0–4 dpf) to the GCs tested resulted in altered immune cell migration, but only at relatively high exposure concentrations. Collectively, our findings show GC exposure impacts embryo-larval zebrafish development, immune function, and skeletal formation, but predominantly at concentrations greater than those currently reported for the aquatic environment. Despite this, however, it is suggested that studies with longer exposure times, and to mixtures of multiple GCs (many GCs act via the same mechanism of action) are warranted before we can confidently assert that these commonly detected contaminants do not pose a risk to fish in the wild.

Dyrk1a is required for craniofacial development in Xenopus laevis.

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

Nuclear receptor Nr5a2 promotes diverse connective tissue fates in the jaw.

Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.

A well-preserved 'placoderm' (stem-group Gnathostomata) upper jaw from the Early Devonian of Mongolia clarifies jaw evolution.

The origin of jaws and teeth remains contentious in vertebrate evolution. 'Placoderms' (Silurian-Devonian armoured jawed fishes) are central to debates on the origins of these anatomical structures. 'Acanthothoracids' are generally considered the most primitive 'placoderms'. However, they are so far known mainly from disarticulated skeletal elements that are typically incomplete. The structure of the jaws-particularly the jaw hinge-is poorly known, leaving open questions about their jaw function and comparison with other placoderms and modern gnathostomes. Here we describe a near-complete 'acanthothoracid' upper jaw, allowing us to reconstruct the likely orientation and angle of the bite and compare its morphology with that of other known 'placoderm' groups. We clarify that the bite position is located on the upper jaw cartilage rather than on the dermal cheek and thus show that there is a highly conserved bite morphology among most groups of 'placoderms', regardless of their overall cranial geometry. Incorporation of the dermal skeleton appears to provide a sound biomechanical basis for jaw origins. It appears that 'acanthothoracid' dentitions were fundamentally similar in location to that of arthrodire 'placoderms', rather than resembling bony fishes. Irrespective of current phylogenetic uncertainty, the new data here resolve the likely general condition for 'placoderms' as a whole, and as such, ancestral morphology of known jawed vertebrates.

Biomechanics of the jaws of spotted ratfish.

Elasmobranch fishes (sharks, skates and rays) consume prey of a variety of sizes and properties, and the feeding mechanism typically reflects diet. Spotted ratfish, Hydrolagus colliei (Holocephali, sister group of elasmobranchs), consume both hard and soft prey; however, the morphology of the jaws does not reflect the characteristics typical of durophagous elasmobranchs. This study investigated the mechanical properties and morphological characteristics of the jaws of spotted ratfish over ontogeny, including strain, stiffness and second moment of area, to evaluate the biomechanical function of the feeding structures. Compressive stiffness of the jaws (E=13.51-21.48 MPa) is similar to that of silicone rubber, a very flexible material. In Holocephali, the upper jaw is fused to the cranium; we show that this fusion reduces deformation experienced by the upper jaw during feeding. The lower jaw resists bending primarily in the posterior half of the jaw, which occludes with the region of the upper jaw that is wider and flatter, thus potentially providing an ideal location for the lower jaw to crush or crack prey. The mechanical properties and morphology of the feeding apparatus of spotted ratfish suggest that while the low compressive stiffness is a material limit of the jaw cartilage, spotted ratfish, and perhaps all holocephalans, evolved structural solutions (i.e. fused upper jaw, shape variation along lower jaw) to meet the demands of a durophagous diet.

Mcrs1 is required for branchial arch and cranial cartilage development

Mcrs1 is a multifunctional protein that is critical for many cellular processes in a wide range of cell types. Previously, we showed that Mcrs1 binds to the Six1 transcription factor and reduces the ability of the Six1-Eya1 complex to upregulate transcription, and that Mcrs1 loss-of-function leads to the expansion of several neural plate genes, reduction of neural border and pre-placodal ectoderm (PPR) genes, and pleiotropic effects on various neural crest (NC) genes. Because the affected embryonic structures give rise to several of the cranial tissues affected in Branchio-otic/Branchio-oto-renal (BOR) syndrome, herein we tested whether these gene expression changes subsequently alter the development of the proximate precursors of BOR affected structures - the otic vesicles (OV) and branchial arches (BA). We found that Mcrs1 is required for the expression of several OV genes involved in inner ear formation, patterning and otic capsule cartilage formation. Mcrs1 knockdown also reduced the expression domains of many genes expressed in the larval BA, derived from either NC or PPR, except for emx2, which was expanded. Reduced Mcrs1 also diminished the length of the expression domain of tbx1 in BA1 and BA2 and interfered with cranial NC migration from the dorsal neural tube; this subsequently resulted in defects in the morphology of lower jaw cartilages derived from BA1 and BA2, including the infrarostral, Meckel's, and ceratohyal as well as the otic capsule. These results demonstrate that Mcrs1 plays an important role in processes that lead to the formation of craniofacial cartilages and its loss results in phenotypes consistent with reduced Six1 activity associated with BOR.

Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition.

The teeth of sharks famously form a series of transversely organized files with a conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here, we use tomographic methods to investigate mandibular structures in several early branching ‘acanthodian’-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckel's cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly.

A Dominant Heterozygous Mutation in COG4 Causes Saul-Wilson Syndrome, a Primordial Dwarfism, and Disrupts Zebrafish Development via Wnt Signaling.

Saul–Wilson syndrome (SWS) is a rare, skeletal dysplasia with progeroid appearance and primordial dwarfism. It is caused by a heterozygous, dominant variant (p.G516R) in COG4, a subunit of the conserved oligomeric Golgi (COG) complex involved in intracellular vesicular transport. Our previous work has shown the intracellular disturbances caused by this mutation; however, the pathological mechanism of SWS needs further investigation. We sought to understand the molecular mechanism of specific aspects of the SWS phenotype by analyzing SWS-derived fibroblasts and zebrafish embryos expressing this dominant variant. SWS fibroblasts accumulate glypicans, a group of heparan sulfate proteoglycans (HSPGs) critical for growth and bone development through multiple signaling pathways. Consistently, we find that glypicans are increased in zebrafish embryos expressing the COG4p.G516R variant. These animals show phenotypes consistent with convergent extension (CE) defects during gastrulation, shortened body length, and malformed jaw cartilage chondrocyte intercalation at larval stages. Since non-canonical Wnt signaling was shown in zebrafish to be related to the regulation of these processes by glypican 4, we assessed wnt levels and found a selective increase of wnt4 transcripts in the presence of COG4p.G516R. Moreover, overexpression of wnt4 mRNA phenocopies these developmental defects. LGK974, an inhibitor of Wnt signaling, corrects the shortened body length at low concentrations but amplifies it at slightly higher concentrations. WNT4 and the non-canonical Wnt signaling component phospho-JNK are also elevated in cultured SWS-derived fibroblasts. Similar results from SWS cell lines and zebrafish point to altered non-canonical Wnt signaling as one possible mechanism underlying SWS pathology.

Defective Neuronal Positioning Correlates With Aberrant Motor Circuit Function in Zebrafish.

Precise positioning of neurons resulting from cell division and migration during development is critical for normal brain function. Disruption of neuronal migration can cause a myriad of neurological disorders. To investigate the functional consequences of defective neuronal positioning on circuit function, we studied a zebrafish frizzled3a (fzd3a) loss-of-function mutant off-limits (olt) where the facial branchiomotor (FBM) neurons fail to migrate out of their birthplace. A jaw movement assay, which measures the opening of the zebrafish jaw (gape), showed that the frequency of gape events, but not their amplitude, was decreased in olt mutants. Consistent with this, a larval feeding assay revealed decreased food intake in olt mutants, indicating that the FBM circuit in mutants generates defective functional outputs. We tested various mechanisms that could generate defective functional outputs in mutants. While fzd3a is ubiquitously expressed in neural and non-neural tissues, jaw cartilage and muscle developed normally in olt mutants, and muscle function also appeared to be unaffected. Although FBM neurons were mispositioned in olt mutants, axon pathfinding to jaw muscles was unaffected. Moreover, neuromuscular junctions established by FBM neurons on jaw muscles were similar between wildtype siblings and olt mutants. Interestingly, motor axons innervating the interhyoideus jaw muscle were frequently defasciculated in olt mutants. Furthermore, GCaMP imaging revealed that mutant FBM neurons were less active than their wildtype counterparts. These data show that aberrant positioning of FBM neurons in olt mutants is correlated with subtle defects in fasciculation and neuronal activity, potentially generating defective functional outputs.

Exposure to hypergravity during zebrafish development alters cartilage material properties and strain distribution.

