Autonomy in specification of primordial germ cells and their passive translocation in the sea urchin

Abstract

SUMMARYThe process of germ line determination involves many conserved genes, yet is highly variable. Echinoderms are positioned at the base of Deuterostomia and are crucial to understanding these evolutionary transitions, yet the mechanism of germ line specification is not known in any member of the phyla. Here we demonstrate that small micromeres (SMics), which are formed at the fifth cell division of the sea urchin embryo, illustrate many typical features of primordial germ cell (PGC) specification. SMics autonomously express germ line genes in isolated culture, including selective Vasa protein accumulation and transcriptional activation of nanos; their descendants are passively displaced towards the animal pole by secondary mesenchyme cells and the elongating archenteron during gastrulation; Cadherin (G form) has an important role in their development and clustering phenotype; and a left/right integration into the future adult anlagen appears to be controlled by a late developmental mechanism. These results suggest that sea urchin SMics share many more characteristics typical of PGCs than previously thought, and imply a more widely conserved system of germ line development among metazoans. KEY WORDS: Vasa, PGC, Germ line, Cadherin, Sea urchin, Strongylocentrotus purpuratusAutonomy in specification of primordial germ cells and their passive translocation in the sea urchin Mamiko Yajima* and Gary M. Wessel* DEVELOPMENT 3787 RESEARCH ARTICLE PGC specification in sea urchin specification and clustering, and we conclude that several overarching mechanisms appear conserved between the SMic lineage and the more widely studied PGCs, such as Drosophila pole cells. MATERIALS AND METHODS Animals, embryos and larval cultureS. purpuratus were collected in Long Beach, CA, USA, and housed in aquaria containing artificial seawater (ASW; Coral Life Scientific Grade Marine Salt; Energy Savers Unlimited, Carson, CA, USA) at 16°C. Gametes were acquired by 0.5 M KCl injection. Eggs were collected in ASW and sperm were collected dry. To obtain embryos, fertilized eggs were cultured in ASW or Millipore-filtered seawater (MFSW) at 16°C. When early stage embryos were required for blastomere labeling, fertilization was performed in the presence of 1 mM 3-aminotriazol (Sigma, St Louis, MO, USA) to inhibit cross-linking of the fertilization envelope. Before labeling, envelopes were removed by gentle pipetting. Chemical treatment and immunolabeling

Deadenylase depletion protects inherited mRNAs in primordial germ cells

“…The sMic-enriched transcripts included Nanos and Delta, which have previously been identified as sMic-localized, as well as SpGcadherin, which is required for sMic fate in S. purpuratus and is enriched in the sMics of Lytechinus variegatus (Juliano et al, 2010;Miller and McClay, 1997;Oliveri et al, 2002;Yajima and Wessel, 2012). The sMic-enriched transcripts fell into diverse functional categories, but transcriptional regulation and RNA binding were overrepresented according to a gene set enrichment analysis (supplementary material Table S1).…”

Migration of sea urchin primordial germ cells

“…It was previously noted that the translocation of small micromeres in early gastrulation is passive and requires their adhesion to one another with G-cadherin (Yajima and Wessel, 2012). Here, we observe a transition beginning late in gastrulation at 43–49 HPF when the cells move from a tightly packed ball of cells to a loosely associated single line, potentially indicating the onset of their motility along the left/right axis.…”

“…Consistent with this hypothesis, small micromeres isolated from embryos at 43 and 48 HPF also bleb and extend filopodia. Remarkably, when small micromeres are isolated from the embryo much earlier in development and cultured with their sister cell lineage, the primary mesenchymal cells, they autonomously express germline genes, including vasa and nanos , yet did not activate a migratory program or produce cortical blebs (Yajima and Wessel, 2012). These results might indicate that the initiation of small micromere motility requires initial contact with neighboring cells or secreted signals but that it is self-maintaining once initiated.…”

“…G-cadherin is expressed at high levels in small micromeres (Miller and McClay, 1997). Morpholino knockdown of G-cadherin causes small micromeres to either trans-differentiate into PMCs or die (Yajima and Wessel, 2012). G-cadherin might be required for small micromeres to re-join the blastoderm before they migrate.…”

“…A previous study suggested that small micromeres translocate from the vegetal to the animal pole during early gastrulation by the movement of the elongating archenteron (Yajima and Wessel, 2012). Here we investigated whether autonomous small micromere migration could also play a role in their positioning within the embryo.…”