Molecular evidence of anteroposterior patterning in adult echinoderms

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Abstract

The origin of the pentaradial body plan of echinoderms from a bilateral ancestor is one of the most enduring zoological puzzles. Since echinoderms are defined by morphological novelty, even the most basic axial comparisons with their bilaterian relatives are problematic. Here, we used conserved antero-posterior (AP) axial molecular markers to determine whether the highly derived adult body plan of echinoderms masks underlying patterning similarities with other deuterostomes. To revisit this classical question, we used RNA tomography and in situ hybridizations in the sea star Patiria miniata to investigate the expression of a suite of conserved transcription factors with well-established roles in the establishment of AP polarity in bilaterians. We find that the relative spatial expression of these markers in P. miniata ambulacral ectoderm shows similarity with other deuterostomes, with the midline of each ray representing the most anterior territory and the most lateral parts exhibiting a more posterior identity. Interestingly, there is no ectodermal territory in the sea star that expresses the characteristic bilaterian trunk genetic patterning program. This suggests that from the perspective of ectoderm patterning, echinoderms are mostly head-like animals, and prompts a reinterpretation of the evolutionary trends that made echinoderms the most derived animal group.

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  1. Excerpt

    Spatial transcriptomics sheds light on the echinoderm body plan! The bilaterian antero- lateral axis shifts to medio-lateral in the ectoderm of adult echinoderms.

  2. c’-f’, Hox genes primarily expressed in the coeloms. g’-j’, Hox genes primarily expressed in the digestive tract. In h’-j’ white asterisks indicate the position of the developing intestinal tract.

    For genes primarily expressed in the mesoderm, would it be possible to display an orthogonal view of the confocal Z-stack? Perhaps as a supplement? It appears that some of these images are of a single slice or projection of a subslice of a full sample.

  3. In P. miniata, we suggest that there is no ectoderm equivalent to a trunk region, because wnt3 is expressed at the edge of the ambulacral region, and because hox1 is the only Hox gene expressed in the ectoderm. Therefore, the deployment of the AP patterning system in P. miniata seems to be limited to the ambulacral region and its boundary.

    Most of trunk and posterior marker genes used for HCR in this study were Hox genes. Hox expression also appears to be restricted to mesoderm in a variety of other echinoderms, so perhaps it would be expected that these genes would not be expressed in the ectoderm of this organism. Are there other non-Hox trunk ectodermal markers – perhaps homologs of chordate or hemichordate genes – which would be strong markers for trunk ectoderm?

    The claim that AP patterning is restricted to the ambulacral region seems to depend strongly on wnt3 being a terminal or posterior marker. Moreover, pax2/5/8 appears to be expressed more laterally than wnt3, which would correspond with a more "trunk-like" identity based on the corresponding expression of chordate/hemichordate homologs. Additional ectoderm-expressed marker genes showing a similar boundary to wnt3 would strengthen the argument that there is no ectoderm equivalent to a trunk region.

  4. c, Experimental design of the RNA tomography.

    For ease of interpretation and consistency with other figures, it might be useful to replace "Left-right" with "Medial-lateral".

  5. d, DAPI-stained (grey) specimen colored to highlight the main anatomical regions of the oral side of a post-metamorphic juvenile. The ambulacral ectoderm (outlined by the green dotted line) comprises two main regions: the medial ambulacral ectoderm (blue) and the podia epidermis (cyan). In some parts of the specimen internal germ layers are apparent through the confocal z-stack projection, such as the pharynx and the terminal ends of then hydrocoel (yellow).

    It might be helpful to have this figure also elaborated as a schematic with corresponding colors, such as in Supp. Fig. 3. As someone without familiarity with the anatomy, it's a little difficult to wrap my head around this image. It would also be helpful to indicate where the ambulacral boundary is with respect to the other features, to be able to more easily interpret panels u-b'. Adding schematics of the classified expression pattern to the left of each row of images could help ease interpretation.

