In many regenerating systems there are three pivotal processes that need to occur: i) cell proliferation to provide a cellular source for regeneration (except in those cases in which there can be a direct transdifferentiation or dedifferentiation and redifferentiation in the absence of proliferation); ii) these new cells will need to differentiate into the missing cell types; and iii) the new tissues, organs and structures derived from those cells will need to be proper patterned and integrate within the pre-existing tissues, organs or structures. Even though is relatively known how these different processes occur and are regulated in different systems, less known is how these 3 events are coordinated with each other.
Two recent papers in Development from the laboratories of José Luis de la Pompa (http://www.ncbi.nlm.nih.gov/pubmed/23344707) and Gilbert Weidinger (http://www.ncbi.nlm.nih.gov/pubmed/23462472) report on the role of Notch signalling pathway in coordinating cell proliferation and differentiation within the blastema during fin regeneration in zebrafish. In these animals fin regeneration goes through 3 stages: i) wound healing, ii) blastema formation (by about 48h post amputation), and iii) regenerative outgrowth (from 48hpa). During the growth of the new fin, the blastema (defined as a mass of proliferating undifferentiated cells) restricts to the distal tip (below the wound) of the new fin whereas a proximo-distal gradient of cell differentiation is established. Thus, the most proximal region close to the amputation plane contains differentiated cells with a very reduced proliferation (“differentiation zone”), whereas the distal proliferative blastema bears mainly precursor cells. Following loss-of-function and gain-of-function of Notch signalling both papers nicely show how this pathway maintains blastema cells in an undifferentiated, proliferative state
After amputation Notch pathway is activated in the proliferative blastema (2dpa) and, as regeneration proceeds, it is mainly restricted in this distal undifferentiated region (as opposed to the proximal “differentiation zone”). The inhibition of the Notch pathway (mediated by different drugs or using specific morpholinos against some elements of this pathway) impairs fin regeneration. Apparently, wound healing and the initial blastema formation are not affected after inhibiting Notch pathway, however from the regenerative outgrowth stage the proliferation of the blastema cells significantly decreases. This suggests that Notch is required to regulate proliferation within the fin blastema. Moreover, Notch seems also necessary to maintain blastemal cells.
Next, both papers used transgenic lines and the Gal4-UAS system to ectopically express the Notch1a intracellular domain (NICD), which translocates to the nucleus and modifies the expression of its target genes. Remarkably, the overexpression of Notch activity also impairs fin regeneration. During the first 3 days after amputation no significant differences with controls are observed, however, from day 5 the regenerative outgrowth is significantly reduced. Also, and compared to controls, treated blastemas appear wider and with obvious alterations in the density and organization of cells in their mesenchymal regions. By checking the proliferative rates along the regenerated fin as wells as the expression of different markers specific of blastema cells, both studies show how proliferation is significantly increased in the proximal differentiation zone and there is also a proximal expansion of the blastema. Interestingly, this proximal expansion of the proliferative blastema goes together with a significant decrease in cell differentiation. Both papers analyse bone regeneration after the overactivaton of Notch pathway by using specific markers of the skeletogenic lineage: pre-osteoblasts, committed osteoblasts and differentiated osteoblasts. In controls these different cell populations are differentially located along the proximo-distal axis of the regenerated fins with differentiated cells in proximal regions and precursors cells more distal. After activating the Notch pathway there is a significant decrease in the number of differentiated osteoblasts at the same time that early precursors, normally restricted to distal regions, are expanded also to more proximal regions.
Thus, Notch signalling appears to be necessary to maintain blastema cells into an undifferentiated proliferative state. The function of Notch in supressing bone differentiation during fin regeneration in zebrafish parallels the role of Notch during bone development in mammalian embryos in which this pathway maintains osteoblast progenitors undifferentiated. In summary, both studies show how the Notch pathway plays a pivotal role in coordinating cell proliferation and differentiation in a regenerative context.