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Evodevo
2012 Aug 09;31:17. doi: 10.1186/2041-9139-3-17.
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Development of an embryonic skeletogenic mesenchyme lineage in a sea cucumber reveals the trajectory of change for the evolution of novel structures in echinoderms.
McCauley BS
,
Wright EP
,
Exner C
,
Kitazawa C
,
Hinman VF
.
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BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.
Figure 1. Orthologous genes are expresed in the mesoderm of sea cucumbers, sea stars and sea urchins. Whole mount in situ hybridization (WMISH) using antisense DIG-labeled probes revealed that sea cucumber orthologs of erg (A), foxn2/3 (B), gata1/2/3 (C), gata4/5/6 (D), tbr (E) and tgif (F) are expressed throughout the mesoderm of sea cucumbers. We also examined the expression of foxa, which is expressed in the endoderm of many animals. (G) foxa is expressed in a ring that likely also marks the endodermally-fated tissue in sea cucumbers. Two-color fluorescent in situs with the endoderm marker foxa confirm that gata4/5/6 (H) and tbr (I) are expressed within the foxa domain and, therefore, are likely expressed throughout the mesoderm. WMISH also shows that sea star ortholog of gata4/5/6, PmGata4/5/6, is expressed within the central mesodermal fated territory (J).
Figure 2. The sea cucumbers skeletogenic mesenchyme arises from the vegetal pole mesoderm during early development. WMISH shows that sea cucumber alx1 transcripts are localized to a population of four cells in the vegetal pole of blastulae (A). During this stage, PpAlx1 is co-expressed with other factors in the mesoderm, as shown here by two-color fluorescent in situ with probes antisense to PpTbr (B) and PpGata4/5/6 (C). Inset is higher magnification view (B and C). The PpAlx1+ cells are among the earliest to ingress at the onset of gastrulation (D). During gastrulation, PpAlx transcripts are localized to the first cells to migrate away from the archenteron (E) and transcripts are later localized in a ring of cells on the dorsal side of the embryo (F, G). In larvae, alx1 transcripts are detected specifically in a localized cluster of cells that corresponds to the position of the skeletal spicule spicule (H; cell cluster is inset). Knockdown of PpAlx1 results specifically in the loss of the skeletal element (arrows) without drastic changes in larval morphology (I vs. J; high magnification shown in K and L). mes. blastula - mesenchyme blastula; EG-early gastrula; d-day; vv-vegetal pole view; control - control MASO injected; KD – MASO knock down.
Figure 3. PmAlx1is expressed in the sea star epithelial mesoderm, but induces skeletogenesis in sea urchins. WMISH using a probe against PmAlx1 demonstrates that transcripts are localized to the central vegetal pole mesoderm of sea star blastulae (A); in later development, transcripts are detected in the coelomic mesoderm, but never in the mesenchyme, as seen by two-color in situ hybridization with PmEts1 (B). In larvae, staining is detected in the lateral aspects of the anterior coeloms (filled arrows in C) as well as in the posterior coelom (arrow in C). To confirm that the absence of a larval skeleton in sea stars is not due to a difference in the sea star PmAlx1 protein, we overexpressed PmAlx1 in sea urchins. (D-F) Ectopic expression of PmAlx1 mRNA in sea urchin embryos results in increased skeletogenesis, as has been observed for overexpression of sea urchin alx1 mRNA [41]. Control embryos were injected with mRNA encoding the RFP variant mCherry. Representative sea urchin gastrula are shown (D-F). Ectopic skeletal spicules were observed upon overexpression of PmAlx1 (E, F). Numbers of embryos showing the illustrated phenotype is shown in the lower right corner (from a total of 40 embryos for the control RFP, and 80 embryos in the PmAlx1 injected embryos); D shows normal skeletal formation, E shows increase in number of skeletal forming centers, and F and dramatic increase in skeleton.
Figure 4. The skeletogenic centers of sea cucumbers do not expressgata4/5/6,gata1/2/3,tbr,tgiforfoxn2/3. WMISH performed against gastrula stage embryos (A-F) show localization of only PpErg and PpAlx1 transcripts in the skeletogenic mesenchyme (A). A probe designed against PpGata4/5/6 is localized to other mesenchyme cells (B). No other factor identified in this study showed expression in mesenchyme. WMISH with probes antisense to PpGata1/2/3 and PpTbr are showed localized staining at the tip of the archenteron in gastrulae (C, D), while PpFoxn2/3 antisense probes stain only within the ectoderm (E) and those antisense to PpTgif in the endoderm (F). In larvae, PpErg antisense probes are localized to the SM (G); and see higher magnification in inset. Probes against PpGata4/5/6 stain within the midgut (H), while those against PcGata1/2/3 (I) are present in the posterior coelom. No staining is found for PpTbr at this stage (J), see higher magnification of SM cells in insert that show no staining. Probes against PpFoxn2/3 (K) ultimately show localization in the posterior foregut. PpTgif antisense probes are localized to the midgut of larvae (L).
Figure 5. Model describing regulatory reorganization of the echinoderm mesoderm and evolution of the larval skeletogenic mesenchyme. (A) Regulatory states for the mesoderm of sea urchins, sea cucumbers and sea stars are shown in the top row of the schematic. The dark blue ring represents the endodermal territory that surrounds the mesoderm. Orange regions indicate derived alx1 expression. Genes expressed in each territory are listed. (B) In later development, these territories will form two types of mesenchyme: the alx1+ SM and the blastocoelar cells (orange and purple, respectively). Gene expression patterns not described in this work have been previously reported [20-30,32-34,39]. Of the organisms studied, only sea cucumbers and sea urchins are known to make skeletogenic mesenchyme, though brittle stars also form a larval skeleton. By comparing gene expression in the mesodermal territories of sea urchins, sea cucumbers and sea stars, we extrapolated the regulatory state of the mesoderm of the echinoderm ancestor (the proto-mesoderm), shown at the base of the tree (phylogeny after [11,14,15]). The broad expression of most TFs within the mesoderm of sea cucumbers and sea stars supports the hypothesis that there was a pan proto-mesoderm during blastula stages. Subsequent regulatory changes within the mesoderm in the lineage leading to at least sea urchins and sea cucumbers created a population of cells, marked by their expression of alx1, that go on to form the larval skeleton.
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