Genetic Requirement for Epidermal Growth Factor Receptor (EGFR) signaling in Progenitor Cell Responses in a Mammalian Limb Regeneration Model

The Epidermal Growth Factor Receptor (EGFR) family of tyrosine kinase receptors plays an important role in cell proliferation, migration, survival and differentiation in multiple tissue types. In skeletal tissue, EGFR family signaling is required for differentiation of skeletal progenitors, and for development and homeostasis of cartilage and bone tissue. Our prior studies have also shown that EGFR family signaling is required for mammalian limb regeneration responses. We found that the spontaneous regeneration response that normally occurs following distal amputation of the neonatal mouse digit tip is impaired in transgenic mice with conditional limb targeted deletion of the EGFR or its related receptor, ErbB3. The goal of this project is to understand the molecular and cellular mechanisms that mediate the genetic requirement for EGFR and ErbB3 for limb regeneration responses in the mouse digit tip. Our study demonstrates that bone formation (volume and length) of the distal most digit phalanx, as revealed by Micro Computed Tomography (uCT) analysis, are significantly reduced in the digit tips of mice with conditional loss of EGFR (EGFR-cko mice) or ErbB3 (ErbB3-cko mice) 6 weeks after amputation compared to littermate control wild type digits. In addition, we found that cell proliferation, as determined by Ki67 immunostaining, is significantly reduced among mesenchymal cells that accumulate at the distal edge of the digit stumps (the putative blastema cells) of ErbB3-cko digits 7 and 11 days after amputation. Cell proliferation in the same region of EGFR-cko digit stumps was unchanged. Expression of the blastema marker msx1 was significantly decreased in the distal mesenchyme of EGFR-cko digit stumps but not in ErbB3-cko digit stumps 11 days after amputation. These results confirm that the spontaneous regeneration response of normal neonatal mouse digit tips amputated at a distal level is impaired by either loss of EGFR or ErbB3. However, our results demonstrate that the mechanism by which genetic loss of EGFR or ErbB3 impairs the spontaneous digit tip regeneration response in amputated neonatal digit tips is distinct. We suggest that genetic loss of ErbB3 impairs digit tip regeneration by reducing blastema cell proliferation, such that insufficient numbers of progenitor cells are available to re-form the amputated digit tip. In contrast, we suggest that genetic loss of EGFR impairs spontaneous digit tip regeneration by suppressing the differentiation of blastema cell progenitors into the skeletal lineage. Based on these results, we propose that the function of ErbB3 signaling during mouse digit tip regeneration is to maintain the proliferation of blastema progenitor cells, while the function of EGFR is to promote the re-differentiation of progenitor cells into the skeletal lineage. These studies suggest that stimulation of distinct EGFR family signals may have therapeutic utility in the future in promoting progenitor cell responses important for limb regeneration in humans.