Ortiz-Pineda PA et al. (2009), Gene expression profiling of intestinal regener...

ECB-ART-41174

BMC Genomics 2009 Jun 08;10:262. doi: 10.1186/1471-2164-10-262.

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Gene expression profiling of intestinal regeneration in the sea cucumber.

Ortiz-Pineda PA , Ramírez-Gómez F , Pérez-Ortiz J , González-Díaz S , Santiago-De Jesús F , Hernández-Pasos J , Del Valle-Avila C , Rojas-Cartagena C , Suárez-Castillo EC , Tossas K , Méndez-Merced AT , Roig-López JL , Ortiz-Zuazaga H , García-Arrarás JE .

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BACKGROUND: Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals that can quickly replace most injured organs. In particular, sea cucumbers are excellent models for studying organ regeneration since they regenerate their digestive tract after evisceration. However, echinoderms have been sidelined in modern regeneration studies partially because of the lack of genome-wide profiling approaches afforded by modern genomic tools.For the last decade, our laboratory has been using the sea cucumber Holothuria glaberrima to dissect the cellular and molecular events that allow for such amazing regenerative processes. We have already established an EST database obtained from cDNA libraries of normal and regenerating intestine at two different regeneration stages. This database now has over 7000 sequences. RESULTS: In the present work we used a custom-made microchip from Agilent with 60-mer probes for these ESTs, to determine the gene expression profile during intestinal regeneration. Here we compared the expression profile of animals at three different intestinal regeneration stages (3-, 7- and 14-days post evisceration) against the profile from normal (uneviscerated) intestines. The number of differentially expressed probes ranged from 70% at p < 0.05 to 39% at p < 0.001. Clustering analyses show specific profiles of expression for early (first week) and late (second week) regeneration stages. We used semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) to validate the expression profile of fifteen microarray detected differentially expressed genes which resulted in over 86% concordance between both techniques. Most of the differentially expressed ESTs showed no clear similarity to sequences in the databases and might represent novel genes associated with regeneration. However, other ESTs were similar to genes known to be involved in regeneration-related processes, wound healing, cell proliferation, differentiation, morphological plasticity, cell survival, stress response, immune challenge, and neoplastic transformation. Among those that have been validated, cytoskeletal genes, such as actins, and developmental genes, such as Wnt and Hox genes, show interesting expression profiles during regeneration. CONCLUSION: Our findings set the base for future studies into the molecular basis of intestinal regeneration. Moreover, it advances the use of echinoderms in regenerative biology, animals that because of their amazing properties and their key evolutionary position, might provide important clues to the genetic basis of regenerative processes.

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Genes referenced: LOC100887844 LOC100893907 LOC115919910 LOC115922213 LOC583082 LOC590297 mmp7 polr3a

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	Figure 3. Microarrays volcano plots. Distribution of sequence expression between normal and normal injected with LPS, 3-, 7- and 14-dpe regenerating stages. Each plot shows the logarithm of the probability of the t-test as a function of the logarithm of fold change for each EST probe. Horizontal lines in each plot represent the nominal significant level of 0.001 for the t-student under the assumption that each gene has a unique variance. The vertical lines represent the limit of significance of the change in expression (fold change >2). Differentially expressed genes are located on the right (over-expressed) and left (under-expressed) top quadrants.
	Figure 4. RT-PCR profiles of Wnt-9, Hox12, Tenascin-R, MMP-11, MMP-14, MMP-15, Actin 1, Actin 2 and Actin 3 in normal and regenerating intestines. The values were normalized against NADH. This set of genes showed up-regulation in the first week of regeneration except for Actin 3 that showed down-regulation during regeneration. Interestingly, all extracellular matrix (ECM) related genes showed the peak of expression at 7-dpe. Moreover, Wnt-9, a developmental gene, was up-regulated during all stages. Values represent the mean ± S.E.M. of 7 biological samples (individual animals) per stage. Paired t-test for mean comparison was used. Significance levels were :p < 0.05, and *:p < 0.01. Images at the base of the bar are RT-PCR pictures.

	Figure 6. Cluster analysis of the microarray expression profiles for 15208 probes (All minus 536 controls). The 5 columns show the following comparisons: LPS (Red: "R") vs. Normal (Green: "G"): 3dpe(R) vs. Normal(G); 7dpe(R) vs. Normal(G); 7dpe(R) vs. 3dpe(G); 14dpe(R) vs. 7dpe(G). The number of genes in each cluster is shown above the bars. Since arrays were normalized against the LPS vs Normal results, the first column appears empty. Colors represent the level of expression of the gene, red for up-regulated and green for down-regulated; White spots represent the average of all the genes per cluster with "y" axis as a relative measure of expression levels.
	Figure 7. Distribution of gene function (GO) for ESTs analyzed in the microarrays. GO values of all ESTs printed on the microarray (A). Percentage of ESTs differentially expressed at 3-dpe (B) or 7-dpe (C). When compared with all the GOs, the 3- and 7-dpe distribution GOs showed no significant differences other than a small decrease in the number of GO results. The number of GOs results for each particular stage is shown in the bottom-left corner. The color code starts at 12 o'clock in the list order.
	Figure 8. Microarray experimental design. Normal (Nml) and regenerating intestines at 3, 7 and 14 dpe (days post-evisceration) were compared among them. Each experiment was replicated as a dye swap with different biological samples. LPS treated tissues where used to normalize the expression for immune activated genes. Arrows show direct comparisons.

References [+] :

Andreasen, Aryl hydrocarbon receptor activation impairs extracellular matrix remodeling during zebra fish fin regeneration. 2007, Pubmed