Viera-Vera J and García-Arrarás JE (2019), Retinoic Acid Signaling Is Associated with Cell...

ECB-ART-48742

Biomolecules 2019 Dec 13;912:. doi: 10.3390/biom9120873.

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Retinoic Acid Signaling Is Associated with Cell Proliferation, Muscle Cell Dedifferentiation, and Overall Rudiment Size during Intestinal Regeneration in the Sea Cucumber, Holothuria glaberrima.

Viera-Vera J , García-Arrarás JE .

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Almost every organism has the ability of repairing damaged tissues or replacing lost and worn out body parts, nevertheless the degree of the response substantially differs between each species. Adult sea cucumbers from the Holothuria glaberrima species can eviscerate various organs and the intestinal system is the first one to regenerate. This process involves the formation of a blastema-like structure that derives from the torn mesentery edges by the intervention of specific cellular processes (e.g., cell dedifferentiation and division). Still, the genetic networks controlling the regenerative response in this model system are just starting to be unraveled. In this work we examined if and how the retinoic acid (RA) signaling pathway is involved in the regenerative response of this deuterostome. We first identified and characterized the holothurian orthologs for short chain dehydrogenase/reductase 7 (SDR7) and aldehyde dehydrogenase family 8A1 (ALDH8A1), two enzymes respectively associated with retinaldehyde and RA anabolism. We then showed that the SDR7 transcript was differentially expressed during specific stages of intestinal regeneration while ALDH8A1 did not show significant differences in regenerating tissues when compared to those of normal (non-eviscerated) organisms. Finally, we investigated the consequences of modulating RA signaling during intestinal regeneration using pharmacological tools. We showed that application of an inhibitor (citral) of the enzyme synthesizing RA or a retinoic acid receptor (RAR) antagonist (LE135) resulted in organisms with a significantly smaller intestinal rudiment when compared to those treated with DMSO (vehicle). The two inhibitors caused a reduction in cell division and cell dedifferentiation in the new regenerate when compared to organisms treated with DMSO. Results of treatment with tazarotene (an RAR agonist) were not significantly different from the control. Taken together, these results suggest that the RA signaling pathway is regulating the cellular processes that are crucial for intestinal regeneration to occur. Thus, RA might be playing a role in echinoderm regeneration that is similar to what has been described in other animal systems.

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Genes referenced: aldh8a1 echs1 LOC100887844 LOC115919910 LOC587800 rarb