Vertebrates have adapted to life on Earth and its constant gravitational field, which exerts load on the body and influences the structure and function of tissues. While the effects of microgravity on muscle and bone homeostasis are well described, with sarcopenia and osteoporosis observed in astronauts returning from space, the effects of shorter exposures to increased gravitational fields are less well characterized. We aimed to test how hypergravity affects early cartilage and skeletal development in a zebrafish model. We exposed zebrafish to 3 g and 6 g hypergravity from three to five days post-fertilization, when key events in jaw cartilage morphogenesis occur. Following this exposure, we performed immunostaining along with a range of histological stains and transmission electron microscopy (TEM) to examine cartilage morphology and structure, atomic force microscopy (AFM) and nanoindentation experiments to investigate the cartilage material properties, and finite element modelling to map the pattern of strain and stress in the skeletal rudiments. We did not observe changes to larval growth, or morphology of cartilage or muscle. However, we observed altered mechanical properties of jaw cartilages, and in these regions we saw changes to chondrocyte morphology and extracellular matrix (ECM) composition. These areas also correspond to places where strain and stress distribution are predicted to be most different following hypergravity exposure. Our results suggest that altered mechanical loading, through hypergravity exposure, affects chondrocyte maturation and ECM components, ultimately leading to changes to cartilage structure and function. Cite this article: Bone Joint Res2021;10(2):137-148.

Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes.

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py3-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py3-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py3-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py3-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py3-FITC solution. Therefore, Py3-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Application of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCRRFLP) Technique in the Analysis of MYO1H Single Nucleotide Polymorphism in Malay Mandibular Prognathism Patients

Genetic studies have reported the association between polymorphism in MYO1H with mandibular prognathism. MYO1H is found in skeletal muscle sarcomeres and is expressed in the mandibular jaw cartilage signifying its importance during craniofacial development. This study aimed to characterise the genotype and allele of MYO1H single nucleotide polymorphism (SNP) (rs3825393) and to associate the SNP with mandibular prognathism in Class III skeletal malocclusion. This was a case-control study, which involved 57 Malay subjects with 30 Class I (control) and 27 Class III skeletal base patients (case). Cephalometric measurements were taken prior to collection of saliva samples. MYO1H SNP (rs3825383) was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Chi-square (χ2) test was used to compare genotype and allele frequencies between the groups while Hardy-Weinberg Equilibrium (HWE) was applied to assess distribution of genotype frequency in both classes. MYO1H SNP (rs3825393) did not yield significant association with mandibular prognathism with p = 0.33; OR = 0.66; 95% CI = 0.289~1.518, that was reflected by no significant difference in allele (p > 0.05) and genotype (p > 0.05) frequency between control and study group. Nevertheless, AA genotype depicted the highest frequency in both groups. The genotype distribution in both groups was in concordance with HWE (p > 0.05). Our data showed no association of MYO1H SNP (rs3825393) with mandibular prognathism. Interestingly, we observed Allele A representing the major allele in Malay population. Presence of MYO1H SNP (rs3825393) was detected in samples analysed. Larger number of samples is required to confirm the involvement of MYO1H polymorphisms in mandibular prognathism.

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.

Expression dynamics of genes in the hypothalamic-pituitary-thyroid (HPT) cascade and their responses to 3,3′,5-triiodo-l-thyronine (T3) highlights potential vulnerability to thyroid-disrupting chemicals in zebrafish (Danio rerio) embryo-larvae

Some chemicals in the environment disrupt thyroid hormone (TH) systems leading to alterations in organism development, but their effect mechanisms are poorly understood. In fish, this has been limited by a lack of fundamental knowledge on thyroid gene ontogeny and tissue expression in early life stages. Here we established detailed expression profiles for a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis of zebrafish (Danio rerio) between 24–120 h post fertilisation (hpf) and quantified their responses following exposure to 3,3’,5-triiodo-L-thyronine (T3) using whole mount in situ hybridisation (WISH) and qRT-PCR (using whole-body extracts). All of the selected genes in the HPT axis demonstrated dynamic transcript expression profiles across the developmental stages examined. The expression of thyroid receptor alpha (thraa) was observed in the brain, gastrointestinal tract, craniofacial tissues and pectoral fins, while thyroid receptor beta (thrb) expression occurred in the brain, otic vesicles, liver and lower jaw. The TH deiodinases (dio1, dio2 and dio3b) were expressed in the liver, pronephric ducts and brain and the patterns differed depending on life stage. Both dio1 and dio2 were also expressed in the intestinal bulb (96–120 hpf), and dio2 expression occurred also in the pituitary (48–120 hpf). Exposure of zebrafish embryo-larvae to T3 (30 and 100 μg L−1) for periods of 48, 96 or 120 hpf resulted in the up-regulation of thraa, thrb, dio3b, thyroid follicle synthesis proteins (pax8) and corticotropin-releasing hormone (crhb) and down-regulation of dio1, dio2, glucuronidation enzymes (ugt1ab) and thyroid stimulating hormone (tshb) (assessed via qRT-PCR) and responses differed across life stage and tissues. T3 induced thraa expression in the pineal gland, pectoral fins, brain, somites, gastrointestinal tract, craniofacial tissues, liver and pronephric ducts. T3 enhanced thrb expression in the brain, jaw cartilage and intestine, while thrb expression was suppressed in the liver. T3 exposure suppressed the transcript levels of dio1 and dio2 in the liver, brain, gastrointestinal tract and craniofacial tissues, while dio2 signalling was also suppressed in the pituitary gland. Dio3b expression was induced by T3 exposure in the jaw cartilage, pectoral fins and brain. The involvement of THs in the development of numerous body tissues and the responsiveness of these tissues to T3 in zebrafish highlights their potential vulnerability to exposure to environmental thyroid-disrupting chemicals.

