A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies

Elements of divergence in germline determination in closely related species

Summary: Evolutionary transitions are particularly important in development of the germ line, cells which directly impact sexual reproduction. Differences in the primordial germ cells (PGCs) of two sea urchin species were examined here by stage-matched, integrated, single cell RNA-seq (scRNA-seq) datasets. Even though both species rely on inherited mechanisms to specify their germ line, this analysis revealed a variety of differences in germline gene expression, including a broader expression of the germline factor Nanos2 (Nan2) in Lytechinus variegatus (Lv) compared to Strongylocentrotus purpuratus (Sp). In Sp, Nan2 mRNA expression is highly restricted to the PGCs by a lability element in its 3′UTR, which is lacking in the mRNA of Lv-Nan2, thus explaining the difference. We discovered that the Lv-Nan2 3′UTR instead leads to its specific translation in the PGCs. The results emphasize that regulatory mechanisms resulting in germline specification rely greatly on post-transcriptional restrictions of key gene products

Multidrug-resistant transport activity protects oocytes from chemotherapeutic agents and changes during oocyte maturation

Objective To determine the multidrug-resistant transporter (MDR) activity in oocytes and their potential role in oocyte susceptibility to chemotherapy. Design Experimental laboratory study. Setting University and academic center for reproductive medicine. Subject(s) Women with eggs retrieved for intracytoplasmic sperm injection cycles and adult female FVBN and B6C3F1 mouse strains. Intervention(s) Inhibition of MDR activity in oocytes. Main Outcome Measure(s) Efflux activity of MDRs with the use of quantitative fluorescent dye efflux, and oocyte cell death when exposed to chemotherapy. Result(s) Oocytes effluxed fluorescent reporters, and this activity was significantly reduced in the presence of the MDR inhibitor PSC 833. Geminal vesicle oocytes were more efficient at efflux than metaphase 2 oocytes. Human oocytes exposed to cyclophosphamide and PSC 833 showed cell death with the use of two different viability assays compared with control samples and those exposed to cyclophosphamide alone. Immunoblots detected MDR-1 in all oocytes, with the greatest accumulation in the geminal vesicle stage. Conclusion(s) Oocytes have a vast repertoire of active MDRs. The implications of this study are that these protective mechanisms are important during oogenesis and that these activities change with maturation, increasing susceptibility to toxicants. Future directions may exploit the up-regulation of these transporters during gonadotoxic therapy. © 2013 by American Society for Reproductive Medicine

Distinct transcriptional regulation of Nanos2 in the germ line and soma by the Wnt and delta/notch pathways

Specification of the primordial germ cells (PGCs) is essential for sexually reproducing animals. Although the mechanisms of PGC specification are diverse between organisms, the RNA binding protein Nanos is consistently required in the germ line in all species tested. How Nanos is selectively expressed in the germ line, however, remains largely elusive. We report that in sea urchin embryos, the early expression of Nanos2 in the PGCs requires the maternal Wnt pathway. During gastrulation, however, Nanos2 expression expands into adjacent somatic mesodermal cells and this secondary Nanos expression instead requires Delta/Notch signaling through the forkhead family member FoxY. Each of these transcriptional regulators were tested by chromatin immunoprecipitation analysis and found to directly interact with a DNA locus upstream of Nanos2. Given the conserved importance of Nanos in germ line specification, and the derived character of the micromeres and small micromeres in the sea urchin, we propose that the ancestral mechanism of Nanos2 expression in echinoderms was by induction in mesodermal cells during gastrulation

Exploitation of the chick embryo chorioallantoic membrane (CAM) as a platform for anti-metastatic drug testing

International audienceThe establishment of clinically relevant models for tumor metastasis and drug testing is a major challenge in cancer research. Here we report a physiologically relevant assay enabling quantitative analysis of metastatic capacity of tumor cells following implantation into the chorioallantoic membrane (CAM). Engraftment of as few as 10 3 non-small cell lung cancer (NSCLC) and prostate cancer (PCa) cell lines was sufficient for both primary tumor and metastasis formation. Standard 2D-imaging as well as 3D optical tomography imaging were used for the detection of fluorescent metastatic foci in the chick embryo. H2228- and H1975-initiated metastases were confirmed by genomic analysis. We quantified the inhibitory effect of docetaxel on LNCaP, and that of cisplatin on A549- and H1299-initiated metastatic growths. The CAM assay also mimicked the sensitivity of ALK -rearranged H2228 and EGFR- mutated H1975 NSCLC cells to tyrosine kinase inhibitors crizotinib and gefitinib respectively, as well as sensitivity of LNCaP cells to androgen-dependent enzalutamide therapy. The assay was suggested to reconstitute the bone metastatic tropism of PCa cells. We show that the CAM chick embryo model may be a powerful preclinical platform for testing and targeting of the metastatic capacity of cancer cells