Morphologic, Histologic And Cellular Analysis Of Tail Regeneration And Study Of BMP2/4Expression Pattern In Tail Regeneration In Amphioxus

Regeneration, in biology, is the process by which some organisms replace orrestore lost or amputated body parts. The relative research concentrated on severalmodel animals. However, little is known to date about regeneration in thecephalochordate amphioxus Branchiostoma japonicum. This paper investigatesQingdao amphioxus tail regeneration process in detail, invoved in morphological,cellular and histological analyses. Meanwhile, the BMP2/4expression patterns atdifferent stages of tail regeneration in amphioxus were studied.First of all, amphioxus tail regeneration process was observed under asteromicroscope after excision of their tail tips close to anus. The whole processof amphioxus tail regeneration takes about4months. Cellular and histologicalanalyses of tail regeneration were carried out by histologic section andhematoxylin-eosin staining method. It was found that amphioxus tail regenerationcan be generally, but not absolutely, devided into three stages. The first stage canbe termed wound healing stage, in which a layer of cells is formed and coveredthe entire wound gradually through the nigration and proliferation of epidermiccells close to the tail amputation plate. The second stage is blastema stage (3weeks after amputation), in which mesenchymal cell-like cells clustered close tothe amputation plate and gradually appeared a convex structure, namedregeneration blastema. The third stage is cell differentiation and pattern formation,in which all kinds of cells and tissues is formed through cell differentiation andpattern formation; newly formed notochord and muscle fiber, for example, areclearly visible after7weeks amputation. Moreover, cell apoptosis during theregeneration was examined by TUNEL assay. It was found that apoptosis mainly occurred in early regeneration stage and strong positive signals for apoptosis weredetected close to the incision.We also studied the expression of amphioxus BMP2/4gene in theregeneration process. Firstly, the spatial and temporal expression patterns ofBMP2/4were studied by in situ hybridization technique. Results showed thatBMP2/4were maily expressed close to incision at early regeneration stage (from1to14days after amputation); whereas its expression decreased in the wake ofregeneration and only very weak or even no expression signals can be detected atlate regeneration stage (14days after amputation). Secondly, BMP2/4expressionlevels at different regeneration periods were examined by real-Time PCR method.It was shown that BMP2/4expression is up-regulated to4times at1st day afteramputation, compared with its expression levels in normal tails, used as controls.Subsequently, the BMP2/4expression levels decreased sharply. Obviously, theresults from the q-PCR and in situ hybridization assays support each other. Thesesuggested that BMP2/4play an important role in the tail regeneration, especiallyin the early stages of regeneration.In summary, this paper detailed the process of amphioxus tail regeneration,involving morphological, histological and cellular charicteristics analysis.Furthermore, BMP2/4expression patterns in the regenerative process wereclarified. All these provide valuble data for the study of evolutionary regeneration.