Evolution of the angiopoietin-like gene family in teleosts and their role in skin regeneration
Genome and EST database searches
Homologues of human angiopoietin-like (ANGPTL) family members were procured in 15 fish genome assemblies (Additional file 1: Table S1). Using as queries the deduced mature protein sequences of human ANGPTLs, ten teleost genomes were explored, nine of which were available from Ensembl [44], accessed in May 2015, and included: two puffer fishes (Tetraodon nigroviridis, Takifugu rubripes), stickleback (Gasterosteus aculeatus), Nile tilapia (Oreochromis niloticus), medaka (Oryzias latipes), platyfish (Xiphophorus maculatus), Atlantic cod (Gadus morhua), cavefish (Astyanax mexicanus) and zebrafish (Danio rerio) and the sea bass (Dicentrarchus labrax) assessed from the sea bass genome assembly [45]. Searches were complemented by mining additional fish genomes at Ensembl [44], accessed in May 2015, a basal ray-finned fish, the spotted gar (Lepisosteus oculatus), the coelacanth (Latimeria chalumnae) that is basal to the tetrapod lineage and a jawless fish, the marine lamprey (Petromyzon marinus). The genome of two cartilaginous fishes the elephant shark (Callorhinchus milii, http://esharkgenome.imcb.a-star.edu.sg/) and little skate (Leucoraja erinacea, http://skatebase.org/) were also analysed.
To assess angptl gene family evolution, searches were extended to genomes of terrestrial vertebrates and invertebrates (early deuterostomes, protostomes and early metazoan). This included 4 terrestrial vertebrates (the amphibian Xenopus tropicalis, the reptile the Anole lizard, Anolis carolinensis, the chicken, Gallus gallus and two mammalians: the marsupial opossum Monodelphis domestica and the placental mouse, Mus musculus available from Ensembl [44] and accessed in May 2015); 4 early deuterostomes (the hemichordate acorn worm, Saccoglossus kowalevskii [46], accessed in May 2015; the echinoderm sea urchin, Strongylocentrotus purpuratus [47], accessed in May 2015; the cephalochordate amphioxus, Branchiostoma floridae [48], accessed in May 2015; and the urochordate Ciona, Ciona intestinalis [44], accessed in May 2015); 11 protostomes (two annelids, Capitela teleta and Helobdella robusta; two molluscs Crassostrea gigas and Lottia gigantea; 5 arthropods the Daphnia pulex, Ixodes scapularis, Tribolium castaneum, Drosophila melanogaster, Anopheles gambiae, the nematode Caernohabditis elegans and the platyhelminth Schistosoma mansoni) and 2 early metazoans (the cnidarian, Nematostella vectensis and the porifera, Amphimedon queenslandica) were accessed from the Ensembl genomes database [44], accessed in May 2015. Searches for putative angptl-like transcripts for the target invertebrate species were also performed at the NCBI database [49] using the deduced protein of human ANGPTL against the species-specific nucleotide collections (nr/nt). The identity of all retrieved sequences as ANGPTL family members was confirmed by reverse blast searches against the human NCBI non-redundant protein sequence [45] database.
To aid in the identification of angptl candidates with a functional role in fish skin, the deduced sea bass Angptl protein sequences were used to identify angptl transcripts isolated from skin EST libraries using a tblastn query against the teleost EST collection [50] (taxid:32443). The EST sequence hits with e???70 score were retained and their identity was confirmed by reverse blast against the human genome. Microarray probes modified in a sea bream skin/scale regeneration experiment [8] and a transcriptome assembly of sea bass skin (Patricia Pinto, personal communication) were also analysed for skin angptl candidates. For the skin expression studies, the angptl family members from the gilthead sea bream (Sparus aurata) were identified from the species-specific NCBI EST database subset [50] (taxid:8175) and a sea bream transcriptome assembly prepared from multiple tissues [51].