Germ Layer Restricted Wnt and Hedgehog Signaling Regulate Digit Tip Regeneration
Zeshaan N. Maan1, Yuval Rinkevich, PhD2, Michael Januszyk, MD, PhD1, Irving Weissman, MD1, Geoffrey C. Gurtner, MD, FACS1.
1Stanford University School of Medicine, Stanford, CA, USA, 2Helmholtz Zentrum München, Munich, Germany.
The human hand plays an essential role in our normal daily activities. The anatomical complexity of the hand, and critical functions of each of its component tissues, highlights the need to understand and effectively treat injuries to this vital multi-tissue structure. Surgical repair is often complicated by the scarcity of local soft tissue and loss of functional capacity. Reports of "salamander-like" spontaneous regeneration in human fingertips and recent advances using mouse models of digit tip regeneration raise the possibility of restorative/regenerative rather than reparative approaches for traumatic hand injuries. These mouse studies demonstrate the various embryonic origins of mammalian limb tissue, including ectoderm, mesoderm, and neural crest, the role of lineage-restricted stem and progenitor cells in mammalian digit regeneration and some of the signaling pathways involved. The discrete signaling mechanisms governing this regenerative process, however, remain poorly understood.
Tissue harvested from murine digit tips 7 days after amputation at the regenerative plane (distal to the germinal matrix) or non-regenerative plane (proximal to the germinal matrix) was compared using microarray analysis. This comparison detected only genes differentially expressed in regeneration, and allowed exclusion of genes related to typical non-regenerative, fibrotic wound healing. Viral knockdown of candidate genes identified by microarray analysis was used to confirm a functional role for these signaling networks in vivo. Tissue specific expression and response patterns of target genes were assessed using gene specific reporter mice.
Microarray analysis identified 287 genes differentially regulated in regenerating v. non-regenerating digit tips. Ingenuity pathway analysis identified the top associated gene networks, the highest ranked of which were related to Wnt and Hedgehog (HH) signaling and suggested cross-talk between these signaling pathways (Figure 1). Dkk1 adenovirus, which suppresses Wnt signaling, significantly impaired digit tip regeneration compared to control adenovirus. Wnt overexpression did not accelerate regeneration, but did lead to nail overgrowth. Gli knockdown, which results in reduced HH signaling, significantly impaired digit regeneration. Interestingly, HH overexpression using an adenovirus impaired epidermal closure, which is typically driven by Wnt, suggesting cross-talk between these pathways. Using a variety of reporter mice for Wnt and HH expression and responsiveness, we demonstrated that Wnt signlaling originated in the ectodermal layer and Wnt responsiveness was also limited to the ectodermal layer. Hedgehog signaling also originated in the ectodermal layer, but HH responsiveness occurred in the mesodermal layer.
Unbiased microarray analysis combined with in vivo knockdown studies identify a central role for Wnt and HH signaling in digit tip regeneration. Ectodermal Wnt signaling targeting the ectoderm interacts with ectodermal Hedgehog signaling targeting the mesoderm to critically regulate regeneration of the mouse digit tip. Understanding and targeting the cross-talk between these pathways could enable regeneration in normally non-regenerative extremity injuries.
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