Kornthong N et al. (2021), Identification and localization of growth facto...

ECB-ART-50665

Heliyon 2021 Nov 13;711:e08370. doi: 10.1016/j.heliyon.2021.e08370.

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Identification and localization of growth factor genes in the sea cucumber, Holothuria scabra.

Kornthong N , Phanaksri T , Saetan J , Duangprom S , Lekskul B , Vivattanasarn T , Songkoomkrong S , Jattujan P , Cummins SF , Sobhon P , Suwansa-Ard S .

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The sea cucumber Holothuria scabra is both an economically important species in Asian countries and an emerging experimental model for research studies in regeneration and medicinal bioactives. Growth factors and their receptors are known to be key components that guide tissue repair and renewal, yet validation of their presence in H. scabra has not been established. We performed a targeted in silico search of H. scabra transcriptome data to elucidate conserved growth factor family and receptor genes. In total, 42 transcripts were identified, of which 9 were validated by gene cloning and sequencing. The H. scabra growth factor genes, such as bone morphogenetic protein 2A (BMP 2A), bone morphogenetic protein 5-like (BMP5-like), neurotrophin (NT) and fibroblast growth factor 18 (FGF18), were selected for further analyses, including phylogenetic comparison and spatial gene expression using RT-PCR and in situ hybridization. Expression of all genes investigated were widespread in multiple tissues. However, BMP 2A, BMP5-like and NT were found extensively in the radial nerve cord cells, while FGF18 was highly expressed in connective tissue layer of the body wall. Our identification and expression analysis of the H. scabra growth factor genes provided the molecular information of growth factors in this species which may ultimately complement the research in regenerative medicine.

