Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants.

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including µCT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRµCT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2 -/- mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2 -/- mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2 -/- zebrafish, and demonstrate the promise of DICE-PPC-SRµCT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.

A novel cis-regulatory element drives early expression of Nkx3.2 in the gnathostome primary jaw joint.

The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in most gnathostomes but undetectable in the jawless hagfish and lamprey. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 results in a significant reduction of early gene expression of nkx3.2 and leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

The role of Gdf5 in the development of the zebrafish fin endoskeleton.

BACKGROUND: The development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota. RESULTS: We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. CONCLUSIONS: Our study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.

The broad role of Nkx3.2 in the development of the zebrafish axial skeleton.

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements.

Embryonic expression patterns and phylogenetic analysis of panarthropod sox genes: insight into nervous system development, segmentation and gonadogenesis.

BACKGROUND: Sox (Sry-related high-mobility-group box) genes represent important factors in animal development. Relatively little, however, is known about the embryonic expression patterns and thus possible function(s) of Sox genes during ontogenesis in panarthropods (Arthropoda+Tardigrada+Onychophora). To date, studies have been restricted exclusively to higher insects, including the model system Drosophila melanogaster, with no comprehensive data available for any other arthropod group, or any tardigrade or onychophoran. RESULTS: This study provides a phylogenetic analysis of panarthropod Sox genes and presents the first comprehensive analysis of embryonic expression patterns in the flour beetle Tribolium castaneum (Hexapoda), the pill millipede Glomeris marginata (Myriapoda), and the velvet worm, Euperipatoides kanangrensis (Onychophora). 24 Sox genes were identified and investigated: 7 in Euperipatoides, 8 in Glomeris, and 9 in Tribolium. Each species possesses at least one ortholog of each of the five expected Sox gene families, B, C, D, E, and F, many of which are differentially expressed during ontogenesis. CONCLUSION: Sox gene expression (and potentially function) is highly conserved in arthropods and their closest relatives, the onychophorans. Sox B, C and D class genes appear to be crucial for nervous system development, while the Sox B genes Dichaete (D) and Sox21b likely play an additional conserved role in panarthropod segmentation. The Sox B gene Sox21a likely has a conserved function in foregut and Malpighian tubule development, at least in Hexapoda. The data further suggest that Sox D and E genes are involved in mesoderm differentiation, and that Sox E genes are involved in gonadal development. The new data expand our knowledge about the expression and implied function of Sox genes to Mandibulata (Myriapoda+Pancrustacea) and Panarthropoda (Arthropoda+Onychophora).