Evolutionary rewiring of gene regulatory network linkages at divergence of the echinoid subclasses

Evolution of animal body plans occurs with changes in the encoded genomic programs that direct development, by alterations in the structure of encoded developmental gene-regulatory networks (GRNs). However, study of this most fundamental of evolutionary processes requires experimentally tractable, phylogenetically divergent organisms that differ morphologically while belonging to the same monophyletic clade, plus knowledge of the relevant GRNs operating in at least one of the species. These conditions are met in the divergent embryogenesis of the two extant, morphologically distinct, echinoid (sea urchin) subclasses, Euechinoidea and Cidaroidea, which diverged from a common late Paleozoic ancestor. Here we focus on striking differences in the mode of embryonic skeletogenesis in a euechinoid, the well-known model Strongylocentrotus purpuratus (Sp), vs. the cidaroid Eucidaris tribuloides (Et). At the level of descriptive embryology, skeletogenesis in Sp and Et has long been known to occur by distinct means. The complete GRN controlling this process is known for Sp. We carried out targeted functional analyses on Et skeletogenesis to identify the presence, or demonstrate the absence, of specific regulatory linkages and subcircuits key to the operation of the Sp skeletogenic GRN. Remarkably, most of the canonical design features of the Sp skeletogenic GRN that we examined are either missing or operate differently in Et. This work directly implies a dramatic reorganization of genomic regulatory circuitry concomitant with the divergence of the euechinoids, which began before the end-Permian extinction

Conserved regulatory state expression controlled by divergent developmental gene regulatory networks in echinoids

Evolution of the animal body plan is driven by changes in developmental gene regulatory networks (GRNs), but how networks change to control novel developmental phenotypes remains, in most cases, unresolved. Here, we address GRN evolution by comparing the endomesoderm GRN in two echinoid sea urchins, Strongylocentrotus purpuratus and Eucidaris tribuloides, with at least 268 million years of independent evolution. We first analyzed the expression of twelve transcription factors and signaling molecules of the S. purpuratus GRN in E. tribuloides embryos, showing that orthologous regulatory genes are expressed in corresponding endomesodermal cell fates in the two species. However, perturbation of regulatory genes revealed that important regulatory circuits of the S. purpuratus GRN are significantly different in E. tribuloides. For example, mesodermal Delta/Notch signaling controls exclusion of alternative cell fates in E. tribuloides but controls mesoderm induction and activation of a positive feedback circuit in S. purpuratus. These results indicate that the architecture of the sea urchin endomesoderm GRN evolved by extensive gain and loss of regulatory interactions between a conserved set of regulatory factors that control endomesodermal cell fate specification

Reorganization of sea urchin gene regulatory networks at least 268 million years ago as revealed by oldest fossil cidaroid echinoid

Echinoids, or sea urchins, are rare in the Palaeozoic fossil record, and thus the details regarding the early diversification of crown group echinoids are unclear. Here we report on the earliest probable crown group echinoid from the fossil record, recovered from Permian (Roadian-Capitanian) rocks of west Texas, which has important implications for the timing of the divergence of crown group echinoids. The presence of apophyses and rigidly sutured interambulacral areas with two columns of plates indicates this species is a cidaroid echinoid. The species, Eotiaris guadalupensis, n. sp. is therefore the earliest stem group cidaroid. The occurrence of this species in Roadian strata pushes back the divergence of cidaroids and euechinoids, the clades that comprise all living echinoids, to at least 268.8 Ma, ten million years older than the previously oldest known cidaroid. Furthermore, the genomic regulation of development in echinoids is amongst the best known, and this new species informs the timing of large-scale reorganization in echinoid gene regulatory networks that occurred at the cidaroid-euechinoid divergence, indicating that these changes took place by the Roadian stage of the Permian

Notch-mediated lateral inhibition is an evolutionarily conserved mechanism patterning the ectoderm in echinoids