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	Figure 1. Occurrence of major growth factors in bilaterians. The occurrence of growth factors in TGF-β superfamily, NT family and FGF8/17/18 family in different taxa of deuterostomes and protostomes. Green and white boxes indicate, respectively, the presence and absence of a given growth factor in a taxonomic group. Asterisks indicate the H. scabra growth factors that were identified in this study. Accession numbers of all growth factors presented are provided in Supplement data 3.
	Figure 2. Comparative analysis of the H. scabra TGF-β2 protein sequence with other species. The multiple alignment of TGF-β2 of H. scabra proteins with TGF-β2 proteins in other species from chordates, hemichordates, molluscs, and other echinoderms. Black shading in the multiple alignment indicates conserved amino acids while grey shading indicates similar amino acids. The asterisks indicate H. scabra TGF-β2 found in this study. The sequence logo above the alignment shows the conservation of amino acid residues. Species abbreviations: Homsa, Homo sapiens; Musmu, Mus musculus; Sacko, Saccoglossus kowalevskii; Lotgi, Lottia gigantea; Apoja, Apostichopus japonicus; Lytva, Lytechinus variegatus; Strpu, Strongylocentrotus purpuratus; Acapl. Acanthaster planci; Holgl, Holothuria glaberrima; Holsc, Holothuria scabra. Accession numbers of TGF-β2 protein sequences presented are provided in Supplement data 4.
	Figure 3. Comparative analysis of the H. scabra BMP2A, BMP2/4 and BMP5-like protein sequences with other species. (A) Comparative analysis of the H. scabra BMP2A and BMP2/4 protein sequences with other species. The multiple alignment of BMP2A and BMP2/4 of H. scabra proteins with BMP2, BMP4, BMP2/4 and BMP2A proteins in other species from chordates, hemichordates, molluscs, insects, crustaceans, and echinoderms. (B) Comparative analysis of the H. scabra BMP5-like protein sequences with other species. The multiple alignment of BMP5-like of H. scabra proteins with BMP, BMP5, BMP5/8 and BMP7 proteins in other species from chordates, molluscs, crustaceans, and echinoderms. Black shading in the multiple alignment indicates conserved amino acids while grey shading indicates similar amino acids. The asterisks indicate H. scabra BMP2A BMP2/4 and BMP5-like that were found in this study. The sequence logo above the alignment shows the conservation of amino acid residues. Species abbreviations: Homsa, Homo sapiens; Musmu, Mus musculus; Xenla, Xenopus laevis; Salsa, Salmo salar; Anagr, Anabarilius graham; Brabe, Branchiostoma belcheri; Brafl, Branchiostoma floridae; Aplca, Aplysia californica; Cryse, Cryptotermes secundus; Armna, Armadillidium nasatum; Sacko, Saccoglossus kowalevskii; Cryse, Cryptotermes secundus; Lytva, Lytechinus variegatus; Strpu, Strongylocentrotus purpuratus; Parli, Paracentrotus lividus; Apoja, Apostichopus japonicus; Funhe, Fundulus heteroclitus; Xentr, Xenopus tropicalis; Mytco, Mytilus coruscus; Erisi, Eriocheir sinensis; Holgl, Holothuria glaberrima; Holsc, Holothuria scabra. Accession numbers of BMP protein sequences presented are provided in Supplement data 5.
	Figure 4. Comparative analysis of the H. scabra activin/inhibin and myostatin protein sequences with other species. (A) Comparative analysis of the H. scabra activin and inhibin protein sequences with other species. The multiple alignment of activin and inhibin of H. scabra proteins with activin and inhibin proteins in other species from chordates, hemichordates, molluscs, insects, crustaceans, and echinoderms. (B) Comparative analysis of the H. scabra myostatin protein sequence with other species. The multiple alignment of myostatin of H. scabra proteins with myostatin proteins in other species from chordates, hemichordates, molluscs, insects, crustaceans, and echinoderms. Black shading in the multiple alignment indicates conserved amino acids while grey shading indicates similar amino acids. The asterisks indicate H. scabra activin, inhibin and myostatin that were found in this study. The sequence logo above the alignment shows the conservation of amino acid residues. Species abbreviations: Homsa, Homo sapiens; Musmu, Mus musculus; Sacko, Saccoglossus kowalevski; Drome, Drosophila melanogaster; Dromi, Drosophila miranda; Armna, Armadillidium nasatum; Zoone, Zootermopsis nevadensis; Parli, Paracentrotus lividus; Apoja, Apostichopus japonicus; Saau, Sparus aurata; Pench, Penaeus chinensis; Feaoc, Frankliniella occidentalis; Pinim, Pinctada imbricata; Strpu, Strongylocentrotus purpuratus; Holgl, H. glaberrima; Holsc, Holothuria scabra. Accession numbers of activin, inhibin and myostatin protein sequences presented are provided in Supplement data 6.
	Figure 5. Phylogenetic tree analysis of TGF-β superfamily proteins from H. scabra and other species from different phyla. The phylogenetic tree was constructed based on the Neighbor-Joining method (number of amino acid substitution model). The number at the nodes represent bootstrap values at an approximate level of percent confidence from 1000 bootstrap replicates. The asterisks indicate H. scabra TGF-β2, myostatin, inhibin, activin, BMP2, BMP2/4 and BMP5-like that were found in this study.
