Emergence of a Thrombospondin Superfamily at the Origin of Metazoans

Abstract

Extracellular matrix (ECM) is considered central to the evolution of metazoan multicellularity; however, the repertoire of ECM proteins in nonbilaterians remains unclear. Thrombospondins (TSPs) are known to be well conserved from cnidarians to vertebrates, yet to date have been considered a unique family, principally studied for matricellular functions in vertebrates. Through searches utilizing the highly conserved C-terminal region of TSPs, we identify undisclosed new families of TSP-related proteins in metazoans, designated mega-TSP, sushi-TSP, and poriferan-TSP, each with a distinctive phylogenetic distribution. These proteins share the TSP C-terminal region domain architecture, as determined by domain composition and analysis of molecular models against known structures. Mega-TSPs, the only form identified in ctenophores, are typically >2,700 aa and are also characterized by N-terminal leucine-rich repeats and central cadherin/immunoglobulin domains. In cnidarians, which have a well-defined ECM, Mega-TSP was expressed throughout embryogenesis in Nematostella vectensis , with dynamic endodermal expression in larvae and primary polyps and widespread ectodermal expression in adult Nematostella vectensis and Hydra magnipapillata polyps. Hydra Mega-TSP was also expressed during regeneration and siRNA-silencing of Mega-TSP in Hydra caused specific blockade of head regeneration. Molecular phylogenetic analyses based on the conserved TSP C-terminal region identified each of the TSP-related groups to form clades distinct from the canonical TSPs. We discuss models for the evolution of the newly defined TSP superfamily by gene duplications, radiation, and gene losses from a debut in the last metazoan common ancestor. Together, the data provide new insight into the evolution of ECM and tissue organization in metazoans.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

“…We next asked whether HAS-7 depletion perturbs normal axis formation as a consequence of diminished HyWnt3 processing. siRNA gene expression knockdown by electroporation of adult polyps has recently been established as a robust method in Hydra [12,26,27]. We designed siRNAs targeting conserved motifs in the HAS-7 pro-domain (siRNA1), catalytic domain (siRNA2) and a less conserved region in the C-terminal ShKT domain (siRNA3).…”

A Wnt-specific astacin proteinase controls head formation inHydra

“…We next asked whether HAS-7 depletion perturbs normal axis formation as a consequence of diminished HyWnt3 processing. siRNA gene expression knockdown by electroporation of adult polyps has recently been established as a robust method in Hydra [12,25,26]. We designed siRNAs targeting conserved motifs in the HAS-7 prodomain (siRNA1), catalytic domain (siRNA2) and a less conserved region in the Cterminal ShKT domain (siRNA3) (sequences are given in Additional file 7: Table S4).…”

“…To be able to monitor the electroporation efficiency in the endoderm we used an endodermal actin::GFP/ecdodermal actin::RFP transgenic strain (reverse watermelon) and applied siGFP in combination with target gene siRNAs [12,25]. Although the knockdown phenotype for the strongly expressed GFP transgene is largely half-sided, we have evidence from previous studies that for native target genes the effect can be systemic [26]. To monitor the gene knockdown at protein level, we raised a polyclonal antibody against a unique epitope in the HAS-7 primary sequence located between the catalytic domain and the N-terminal ShKT domain (APPTAGPTISPT).…”

Modelling the early evolution of extracellular matrix from modern Ctenophores and Sponges

“…Williams et al [13] established that three species of these protists express distinct sets of predicted secreted proteins (identified by presence of a N-terminal secretory signal peptide and no transmembrane domain), none of which have a domain composition equivalent to a metazoan ECM protein, although individual domains common in ECM proteins are present. Specific examples are the separate domains of fibrillar collagens [15] or thrombospondins [16]. These data suggest that gene rearrangement and domain shuffling had an important role in the emergence of the large, multidomain, secreted proteins that characterise the metazoan 4 ECM.…”

“…In contrast, poriferans were confirmed to encode a wider repertoire of ECM proteins including fibrillar-like collagens of various domain architectures and fibrillin, as well as SPARC and one or more thrombospondin superfamily members (see [16] for details of the thrombospondin superfamily). Collagen Silecateins were identified as expected in the demosponge A. queenslandica and also in the other species examined ( Table 3).…”