Expression of univin, a TGF-beta growth factor, requires ectoderm-ECM interaction and promotes skeletal growth in the sea urchin embryo

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-beta superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis.     

Duplication and distinct expression patterns of two thrombospondin-1 isoforms in teleost fishes

Two types of thrombospondin-1 (named TSP-1a and TSP-1b) were cloned from two species of teleosts, the Nile tilapia and medaka. Phylogenetic analysis of these TSP-1 sequences, together with those available from other vertebrates further demonstrated that two types of TSP-1 exist only in teleosts, extending the finding in fugu and tetraodon to two additional fish species. The expression of both genes was examined using tilapia at various developmental stages. Tilapia TSP-1a and TSP-1b were each expressed in a wide range of tissues examined. The early expression of TSP-1b in both XX and XY gonads from 5 dah (day after hatching) onwards suggested an important role in the formation of gonads, while the expression of TSP-1a only in ovaries during later stages of development (from 120 dah onwards) may suggest that TSP-1a is involved in oogenesis. During the 14-day spawning cycle, the expression of both types of TSP-1 exhibited distinct peaks at day 5 (peak of vitellogenesis) and day 12 (oocyte maturation). In situ hybridization analyses revealed differential expression, with TSP-1a occurring in granulosa cells and TSP-1b in theca cells. Furthermore, both TSP-1a and -1b were expressed in skeletal tissues but with clear temporal and spatial differences. In contrast, only TSP-1b was found in the myosepta. The positive signals of both TSP-1a and TSP-1b were also detected in the heart and spleen, and TSP-1a in brain and intestine by both RT-PCR and in situ hybridization.     

A switch in the cellular basis of skeletogenesis in late-stage sea urchin larvae

Primary mesenchyme cells (PMCs) are solely responsible for the skeletogenesis during early larval development of the sea urchin, but the cells responsible for late larval and adult skeletal formation are not clear. To investigate the origin of larval and adult skeletogenic cells, I first performed transplantation experiments in Pseudocentrotus depressus and Hemicentrotus pulcherrimus, which have different skeletal phenotypes. When P. depressus PMCs were transplanted into H. pulcherrimus embryos, the donor phenotype was observed only in the early larval stage, whereas when secondary mesenchyme cells (SMCs) were transplanted, the donor phenotype was observed in late and metamorphic larvae. Second, a reporter construct driven by the spicule matrix protein 50 (SM50) promoter was introduced into fertilized eggs and their PMCs/SMCs were transplanted. In the resultant 6-armed pluteus, green fluorescent protein (GFP) expression was observed in both PMC and SMC transplantations, suggesting SMC participation in late skeletogenesis. Third, transplanted PMCs or SMCs tagged with GFP were analyzed by PCR in the transgenic chimeras. As a result, SMCs were detected in both larval and adult stages, but GFP from PMCs was undetectable after metamorphosis. Thus, it appears that SMCs participate in skeletogenesis in late development and that PMCs disappear in the adult sea urchin, suggesting that the skeletogenesis may pass from PMCs to SMCs during the late larval stage.     

UVB radiation prevents skeleton growth and stimulates the expression of stress markers in sea urchin embryos

Ozone depletion results in an increased flux of biologically damaging radiations reaching the earth. Although ultraviolet (UV) penetration is attenuated by the seawater, harmful effects can be still observed at low depths where sea urchin embryos are living. We have used Paracentrotus lividus embryos to study the impacts of UV radiation on their development. Blastula cultures were exposed to different doses of UVB (312 nm) radiations and the resulting endpoint effects were evaluated in terms of embryonic morphological abnormalities, variations in specific gene expression, and changes in the levels of stress proteins. We found that embryos were moderately sensitive to 50 J/m² UVB radiation; an increase in the number of developmentally delayed and malformed embryos was detected when increasing doses, up to 1000 J/m², were used. Major developmental defects, observed 24 and 48 h after exposure, consisted in the failure of skeleton elongation and patterning. Accordingly, we found a reduction in the number of primary mesenchyme cells that expressed Pl-SM30, a gene coding for one of the specific matrix proteins of the skeleton. The morphological effects observed 1, 24, and 48 h after exposure were correlated with a dose-dependent increase in the level and in the activation of two recognized stress markers, namely hsp70 and p38 MAPk, respectively, consistent with their role in mediating cellular response to stress and suggesting a function in embryo survival.     

CCN proteins in the musculoskeletal system: current understanding and challenges in physiology and pathology

The acronym for the CCN family was recently revised to represent “cellular communication network”. These six, small, cysteine-enriched and evolutionarily conserved proteins are secreted matricellular proteins, that convey and modulate intercellular communication by interacting with structural proteins, signalling factors and cell surface receptors. Their role in the development and physiology of musculoskeletal system, constituted by connective tissues where cells are interspersed in the cellular matrix, has been broadly studied. Previous research has highlighted a crucial balance of CCN proteins in mesenchymal stem cell commitment and a pivotal role for CCN1, CCN2 and their alter ego CCN3 in chondrogenesis and osteogenesis; CCN4 plays a minor role and the role of CCN5 and CCN6 is still unclear. CCN proteins also participate in osteoclastogenesis and myogenesis. In adult life, CCN proteins serve as mechanosensory proteins in the musculoskeletal system providing a steady response to environmental stimuli and participating in fracture healing. Substantial evidence also supports the involvement of CCN proteins in inflammatory pathologies, such as osteoarthritis and rheumatoid arthritis, as well as in cancers affecting the musculoskeletal system and bone metastasis. These matricellular proteins indeed show involvement in inflammation and cancer, thus representing intriguing therapeutic targets. This review discusses the current understanding of CCN proteins in the musculoskeletal system as well as the controversies and challenges associated with their multiple and complex roles, and it aims to link the dispersed knowledge in an effort to stimulate and guide readers to an area that the writers consider to have significant impact and relevant potentialities.     

TALEN-mediated mutagenesis in zebrafish reveals a role for r-spondin 2 in fin ray and vertebral development

R-spondin (Rspo) encodes a multi-domain protein that modulates the Wnt-signaling pathway. Two distinct rspo2 zebrafish mutants were generated by TALEN-mediated mutagenesis: a null mutant, rspo2^null, lacking all functional domains, and a hypomorphic mutant, rspo2^tsp, lacking the two N-terminal domains. Mutants were analyzed mainly for abnormalities in the skeletal system. Fin ray skeletons were formed normally in the rspo2^tsp mutants, but were absent from the rspo2^null mutants. Hypoplasia of the neural/hemal arches and ribs was observed in both mutants. Thus, the two rspo2 mutants help to identify the functions of Rspo2 in skeletogenesis, as well as functional differences among multiple Rspo2 domains.