A START-domain-containing protein is a novel marker of nervous system components of the sea cucumber Holothuria glaberrima

Abstract

One of the main challenges faced by investigators studying the nervous system of members of the phylum Echinodermata is the lack of markers to identify nerve cells and plexi. Previous studies have utilized an antibody, RN1, that labels most of the nervous system structures of the sea cucumber Holothuria glaberrima and other echinoderms. However, the antigen recognized by RN1 remained unknown. In the present work, the antigen has been characterized by immunoprecipitation, tandem mass spectrometry, and cDNA cloning. The RN1 antigen contains a START lipid-binding domain found in Steroidogenic Acute Regulatory (StAR) proteins and other lipid-binding proteins. Phylogenetic tree assembly showed that the START domain is highly conserved among echinoderms. We have named this antigen HgSTARD10 for its high sequence similarity to the vertebrate orthologs. Gene and protein expression analyses revealed an abundance of HgSTARD10 in most H. glaberrima tissues including radial nerve, intestine, muscle, esophagus, mesentery, hemal system, gonads and respiratory tree. Molecular cloning of HgSTARD10, consequent protein expression and polyclonal antibody production revealed the STARD10 ortholog as the antigen recognized by the RN1 antibody. Further characterization into this START domain-containing protein will provide important insights for the biochemistry, physiology and evolution of deutorostomes.

Intestine Explants in Organ Culture: A Tool to Broaden the Regenerative Studies in Echinoderms

“…Overall, the gut explant culture method described here is suitable for short-term studies on critical aspects of the intestine regeneration process. The presence of mesothelial, neural, and extracellular matrix components in cultured explants similar to those observed in non-cultured explants and in vivo tissues [16][17][18][19][20][21][22] is a remarkable finding. Explants retain their morphology and histoarchitecture for up to 10 days, with minor changes.…”

“…Then the tissues were blocked with normal goat serum, and subsequently they were incubated ON at room temperature in a humid chamber with the primary antibodies shown in Table 1. These antibodies have been widely used to identify different cell populations in holothurian tissues in in vivo studies [16][17][18][19][20][21][22]. Next day, the slides were washed three times with PBS for 15 min each wash. Then, the tissue sections were incubated with the secondary antibody goat anti-mouse labeled with Cy3 (GAM-Cy3, 1:1000) or goat anti-rabbit labeled with Cy3 (GAR-Cy3, 1:1000) from Jackson ImmunoResearch Labs (West Grove, PA, USA) for an hour at room temperature.…”

Single-cell RNA sequencing of the holothurian regenerating intestine reveals the pluripotency of the coelomic epithelium

“…These cells are shown to be expressing neuroepithelial or neuronal genes such as neurotrypsin ( NETR ), potassium gated-voltage channels, PRD10 (PRDM10 - PR domain zinc finger protein 10 ) , ELAV2 and STA10 (STARD10 - START domain-containing protein 10). The latter is a protein that we have characterized as being expressed by enteric neurons and nerve bundles (Rosado-Olivieri et al, 2017) (Fig 6A). The holothurian STA10 (STARD10) is recognized by our monoclonal antibody RN1.…”

“…An additional population in our dataset, is the neuroepithelial population (C9). This population has STARD10 , among its top expressed genes, known to be a phospholipid transfer protein present in a neural cell population localized in the coelomic epithelium of the intestine (García-Arrarás et al, 2019; Rosado-Olivieri et al, 2017). Furthermore, cells of this cluster have high expression of other genes reported to be expressed by neuronal cells of regenerative and developmental tissues, such as beta-tubulin in developing human gut and sea urchin larva, and synapsin in planaria regenerating tissue (Cocurullo et al, 2023; Elmentaite et al, 2020; King et al, 2024).…”

“…An additional population in our dataset, is the neuroepithelial population (C9). This population has STARD10, among its top expressed genes, known to be a phospholipid transfer protein present in a neural cell population localized in the coelomic epithelium of the intestine (García-Arrarás et al, 2019;Rosado-Olivieri et al, 2017).…”