The development of the sea cucumber, Parastichopus parvimensis
Although no sea cucumber has been developed as a model organism, respective have been the subjugate of embryological studies ( reviewed in [ 10 ] ). respective papers reported the development of Stichopus ( now referred to as Apostichopus, Parastichopus and Stichopus ) species [ 35 – 37 ]. In particular, the early on growth of S. tremulus was previously described [ 35 ]. early development in S. tremulus is very exchangeable to what we observe in P. parvimensis ( shown in Additional file 1 ). early cleavage is equal and little cell-cell attachment 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 embryo elongate along the animal-vegetal axis, and a node is observed at the vegetal terminal, termed the vegetal plate. Mesenchyme cells ingres from the vegetal plate before invagination of the archenteron occurs. At the mid-gastrula degree around 48 hours of development, the mesenchyme has migrated. By 72 hours of development, the mouthpiece has formed, and the embryo is now an early on auricularia larva. presumptive muscle cells are seen associated with the foregut, and a thicken ciliary ring is discernible in the oral hood and looping above the anus. A small bony element is situated at the back tooth end of the larva, under the back tooth coleom .
The ancestral echinoderm mesodermal regulatory state
We sought to determine the commonalities in mesoblastic regulative submit that exist between sea stars, sea urchins and sea cucumbers. The transcription factors erg, alx1, foxa, foxn2/3, gata1/2/3, gata4/5/6, tbr and tgif ( named here PpErg, PpAlx1, PpFoxa, PpFoxn2/3, PpGata1/2/3, PpGata4/5/6, PpTbr and PpTgif, respectively ) were selected for this survey as these are the orthologs of regulative genes that operate within the well characterized ocean urchin GRN for mesoblastic weave specification. phylogenetic analysis confirms the orthology of each gene ( Additional file 2 ). PpErg, PpFoxn2/3, PpGata1/2/3, PpGata4/5/6, PpTbr and PpTgif are all expressed throughout the presumptive mesoderm at the central vegetal pole of sea cucumber embryo ( number 1 A-F ). We confirmed this using a series of two-color in situ hybridizations, indicate, for case, that both PpTbr and PpGata4/5/6 are expressed throughout a territory that is bounded by PpFoxa expression ( figure 1 G-I ). foxa, which is expressed in a torus above the central vegetal plate, probable marks the gang of future endoderm in sea cucumbers as it does in sea urchins and ocean stars [ 33, 38 ]. It has besides previously been shown that the ets1 transcription component is expressed in the vegetal pole of a closely-related species of sea cucumber [ 39 ]. expression of ocean star orthologs of these transcription factors has been reported [ 32 – 34, 40 ]. We re-examined the expression of the ocean star PmGata4/5/6 gene. At an early blastula stagecoach, PmGata4/5/6 is detected in the cardinal vegetal perch mesoblastic precursors ( Figure 1 J ), which was not previously reported [ 40 ] .Figure 1
Orthologous genes are expresed in the mesoderm of sea cucumbers, sea stars and sea urchins. Whole saddle horse in situ hybridization ( WMISH ) using antisense DIG-labeled probes revealed that ocean 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 besides examined the expression of foxa, which is expressed in the endoderm of many animals. ( G ) foxa is expressed in a ring that probably besides 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 knowledge domain and, therefore, are likely expressed throughout the mesoderm. WMISH besides shows that ocean star ortholog of gata4/5/6, PmGata4/5/6, is expressed within the cardinal mesoblastic fated territory ( J ). Full size image
consequently, the sea cucumber, like the sea asterisk, forms a cardinal world 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 fix of regulative genes that has been maintained in a single mesoblastic sphere of blastula since sea cucumbers and sea stars final shared a coarse ancestor some 450 million years ago [ 11 ]. This suggests that there has been strong survival for this mesoblastic regulative express in the auricularia-like larva of these two classes of echinoderms. This pleisiomorphic mesoderm is a mix of the regulative states that are now segregated into divide mesoblastic lineages in modern sea urchins. Of course, recording 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
Despite the overall similarity of regulative gene construction between the ocean cucumber and sea star, we show for the first base time that the sea cucumber besides develops an SM linage and that this population is most likely homologous to the ocean urchin SM descent. It has been known for some time that sea cucumber larva late produce a small spicule, morphologically quite unlike the ocean urchin larval skeleton [ 10 ], but the embryonic origin of this bony element has not been determined. In sea urchins, SpAlx1 is one of the first transcription factors activated within the SM linage and is needed for the specification of these cells [ 21 ]. In sea cucumber blastula, PpAlx1 is expressed in merely four cells in the vegetal pole mesoderm ( Figure 2 A ). initially, this gene is co-expressed with other TFs expressed in the mesoderm, as shown by two-color in situ hybridization with PpTbr and PpGata4/5/6 ( Figure 2 B, C ). The PpAlx1 + cells are among the first to ingress at the attack of gastrulation and are the first to migrate away from the archenteron ( Figure 2 D, E ). These cells form a chain on the abaxial english of the embryo ( Figure 2 F, G ), a lot like that seen with the sea urchin SM, and then are found as a bunch of approximately eight cells localized underneath the dorsal ectoderm ( Figure 2 H ). This cell bunch appears to be in the lapp location as the bony spicule. To far confirm that the PpAlx1 + cells are the skeletogenic cells of sea cucumber larva, we inhibited transformation of PpAlx1 by injection of an antisense morpholino-substituted oligonucleotide. As shown in Figure 2 I-L, such morphants are morphologically identical normal, except they specifically lack the bunch of cells that secretes the spicule. thus, phenomenologically, the behavior of the PpAlx1 -expressing cells in ocean cucumbers is inordinately like to that of the SpAlx1 -expressing SM in sea urchins. Both populations undergo early ingression, directed abaxial migration to form a ring of cells, and bunch to secrete the larval skeleton. We conclude that the PpAlx1 -expressing cells in ocean cucumbers are homologous to the SM descent of ocean urchins .