| Tissue regeneration has been an important topic in many biological researchers. Realization of tissue and organ regeneration is the human dream for a long time. There has been growing interest in applying tissue engineering to stem cell-based regeneration therapies. The most attractive strategy is to convert the available cells into the desired cell types. In order to achieve this aim, we need to know the molecular mechanism of tissue regeneration of the animals like invertebrate species, certain urodeles, and fish.It could provide some theoretical support to induce stem cell differentiation and induce tissue regeneration in situ.Zebrafish(Danio rerio) has a dramatic regenerative capability. Previous research has reported that zebrafish can faithfully regenerate complicated tissue structures through blastemal cell type conversions and tissue reorganization. The process of the lower jaw regeneration is similar to the previous report. The wound bed is completely covered in the first day after injury. Following the new epithelium formation, fibroblast-like spindle cells proliferate in the subcutaneous tissue on day seven after injury, cartilage islands grow and are gradually replaced by bone ossification within two months. We identify four time points corresponding to preamputation(0hpa), reepitheliation(2hpa), blastemal formation(2dpa), and respecification(5dpa). However it is not clear that the expression pattern of genes during lower jaw regeneration, and how they regulated cell transformation andregeneration. This study is designed to further clarify inter-molecular interactions involved in complicated tissue regeneration of zebrafish lower jawAfter a transverse removal of one-third of the mandible and associated epidermis, dermis, hypodermis, mandibular symphysis, and part of intermandibularis anterior epidermis reconstitution was initiated following homeostasis. At 24 hpa, the newly formed epithelium consisted of epidermal and intermediate cells. The intermediate cells displayed nuclear condensation and cytoplasmic shrinkage or vacuolation, and were mo rphologically between mesenchymal cells/nbc and epidermal keratinocytes /mucous cell s. We attribute the complete reepitheliation to cell-type conversions to tissue interaction s between ectodermal-mesodermal components.To unveil the regenerative factors and engineering arts of blastemal regeneration, we conducted transcriptomal analyses at four time points corresponding to preamputation, reepitheliation, blastemal formation, and respecification. Analysis of differentially expressed genes, 5118 genes were significantly deregulated(q-value cut-off 0.05) in time between all four time points. Except for 67 genes that were regulated at all stages, wound healing and regeneration temporally altered the expression of 2800 genes between 2 hpa and 0 hpa, 533 genes between 2dpa and 2hpa, and 240 genes between 5dpa and 2dpa. We compared this map with ten major intracellular signaling pathways previously reported to be key players in initiating and maintaining the regeneration:MAPK, Erb B, Wnt, Notch, Hedgehog, TGF-beta, VEGF, ECM-receptor interaction, Cell adhesion molecules, and Jak-STAT signaling pathways. All of these pathways were successfully identified in our time-lapse transcriptomal sequences of lower jaw regeneration.However, statistical analysis revealed that only four of the ten pathways weresignificantly enriched, suggesting distinct regenerative mechanisms responsible for lower jaw regeneration.GO classification and enrichment not only confirmed the GO terms associated with RNA processing, transcription regulation, GTP binding protein kinase, transmembrane transporters, and calcium ion binding, but also functionally assigned the clustered genes to the superfamily level. We found that 10 superfamilies of Fox, Sox, Pax, POU, Six, Hox, Wnt, Bmp, Fgf, and Collagen enriched 101 deregulated genes. Thus, integration of functional classification into hierarchical GO trees permitted us to focus more on the GOs relevant to oxygen metabolism, angiogenesis, transmembrane transporters, GTPase-tyrosine kinase activity, DNA replication, neural crest cell migration, cell differentiation, axon extension, and epidermal and muscle development.By combining the hierarchical Gene Ontology(GO) term network, the DAVID annotation system,and Euclidean distance clustering, we identified four signaling pathways: foxi1-foxo1b-pou3f1, pax3a-mant3a-col11/col2, pou5f1-cdx4-kdrl, and isl1-wnt11 PCP-sox9 a. Results from immunohistochemical staining and promoter-driven transgenic fish suggest that these pathways respectively define wound epidermis reconstitution, cell type conversions, blastemal angiogenesis/vasculogenesis, and cartilage matrix-orientation. Foxi1 morpholino-knockdown caused expansions of Foxo1b-and Pax3a-expression in the basal layer-blastemal junction region. Moreover, foxi1 morphants displayed increased sox9 a and hoxa2 b transcripts in the embryonic pharyngeal arches. Thus, a Foxi1 signal switch is required to establish correct tissue patterns, including reepitheliation and blastema formation. This study provides novel insight into a blastema regeneration trategy devised by epithelial cell transdifferentiation, blood vessel engineering, and cartilage matrix deposition. |
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