Notch signaling is a crucial cog in early development of euechinoid sea urchins, specifying both non-skeletogenic mesodermal lineages and serotonergic neurons in the apical neuroectoderm. Here, the spatial distributions and function of delta, gcm, and hesc, three genes critical to these processes in euechinoids, are examined in the distantly related cidaroid sea urchin Eucidaris tribuloides. Spatial distribution and experimental perturbation of delta and hesc suggest that the function of Notch signaling in ectodermal patterning in early development of E. tr ibuloides is consistent with canonical lateral inhibition. Delta transcripts were observed in t he archenteron, apical ectoderm, and lateral ectoderm in gastrulating e mbryos of E. tribuloides. Perturbation of Notch signaling by either delta morpholino or treatment of DAPT downregulated hesc and upregulated delta and gcm, resulting in ectopic expression of delta and gcm. Similarly, hesc perturbation mirrored the effects of delta perturbation. Interestingly, perturbation of delta or hesc resulted in more cells expressing gcm and supernumerary pigment cells, suggesting that pigment cell proliferation is regulated by Notch in E. tribuloides. These results are consistent with an evolutionary scenario whereby, in the echinoid ancestor, Notch signaling was deployed in the ectoderm to specify neurogenic progenitors and controlled pigment cell proliferation in the dorsal ectoderm

Notch-mediated lateral inhibition is an evolutionarily conserved mechanism patterning the ectoderm in echinoids

Notch signaling is a crucial cog in early development of euechinoid sea urchins, specifying both non-skeletogenic mesodermal lineages and serotonergic neurons in the apical neuroectoderm. Here, the spatial distributions and function of delta, gcm, and hesc, three genes critical to these processes in euechinoids, are examined in the distantly related cidaroid sea urchin Eucidaris tribuloides. Spatial distribution and experimental perturbation of delta and hesc suggest that the function of Notch signaling in ectodermal patterning in early development of E. tr ibuloides is consistent with canonical lateral inhibition. Delta transcripts were observed in t he archenteron, apical ectoderm, and lateral ectoderm in gastrulating e mbryos of E. tribuloides. Perturbation of Notch signaling by either delta morpholino or treatment of DAPT downregulated hesc and upregulated delta and gcm, resulting in ectopic expression of delta and gcm. Similarly, hesc perturbation mirrored the effects of delta perturbation. Interestingly, perturbation of delta or hesc resulted in more cells expressing gcm and supernumerary pigment cells, suggesting that pigment cell proliferation is regulated by Notch in E. tribuloides. These results are consistent with an evolutionary scenario whereby, in the echinoid ancestor, Notch signaling was deployed in the ectoderm to specify neurogenic progenitors and controlled pigment cell proliferation in the dorsal ectoderm

A Conserved Role for VEGF Signaling in Specification of Homologous Mesenchymal Cell Types Positioned at Spatially Distinct Developmental Addresses in Early Development of Sea Urchins

Comparative studies of early development in echinoderms are revealing the tempo and mode of alterations to developmental gene regulatory networks and to the cell types they specify. In euechinoid sea urchins, skeletogenic mesenchyme (SM) ingresses prior to gastrulation at the vegetal pole and aligns into a ring-like array with two bilateral pockets of cells, the sites where spiculogenesis will later occur. In cidaroid sea urchins, the anciently diverged sister clade to euechinoid sea urchins, a homologous SM cell type ingresses later in development, after gastrulation has commenced, and consequently at a distinct developmental address. Thus, a heterochronic shift of ingression of the SM cell type occurred in one of the echinoid lineages. In euechinoids, specification and migration of SM are facilitated by vascular endothelial growth factor (VEGF) signaling. We describe spatiotemporal expression of vegf and vegfr and experimental manipulations targeting VEGF signaling in the cidaroid Eucidaris tribuloides. Spatially, vegf and vegfr mRNA localizes similarly as in euechinoids, suggesting conserved deployment in echinoids despite their spatially distinct development addresses of ingression. Inhibition of VEGF signaling in E. tribuloides suggests its role in SM specification is conserved in echinoids. Temporal discrepancies between the onset of vegf expression and SM ingression likely result in previous observations of SM “random wandering” behavior. Our results indicate that, although the SM cell type in echinoids ingresses into distinct developmental landscapes, it retains a signaling mechanism that restricts their spatial localization to a conserved developmental address where spiculogenesis later occurs

Reorganization of sea urchin gene regulatory networks at least 268 million years ago as revealed by oldest fossil cidaroid echinoid