  6. an extensive interambulacral domain that wraps around the aboral side of the animal and displays uncertain axial identity, without coherent deployment of the ancestral AP patterning program

    It would be very interesting to look at the oral-aboral tomography data to look for genes that are more highly expressed on the aboral side - perhaps those genes could help clarify the axial identity of this unplaced tissue.

  7. This manuscript explores how the AP axis is patterned in the derived body plan of echinoderms. The authors use RNA tomography in multiple sequential sections of juvenile starfish limbs to evaluate the expression of canonical groups of AP genes with respect to the proximal-distal, oral-aboral, and medial-lateral axes. The authors visualize the gene expression patterns of canonical AP marker genes using HCR in starfish juveniles.

    Based on these data, the authors build a model of the starfish body plan that places anterior genes in the ambulacral region of the ectoderm (the ambulacral-anterior hypothesis). The authors show that a large portion of the starfish ectoderm – the interambulacral region – does not appear to express canonical ectodermal markers of the AP axis. Most of the trunk markers used in this study were Hox genes, which in this study and other echinoderm studies appear to be primarily restricted to the mesoderm. HCR of additional ectodermal trunk markers would help clarify the identity of the interambulacral region.

    It will be very interesting to understand what genes are expressed in the interambulacral region to determine whether starfish are indeed "mostly head-like animals."

  8. This suggests that from the perspective of ectoderm patterning, echinoderms are mostly head-like animals, and prompts a reinterpretation of the evolutionary trends that made echinoderms the most derived animal group.

    This is a really exciting idea - I was hoping to read more about why you felt like this was the key take-away based on the data of the patterning system of the ambulacral ectoderm in the discussion section? Also, this is reminiscent of tardigrades as head-like animals, missing the Hox trunk genes from Bob Goldstein's lab and I think some of Andi Hejnol's work on many spiralian taxa - it feels like something important evolutionary is going on here with a many metazoan taxa utilizing the deuterostome/vertebrate "head" patterning system, though perhaps we should be thinking about the deuterostome/vertebrate co-option of this system???

  9. These marker genes included transcription factors

    Did you find any new markers with regional/cell-type specificity from your spatial transcriptomics (maybe this is the basis for future work and if so, great!)

  10. The uncoupling of an ectodermal head and trunk programs is not unique to P. miniata and has been demonstrated in both larval echinoderms and hemichordates39,47, and recently in annelid larvae43, suggesting that these regulatory programs can be uncoupled over macroevolutionary time frames.

    from my comment in the abstract - this seems like the perfect place to talk a little more about the "adult sea stars are heads" hypothesis

  11. These marker genes included transcription factors

    Did you find any new markers with regional/cell-type specificity from your spatial transcriptomics (maybe this is the basis for future work and if so, great!)

  12. The uncoupling of an ectodermal head and trunk programs is not unique to P. miniata and has been demonstrated in both larval echinoderms and hemichordates39,47, and recently in annelid larvae43, suggesting that these regulatory programs can be uncoupled over macroevolutionary time frames.

    from my comment in the abstract - this seems like the perfect place to talk a little more about the "adult sea stars are heads" hypothesis

  13. This suggests that from the perspective of ectoderm patterning, echinoderms are mostly head-like animals, and prompts a reinterpretation of the evolutionary trends that made echinoderms the most derived animal group.

    This is a really exciting idea - I was hoping to read more about why you felt like this was the key take-away based on the data of the patterning system of the ambulacral ectoderm in the discussion section? Also, this is reminiscent of tardigrades as head-like animals, missing the Hox trunk genes from Bob Goldstein's lab and I think some of Andi Hejnol's work on many spiralian taxa - it feels like something important evolutionary is going on here with a many metazoan taxa utilizing the deuterostome/vertebrate "head" patterning system, though perhaps we should be thinking about the deuterostome/vertebrate co-option of this system???

