Germ lineage properties in the urochordate Botryllus schlosseri – From markers to temporal niches

Abstract

The primordial germ cells (PGCs) in the colonial urochordate Botryllus schlosseri are sequestered in late embryonic stage. PGC-like populations, located at any blastogenic stage in specific niches, inside modules with curtailed lifespan, survive throughout the life of the colony by repeated weekly migration to newly formed buds. This cyclical migration and the lack of specific markers for PGC-like populations are obstacles to the study on PGCs. For that purpose, we isolated the Botryllus DDX1 (BS-DDX1) and characterized it by normal expression patterns and by specific siRNA knockdown experiments. Expression of BS-DDX1 concurrent with BS-Vasa, γ-H2AX, BS-cadherin and phospho-Smad1/5/8, demarcate PGC cells from soma cells and from more differentiated germ cells lineages, which enabled the detection of additional putative transient niches in zooids. Employing BS-cadherin siRNA knockdown, retinoic acid (RA) administration or β-estradiol administration affirmed the BS-Vasa(+)BS-DDX1(+)BS-cadherin(+)γ-H2AX(+)phospho-Smad1/5/8(+) population as the B. schlosseri PGC-like cells. By striving to understand the PGC-like cells trafficking between transient niches along blastogenic cycles, CM-DiI-stained PGC-like enriched populations from late blastogenic stage D zooids were injected into genetically matched colonial ramets at blastogenic stages A or C and their fates were observed for 9 days. Based on the accumulated data, we conceived a novel network of several transient and short lived 'germ line niches' that preserve PGCs homeostasis, protecting these cells from the weekly astogenic senescence processes, thus enabling the survival of the PGCs throughout the organism's life.

The involvement of three signal transduction pathways in botryllid ascidian astogeny, as revealed by expression patterns of representative genes

“…The p-Smad1/5/8 expression pattern has already been studied in B. schlosseri with emphasis on the germ lineage (Rosner et al, 2013). In the present study, extremely faint staining with p-Smad1/5/8 was detected within the embryonic tissues (white arrows; Figs.…”

“…Inhibition of TGF-b/Activin pathways reduced p-Smad2 staining, also resulting in malformation in the primary buds along the A/P axis: degenerated/malformed tissue at the anterior part of the buds (endostyle, branchial sac), with healthy looking tissues at the posterior part (stomach).The L/R axis was also affected as the stomach and intestine shifted to the right following the formation of large cavities. On the colonial level, system unit formation was lost and replaced by a chaotic distribution of the zooids, resembling changes that were induced in B. schlosseri colonies by retinoic acid treatment (Rosner et al, 2013).…”

“…(Rosner et al, 2013). The previously undetermined specificities of the other antibodies were resolved in this study; We've shown a high sequence similarity between putative human derived immunogens (the exact sequences are not provided by the manufacturers), which served to elicit antibodies for each of the antigens p-Smad2, p-Mek1/2 or b-catenin and corresponding B. schlosseri orthologues (Fig.…”

“…In the germ lineage, cytoplasmic and nuclear expressions were detected in the PGC-like (primordial germ cells; Rosner et al, 2013) or gonia cells (Fig. 3P).…”

“…Total proteins were extracted as described by Rosner et al, (2013). Tissues were boiled for 3 min before being loaded on SDS-PAGE.…”