	Figure 6. Comparative analysis of the H. scabra neurotrophin (NT) with other species. (A) The alignment of H. scabra NT protein with the ligands of NT family, including NGF, BDNF, and NT from other species of different phyla. Black shading in the multiple alignment indicates conserved amino acids while grey shading indicates similar amino acids. The blue letter C's indicate the conserved cysteine residues in the multiple sequence alignment. The sequence logo above the alignment shows the conservation of amino acid residues. (B) Phylogenetic tree analysis of H. scabra NT and the ligands of NT family of other species from different phyla. The phylogenetic tree was constructed based on the Neighbor-Joining method (number of amino acid substitution model). The number at the nodes represent bootstrap values at an approximate level of percent confidence from 1000 bootstrap replicates. The asterisks indicate H. scabra NT found in this study. Species abbreviations: Homsa, Homo sapiens; Musmu, Mus musculus; Ratno, Rattus norvegicus; Galga, Gallus gallus; Xentr, Xenopus tropicalis; Sacko, Saccoglossus kowalevskii; Aplca, Aplysia californica; Penmo, Penaeus monodon; Strpu, Strongylocentrotus purpuratus; Holgl, Holothuria glaberrima; Holsc, Holothuria scabra. Accession numbers of NGF, BDNF, and NT protein sequences presented are provided in Supplement data 7.
	Figure 7. Comparative analysis of H. scabra fibroblast growth factor 18 (FGF18). (A) The alignment of H. scabra FGF protein with the ligands of FGF family, including FGF, FGF8, FGF17, FGF18, FGF8/17/18 and FGF18-like from other species of different phyla. Black shading in the multiple alignment indicates conserved amino acids while grey shading indicates similar amino acids. The sequence logo above the alignment shows the conservation of amino acid residues. (B) Phylogenetic tree analysis of H. scabra FGF18 and the ligands of FGF family of other species from different phyla. The phylogenetic tree was constructed based on the Neighbor-Joining method (number of amino acid substitution model). The number at the nodes represent bootstrap values at an approximate level of percent confidence from 1000 bootstrap replicates. The asterisks indicate H. scabra FGF18. Species abbreviations: Homsa, Homo sapiens; Musmu, Mus musculus; Galga, Gallus gallus; Brala, Branchiostoma lanceolatum; Brafl, Branchiostoma floridae; Lytva, Lytechinus variegatus; Ampho, Amphipholis kochii; Mizye, Mizuhopecten yessoensis; Strpu, Strongylocentrotus purpuratus; Astru, Asterias rubens; Acapl, Acanthaster planci; Holgl, Holothuria glaberrima; Holsc, Holothuria scabra. Accession numbers of FGF protein sequences presented are provided in Supplement data 8.
	Figure 8. (A) Tissue-specific expression of H. scabra BMP2A, BMP2/4, BMP5-like, myostatin, inhibin, activin, TGF-β2, NT, and FGF18 mRNA using RT-PCR (amplicon sizes about 150–350 bp). The 16s rRNA expression was used as a positive control. The non-adjusted agarose gel images of H. scabra growth factors and 16s rRNA RT-PCR were provided in Supplement data 9. (B) Schematic diagram of H. scabra anatomy and the presence of various growth factors in each organ. The color code of circles in (B) represents different growth factors, which corresponds to those in (A). Abbreviations: radial nerve cord (RN), nerve ring (NR), inner part of the body wall (IW), testis (TT), ovary (OV), respiratory tree (RT), intestine (IN) and longitudinal muscle (LM). Negative controls (no template control; NTC).
	Figure 9. Histology and in situ hybridization of H. scabra BMP 2A, BMP5-like, NT, and FGF18 in H. scabra radial nerve cord and other structures of the inner part body wall. (A) Cross-section of the radial nerve cord (RN) with the inner part of the body wall (IW) stained with H&E, showing the anatomy of the RN, the longitudinal muscle band (LM), the water vascular canal (WVC), the hemal lacuna (H) and the connective tissue layer (CTL) of the body wall. (B) High magnification of the box in (A). (C) Negative control micrograph using a DIG-labeled sense-strand riboprobe showing no positive signal. (D-F) An anti-sense H. scabra BMP2A riboprobe showing positive signals in the area of RN, ectoneural neuroepithelium (EN), CTL and H, the positive staining the cells of EN indicated by the arrows. (G-I) An anti-sense H. scabra BMP5-like riboprobe showing the positive signals in the RN, NR and the connective tissue connecting the LM, the positive staining the cells of EN and NR indicated by the arrows. (J-L) An anti-sense H. scabra NT riboprobe showing positive signals in the RN and CTL; the positive staining in cells of the EN are indicated by arrows. (M-O) Low to high magnifications of anti-sense H. scabra FGF18 showing positive signals in the RN, H and CTL. Scale bars: 500 μm in A, D, J, and M; 200 μm in B, C, E, G, K and N; 100 μm in F, H, I, L and O.

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