Reinardy HC et al. (2015), Tissue regeneration and biomineralization in se...

ECB-ART-44175

PLoS One 2015 Jan 01;108:e0133860. doi: 10.1371/journal.pone.0133860.

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Tissue regeneration and biomineralization in sea urchins: role of Notch signaling and presence of stem cell markers.

Reinardy HC , Emerson CE , Manley JM , Bodnar AG .

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Echinoderms represent a phylum with exceptional regenerative capabilities that can reconstruct both external appendages and internal organs. Mechanistic understanding of the cellular pathways involved in regeneration in these animals has been hampered by the limited genomic tools and limited ability to manipulate regenerative processes. We present a functional assay to investigate mechanisms of tissue regeneration and biomineralization by measuring the regrowth of amputated tube feet (sensory and motor appendages) and spines in the sea urchin, Lytechinus variegatus. The ability to manipulate regeneration was demonstrated by concentration-dependent inhibition of regrowth of spines and tube feet by treatment with the mitotic inhibitor, vincristine. Treatment with the gamma-secretase inhibitor DAPT resulted in a concentration-dependent inhibition of regrowth, indicating that both tube feet and spine regeneration require functional Notch signaling. Stem cell markers (Piwi and Vasa) were expressed in tube feet and spine tissue, and Vasa-positive cells were localized throughout the epidermis of tube feet by immunohistochemistry, suggesting the existence of multipotent progenitor cells in these highly regenerative appendages. The presence of Vasa protein in other somatic tissues (e.g. esophagus, radial nerve, and a sub-population of coelomocytes) suggests that multipotent cells are present throughout adult sea urchins and may contribute to normal homeostasis in addition to regeneration. Mechanistic insight into the cellular pathways governing the tremendous regenerative capacity of echinoderms may reveal processes that can be modulated for regenerative therapies, shed light on the evolution of regeneration, and enable the ability to predict how these processes will respond to changing environmental conditions.

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Species referenced: Echinodermata
Genes referenced: coel1 ddx4 gata3 gcml hes hey1 LOC100887844 LOC100893859 LOC115919910 LOC583082 LOC592057 rpl8a

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	Fig 1. Sea urchin regeneration assay.A single strip of tube feet and adjacent spines from one ambulacral section were cut at the base (A, 1 day post amputation, dpa). Regrowth of spines was measured directly with calipers, and tube feet length was measured by image analysis of underwater photographs (B, 8 dpa), with the scale set from a ruler placed adjacent to cut section (visible on right, panel A). Panels C–E indicate regrowth in a control animal after 8, 15, and 22 dpa, respectively. Panels F–H indicate regrowth in an animal treated with 3 μg/g DAPT after 8, 15, and 22 dpa, respectively.
	Fig 2. Spine and tube feet regeneration following treatment with the mitotic inhibitor, vincristine.Regeneration (% of full-length, uncut appendages) in spines (A) and tube feet (B) after treatment with 0 (black bars), 0.2 μg/g (grey bars), and 0.6 μg/g (white bars) vincristine. Data are means ± s.e.m., n = 4 individuals (except 29 dpa, n = 3, 0 μg/g, and n = 2, 0.6 μg/g due to mortality prior to measurement). *Significant reduction in regeneration with concentration of vincristine (arcsine-transformed, One-way ANOVA, post-hoc MRT, p<0.05).
	Fig 3. Spine and tube feet regeneration following treatment with DAPT.Regeneration (% of full-length, uncut appendages) in spines (A) and tube feet (B) after treatment with 0 (black bars), 1 μg/g (grey bars), and 3 μg/g (white bars) DAPT. Data are means ± s.e.m., n = 4 individuals (except tube feet from 0 μg/g, 29 dpa, n = 3 due to mortality prior to measurement). *Significant reduction in regeneration with concentration of DAPT (arcsine-transformed, One-way ANOVA, post-hoc MRT, p<0.05).
	Fig 4. Inhibition of Notch signaling in regenerating sea urchins treated with DAPT.Tube feet sampled 24 hours after final treatment with DAPT, 29 days post amputation. Gene expression (qRT-PCR) of selected Notch target genes (hey, gataC, hes, gcm), compared with the geometric mean of three most stable control genes (cyclophilin7, rpl8, profilin), data are geomeans ± s.e.m., n = 3 animals, *significant down-regulation (p < 0.05).
	Fig 5. Stem cell marker, Vasa, in sea urchin adult tissues.Immunohistochemistry of tube foot [A and B (detail); Mu = muscle, Ep = epidermis, CT = connective tissue, Lu = lumen], esophagus (C), radial nerve (D), Aristotle’s lantern muscle (E), and coelomocytes (F) stained with DAPT (i), antibody to vasa visualized with DyLight 488 secondary antibody (ii), vasa/DAPI image overlay (iii). Representative images from n = 6 (tube feet) or n = 4 (other tissues) individuals. Scale bar is 100 μm.

References [+] :

Beck, Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate. 2003, Pubmed