Single cell RNA seq of the major cell types in the larva of the sea star, Patiria miniata

Echinoderms are invertebrate deuterostomes closely related to chordates and have become a tractable model for study of the evolution of mechanisms involved in development, primordial germ cell specification, and regeneration. Sea urchins rely on inherited mechanisms for germline formation while sea stars rely instead on cell-cell inductive signaling mechanisms. Here, we present a single cell RNA sequencing of the sea star Patiria miniata day3 larva, and its integration into the datasets of the first four days of development. We identified each cell cluster of the larva using marker genes for in situ RNA hybridization and found that, surprisingly, the primordial germ cells share many gene expression profiles with cells in the coelomic pouches, and that the ectodermal epithelium is quite heterogenous. This dataset from the sea star provides a developmental trajectory of gene expression leading to each major cell type in the larva, providing a foundation for comparative analysis with other echinoderm species in parsing out mechanisms of developmental specification, regeneration, and germ line formation. Overall design: Adult P. miniata animals were collected by Peter Halmay (San Diego Fishermen's Working Group). Embryos were cultured as described previously (Fresques et al., 2016). Embryos were cultured in filtered (0.2 µm) sea water collected at the Marine Biological Laboratories in Woods Hole, MA, USA, until the appropriate stage for dissociation. All embryos used in the study resulted from the mating of one male and one female to ensure complete comparative capability. Multiple fertilizations were initiated in this study and timed such that the appropriate stages of embryonic development were reached at a common endpoint. The embryos were then collected and washed twice with calcium-free sea water, and then suspended in hyalin-extraction media (HEM) for 10-15 min, depending on the stage of dissociation. When cells were beginning to dissociate, the embryos were collected and washed in 0.5 M NaCl, gently sheared with a pipette, run through a 40 µm Nitex mesh, counted on a hemocytometer and diluted to reach the appropriate concentration for the scRNA-seq protocol. Equal numbers of embryos were used in each time point and at no time were cells or embryos pelleted in a centrifuge.