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Conservation and contrast in cell states of echinoderm ovaries

ABSTRACT: In contrast to women, echinoderms have the amazing ability to keep producing functional gametes throughout their lifespan, in some cases exceeding 200 years. The histology and ultrastructure of echinoderm ovaries has been described but how these ovaries function and maintain the production of high-quality gametes is still a mystery. Here, we present the first single cell RNA sequencing (scRNAseq) datasets of two sea urchin species (Strongylocentrotus purpuratus and Lytechinus variegatus) and one sea star species (Patiria miniata). We find 14 cell states in the Sp ovary, 16 cell states in the Lv ovary and 13 cell states in the ovary of the sea star. This resource is essential to understand the structure and functional biology of the ovary in echinoderms, and better informs decisions in the utilization of in situ RNA hybridization probes selective for various cell types. We link key genes with cell clusters of the feature plots in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production, is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum. Highly selective gene expression revealed by this dataset also divulges gene targets of highest priority for interrogating gene activities by Cas9-targeted gene knock-out and knock-in approaches and in dissociated ovarian cell cultures to test the function of each cell type identified.

ORGANISM(S): Strongylocentrotus purpuratus Lytechinus variegatus Patiria miniata

PROVIDER: GSE246430 | GEO | 2023/11/29

REPOSITORIES: GEO

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Project description:Purpose: The Tbrain transcription factor has demonstrated an evolved preference for low-affinity, secondary site binding motifs between the sea star and sea urchin orthologs. We sought to identify targets of sea urchin and sea star orthologs of Tbr. Because less is known about the function of Tbr during sea star development, we used RNA-seq in conjuction with ChIP-seq studies (GEO:xxxx) to determine the targets of sea star Tbr in early development. Methods: Sea star (Patiria miniata) embryos were injected with translation-blocking morpholino antisense oligonucleotides to knock-down PmTbr expression, as described previously. Control morpholinos were injected into sibling embryos. Embryos were allowed to develop until hatching (30-36 hpf) at which point injected embryos were collected and RNA was extracted. RNA-seq libraries were prepared, sequenced, and analyzed using standard protocols. Results: There are 2,562 genes that are significantly differentially expressed relative to control morpholino inected embryos (FDR < 0.05). There are roughly equivalent numbers of genes down-regulated (1,041) and up-regulated (1,521) by Pm-tbr knockdown, suggesting that PmTbr may act as both a transcriptional activator and repressor. 1,165 differentially expressed genes are located within 75 kb of a PmTbr binding site determined using ChIP-seq, and this set is used as a basis for comparison between sea star and sea urchin binding sites. Conclusions: 1,165 targets of the PmTbr transcription factor were identified based on differential expression following knockdown and the presence of transcription factor binding sites proximal to differentially expressed genes. There are an equal number of up- and down-regulated targets, suggesting Tbr may function as a transcriptional activator and repressor, depending on context and target gene. There was no clear association of motif utilization with either the direction of differential expression or ontological category of the target gene. There are only a small fraction of target genes (approximately 10%) that are in common between the sea star and sea urchin sets.

Project description:Luteinizing hormone (LH) activation of the EGF receptor/RAS/ERK1/2 pathway is essential for ovulation and luteinization because granulosa cell depletion of Erk1/2 (Erk1/2gc-/- mice) renders mice completely infertile. Because of their potential as mediators of ERK1/2-dependent granulosa cell differentiation, we disrupted genes encoding the CCAAT/enhancer-binding proteins, (C/EBP) a and C/EBPb. Female Cebpbgc-/- mutant mice but not Cebpagc-/- mice were subfertile whereas Cebpa/bgc-/- double mutant females were sterile. Follicles failed to ovulate, ovaries were devoid of corpora lutea, luteal cell marker genes (Lhcgr, Prlr, Ptgfr, Cyp11a1 and Star) were absent and serum progesterone levels were low. Microarray analyses identified numerous C/EBPa/b target genes in eCG-hCG treated mice. At 4h post-hCG, a subset (19%) of genes altered in the Cebpa/b depleted cells was also altered in Erk1/2 depleted cells; hence they are common effectors of ERK1/2. Additional genes down-regulated in the Cebpa/b depleted cells at 8 and 24h post-hCG include known (Akr1b7, Runx2, Star, Saa3) and novel (Abcb1b, Apln, Igfbp4, Prlr, Ptgfr Timp4) C/EBP target genes including effectors of vascular cell development. Bhmt, a gene controlling methionine metabolism and expressed exclusively in liver and kidney, was high in WT luteal cells but totally absent in Cebpa/b mutant cells. Because numerous genes potentially associated with vascular development were suppressed in the mutant cells, C/EBPa/b appear to dictate the luteinization process by also controlling genes that regulate the formation of the extensive vascular network required to sustain luteal cells. Thus, C/EBPa/b mediate several aspects of granulosa cell differentiation as well as the complex process of luteinization. Five granulosa cell treatments were included: CEBPM-NM-1/M-NM-2 conditional KO ovary, eCG-treated 48h, hCG 4h wild type ovary, eCG-treated 48h, hCG 8h CEBPM-NM-1/M-NM-2 conditional KO ovary, eCG-treated 48h, hCG 8h wild type ovary, eCG-treated 48h, hCG 24h CEBPM-NM-1/M-NM-2 conditional KO ovary, eCG-treated 48h, hCG 24h

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Conservation and contrast in cell states of echinoderm ovaries

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