Cary GA et al. (2019), Analysis of sea star larval regeneration reveal...

ECB-ART-46993

BMC Biol 2019 Feb 22;171:16. doi: 10.1186/s12915-019-0633-9.

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Analysis of sea star larval regeneration reveals conserved processes of whole-body regeneration across the metazoa.

Wolff A , Zueva O , Pattinato J , Hinman VF .

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BACKGROUND: Metazoan lineages exhibit a wide range of regenerative capabilities that vary among developmental stage and tissue type. The most robust regenerative abilities are apparent in the phyla Cnidaria, Platyhelminthes, and Echinodermata, whose members are capable of whole-body regeneration (WBR). This phenomenon has been well characterized in planarian and hydra models, but the molecular mechanisms of WBR are less established within echinoderms, or any other deuterostome system. Thus, it is not clear to what degree aspects of this regenerative ability are shared among metazoa. RESULTS: We characterize regeneration in the larval stage of the Bat Star (Patiria miniata). Following bisection along the anterior-posterior axis, larvae progress through phases of wound healing and re-proportioning of larval tissues. The overall number of proliferating cells is reduced following bisection, and we find evidence for a re-deployment of genes with known roles in embryonic axial patterning. Following axial respecification, we observe a significant localization of proliferating cells to the wound region. Analyses of transcriptome data highlight the molecular signatures of functions that are common to regeneration, including specific signaling pathways and cell cycle controls. Notably, we find evidence for temporal similarities among orthologous genes involved in regeneration from published Platyhelminth and Cnidarian regeneration datasets. CONCLUSIONS: These analyses show that sea star larval regeneration includes phases of wound response, axis respecification, and wound-proximal proliferation. Commonalities of the overall process of regeneration, as well as gene usage between this deuterostome and other species with divergent evolutionary origins reveal a deep similarity of whole-body regeneration among the metazoa.

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Species referenced: Echinodermata
Genes referenced: dach1 egr3 elk1 fzd5 fzd9 gli3 klf2 LOC100887844 LOC100893907 LOC115919910 LOC583082 mcm2 pole runx2

