Evolution of the extracellular matrix in deuterostomes and the influence of calcitropic hormones

Tese de dout., Biologia (Biologia Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

The extracellular matrix (ECM) is an essential component of all multicellular organisms. The present thesis characterized calcified tissue and its homeostasis considering ECM proteins and endocrine factors. From an evolutionary perspective homologues of mammalian calcified ECM genes were identified in teleost fish and aquatic bivalves (Crassostrea gigas and Laternula elliptica). Transcripts for collagens, fibronectin and thrombospondins and some factors with a possible role in calcium homeostasis and shell formation, e.g. calmodulin, bone morphogenetic protein (BMP) and calcitonin receptor-like (CLR) were identified in the bivalves. In the teleosts, sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) ECM constituents, like collagens and adhesive proteins were identified as well as homologues of genes involved in human skeletal metabolism and 3 CLR were identified. The role of CLR in fish is ambiguous and in silico promoter analysis was used to infer possible function and suggested teleost CLR1 is most linked to the skeleton and CLR2 to reproduction. Experimental models to explore calcified tissue homeostasis included vertebra, jaws, and branchial arches. Treatment of sea bream with the Selective Estrogen-Receptor Modulator (SERM), raloxifene caused down-regulation of skeletal related transcripts (Alkaline Phosphatase (ALP), Core binding factor alpha 1(Cbfa1) and Cartilage Intermediate Layer protein (CILP)) in the branchial arches but not in vertebra and jaws. Regeneration of the integument (skin and scales) was studied with a sea bream oligo array. The wound rapidly repaired and within 3 days re-epithelization had occurred and the scale pocket was sealed and 7 days after scale removal a thin regenerated scale was visible. Major alterations of the transcriptome occurred at day 3 after scale removal and it had almost returned to basal levels by day 7. Global transcriptome changes during regeneration grouped under biological processes like cell cycle, tissue morphogenesis and immune physiology. Overall, the results provide knowledge to guide future functional studies of calcified tissue homeostasis in invertebrates and vertebrates.