Development of an embryonic skeletogenic mesenchyme lineage in a sea cucumber reveals the trajectory of change for the evolution of novel structures in echinoderms
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The mechanisms by which the conserved genetic “toolkit” for development generates phenotypic disparity across metazoans is poorly understood. Echinoderm larvae provide a great resource for understanding how developmental novelty arises. The sea urchin pluteus larva is dramatically different from basal echinoderm larval types, which include the auricularia-type larva of its sister taxon, the sea cucumbers, and the sea star bipinnaria larva. In particular, the pluteus has a mesodermally-derived larval skeleton that is not present in sea star larvae or any outgroup taxa. To understand the evolutionary origin of this structure, we examined the molecular development of mesoderm in the sea cucumber, Parastichopus parvimensis.
By comparing gene expression in sea urchins, sea cucumbers and sea stars, we partially reconstructed the mesodermal regulatory state of the echinoderm ancestor. Surprisingly, we also identified expression of the transcription factor alx1 in a cryptic skeletogenic mesenchyme lineage in P. parvimensis. Orthologs of alx1 are expressed exclusively within the sea urchin skeletogenic mesenchyme, but are not expressed in the mesenchyme of the sea star, which suggests that alx1+ mesenchyme is a synapomorphy of at least sea urchins and sea cucumbers. Perturbation of Alx1 demonstrates that this protein is necessary for the formation of the sea cucumber spicule. Overexpression of the sea star alx1 ortholog in sea urchins is sufficient to induce additional skeleton, indicating that the Alx1 protein has not evolved a new function during the evolution of the larval skeleton.
The proposed echinoderm ancestral mesoderm state is highly conserved between the morphologically similar, but evolutionarily distant, auricularia and bipinnaria larvae. However, the auricularia, but not bipinnaria, also develops a simple skelotogenic cell lineage. Our data indicate that the first step in acquiring these novel cell fates was to re-specify the ancestral mesoderm into molecularly distinct territories. These new territories likely consisted of only a few cells with few regulatory differences from the ancestral state, thereby leaving the remaining mesoderm to retain its original function. The new territories were then free to take on a new fate. Partitioning of existing gene networks was a necessary pre-requisite to establish novelty in this system.
KeywordsSea cucumber Echinoderm Evolution of novelty Co-option Skeletogenesis Alx1
Gene regulatory network
Whole mount in situ hybridization.
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, 4, 5, 6, 7, 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, 21, 22, 23, 24, 25, 26, 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, 33, 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.
Results and discussion
The development of the sea cucumber, Parastichopus parvimensis
Although no sea cucumber has been developed as a model organism, several have been the subject of embryological studies (reviewed in ). Several papers reported the development of Stichopus (now referred to as Apostichopus, Parastichopus and Stichopus) species [35, 36, 37]. In particular, the early development of S. tremulus was previously described . Early development in S. tremulus is very similar to what we observe in P. parvimensis (shown in Additional file 1). Early cleavage is equal and little cell-cell adhesion is seen between blastomeres. Blastulae are formed by 16 hours after fertilization and hatch from the fertilization envelope around 26 hours. Prior to gastrulation, the embryos elongate along the animal-vegetal axis, and a thickening is observed at the vegetal pole, termed the vegetal plate. Mesenchyme cells ingress from the vegetal plate before invagination of the archenteron occurs. At the mid-gastrula stage around 48 hours of development, the mesenchyme has migrated. By 72 hours of development, the mouth has formed, and the embryo is now an early auricularia larva. Presumptive muscle cells are seen associated with the foregut, and a thickened ciliary band is evident in the oral hood and looping above the anus. A small skeletal element is situated at the posterior end of the larva, under the posterior coleom.
The ancestral echinoderm mesodermal regulatory state
Therefore, the sea cucumber, like the sea star, forms a central domain of mesodermally fated cells in which orthologs of erg, ets1, gata4/5/6, foxn2/3, tbr, tgif and gata1/2/3 are co-expressed. As such, these transcription factors, at least, represent the core set of regulatory genes that has been maintained in a single mesodermal domain of blastulae since sea cucumbers and sea stars last shared a common ancestor some 450 million years ago . This suggests that there has been strong selection for this mesodermal regulatory state in the auricularia-like larvae of these two classes of echinoderms. This pleisiomorphic mesoderm is a mixture of the regulatory states that are now segregated into separate mesodermal lineages in modern sea urchins. Of course, transcription factors uniquely expressed in the mesoderm of one organism or another have been identified and more are likely to be found.
The sea cucumber, like the sea urchin, has an SM lineage that arises from the mesoderm during early development
Exclusion of TF expression from the sea cucumber SM during gastrulation reveals additional similarities with the sea urchin skeletogenic lineage
The evolution of novelty
A comparison of the regulatory state of embryonic mesenchyme in three classes of echinoderm embryos, those of sea urchins, sea cucumbers and sea stars, provides an extraordinary opportunity to understand the trajectory of change associated with a novel morphology. The sea urchin ingresses skeletogenic, pigment and blastoceolar cells as mesenchyme. Like the sea urchin, the phylogenetically intermediate sea cucumber also ingresses a skeletogenic lineage that is distinct from blastocoelar cells. Thus, the skeletogenic mesenchyme, defined as a population of mesenchyme expressing alx1 that secretes the larval skeleton, is a synapomorphy of at least sea cucumbers and sea urchins. In the sea cucumber SM, however, this lineage secretes only a very small skeletal element in contrast to the dramatic skeleton that defines the morphology of the sea urchin pluteus larva.