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	Fig. 1. Models of whole-body regeneration. a Phylogeny depicting regeneration capacity of various taxa, after [2, 89]. Species from the three taxa marked with a star were considered in this study. b Schematic of a sea star bipinnaria larva indicating the bisection plane (dashed line) and relevant anatomical features including the ciliary band epithelium (green), coelomic pouch epithelium (purple), and enteric organs (blue)
	Fig. 2. Sea star bipinnaria regeneration involves wound healing, re-proportioning, and respecification.(a) DIC images showing larval recovery following bisection (top row) and magnifications of the wound site at each stage (bottom row). Important anatomical features are highlighted in the magnified images including the wound site (arrowheads), opening to the gut lumen (dotted lines), and new ciliary bands (asterisks). Scale bar = 100â€‰Î¼m; applicable to all images in panel. (b) Two serial sections from the same individual showing wound closure (arrowheads) and many free cells within the blastocoelar space (asterisks). (c) Ratios of length from the posterior pole to the top of the post-oral ciliary band to length from the posterior pole to the anterior pole (i.e., total length of the specimen) are plotted along with the difference of the means (i.e., Î” length ratio) and 95% confidence interval. Those timepoints with a ratio found to be significantly different than uncut larvae are indicated by the red line and asterisk (Mann-Whitney U test, p value <â€‰0.001). nâ€‰=â€‰number of individuals measured at each timepoint
	Fig. 3. Cell proliferation decreases and localizes to wound-proximal cells. a EdU staining of S-phase cells in intact and regenerating sea star larvae (1â€“7â€‰days post-bisection [dpb]). EdU-positive cells are shown in green. Nuclei were stained with DAPI and shown in gray. Cell proliferation in uncut larvae is throughout the ciliary band epithelium (cb), mouth (mo), stomach (s), and coelomic pouches (cp). Regenerating anterior fragments (top row) and posterior fragments (bottom row) demonstrate similar initial distributions of proliferation, although the number of EdU+ cells decreased by 3 dpb. Beginning at 6 dpb, EdU+ cells are concentrated near the wound site in both anterior and posterior regenerating fragments in a putative regeneration blastema (bl). b Quantitation of the EdU+ cells shows a steady decline in the number of proliferating cells in both anterior and posterior regenerating fragments. The difference of the means (i.e., Î” EdU+ Cells) is plotted and significance differences are indicated (Mann-Whitney, pâ€‰<â€‰0.05, red asterisk). nâ€‰=â€‰total number of bisected animals counted. c The fraction of EdU+ cells in each of the wound-proximal, middle, and wound-distal thirds of each regenerating larval fragment from panel B is shown. The number of individuals counted is the same as in (b). The difference of the means (i.e., Î” % EdU+ cells) is plotted and significance differences are indicated (Mann-Whitney, pâ€‰<â€‰0.05, red asterisk). d The experimental regimen of the BrdU/EdU pulse-chase experiments is shown. Regenerating larvae (left) or uncut larvae (right) were labeled with BrdU (magenta) for 6â€‰h after which the BrdU was washed out. Larvae are subsequently labeled with a 6â€‰h EdU pulse (green) at the onset of wound-proximal proliferation or after a similar duration for uncut larvae
	Fig. 4. Apoptotic cell death persists and increases in later phases. a TUNEL+ cells (green) in control animals are normally distributed throughout larval tissues and is concentrated within the ciliary band epithelium. Nuclei (gray) stained with DAPI. Regenerating anterior (b) and posterior (c) fragments display similar patterns and numbers of TUNEL+ cells from 3Â h post-bisection (hpb) until 6â€‰days post-bisection (dpb) when there is an increase. d Quantitation of TUNEL+ cells in regenerating anterior and posterior fragments shows that there is no significant difference in the number of TUNEL+ cells until 6 dpb when a significant increase in apoptotic cells are detected. The difference of the means (i.e., Î” TUNEL+ cells) is plotted and significance differences are indicated (Mann-Whitney, pâ€‰<â€‰3â€‰Ã—â€‰10âˆ’4, red asterisk). nâ€‰=â€‰the number of individuals sampled
	Fig. 5. Cluster analysis indicates genes involved in regenerative functions. a The heatmap depicts log fold change values for genes (rows) in anterior (ANT) and posterior (POST) regenerating fragments compared with sibling uncut control (CONT) larvae over the sampled regeneration time points (columns; 3â€‰h post-bisection [hpb], 3â€‰days post-bisection [dpb], and 6 dpb). Green indicates a positive fold change (upregulated with respect to uncut controls), whereas purple indicates a negative fold change (downregulated with respect to control). b Gene ontology (GO) term enrichments for each of the five clusters. The enrichment of each GO term is indicated by a circle where the area corresponds to the fraction of genes annotated with that term are present in the cluster, and the color of the circle corresponds to the corrected hypergeometric p value of term enrichment. Terms marked with an asterisk [*] are from the annotation set generated by mouse gene ortholog prediction (Fig.Â 5, AdditionalÂ fileÂ 1: Figure S3)
	Fig. 6. Evolutionarily similar early regeneration response. (a) These plots show sea star gene log fold change values for genes differentially expressed early in both anterior and posterior regenerating fragments compared with non-bisected sibling control larvae. Genes upregulated in both fragments (top row) correspond to cluster I, and genes downregulated in both fragments (bottom row) correspond to cluster II. All genes assigned to each cluster are plotted in gray. Several genes, either referenced in the text or representative of functions considered, are indicated with colored lines. Next to the key for each gene is an indication (+) of whether an ortholog for that gene was found in an analogous cluster in either the planaria (S.m.) or hydra (H.m.) datasets. Indicators in brackets (e.g., â€œ[+]â€) are those was no overlapping ortholog identified by our analyses, but genes with the same name were implicated by published datasets. Genes plotted with dashed lines are shown by in situ (right). Several additional genes are shown in a supplemental figure (AdditionalÂ fileÂ 1: Figure S9). The expression patterns of Elk (b), Egr (c), and Klf2/4 (d) are shown. (bâ€²â€“dâ€²) are magnifications of the wound site shown in the boxed regions in panels (bâ€“d). Expression patterns in uncut larva are also shown (bâ€³â€“dâ€³)
	Fig. 7. Fragment-specific recovery of appropriate anterior-posterior gene expression. a The expression of genes asymmetrically expressed in either anterior (ANT; solid lines, cluster III) or posterior (POST; dashed lines, cluster IV) sea star larval territories was examined at 3â€‰h post-bisection (hpb), 3â€‰days post-bisection (dpb), and 6 dpb. The log fold change values for each gene in regenerating anterior or posterior fragments compared with non-bisected sibling control larvae is reported for each fragment (ANT/CONT and POST/CONT, respectively) over the regenerating time course sampled. Black lines show the detected expression of Frizz5/8 and Frizz9/10. b Model for recover of genes asymmetrically expressed along the anterior-posterior axis, with Frizz9/10 (blue) and Frizz5/8 (maroon) provided as examples. c Whole-mount fluorescent in situ hybridization illustrating the re-activation of Frizz9/10 (magenta) in the posterior aspect of regenerating anterior fragments beginning at 5 dpb and preceding the concentration of proliferating EdU+ cells (green) near the wound site. d Re-activation of Frizz5/8 (magenta) in the anterior aspect of regenerating posterior fragments beginning at 2 dpb and preceding the concentration of proliferating EdU+ cells near the wound site
	Fig. 8. Shared proliferation-associated genes. a These data show sea star log fold change values for genes differentially expressed at later stages in regenerating fragments compared with non-bisected sibling control larvae (i.e., sea star cluster V). All genes assigned to cluster V are plotted in gray. Several genes, either referenced in the text or representative of functions considered, are indicated with colored lines. Next to the key for each gene is an indication (i.e., â€œ+â€) of whether an ortholog for that gene was found in an analogous cluster in either the planaria (S.m.) or hydra (H.m.) datasets. Indicators in brackets (e.g., â€œ[+]â€) are those where no overlapping ortholog was identified by our analyses, but genes with the same name were implicated by published datasets. Genes plotted with dashed lines are shown by fluorescent in situ hybridization (below). Mcm2 (b), Runt1 (c), GliA (d), and Dach1 (e) are all expressed in the anterior aspects of regenerating fragments at 6 dpb. In many cases, the expression of these genes is coincident with an EdU+ cell, suggesting that these genes are expressed, at least in part, in proliferating cells
	Fig. 9. Summary of similarities between WBR models. The reported features of regeneration at early, middle, and late stages of regeneration, with respect to the datasets considered in this study, are indicated. Features detected in the sea star model in our study that are shared with the other two models are highlighted in red. Some aspects are considered in common based on shared gene expression (e.g., MAPK signaling) whereas others are based on cytological observations (e.g., blastema proliferation)

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