It has been known for many years that most animals rely on the use of the same basic set of regulatory genes during development . The great challenge is to determine how novel structures are specified by orthologous genes. Gene regulatory networks (GRNs) for novel structures can presumably be built “from scratch” (also termed de novo construction), or be re-wired from existing GRNs, which can include the co-option and fusion of GRN subcircuits. In particular, co-option, that is, the reuse of ancestral regulatory programs in novel contexts, might be a major mechanism through which novelties arise during evolution (reviewed in ). For example, this process is thought to have occurred in the formation of butterfly eyespots, patterning appendage outgrowth, the formation of novel insect appendages, and in the evolution of gnathostome oral teeth, among many others [3–8]. Relevant to this study, it has also been suggested that the sea urchin larval skeleton might have arisen via co-option of an adult echinoderm skeletogenic program . However, it is essentially unknown how an ancestral GRN can be rewired to accommodate the acquisition of a co-opted regulatory program in order to produce a viable new state.
The larvae of echinoderms provide a rich source of morphological variation for understanding the evolution of novelty. Broadly, echinoderms have two types of feeding larvae: the pluteus-like larvae of sea urchins and brittle stars, and the auricularia-like larvae of sea cucumbers and sea stars . Interestingly, most sea lilies, which are the earliest-branching echinoderms , have a secondarily-derived non-feeding larva, but at least one species is known to go through an auricularia-like stage . Due to the similarity between the hemichordate tornaria larva and the echinoderm auricularia-type larva, this is considered to be the basal form , possibly to the whole deuterostome clade. Intriguingly, larval morphology does not track with phylogenetic relationships among the echinoderms. Sea urchins and sea cucumbers are sister taxa, while brittle stars either form the outgroup to this clade or are the sister group to sea stars, though the exact phylogeny remains unclear due to the rapid divergence of these classes [11, 14, 15]. Sea lilies are thought to be the most basal echinoderms. Thus, the derived plutei of sea urchins and brittle stars do not group together, but instead are scattered among the ancestral auricularia-like larvae in the echinoderm phylogenetic tree.
There are many differences between echinoderm larval forms, but perhaps the most dramatic and obvious is the larval skeleton that is found in the sea urchin plutei that is not at all present in sea star larvae. The larval skeleton provides the structure that gives the sea urchin the dramatic pluteus morphology. The larval skeleton in modern sea urchins (non-irregular euechinoids, hereafter referred to as simply “sea urchins”) forms from the skeletogenic mesenchyme (SM) that arises from micromeres at the vegetal-most pole (reviewed in ). The remaining mesoderm goes on to produce mesenchymal blastocoelar cells, as well as pigment cells, epithelial coeloms and muscle [17, 18]. The specification of the mesodermal territories is extraordinarily well known in sea urchins, and the SM in particular has, perhaps, the best characterized GRN for any developmental system . Each territory expresses a unique complement of transcription factors during blastula stages, though there is considerable overlap in the expression of individual factors among these lineages. alx1, erg, ets1, foxn2/3, hex, tbr and tgif are expressed in the SM lineage [20–27]. The dorsally-restricted pigment cell precursors express gcm, foxn2/3 and gata4/5/6[26, 28, 29], while the ventrally-localized presumptive blastocoelar cells express erg, gata1/2/3, foxn2/3, ets1 and gata4/5/6[25, 26, 29, 30]. Each of these mesodermal cell types goes on to exhibit distinct behavior during gastrulation. The SM ingresses into the blastocoel at the onset of gastrulation and migrates to form a characteristic ring at the dorsal side of the embryo, which terminates in two ventro-lateral clusters. During late gastrulation, cells of the SM fuse and begin secreting the larval skeleton. The pigment cells are the next population of mesenchyme to ingress from the tip of the archenteron during early gastrulation; they migrate to the dorsal ectoderm, into which they intercalate and begin secreting pigment. Blastocoelar cells do not ingress until the late gastrula stage .
Previous studies have revealed that orthologs of many of the regulatory genes expressed in the sea urchin mesodermal territories are expressed in the presumptive mesoderm of sea stars [32–34], which will give rise to blastocoelar cells and epithelial mesoderm, but not an SM lineage. We, therefore, undertook an analysis of the expression of orthologous transcription factors in the embryos of a phylogenetic intermediate, the sea cucumber, Parastichopus parvimensis, to better correlate changes in regulatory gene expression with morphological novelty. This study is the first comprehensive gene expression analysis performed in any species of sea cucumber and identifies a highly conserved pleisiomorphic mesodermal regulatory state. We show for the first time that sea cucumbers develop a mesodermally-derived skeletogenic mesenchyme from the vegetal pole of blastulae, although the resulting larval skeleton is much less morphologically complex than that of sea urchins. Our results suggest that the evolution of larval skeletogenic cells in echinoderms occurred in a step-wise manner, with just minor changes in the regulatory state of the mesodermal precursors occurring prior to subdivisions of the ancestral gene regulatory network subcircuits.