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	Figure 1. Nucleotide and amino acid sequences of short-chain dehydrogenase reductase found in the sea cucumber, H. glaberrima.
	Figure 2. Multiple sequence alignment of the sea cucumber short-chain dehydrogenase reductase 7 amino acid sequence and those of selected protostomes, deuterostomes, chordates, and vertebrates. Conserved residues are shaded in black when they are 100% similar, dark grey if they are 99–80% similar, light grey when 79–60% similar, or white when less than 60% similar. The characteristic functional sites and interfaces of the protein are depicted herein.
	Figure 3. Predicted 3-D folding pattern of the sea cucumber SDR7 sequence using the Phyre2 program.
	Figure 4. Nucleotide and amino acid organization of aldehyde dehydrogenase 8A1 found in the sea cucumber, H. glaberrima.
	Figure 5. Multiple sequence alignment of the sea cucumber aldehyde dehydrogenase family 8A1 amino acid sequence and those of selected metazoan species. Conserved residues are shaded in black when they are 100% similar, dark grey if they are 99–80% similar, light grey when 79–60% similar, or white when less than 60% similar. The characteristic functional domains and sites of the protein are depicted herein.
	Figure 6. Maximum likelihood phylogram based on the amino acid sequences of the short chain dehydrogenase reductase 7 found in the sea cucumber, Holothuria glaberrima, compared against homologs from multiple species, other SDRs families, and retinol dehydrogenases (RD) from the sea urchin. Every SDR7 localize to the top of the phylogram and is identified by a “.7” suffix while every other SDR localize to the bottom of it and are identified by the specific family number or letter suffix. The retinol dehydrogenases also localize to the bottom of the phylogram and are identified by a “.rd#” suffix. Every sequence used for this analysis is referenced in Supplementary Table S1. Major taxonomic groups are defined by color boxes (chordates = blue; non-chordate deuterostomes = green; Lophotrochozoa = red; Ecdysozoa = beige; and other SDRs or RDs = grey) and by the bootstrap values depicted in a larger font. The numbers at the nodes correspond to the bootstrap proportion expressed as the percentage of 1000 replicates while the length of the branches is proportional to the amount of inferred evolutionary change as defined by the bar in substitutions per site.
	Figure 7. Maximum likelihood phylogram based on the amino acid sequences of the aldehyde dehydrogenase 8A1 (ALDH8A1) found in the sea cucumber, Holothuria glaberrima, compared against its homologs in multiple species, other ALDH8A1 families from the sea urchin, and retinaldehyde dehydrogenases (RALDH) from the mouse. Every ALDH8A1 localize to the bottom of the phylogram and is identified by a “.8” suffix while every other ALDH localize to the top of it and are identified by the specific family number or letter suffix. The RALDHs also localize to the top of the phylogram and are identified by a “.R#” suffix. Every sequence used for this analysis is referenced in Table S1. Major taxonomic groups are defined by color boxes (chordates = blue; non-chordate deuterostomes = green; Lophotrochozoa = red; Ecdysozoa = beige, Cnidaria = purple; and other ALDHs or RALDHs = grey) and by the bootstrap values depicted in a larger font. The numbers at the nodes correspond to the bootstrap proportion expressed as the percentage of 1000 replicates while the length of the branches is proportional to the amount of inferred evolutionary change as defined by the bar in substitutions per site.
	Figure 8. mRNA expression profiles of short chain dehydrogenase reductase 7 includes different regenerative stages (3, 5, 7, and 14 days post evisceration) and normal intestinal tissue. Semi-quantitative RT-PCR of the SDR7 transcript along the (a) anterior, (b) medial, and (c) posterior segments of the intestine. Three biological replicates were analyzed at each regenerating interval and four in the normal gut. Ordinary one-way ANOVA showed differences among means statistically significant in graphs b (p = 0.02), F = 3.5, df(treatment) = 4, df(residual) = 10 and c (p = 0.01), F = 5.3, df(treatment) = 4, df(residual) = 11. A multiple comparison test (Tukey) showed significant difference (p < 0.5) between groups bearing different letters in these two graphs. Those groups bearing two letters are not statistically different to those with any of the letters. n = biological replicates.
	Figure 9. mRNA expression profiles of aldehyde dehydrogenase family 8A1 includes different regenerative stages (3, 5, 7, and 14 days post evisceration) and normal intestinal tissue. Semi-quantitative RT-PCR of the ALDH8A1 transcript along the (a) anterior, (b) medial, and (c) posterior segments of the intestine. Three biological replicates were analyzed at each regenerating interval and four in the normal gut. Ordinary one-way ANOVA showed no significant difference among the groups in each graph (p = 0.2), F = 1.5, df (treatment) = 4, df(residual) = 10. n = biological replicates.
	Figure 10. Quantification of the intestinal rudiment area of regenerating individuals. (a) The area of the tissue section was measured on individuals treated with (b) DMSO—vehicle, (c) tazarotene—RAR agonist, (d) citral—RALDH inhibitor, or (e) LE135—RAR antagonist following six days post evisceration. Five technical replicates (different slide sections of the same animal) and at least four biological replicates were performed per group: DMSO n = 10, tazarotene n = 4, citral n = 10, LE135 n = 4. Ordinary one-way ANOVA showed differences among means statistically significant with a p value < 0.0001, F = 15.87, df(treatment) = 3, df(residual) = 22. A multiple comparison test (Tukey) in these showed significant difference when comparing DMSO against citral or LE135 (p < 0.001) or tazarotene against citral or LE135 (p < 0.01). Statistically significant difference between groups is represented by different letters atop of the bars. Arrows signal the boundary between the mesentery and the new intestinal rudiment. n = biological replicates.
	Figure 11. Cell proliferation along the regenerating blastema of the intestinal rudiment. (a) The percentage of dividing cells along the rudiment’s mesothelium was established from tissue sections labeled with an antibody against BrdU (red) on individuals treated with (b) DMSO—vehicle, (c) tazarotene – RAR agonist, (d) citral—RALDH inhibitor, or (e) LE135—RAR antagonist following 6 days post evisceration. Between three and five technical replicates and at least three biological replicates were performed per group: DMSO n = 8, tazarotene n = 3, citral n = 10, LE135 n = 4. Ordinary one-way ANOVA showed differences among means statistically significant with a p value < 0.0001, F = 15.28, df(treatment) = 3, df(residual) = 22. A multiple comparison test (Tukey) in these showed significant difference when comparing DMSO against citral or LE135 (p < 0.001) and tazarotene against citral or LE135 (p < 0.01) that is represented by different letters atop of the bars. Arrows signal the boundary between the mesentery and the new intestinal rudiment. n = biological replicates.
	Figure 12. Formation of spindle-like structures by muscle cells during intestinal regeneration. (a) The percentage of SLS along the mesenteries near the blastema was established form animals treated with (b) DMSO—vehicle, (c) tazarotene—RAR agonist, (d) citral—RALDH inhibitor, or (e) LE135—RAR antagonist following 6 days post evisceration. Between three and five technical replicates and at least three biological replicates were performed per group: DMSO n = 9, tazarotene n = 4, citral n = 10, LE135 n = 3. Ordinary one-way ANOVA showed differences among means statistically significant with a p value < 0.0001 F = 29.55, df(treatment) = 3, df(residual) = 22. A multiple comparison test (Tukey) in these showed significant difference when comparing DMSO against citral or LE135 (p < 0.001) and tazarotene against citral or LE135 (p < 0.01) that is represented by different letters atop of the bars. n = biological replicates.

References [+] :

Al Haj Zen, The Retinoid Agonist Tazarotene Promotes Angiogenesis and Wound Healing. 2016, Pubmed