Dataset Information

Proteomics analysis of the zebrafish skeletal extracellular matrix.

ABSTRACT: The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development.

SUBMITTER: Kessels MY

PROVIDER: S-EPMC3946537 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Proteomics analysis of the zebrafish skeletal extracellular matrix.

Kessels Maurijn Y MY Huitema Leonie F A LF Boeren Sjef S Kranenbarg Sander S Schulte-Merker Stefan S van Leeuwen Johan L JL de Vries Sacco C SC

PloS one 20140307 3

The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular ...[more]

PMID: 24608635

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Project description:Craniofacial and skeletal dysmorphologies account for the majority of birth defects. A number of the disease phenotypes have been attributed to abnormal synthesis, maintenance and composition of extracellular matrix (ECM), yet the molecular and cellular mechanisms causing these ECM defects remain poorly understood. The zebrafish feelgood mutant manifests a severely malformed head skeleton and shortened body length due to defects in the maturation stage of chondrocyte development. In vivo analyses reveal a backlog of type II and type IV collagens in rough endoplasmic reticulum (ER) similar to those found in coat protein II complex (COPII)-deficient cells. The feelgood mutation hinders collagen deposition in the ECM, but trafficking of small cargos and other large ECM proteins such as laminin to the extracellular space is unaffected. We demonstrate that the zebrafish feelgood mutation causes a single amino acid substitution within the DNA-binding domain of transcription factor Creb3l2. We show that Creb3l2 selectively regulates the expression of genes encoding distinct COPII proteins (sec23a, sec23b and sec24d) but find no evidence for its regulation of sec24c expression. Moreover, we did not detect activation of ER stress response genes despite intracellular accumulation of collagen and prominent skeletal defects. Promoter trans-activation assays show that the Creb3l2 feelgood variant is a hypomorphic allele that retains approximately 50% of its transcriptional activity. Transgenic rescue experiments of the feelgood phenotype restore craniofacial development, illustrating that a precise level of Creb3l2 transcriptional activity is essential for skeletogenesis. Our results indicate that Creb3l2 modulates the availability of COPII machinery in a tissue- and cargo-specific manner. These findings could lead to a better understanding of the etiology of human craniofacial and skeletal birth defects as well as adult-onset diseases that are linked to dysregulated ECM deposition, such as arthritis, fibrosis or osteoporosis.

Project description:Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.

Dataset Information

Proteomics analysis of the zebrafish skeletal extracellular matrix.

Publications

Proteomics analysis of the zebrafish skeletal extracellular matrix.

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