Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Abstract

Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.

Single-Cell RNA Sequencing Atlas From a Bivalve Larva Enhances Classical Cell Lineage Studies

“…Similar cluster diversity in neuronal cell types is found in single cell datasets from other metazoans such as the annelid Capitella teleta and the sea urchin Strongylocentrotus purpuratus. All neuronal cell types of these species share a baseline transcriptomic signature, with only enough differences in expression to subdivide the overall cluster into different categories, e.g., 12 neuronal subtypes in the sea urchin and four neural subgroups in C. teleta (Achim et al, 2018;Foster et al, 2020;Paganos et al, 2021). Moreover, when analyzing the cell lineage tree of Dreissena, our data highlights that the underlying transcriptomic signature is similar across lineages that give rise to the same fate (Figure 3C).…”

“…Ciliated cells found in the annelids Platynereis dumerilii, Hydroides elegans, and Capitella teleta similarly express orthologs of Caveolin, Forkhead box J (foxJ), rshp4, dynein heavy chain 9 (DNAH9), tubulin polymerization-promoting family member 3, and tektin4 (Supplementary Figure 4 and Supplementary Tables 1-3; Arenas-Mena et al, 2007;Achim et al, 2018;Sur and Meyer, 2021). Furthermore, genes expressed in ciliary cells of Dreissena are also present in motor cilia of more distantly related organisms such as the acoel Isodiametra pulchra (i.e., Dynein heavy chain and tubulin homologs) and in ciliary band cells of the sea urchin Strongylocentrotus purpuratus (e.g., foxJ) (Duruz et al, 2020;Paganos et al, 2021). This shows that the "ciliary/prototroch" cells in our data possess a distinct transcriptomic signature indicative of the ability to produce cilia, and that at least some of this molecular program was present in the last common ancestor of bivalves, annelids, sea urchins, and xenacoelomorphs.…”

“…Galectin has been reported to promote phagocytosis of pathogens as a defense mechanism present in all cells and in hemocytes in adult stages, playing a role in innate immune responses and intracellular digestion in aquatic mollusks (Vasta et al, 2015). Hnf4 is considered a marker of developing endoderm since its expression has been found in gut stem cells in several metazoans (Achim et al, 2018;Wendt et al, 2020;Paganos et al, 2021;Sur and Meyer, 2021) and was highly expressed exclusively in this cluster (in a limited number of cells only). Hhex is considered a regulator of hepatocyte development in deuterostomes (Wallace et al, 2001;Evseeva et al, 2021;Paganos et al, 2021) and was also found to be expressed in cells of this cluster (Supplementary Figure 4 and Supplementary Tables 1-3).…”

“…Hnf4 is considered a marker of developing endoderm since its expression has been found in gut stem cells in several metazoans (Achim et al, 2018;Wendt et al, 2020;Paganos et al, 2021;Sur and Meyer, 2021) and was highly expressed exclusively in this cluster (in a limited number of cells only). Hhex is considered a regulator of hepatocyte development in deuterostomes (Wallace et al, 2001;Evseeva et al, 2021;Paganos et al, 2021) and was also found to be expressed in cells of this cluster (Supplementary Figure 4 and Supplementary Tables 1-3). Although detection of these transcriptional markers is restricted to few cells, this is not uncommon for transcription factors, and overall the molecular fingerprint of this cluster is coherent with the developing gastrodermis.…”

“…Such studies have resulted in comprehensive lists of genes that are expressed during ontogeny (Tang et al, 2010;Trapnell et al, 2014;Achim et al, 2015;Satija et al, 2015;Trapnell, 2015;Vergara et al, 2017;Svensson et al, 2018;Zhong et al, 2018). Accordingly, recent studies have uncovered the presence of previously uncharacterized cell types and an unexpected transcriptomic heterogeneity amongst cell populations and have revealed numerous genes with hitherto unknown function (Karaiskos et al, 2017;Achim et al, 2018;Briggs et al, 2018;Farrell et al, 2018;Fincher et al, 2018;Plass et al, 2018;Sebé-Pedrós et al, 2018;Wagner et al, 2018;Foster et al, 2019Foster et al, , 2020Siebert et al, 2019;Duruz et al, 2020;Wendt et al, 2020;García-Castro et al, 2021;Paganos et al, 2021;Sur and Meyer, 2021).…”