Time Point-Based Integrative Analyses of Deep-Transcriptome Identify Four Signal Pathways in Blastemal Regeneration of Zebrafish Lower Jaw

Abstract

There has been growing interest in applying tissue engineering to stem cell-based regeneration therapies. We have previously reported that zebrafish can faithfully regenerate complicated tissue structures through blastemal cell type conversions and tissue reorganization. To unveil the regenerative factors and engineering arts of blastemal regeneration, we conducted transcriptomal analyses at four time points corresponding to preamputation, re-epitheliation, blastemal formation, and respecification. By combining the hierarchical gene ontology 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 PCPsox9a. 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 morpholinoknockdown caused expansions of Foxo1b-and Pax3a-expression in the basal layer-blastemal junction region. Moreover, foxi1 morphants displayed increased sox9a and hoxa2b transcripts in the embryonic pharyngeal arches. Thus, a Foxi1 signal switch is required to establish correct tissue patterns, including re-epitheliation and blastema formation. This study provides novel insight into a blastema regeneration strategy devised by epithelial cell transdifferentiation, blood vessel engineering, and cartilage matrix deposition. STEM CELLS 2015;33:806-818

Tissue Extract Fractions from Starfish Undergoing Regeneration Promote Wound Healing and Lower Jaw Blastema Regeneration of Zebrafish

“…As previously reported5, complete reepitheliation and transition from mesenchymal blastema to chondrogenic blastema were clearly observed in DMSO control specimens (Fig. 5).…”

“…2b–d). These genes (supplemental Table 1) have been previously found to be deregulated during zebrafish lower jaw regeneration5. Compared with GCV treatment, two genes were simultaneously induced by all A. typicus extract fractions.…”

“…It is unclear whether the injured tissues or water vascular canal area release bioactive substances that direct cell growth and behavior. Because lower jaw amputation induces extensive responses in multiple zebrafish tissues as early as 2 hpa5 (data not shown), we cut the arm-tips to induce tissue regeneration, and then collected the entire remaining starfish for extraction. Cell viability and proliferation was determined with an MTT assay in the presence of extract fractions from A. typicus .…”

“…They can regenerate entire arms after both autotomic and traumatic amputation2. Unlike most vertebrates in which the regenerative capacity is generally limited to the healing of wounds, zebrafish can regenerate lost organs and restore complex tissue structures345.…”

Zebrafish Craniofacial Development

“…Time-lapse imaging in adults has beautifully demonstrated this unusual dedifferentiation process in real time (Knopf et al, 2011), with more recent work showing a similar type of regeneration in the calvarial bones of fish (Geurtzen et al, 2014). In contrast, regeneration of the lower jaw proceeds through a cartilage intermediate (S. Paul & G. D. Crump, unpublished; Wang et al, 2012; Zhang et al, 2015), much like fracture repair in humans. In the future, it will be interesting to examine the extent to which zebrafish skeletal tissues regenerate better than their mammalian counterparts, as well as the genetic and cellular basis of such regeneration.…”