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The developmental gene regulatory networks (dGRNs) of two sea urchin species,Lytechinus variegatus (Lv)andStrongylocentrotus purpuratus (Sp),have remained remarkably similar despite about 50 million years since a common ancestor. Hundreds of parallel experimental perturbations of transcription factors with similar outcomes support this conclusion. A recent scRNA-seq analysis suggested that the earliest expression of several genes within the dGRNs differs betweenLvandSp. Here, we present a careful reanalysis of the dGRNs in these two species, paying close attention to timing of first expression. We find that initial expression of genes critical for cell fate specification occurs during several compressed time periods in both species. Previously unrecognized feedback circuits are inferred from the temporally corrected dGRNs. Although many of these feedbacks differ in location within the respective GRNs, the overall number is similar between species. We identify several prominent differences in timing of first expression for key developmental regulatory genes; comparison with a third species indicates that these heterochronies likely originated in an unbiased manner with respect to embryonic cell lineage and evolutionary branch. Together, these results suggest that interactions can evolve even within highly conserved dGRNs and that feedback circuits may buffer the effects of heterochronies in the expression of key regulatory genes.

 

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to ‘stitch’ together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate.

 

Abstract Lytechinus variegatus is a camarodont sea urchin found widely throughout the western Atlantic Ocean in a variety of shallow-water marine habitats. Its distribution, abundance, and amenability to developmental perturbation make it a popular model for ecologists and developmental biologists. Here, we present a chromosomal-level genome assembly of L. variegatus generated from a combination of PacBio long reads, 10× Genomics sequencing, and HiC chromatin interaction sequencing. We show L. variegatus has 19 chromosomes with an assembly size of 870.4 Mb. The contiguity and completeness of this assembly are reflected by a scaffold length N50 of 45.5 Mb and BUSCO completeness score of 95.5%. Ab initio and transcript-informed gene modeling and annotation identified 27,232 genes with an average gene length of 12.6 kb, comprising an estimated 39.5% of the genome. Repetitive regions, on the other hand, make up 45.4% of the genome. Physical mapping of well-studied developmental genes onto each chromosome reveals nonrandom spatial distribution of distinct genes and gene families, which provides insight into how certain gene families may have evolved and are transcriptionally regulated in this species. Lastly, aligning RNA-seq and ATAC-seq data onto this assembly demonstrates the value of highly contiguous, complete genome assemblies for functional genomics analyses that is unattainable with fragmented, incomplete assemblies. This genome will be of great value to the scientific community as a resource for genome evolution, developmental, and ecological studies of this species and the Echinodermata.