  14. c, Experimental design of the RNA tomography.

    For ease of interpretation and consistency with other figures, it might be useful to replace "Left-right" with "Medial-lateral".

  15. This manuscript explores how the AP axis is patterned in the derived body plan of echinoderms. The authors use RNA tomography in multiple sequential sections of juvenile starfish limbs to evaluate the expression of canonical groups of AP genes with respect to the proximal-distal, oral-aboral, and medial-lateral axes. The authors visualize the gene expression patterns of canonical AP marker genes using HCR in starfish juveniles.

    Based on these data, the authors build a model of the starfish body plan that places anterior genes in the ambulacral region of the ectoderm (the ambulacral-anterior hypothesis). The authors show that a large portion of the starfish ectoderm – the interambulacral region – does not appear to express canonical ectodermal markers of the AP axis. Most of the trunk markers used in this study were Hox genes, which in this study and other echinoderm studies appear to be primarily restricted to the mesoderm. HCR of additional ectodermal trunk markers would help clarify the identity of the interambulacral region.

    It will be very interesting to understand what genes are expressed in the interambulacral region to determine whether starfish are indeed "mostly head-like animals."

  16. In P. miniata, we suggest that there is no ectoderm equivalent to a trunk region, because wnt3 is expressed at the edge of the ambulacral region, and because hox1 is the only Hox gene expressed in the ectoderm. Therefore, the deployment of the AP patterning system in P. miniata seems to be limited to the ambulacral region and its boundary.

    Most of trunk and posterior marker genes used for HCR in this study were Hox genes. Hox expression also appears to be restricted to mesoderm in a variety of other echinoderms, so perhaps it would be expected that these genes would not be expressed in the ectoderm of this organism. Are there other non-Hox trunk ectodermal markers – perhaps homologs of chordate or hemichordate genes – which would be strong markers for trunk ectoderm?

    The claim that AP patterning is restricted to the ambulacral region seems to depend strongly on wnt3 being a terminal or posterior marker. Moreover, pax2/5/8 appears to be expressed more laterally than wnt3, which would correspond with a more "trunk-like" identity based on the corresponding expression of chordate/hemichordate homologs. Additional ectoderm-expressed marker genes showing a similar boundary to wnt3 would strengthen the argument that there is no ectoderm equivalent to a trunk region.

  17. c’-f’, Hox genes primarily expressed in the coeloms. g’-j’, Hox genes primarily expressed in the digestive tract. In h’-j’ white asterisks indicate the position of the developing intestinal tract.

    For genes primarily expressed in the mesoderm, would it be possible to display an orthogonal view of the confocal Z-stack? Perhaps as a supplement? It appears that some of these images are of a single slice or projection of a subslice of a full sample.

  18. d, DAPI-stained (grey) specimen colored to highlight the main anatomical regions of the oral side of a post-metamorphic juvenile. The ambulacral ectoderm (outlined by the green dotted line) comprises two main regions: the medial ambulacral ectoderm (blue) and the podia epidermis (cyan). In some parts of the specimen internal germ layers are apparent through the confocal z-stack projection, such as the pharynx and the terminal ends of then hydrocoel (yellow).

    It might be helpful to have this figure also elaborated as a schematic with corresponding colors, such as in Supp. Fig. 3. As someone without familiarity with the anatomy, it's a little difficult to wrap my head around this image. It would also be helpful to indicate where the ambulacral boundary is with respect to the other features, to be able to more easily interpret panels u-b'. Adding schematics of the classified expression pattern to the left of each row of images could help ease interpretation.

  19. an extensive interambulacral domain that wraps around the aboral side of the animal and displays uncertain axial identity, without coherent deployment of the ancestral AP patterning program

    It would be very interesting to look at the oral-aboral tomography data to look for genes that are more highly expressed on the aboral side - perhaps those genes could help clarify the axial identity of this unplaced tissue.