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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 threedimensional (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 phasecontrast 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 boneassociated soft tissues in small-fish skeletal disease models and developmental studies more broadly. [ABSTRACT FROM AUTHOR]

Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis. [ABSTRACT FROM AUTHOR]

Enamel, the hardest vertebrate tissue, covers the teeth of almost all sarcopterygians (lobe-finned bony fishes and tetrapods) as well as the scales and dermal bones of many fossil lobe-fins (1-5). [...]

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. Key Findings: Generation of zebrafish knockout mutant for the gdf5 gene.The endoskeletal disc segmentation is disrupted, causing posterior radials in the pectoral fins to be lost.Endoskeletal elements in the median fins are disorganized and reduced in length.The severity of the effect increases from anterior to posterior elements. [ABSTRACT FROM AUTHOR]

Abstract Background The dog is an important model organism and it is considered to be closer to humans than rodents regarding metabolism and responses to drugs. The close relationship between humans and dogs over many centuries has lead to the diversity of the canine species, important genetic discoveries and an appreciation of the effects of old age in another species. The superfamily of G protein-coupled receptors (GPCRs) is one of the largest gene families in most mammals and the most exploited in terms of drug discovery. An accurate comparison of the GPCR repertoires in dog and human is valuable for the prediction of functional similarities and differences between the species. Results We searched the dog genome for non-olfactory GPCRs and obtained 353 full-length GPCR gene sequences, 18 incomplete sequences and 13 pseudogenes. We established relationships between human, dog, rat and mouse GPCRs resolving orthologous pairs and species-specific duplicates. We found that 12 dog GPCR genes are missing in humans while 24 human GPCR genes are not part of the dog GPCR repertoire. There is a higher number of orthologous pairs between dog and human that are conserved as compared with either mouse or rat. In almost all cases the differences observed between the dog and human genomes coincide with other variations in the rodent species. Several GPCR gene expansions characteristic for rodents are not found in dog. Conclusion The repertoire of dog non-olfactory GPCRs is more similar to the repertoire in humans as compared with the one in rodents. The comparison of the dog, human and rodent repertoires revealed several examples of species-specific gene duplications and deletions. This information is useful in the selection of model organisms for pharmacological experiments.

Abstract Background The Adhesion G protein-coupled receptors (GPCRs) are membrane-bound receptors with long N termini. This family has 33 members in humans. Several Adhesion GPCRs are known to have important physiological functions in CNS development and immune system response mediated by large cell surface ligands. However, the majority of Adhesion GPCRs are still poorly studied orphans with unknown functions. Results In this study we performed the extensive tissue localization analysis of the entire Adhesion GPCR family in rat and mouse. By applying the quantitative real-time PCR technique we have produced comparable expression profile for each of the members in the Adhesion family. The results are compared with literature data and data from the Allen Brain Atlas project. Our results suggest that the majority of the Adhesion GPCRs are either expressed in the CNS or ubiquitously. In addition the Adhesion GPCRs from the same phylogenetic group have either predominant CNS or peripheral expression, although each of their expression profile is unique. Conclusion Our findings indicate that many of Adhesion GPCRs are expressed, and most probably, have function in CNS. The related Adhesion GPCRs are well conserved in their structure and interestingly have considerable overlap in their expression profiles, suggesting similarities among the physiological roles for members within many of the phylogenetically related clusters.

Abstract Background The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey. Results We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle. Conclusion This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.

Glycosaminoglycans are sulfated polysaccharide molecules, essential for many biological processes. The 6-O sulfation of glycosaminoglycans is carried out by carbohydrate 6-O sulfotransferases (C6OSTs), previously named Gal/GalNAc/GlcNAc 6-O sulfotransferases. Here, for the first time, we present a detailed phylogenetic reconstruction, analysis of gene synteny conservation and propose an evolutionary scenario for the C6OST family in major vertebrate groups, including mammals, birds, nonavian reptiles, amphibians, lobe-finned fishes, ray-finned fishes, cartilaginous fishes, and jawless vertebrates. The C6OST gene expansion likely started early in the chordate lineage, giving rise to four ancestral genes after the divergence of tunicates and before the emergence of extant vertebrates. The two rounds of whole-genome duplication in early vertebrate evolution (1R/2R) only contributed two additional C6OST subtype genes, increasing the vertebrate repertoire from four genes to six, divided into two branches. The first branch includes CHST1 and CHST3 as well as a previously unrecognized subtype, CHST16 that was lost in amniotes. The second branch includes CHST2 , CHST7 , and CHST5. Subsequently, local duplications of CHST5 gave rise to CHST4 in the ancestor of tetrapods, and to CHST6 in the ancestor of primates. The teleost-specific gene duplicates were identified for CHST1 , CHST2 , and CHST3 and are result of whole-genome duplication (3R) in the teleost lineage. We could also detect multiple, more recent lineage-specific duplicates. Thus, the vertebrate repertoire of C6OST genes has been shaped by gene duplications and gene losses at several stages of vertebrate evolution, with implications for the evolution of skeleton, nervous system, and cell–cell interactions. [ABSTRACT FROM AUTHOR]