Identifying cellular and molecular events during adult mouse digit tip regeneration
Description
Non-lethal amputation injuries following a typical wound healing response, in human beings may result in lifelong disability. Therefore, the ability to induce regeneration of amputated limbs, similar to axolotls, is of primary interest of regenerative medicine. The only regions in mammals that demonstrate epimorphic regeneration is restricted to the terminal phalanx. Amputation of the distal elements of the terminal phalanx of mice causes an initial wound healing response followed by a complete regeneration of the nail, bone and connective tissue. Thus far, most functional and genetic studies of digit regeneration have been performed in embryos or neonates, however, few studies have examined digit regeneration in the adult mouse Here, the first chapter encompasses a comprehensive, morphological, histological and volumetric analysis of the stages of adult digit regeneration. We found that amputation of approximately one third of the length of the digit led to an initial wound closure and an accumulation of a zone of mesenchymal cells that resembled a blastema at 10-12 days post-amputation. Next we performed a region-specific immunohistochemical analysis of the regenerating connective tissue using antibodies specific for Vimentin, a common mesenchymal marker, SCA-1, a putative stem-cell marker, and CD-31, an endothelial marker. This analysis revealed the ubiquitous expression of Vimentin in the blastema. However, only 7% of cells expressed SCA-1, and only 10% of cells expressed CD-31. Interestingly, 94% of SCA-1+ cells in the blastema were also CD-31 +. Compared to digits that were newly amputated, the total SCA-1 +, CD-31+, and SCA-1+/CD-31 + cells in the bone marrow and connective tissue were significantly lower when compared to the regenerate. According to our knowledge, this is the first molecular phenotypic expression analysis of the regenerating blastema tissue during adult digit regeneration The cellular origins of the adult mouse blastema are unknown. A lack of a means to analyze the origins of blastema cells has slowed our progress understanding the fundamentals of digit tip regeneration. In order to analyze the cellular origin of the blastema, the second chapter encompasses on developing methods that involve in vivo injection/electroporation of plasmids, and in vivo DiI injection and subsequent in vivo imaging of adult mouse digit tips. Using these methods, we demonstrate the movement of existing vimentin-positive local dermal connective tissue cells that contribute to the blastema during regeneration of the terminal phalanx In most adult mammalian amputated appendages, the wound healing responses lead to scaring and regeneration failure. However, the wound healing events of the amputated distal portions of the adult mouse digit tip leads to regeneration. This provides a rare opportunity to study the wound healing mechanisms present at an injury conducive for adult mammalian appendage regeneration. However, the genetic mechanisms related to adult mouse digit tip wound healing are yet to be explored. In order to generate a database of genes related to early wound healing, we conducted microarray analysis that revealed up regulation of 181 genes and down regulation of 286 genes during an early wound healing phase of regeneration. We used semi quantitative RTPCR, and immunohistochemistry and verified the upregulation of genes that include Mmp9, CathepsinK, and Tgfbeta1 three genes identified by the microarray. Due to functional significance of Tgfbeta signaling in other regeneration competent vertebrates systems, we decided to further analyze the expression pattern of a downstream target of Tgfbeta signaling, PSmad2, and found a similar expression pattern to that of Tgfbetar1. Injection of a Tgfbetar1 inhibitor, SB431542, during the wound healing phase inhibited blastema formation, thus suggesting the possibility that early wound healing events that lead to regeneration competence of the digit tip may proceed via Tgfbeta signaling