Echinoids, or sea urchins, are rare in the Palaeozoic fossil record and thus the details regarding the early diversification of crown group echinoids are unclear. Here we report on the earliest probable crown group echinoid from the fossil record, recovered from Permian (Roadian-Capitanian) rocks of west Texas, which has important implications for the timing of the divergence of crown group echinoids. The presence of apophyses and rigidly sutured interambulacral areas with two columns of plates indicates this species is a cidaroid echinoid. The species, Eotiaris guadalupensis, n. sp. is therefore the earliest stem group cidaroid. The occurrence of this species in Roadian strata pushes back the divergence of cidaroids and euechinoids, the clades that comprise all living echinoids, to at least 268.8 Ma, ten million years older than the previously oldest known cidaroid. Furthermore, the genomic regulation of development in echinoids is amongst the best known and this new species informs the timing of large-scale reorganization in echinoid gene regulatory networks that occurred at the cidaroid-euechinoid divergence, indicating that these changes took place by the Roadian stage of the Permian

Ancestral state reconstruction by comparative analysis of a GRN kernel operating in echinoderms

Diverse sampling of organisms across the five major classes in the phylum Echinodermata is beginning to reveal much about the structure and function of gene regulatory networks (GRNs) in development and evolution. Sea urchins are the most studied clade within this phylum, and recent work suggests there has been dramatic rewiring at the top of the skeletogenic GRN along the lineage leading to extant members of the euechinoid sea urchins. Such rewiring likely accounts for some of the observed developmental differences between the two major subclasses of sea urchins—cidaroids and euechinoids. To address effects of topmost rewiring on downstream GRN events, we cloned four downstream regulatory genes within the skeletogenic GRN and surveyed their spatiotemporal expression patterns in the cidaroid Eucidaris tribuloides. We performed phylogenetic analyses with homologs from other non-vertebrate deuterostomes and characterized their spatiotemporal expression by quantitative polymerase chain reaction (qPCR) and whole-mount in situ hybridization (WMISH). Our data suggest the erg–hex–tgif subcircuit, a putative GRN kernel, exhibits a mesoderm-specific expression pattern early in Eucidaris development that is directly downstream of the initial mesodermal GRN circuitry. Comparative analysis of the expression of this subcircuit in four echinoderm taxa allowed robust ancestral state reconstruction, supporting hypotheses that its ancestral function was to stabilize the mesodermal regulatory state and that it has been co-opted and deployed as a unit in mesodermal subdomains in distantly diverged echinoderms. Importantly, our study supports the notion that GRN kernels exhibit structural and functional modularity, locking down and stabilizing clade-specific, embryonic regulatory states

Paleogenomics of echinoids reveals an ancient origin for the double-negative specification of micromeres in sea urchins

Establishing a timeline for the evolution of novelties is a common, unifying goal at the intersection of evolutionary and developmental biology. Analyses of gene regulatory networks (GRNs) provide the ability to understand the underlying genetic and developmental mechanisms responsible for the origin of morphological structures both in the development of an individual and across entire evolutionary lineages. Accurately dating GRN novelties, thereby establishing a timeline for GRN evolution, is necessary to answer questions about the rate at which GRNs and their subcircuits evolve, and to tie their evolution to paleoenvironmental and paleoecological changes. Paleogenomics unites the fossil record and all aspects of deep time, with modern genomics and developmental biology to understand the evolution of genomes in evolutionary time. Recent work on the regulatory genomic basis of development in cidaroid echinoids, sand dollars, heart urchins, and other nonmodel echinoderms provides an ideal dataset with which to explore GRN evolution in a comparative framework. Using divergence time estimation and ancestral state reconstructions, we have determined the age of the double-negative gate (DNG), the subcircuit which specifies micromeres and skeletogenic cells in Strongylocentrotus purpuratus. We have determined that the DNG has likely been used for euechinoid echinoid micromere specification since at least the Late Triassic. The innovation of the DNG thus predates the burst of post-Paleozoic echinoid morphological diversification that began in the Early Jurassic. Paleogenomics has wide applicability for the integration of deep time and molecular developmental data, and has wide utility in rigorously establishing timelines for GRN evolution