Developmental Single-Cell RNA Sequencing in the Sea Urchin Species Lytechinus variegatus and Heliocidaris erythrogramma

The process by which a single cell develops into a complex multicellular organism with specified cell types and well-defined cellular roles is not completely understood, and the evolution of that process is even more enigmatic. In sea urchin embryos, cell fate specification and differentiation of cell types occurs through gene regulatory networks, or circuits of nodes or genes that interact with and regulate each other’s DNA elements. However, what remains unclear is how a highly conserved developmental GRN can change over evolutionary time and how that can result in altered embryonic form and function.To begin to understand these processes, we developed methods to compare two relatively simple, yet complex sea urchin embryos with radically different life history strategies, the planktotrophic (conserved) Lytechinus variegatus and lecithotrophic (derived) Heliocidaris erythrogramma. To do this, single cell RNA sequencing methods were developed and adapted for the two species to address to what degree the developmental gene regulatory network genes were present, or altered when compared with the known planktotrophic dGRN. Developmental GRN information and gene signatures were applied to assign cell identities, and were vital to the identification of prospective co-expressed candidate dGRN nodes that could participate in a developmental context. With developmental atlases completed in the two species and dGRN nodes examined in both, we then compared gene signatures and dGRN nodes directly to identify prospective candidate dGRN nodes that could participate in the evolution of specification processes. We identified various novel candidates for dGRN analysis and found that, despite the obvious morphological differences, most of the dGRN circuits were conserved. In particular, we found that general specification events are delayed in Heliocidaris erythrogramma; this delay is especially prominent in the skeletogenic precursors, and the number of cells present is greatly reduced—by a factor of 10. In addition, the order of cell specification events, as shown by gene signatures, developmental sub clustering, and integrated analyses, indicate that pigment cells are among the first cell types to be specified, a finding in sharp contrast to known planktotrophic developers. Lastly, this work creates a unified framework of sea urchin development using a novel integrated model based on 1:1 gene orthologs. The application of single cell RNA-seq is a highly useful technique; when applied to a single developmental time series, it can yield insight into the genes and networks deployed over developmental time. Further, when applied to a second developmental time course, it becomes possible to uncover information about the evolution of development, as this application allows us sharp discernment into the genes and networks deployed over evolutionary time.