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 blastula ( 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 exaggeration position ( 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 late localized in a call of cells on the abaxial side of the embryo ( F, G ). In larva, alx1 transcripts are detected specifically in a localized bunch of cells that corresponds to the place of the bony spicule spicule ( H ; cell cluster is inset ). Knockdown of PpAlx1 results specifically in the loss of the bony element ( arrows ) without drastic changes in larval morphology ( I vanadium. J ; high magnification shown in K and L ). mes. blastula – mesenchyme blastula ; EG-early gastrula ; d-day ; vv-vegetal pole view ; control – restraint MASO injected ; KD – MASO knock down. Full size image
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In contrast, in situ hybridization ( figure 3 A-C ) reveals that in sea stars, PmAlx1 is expressed throughout the vegetal terminal mesoderm of blastula. As development proceeds, PmAlx1 is continually expressed in mesoblastic derivatives. In gastrula, bespeak is detected in the mesoblastic bulb at the tip off of the archenteron. In larva, PmAlx1 is expressed in the lateral margins of the coelomic pouches, which are derived from the mesoblastic bulb of the gastrula, and in the back tooth celom. Throughout exploitation, this model is in nonindulgent line to what is seen in the sea urchins and ocean cucumbers. Taken together, these data suggest that changes in both the regulation and affair of alx1 underlie the evolution of the fresh echinoderm skeletogenic descent. To confirm that a change in the Alx1 protein itself did not contribute to the evolution of larval skeletogenesis, we overexpressed the sea star ortholog of Alx1, PmAlx1, in sea urchin embryo. PmAlx1 overexpressing embryo, but not embryos with ectopic control RNA ( mCherry ), shape ectopic bony elements ( Figure 3 D-F ). This is reproducible with the phenotype associated with overexpression of ocean urchin alx1 [ 41 ], which suggests that a protein-level exchange in Alx1 did not cause the development of larval skeletogenesis .Figure 3
PmAlx1 is 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 cardinal vegetal pole mesoderm of sea star blastula ( 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 larva, staining is detected in the lateral aspects of the anterior coeloms ( filled arrows in C ) equally well as in the back tooth celom ( arrow in C ). To confirm that the absence of a larval skeleton in ocean stars is not due to a remainder in the sea star PmAlx1 protein, we overexpressed PmAlx1 in ocean urchins. ( D-F ) Ectopic expression of PmAlx1 messenger rna in sea urchin embryo results in increase skeletogenesis, as has been observed for overexpression of sea urchin alx1 messenger rna [ 41 ]. Control embryo were injected with messenger rna encoding the RFP discrepancy mCherry. spokesperson ocean urchin gastrula are shown ( D-F ). Ectopic skeletal spicules were observed upon overexpression of PmAlx1 ( E, F ). Numbers of embryo showing the illustrate phenotype is shown in the lower right corner ( from a full of 40 embryo for the control RFP, and 80 embryo in the PmAlx1 injected embryo ) ; D shows normal bony geological formation, E shows increase in number of skeletal form centers, and F and dramatic increase in skeleton. Full size effigy
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Exclusion of TF expression from the sea cucumber SM during gastrulation reveals additional similarities with the sea urchin skeletogenic lineage
The presence of an SM descent in both sea urchin and sea cucumber embryo provides an opportunity to begin to uncover the minimal complement of regulative genes required for skeleton deposition. therefore, we next carefully examined the saying of regulative genes within the newly discovered sea cucumber SM after ingression. Two-color in situ hybridization clearly shows that in sea cucumber gastrula, while PpAlx1 and PpGata4/5/6 do not localize to the same cells, a subset of PpErg + cells co-express PpAlx1 ( Figure 4 A, B ). similarly, in larva, only PpErg is expressed in the dorsal cluster of skeletogenic cells ( Figure 4 G ). No other TFs we identified are expressed in the mesenchyme ( digit 4 C-F, H-M ). A previous study suggests that a ocean cucumber ets1 ortholog is besides expressed in the spicule forming cells of a associate sea cucumber [ 39 ]. The expression of alx1, erg and ets1, but not gata1/2/3 and gata4/5/6 formulation in these cells is similar to the regulative express of the sea urchin SM during blastula stages in sea urchins. In contrast, SpTbr is needed for skeleton deposition in sea urchins [ 19 ]. The fact that PpTbr is not expressed in the sea cucumber SM suggests that the function of SpTbr in larval skeletogensis is an apomorphy of modern sea urchins. Although less parsimonious, it is besides potential that tbr expression was secondarily lost from the sea cucumber SM .Figure 4
The skeletogenic centers of sea cucumbers do not express gata4/5/6 , gata1/2/3 , tbr , tgif or foxn2/3. WMISH performed against gastrula stage embryo ( A-F ) show localization of function 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 construction in mesenchyme. WMISH with probes antisense to PpGata1/2/3 and PpTbr are showed localize staining at the lean of the archenteron in gastrula ( C, D ), while PpFoxn2/3 antisense probes stain merely within the ectoderm ( E ) and those antisense to PpTgif in the endoderm ( F ). In larva, PpErg antisense probes are localized to the SM ( G ) ; and see higher magnification in gusset. Probes against PpGata4/5/6 stain within the midgut ( H ), while those against PcGata1/2/3 ( I ) are present in the posterior celom. No stain is found for PpTbr at this stage ( J ), see higher magnification of SM cells in tuck that show no tarnish. Probes against PpFoxn2/3 ( K ) ultimately show localization in the later foregut. PpTgif antisense probes are localized to the midgut of larva ( L ). Full size double
