Summary Digit regeneration is regulated by Msx 1 and BMP 4 in fetal mice

The amphibian limb has been the prototypical organ for the study of epimorphic regeneration both in adults and during development. These studies indicate a number of requirements for successful limb regeneration, including: (1) a wound healing response with the formation of an apical epithelial cap (AEC); (2) a cellular contribution, via the de-differentiation of mature tissues and/or stem cells, to form the blastemal mesenchyme; (3) a proliferative response regulated by multiple tissue interactions, including a neurotrophic effect, an AEC effect and a positional effect; and (4) morphogenesis and redifferentiation to restore the limb pattern (reviewed by Stocum, 1995; Tsonis, 1996). In general, the regeneration response can be separated into those events that are injury related and crucial for blastema formation, and those that represent a ‘redevelopment’ response whereby regeneration recapitulates limb development (see Bryant et al., 2002). It is clear that the complexity of the regeneration response lends itself to multiple avenues whereby regeneration can be inhibited, and indeed numerous requirements for limb regeneration have been identified in this way. In parallel, there is a growing database of genes that are likely to play key roles, based on their regulated expression during regeneration, yet in most cases a functional understanding of how these genes act remains largely unexplored (see Gardiner et al., 2002). The regeneration of amputated distal digit/finger tips has been reported in various mammals, including humans and rodents, and represents a mammalian system where successful epimorphic regeneration can occur (see Muller et al., 1999). In humans, regenerative potential is restricted to the distal tip of the finger in a region associated with the nail organ (Douglas, 1972; Illingworth, 1974). Experimental studies using rodent models demonstrate that regenerative capacity is level specific and, similarly, is associated with the proximal extent of the nail forming organ (Borgens, 1982; Zhao and Neufeld, 1995). A role for the nail organ in the regeneration response is supported by experiments showing that excluding the nail organ from the amputation wound results in no regenerative response; however, nail organ grafts result in ectopic bone formation and do not induce regeneration (Zhao and Neufeld, 1995; Mohammad et al., 1999). These experimental results, together with clinical studies in humans, provide evidence that the nail organ plays some role in the stimulation of adult digit tip regeneration. Genes specifically expressed in, or associated with, the nail organ are candidates for regulating this regenerative response. In previous studies, we demonstrated that the homeobox-containing genes Msx1 and Msx2are expressed in association with the nail organ of neonatal digits, and at the apex of developing digits in the nail-forming region (Reginelli et al., 1995). Furthermore, mapping the regenerative ability of embryonic and fetal digit tips demonstrated that regenerative capacity correlated with amputation within the Msx1, but not the Msx2, expression domain in developing digits. These studies suggest a role for MSX genes in the digit regeneration response. In all tetrapod vertebrates, Msx1 and Msx2are co-expressed The regeneration of digit tips in mammals, including humans and rodents, represents a model for organ regeneration in higher vertebrates. We had previously characterized digit tip regeneration during fetal and neonatal stages of digit formation in the mouse and found that regenerative capability correlated with the expression domain of the Msx1gene. Using the stage 11 (E14.5) digit, we now show that digit tip regeneration occurs in organ culture and that Msx1, but not Msx2, mutant mice display a regeneration defect. Associated with this phenotype, we find that Bmp4 expression is downregulated in the Msx1 mutant digit and that mutant digit regeneration can be rescued in a dose-dependent manner by treatment with exogenous BMP4. Studies with the BMP-binding protein noggin show that wild-type digit regeneration is inhibited without inhibiting the expression of Msx1, Msx2or Bmp4. These data identify a signaling pathway essential for digit regeneration, in which Msx1functions to regulate BMP4 production. We also provide evidence that endogenous Bmp4 expression is regulated by the combined activity of Msx1 and Msx2 in the forming digit tip; however, we discovered a compensatory Msx2response that involves an expansion into the wild-type Msx1 domain. Thus, although both Msx1 and Msx2 function to regulate Bmp4expression in the digit tip, the data are not consistent with a model in which Msx1 and Msx2 serve completely redundant functions in the regeneration response. These studies provide the first functional analysis of mammalian fetal digit regeneration and identify a new function for Msx1and BMP4 as regulators of the regenerative response.