Conservation and contrast in cell states of echinoderm ovaries. Conservation and contrast in cell states of echinoderm ovaries

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. Overall design: Adult Strongylocentrotus purpuratus were obtained from Pete Halmay of Pt. Loma Marine Invertebrate Lab (Lakeside, CA, e-mail: [e-mail]) and housed in aquaria with artificial seawater (ASW) at 16°C (Coral Life Scientific Grade Marine Salt; Carson, CA). Adult Lytechinus variegatus were obtained from the Pelagic Corporation ([e-mail]). They were housed in aquaria with ASW at room temperature (Coral Life Scientific Grade Marine Salt; Carson, CA). Adults Patiria miniata were collected by Josh Ross (South Coast Bio-Marine) off the Californian coast housed in aquaria with artificial seawater (ASW) at 16°C. Ovaries were collected from adult echinoderms and washed twice with calcium-free seawater (For 1 L: NaCl 26.5 gm; KCl 0.7 gm; MgSO4-7H2O 11.9 gm; NaHCO3 0.5 gm; pH to 8.0, salinity should be 34 ppt; McClay, 2004), and then suspended in hyalin-extraction media (HEM, for 1 L: NaCl 18.5 gm; KCl 0.7 gm; MgSO4-7H2O 11.9 gm; glycine 22.5 gm; Tris base 1.21 gm; EGTA 0.76 gm; pH to 8.0; 56) for 15 min. When cells were beginning to dissociate, the ovaries were collected and washed in 0.5M NaCl, gently sheared with a pipette, run through a 40 micron Nitex mesh, counted on a hemocytometer, and diluted to reach the appropriate concentration for the scRNA-seq protocol.

与女性不同，棘皮动物拥有令人惊叹的能力，可在终生持续产生功能性配子，部分个体的寿命甚至超过200年。目前学界已对棘皮动物卵巢的组织学与超微结构进行了描述，但这类卵巢如何运作并持续产生高质量配子仍是未解之谜。本研究首次公布了两种海胆物种——紫海胆（Strongylocentrotus purpuratus）与杂色海胆（Lytechinus variegatus），以及一种海星物种——小海燕海星（Patiria miniata）的单细胞RNA测序（single cell RNA sequencing，简称scRNAseq）数据集。我们在紫海胆卵巢中鉴定出14种细胞状态，杂色海胆卵巢中鉴定出16种，海星卵巢中则鉴定出13种。该数据集资源对于解析棘皮动物卵巢的结构与功能生物学特征至关重要，同时可为利用针对不同细胞类型的原位RNA杂交探针提供决策参考。我们将关键基因与特征图中的细胞簇进行关联，以此验证该实验方法的有效性。该资源还有助于鉴定出支持长期持续配子产生的干细胞，为研究年度生殖周期的变化奠定基础，并对理解这一重要动物门类的生殖演化具有关键意义。本数据集揭示的高度选择性基因表达特征，还明确了通过Cas9靶向基因敲除与敲入技术，以及解离卵巢细胞培养实验来探究各鉴定细胞类型功能的最高优先级基因靶点。实验设计概述：实验所用成年紫海胆购自加州莱克赛德市洛马角海洋无脊椎动物实验室的Pete Halmay（邮箱："[e-mail]"），饲养于人工海水（artificial seawater，简称ASW）水族箱中，水温维持在16℃，所用人工海水为Coral Life科研级海洋盐（购自加州卡森市）。成年杂色海胆购自Pelagic Corporation（邮箱："[e-mail]"），饲养于人工海水水族箱中，水温为室温，所用人工海水与前述一致。成年小海燕海星由South Coast Bio-Marine的Josh Ross于加利福尼亚沿海采集，饲养于人工海水水族箱中，水温维持在16℃。从成年棘皮动物体内采集卵巢，用无钙海水清洗两次（配方：每升含NaCl 26.5g、KCl 0.7g、MgSO₄·7H₂O 11.9g、NaHCO₃ 0.5g，pH调至8.0，盐度为34 ppt；参考McClay, 2004），随后置于透明蛋白提取液（hyalin-extraction media，简称HEM）中静置15分钟。当细胞开始解离时，收集卵巢并用0.5M NaCl溶液清洗，用移液管轻柔吹打分散细胞，将细胞悬液通过40微米Nitex滤网过滤，在血细胞计数板上计数细胞浓度，并稀释至适配单细胞RNA测序实验方案的浓度。