Latent TGF-beta binding protein-1 plays an important role in craniofacial development.

Objective:This study aims to replicate the phenotype of Ltbp1 knockout mice in zebrafish, and to address the function of LTBP1 in craniofacial development.Methods:Whole mount in situ hybridization (WISH) of ltbp1 was performed at critical periods of zebrafish craniofacial development to explore the spatial-temporal expression pattern. Furthermore, we generated morpholino based knockdown model of ltbp1 to study the craniofacial phenotype.Results:WISH of ltbp1 was mainly detected in the mandibular jaw region, brain trunk, and internal organs such as pancreas and gallbladder. And ltbp1 colocalized with both sox9a and ckma in mandibular region. Morpholino based knockdown of ltbp1 results in severe jaw malformation. Alcian blue staining revealed severe deformity of Meckel's cartilage along with the absence of ceratobranchial. Three-dimension measurements of ltbp1 morphants jaws showed decrease in both mandible length and width and increase in open mouth distance. Expression of cartilage marker sox9a and muscle marker ckma was decreased in ltbp1 morphants.Conclusions:Our experiments found that ltbp1 was expressed in zebrafish mandibular jaw cartilages and the surrounding muscles. The ltbp1 knockdown zebrafish exhibited phenotypes consistent with Ltbp1 knockout mice. And loss of ltbp1 function lead to significant mandibular jaw defects and affect both jaw cartilages and surrounding muscles.

Nkx3.2 mutant zebrafish accommodate jaw joint loss through a phenocopy of the head shapes of Paleozoic jawless fish.

The vertebrate jaw is a versatile feeding apparatus. To function, it requires a joint between the upper and lower jaws, so jaw joint defects are often highly disruptive and difficult to study. To describe the consequences of jaw joint dysfunction, we engineered two independent null alleles of a single jaw joint marker gene, nkx3.2, in zebrafish. These mutations caused zebrafish to become functionally jawless via fusion of the upper and lower jaw cartilages (ankylosis). Despite lacking jaw joints, nkx3.2 mutants survived to adulthood and accommodated this defect by: (a) having a remodeled skull with a fixed open gape, reduced snout and enlarged branchial region; and (b) performing ram feeding in the absence of jaw-generated suction. The late onset and broad extent of phenotypic changes in the mutants suggest that modifications to the skull are induced by functional agnathia, secondarily to nkx3.2 loss of function. Interestingly, nkx3.2 mutants superficially resemble ancient jawless vertebrates (anaspids and furcacaudiid thelodonts) in overall head shape. Because no homology exists in individual skull elements between these taxa, the adult nkx3.2 phenotype is not a reversal but rather a convergence due to similar functional requirements of feeding without moveable jaws. This remarkable analogy strongly suggests that jaw movements themselves dramatically influence the development of jawed vertebrate skulls. Thus, these mutants provide a unique model with which to: (a) investigate adaptive responses to perturbation in skeletal development; (b) re-evaluate evolutionarily inspired interpretations of phenocopies generated by gene knockdowns and knockouts; and (c) gain insight into feeding mechanics of the extinct agnathans.