An understanding of the evolutionary trajectory of echinoderm larval skeleton formation could be strengthened by a molecular study of the embryology of this process in brittle stars. If the third scenario is upheld, parsimony also supports a single origin of the echinoderm larval skeleton and, therefore, a monophyletic grouping of sea urchins, sea cucumbers and brittle stars, with the sea star as the outgroup that forms no skeleton. Molecular phylogenetic analysis finds equal support for this grouping as for there being a separate brittle star + sea star clade [11, 14, 15].
The other important, and unexpected, observation to arise from this comparison is that there is a loss of gene expression in each of the sea urchin mesodermal lineages relative to the ancestral mesoderm (Figure 5). The sea urchin uses distinct GRNs, linked in part by intercellular signaling, to drive the development of its skeletogenic, pigment and blastocoelar cells. Each of these sub-populations retains some, but not all, features of the ancestral network. Thus, partitioning of an ancestral GRN may have been an important mechanism for allowing evolutionary change.
Culturing and microinjection of embryos
Adult animals were collected off the southern California coast, USA. Spawning was induced in adult sea cucumbers by intra-coelomic injection of 100nM NGLWY-amide followed by heat shock in room temperature sea water after the method of Kato et al.. Freshly shed eggs were mixed with dilute sperm to fertilize, and embryos were cultured in artificial seawater at 15°C until the desired stage. Sea star and sea urchin embryos were prepared according to standard protocols [31, 43] and cultured at 15°C until the desired stage. Zygotes were injected as previously described for sea urchin and sea star [43, 44]. Sea cucumber microinjection was performed following protocols for sea stars.
Cloning sea cucumber and sea star transcription factor orthologs
RNA was isolated from embryos at blastula, gastrula and larva stages using the Total Mammalian RNA Miniprep kit (Sigma, St. Louis, MO, USA). First strand cDNA synthesis was performed with the iScript Select cDNA Synthesis kit (BioRad, Hercules, CA, USA). Degenerate primers were designed against conserved domains and PCR was performed under non-stringent conditions. Once partial sequences were obtained, 5′ and 3′ RACE was performed according to directions (GeneRacer; Invitrogen, Carlsbad, CA, USA). PCR primer sequences are available on request. Sequences were deposited in GenBank under the following accession numbers: PmAlx1, GenBank:JQ740823; PpAlx1, GenBank:JQ740824; PpErg, GenBank:JQ740825; PpFoxa, GenBank:JQ740826; PpFoxn2/3, GenBank:JQ740827; PpGata1/2/3, GenBank:JQ740828; PpGata4/5/6, GenBank:JQ740829; PpGcm, GenBank:JQ740830; PpTbr, GenBank:JQ740831; PpTgif, GenBank:JQ740832.
Whole mount in situ hybridization (WMISH)
Antisense digoxigenin-labeled RNA probes were generated against genes of interest. WMISH was performed as previously described for sea stars . Embryos were photographed using DIC optics on a Leica DMI4000B at 200× magnification using the Leica Application Suite software (Leica, Wetzlar, Germany).
Two-color in situ hybridization
Antisense dinitrophenol-labeled RNA probes were generated against PpAlx1, PpFoxa, PpGcm, and PmEts1. Embryos were fixed as for WMISH, and two-color WMISH was carried out as described by Yankura et al.. In some cases, embryos were counterstained with 1 μM DAPI. Embryos were photographed on a LSM 510 Meta/UV DuoScan Inverted Spectral Confocal Microscope with the ZEN 2009 Imaging suite (Carl Zeiss, Thornwood, NY, USA).
RNA overexpression of PmAlx1 and RFP mRNA in sea urchins and knockdown of PpAlx1
Full-length PmAlx1 and mCherry, an RFP derivative, were cloned into the pCS2+ vector. The resulting plasmids were linearized with HpaI and NotI, respectively. mRNA was synthesized using the mMessage mMachine kit (Ambion; Grand Island, NY, USA). The PpAlx1 morpholino antisense oligonucleotide (MASO) 5′ GGCTCACAAAGTCTGAAATAATCAT 3′ was designed by GeneTool, LLC (Philomath, O, USA). The mRNAs and MASO were injected into embryos in 200 mM KCl containing 0.5 mg/mL fluorescent rhodamine tracer.
The authors would like to thank Kristen Yankura and Alys Cheatle for helpful commentary and discussion. We also thank Hiroshi Wada for discussion of Alx1/4 expression among echinoderms and the helpful comments of three anonymous reviewers. Animals were collected by Patrick Leahy and Peter Halmay or Marinus Inc. under permit number SC-11478 from California Department of Fish and Game to VFH. This research was funded by NSF grant IOS-0844948 and the Winters Foundation.
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