Histocvtological Events And Analysis Of Key Genes During Intestine Regeneration In Sea Cucumber Apostichopus Iaponicus (Selenka)

Sea cucumber Apostichopus japonicus (Selenka) is an economically importantspecies which has remarkable abilities of regeneration. When exposed to the stressfulcircumstance or dangerous conditions, they would instinctively abandon their viscera;Surprisingly, in favorable environment abandoned organs could be restored and stillfunctioned normally. Therefore, sea cucumber serves as a superior model for the studyof tissue and organ regeneration and development. The pattern of evisceration varies withdifferent species of sea cucumbers, so the pattern of visceral regeneration is different fromspecies to species. Similarly, according to all species of sea cucumbers reported, the firstorgan to regenerate is the digestive tract with mesentery being the “center” of visceralregeneration. So far, most studies focus on hisological and celluar events or menchanism ofintestine regeneration, while the study on molecular mechanism needs more efforts.In this paper, we used histology, immunohistochemistry, high-throughputsequencing, RACE, Real-time PCR et al. to study the mechanism of intestineregeneration in sea cucumbers. We explored the histological and celluar events ofintestine regeneration,constructed the transciptome of regeneration, whichto someextent improved the relatively weak status of Nucleic acid information. Wedetermined gene expression profiles through RNA-Seq techniques to explore theglobal gene regulation of intestine regeneration. Furthermore, some key genes duringintestine regeneration like wnt6、Hox6、HMG and RAP were cloned and analyszedMain results for this study are as follows:1. The process of alimentary tract regeneration can be divided into the following five stages according to morphological development: wound healing, formation ofblastema, lumen formation, intestine differentiation, and intestine growing.Ultrastructural observation on the early stage of intestine regeneration showedobvious cell de-differentiation in the esophagus and newly formed intestine. The newlyformed intestine only consisted of serosa and inner connective tissue, and then the serosa, amuscle layer, inner connective tissue, and mucosa were gradually differentiated into. Thecells of new tissue were mainly derived from cell migration (morphallaxis) at the early stageand from cell division (epimorphosis) at the later stage.2.454high-through sequencing technique was used to construct thetranscriptome. We obtained182,473raw reads from regenerative tissue of seacucumbers and clean reads were assembled to71086unigene, which improved theweak status of Nucleic acid information. The genes associated with structuralmolecule activity were notably abundant; and the genes associated with calalyticacitivity decreased in the regeneration sample compared with control sample. Keysignal pathways including Wnt and Notch were activated in regeneration process.3. We used RNA-Seq to construct the dynamic gene expression profile, whichexplored the global dynamic changes occurring during the intestine regeneration in A.japonicus. At the early stage of intestine regeration, gene expression varieddramatically, then with the completion of newly regenerated intestine tract, geneexpression returned to normal levels, which indicated that intestine regeneration is acomplex process determined by multiple genes. Specific “Go terms” revealed that theDEGs (Differentially Expressed Genes) performed an important function at everyregeneration stage. Besides some expected pathways (for example, Ribosome andSpliceosome pathway term), the “Notch signaling pathway”, the “ECM-receptorinteraction” and the “Cytokine-cytokine receptor interaction” were significantlyenriched and worth further research4. We obtained the full-length cDNA of Wnt6and AjHBOX6(Hox6). Real-timePCR analysis demonstrated that the expression of Wnt6was significantly up-regulatedat the early stage and decreased to the normal level at the later sthage, underlying thatWnt6could activate many genes to trigger the intestine regeneration. The expression of AjHBOX6was up-regulated at the middle and later stage and decreased at last,which probably guide intestine differentiate to anterior, middle and posterior intestine.5. We obtained the full-length cDNA of RAP and HMG. The expression level ofRAP was up-regulated by over400fold of the control. Except Saccoglossuskowalevskii, RAP specific regeneration genes.has not been found in other species,HMG is an important cytokine which regulate the cell migration; and its expressionwas up-regulated at the early stage.So far, most studies on intestine regeneration of sea cucumber have beenconducted at the mRNA level. Further studies at the protein and microRNA levels,construction of regulatory network as well as analysis of key genetic function by RNAinterference and antibody blocking technique are needed to explore the mechanism ofintestine regeneration.