Reconstructing Connective Tissue Lineage Diversification at the Single Cell Level in the Zebrafish Face

Assembly of the vertebrate face requires the precisely regulated differentiation of neural crest‐derived cells into a diversity of connective tissue fates, including cartilage, bone, and tendon/ligament cells. In order to better understand spatiotemporal regulation of this process, we are conducting a single‐cell transcriptomic analysis of neural crest‐derived cells from embryonic through adult stages. Our preliminary data reveal different trajectories for permanent cartilage, replacement cartilage, bone, and tendon/ligament cells, as well as diverse populations of undifferentiated cells in the adult face that may represent distinct progenitor populations. In the embryo, we have confirmed the presence of both multipotent and lineage‐committed cells through clonal labeling of sox10:CreER animals treated with a limiting dose of tamoxifen. We have also uncovered novel genes implicated in early lineage decisions. In particular, we find that the nuclear receptor transcription factor Nr5a2 marks cells destined to become lower jaw tendons, which we show by generating an nr5a2:GFP knock‐in transgenic reporter and performing time‐lapse microscopy. In nr5a2 mutants, tendon precursors marked by nr5a2:GFP inappropriately become cartilage cells, which results in an enlarged lower jaw cartilage that fails to properly attach to lower jaw musculature due to loss of tendons. These ongoing studies validate genomics approaches in identifying new regulators of connective tissue fates in the developing face.Support or Funding InformationNIDCR R35 DE027550This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Functional Studies of MYO1H for Mandibular Prognathism

Mandibular prognathism (MP) is regarded as a craniofacial deformity resulting from the combined effects of environmental and genetic factors, while the genetically predetermined component is considered to play an important role to develop MP. Although linkage and association studies for MP have identified multiple strongly associated regions and genes, the causal genes and variants responsible for the deformity remain largely undetermined. To address this, we performed targeted sequencing of 396 genes selected from previous studies as well as genes and pathways related with craniofacial development as primary candidates in 199 MP cases and 197 controls and carried out a series of statistical and functional analyses. A nonsynonymous common variant of MYO1H rs3825393, C>T, p.Pro1001Leu, was identified to be significantly associated with MP. During zebrafish embryologic development, expression of MYO1H orthologous genes were detected at mandibular jaw. Furthermore, jaw cartilage defects were observed in zebrafish knockdown models. Collectively, these data demonstrate that MYO1H is required for proper jaw growth and contributes to MP pathogenesis, expanding our knowledge of the genetic basis of MP.

Paleogene origin of planktivory in the Batoidea

ABSTRACTThe planktivorous mobulid rays are a sister group to, and descended from, rhinopterid and myliobatid rays that possess a dentition showing adaptations consistent with a specialized durophagous diet. Within the Paleocene and Eocene, there are several taxa that display dentitions apparently transitional between these extreme trophic modalities, in particular the genus Burnhamia. The holotype of Burnhamia daviesi was studied through X-ray computed tomography (CT) scanning. Digital renderings of this incomplete but articulated jaw and dentition revealed previously unrecognized characters regarding the jaw cartilages and teeth. In addition, the genus Sulcidens, gen. nov., is erected for articulated dentitions from the Paleocene previously assigned to Myliobatis. Phylogenetic analyses confirm Burnhamia as a sister taxon to the mobulids and the Mobulidae as a sister group to Rhinoptera. Shared dental characters between Burnhamia and Sulcidens likely represent independent origins of planktivory within the rhinopterid-myliobatid clade. The transition from highly specialized durophagous feeding morphologies to the morphology of planktivores is perplexing but was facilitated by a pelagic swimming mode in these rays and, we propose, through subsequent transition from either meiofauna-feeding or pelagic fish-feeding to pelagic planktivory.http://zoobank.org/urn:lsid:zoobank.org:pub:511E9C39-1208-4F56-8DC1-2B92D2EDB4B3Citation for this article: Underwood, C. J., M. A. Kolmann, and D. J. Ward. 2017. Paleogene origin of planktivory in the Batoidea. Journal of Vertebrate Paleontology. DOI: 10.1080/02724634.2017.1293068.

ADAM12-deficient zebrafish exhibit retardation in body growth at the juvenile stage without developmental defects.

ADAM (a disintegrin and metalloprotease) constitutes a family of multi-domain proteins that are involved in development, homeostasis, and disease. ADAM12 plays important roles in myogenesis and adipogenesis in mice; however, the precise physiological mechanisms are not known, and the function of this gene in other vertebrates has not been examined. In this study, we used a simple model vertebrate, the zebrafish, to investigate the functions of ADAM12 during development. Zebrafish adam12 is conserved with those of mammals in the synteny and the amino-acid sequence. We examined adam12 expression in zebrafish embryos by whole mount insitu hybridization and the promoter activity of the adam12 upstream sequence. We found that adam12 is strongly expressed in the cardiovascular system, erythroid progenitors, brain, and jaw cartilage during zebrafish development, and adam12-knockout zebrafish exhibited reduced body size in the juvenile stage without apparent morphological defects. Taken together, these results suggest that adam12 plays a significant role in the regulation of body growth during juvenile stage in zebrafish, although the precise molecular mechanisms await further study.