Cellular Transformation And Molecular Characterization Of Blastema In Zebrafish Lower Jaw Regeneration

Using model organisms is an important approach in life science research. Over thelast decades, the zebrafish has developed into an outstanding model organism inbiomedical research for its many advantages, such as the powerful reproductive capacity,in vitro fertilization and development, the transparency of embryos, and the high geneticsimilarity with human beings especially. In recent years, it has been discovered that thezebrafish has a dramatic regenerative capability in its fin and heart. As a regenerationresearch model, zebrafish has attracted more attention and promoted the development ofstem cells and the regenerative medicine research.Like most animal models of regeneration, zebrafish possess a remarkable ability toregenerate complicated structures by formation of a mass of undifferentiatedmesenchymal cells called blastema. But it is still uncertain about the blastemal cellsorigin and differentiation mechanism, and there is few report about the activity ofrelated genes and crucial factors in tissues regeneration.To understand how the blastema retains the original structural form, we investigatecellular transitions and transcriptional characteristics of cell identity genes during allstages of regeneration of an amputated lower jaw. During zebrafish embryonicdevelopment, the lower jaw formation is regulated by the migration and differentiationof cranial neural crest stem cells, include the skeleton, muscles, pigment cells andconnective tissues. To deepen our understanding of regenerative process in complextissues, this article has described various histological staining methods and underlinedthe cellular transitions after amputating one third of zebrafish lower jaw. The resultsshow that the wound bed is completely covered in the first day after injury. Followingthe new epithelium formation, fibroblast-like spindle cells proliferated in thesubcutaneous tissue on day seven after injury, cartilage islands grew and are graduallyreplaced by bone ossification by two months, concurrently, the other tissueregenerations are basically completed. We find that the first week is the main occurring stage of zebrafish lower jaw regeneration, which containe inflammatory reaction andepithelial tissue regeneration，blastema formation，blastema reformation, and theyhappened at2hpa (hour post amputation)，2dpa (day post amputation) and5dpaseparately. The identification of these three key point times will take an important roleto understand the molecular mechanism of blastema formation and reformation.The immunohistochemistry experiment show that blastema have positiveexpression of CK19and α-SMA in different levels. The blastema may have variouscharacterization of different cell types, that is, the blastema cells are in a pluripotentstate. By qRT-PCR and RNA-Seq, combined with tissue sections analysis, we find thatnucleated blood cells, fibroblasts, damaged muscle cells, and pigment cells are firsttransformed to two origins of blastemal precursors: foxi1-positive neural crest cells andisl1-positive mesoderm progenitors, then followed by blastema mesenchymalization andchondrogenic ossification. Time point-based transcriptomal analysis reveals that five3’-end Hox genes and equal number of5’-end Hox genes are activated largely duringblastema reformation. RNA in situ hybridization demonstrates that hoxa2b is highlyexpressed in the presumptive chondrogenic center and partially overlap with theexpression domain of foxi1and sox9a. In contrast, hoxa11b and pax3a are respectivelyexpressed in blastema mesenchyme and regenerating muscle. These results suggest thatblastema regeneration generally maintain the developmental signaling pathways bywhich two origins of blastemal precursors cooperatively regulate blastema skeletonrespecification and mesodermal segmentation in zebrafish lower jaw regeneration.According to the above experiments, we put forward a new type induced model ofregeneration: a series of regulatory factors are released for the injury irritation, and thenthey regulate the blastema formation and reformation in regeneration.