Laura A. Romano

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged ∼35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

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.

Endo16 is required for gastrulation in the sea urchin Lytechinus variegatus

The Endo16 gene encodes a large extracellular protein with several functional domains that provide some insight into the role of this protein during embryonic development. We isolated the full‐length cDNA sequence from Lytechinus variegatus and utilized morpholinos to further investigate the role of Endo16 during embryonic development in this species. Endo16‐deficient embryos failed to undergo gastrulation and the blastocoele became filled with dissociated cells after 24 h of incubation. Moreover, there was a delay in endoderm differentiation as assayed by staining with an antibody that recognizes Endo1. The differentiation of other cell types including oral ectoderm, primary mesenchymal cells (PMC) and secondary mesenchymal cells (SMC) appeared to be normal, although the patterns of protein expression did not resemble control embryos due to the gross morphological abnormalities elicited by the LvEndo16 morpholino. Microinjection of full‐length EGFP mRNA with the LvEndo16 morpholino‐targeted sequence confirmed that this phenotype can be attributed specifically to the loss of Endo16 protein. Taken together, our data suggest that Endo16 may be required for the cell–extracellular matrix (ECM) interactions that are required for endoderm differentiation in the sea urchin embryo.

Evolutionary analysis of the cis-regulatory region of the spicule matrix gene SM50 in strongylocentrotid sea urchins

An evolutionary analysis of transcriptional regulation is essential to understanding the molecular basis of phenotypic diversity. The sea urchin is an ideal system in which to explore the functional consequence of variation in cis-regulatory sequences. We are particularly interested in the evolution of genes involved in the patterning and synthesis of its larval skeleton. This study focuses on the cis-regulatory region of SM50, which has already been characterized to a considerable extent in the purple sea urchin, Strongylocentrotus purpuratus. We have isolated the cis-regulatory region from 15 individuals of S. purpuratus as well as seven closely related species in the family Strongylocentrotidae. We have performed a variety of statistical tests and present evidence that the cis-regulatory elements upstream of the SM50 gene have been subject to positive selection along the lineage leading to S. purpuratus. In addition, we have performed electrophoretic mobility shift assays (EMSAs) and demonstrate that nucleotide substitutions within Element C affect the ability of nuclear proteins to bind to this cis-regulatory element among members of the family Strongylocentrotidae. We speculate that such changes in SM50 and other genes could accumulate to produce altered patterns of gene expression with functional consequences during skeleton formation.