Molecular Basis for the Nerve Dependence of Limb Regeneration in an Adult Vertebrate

Abstract

The limb blastemal cells of an adult salamander regenerate the structures distal to the level of amputation, and the surface protein Prod 1 is a critical determinant of their proximodistal identity. The anterior gradient protein family member nAG is a secreted ligand for Prod 1 and a growth factor for cultured newt blastemal cells. nAG is sequentially expressed after amputation in the regenerating nerve and the wound epidermis-the key tissues of the stem cell niche-and its expression in both locations is abrogated by denervation. The local expression of nAG after electroporation is sufficient to rescue a denervated blastema and regenerate the distal structures. Our analysis brings together the positional identity of the blastema and the classical nerve dependence of limb regeneration.

Mechanisms of urodele limb regeneration

“…Many growth factors are glycoproteins, several of which are expressed by the AEC and stimulate blastema cell proliferation in vitro and in vivo. Fgf1, Fgf2, Fgf8, and the anterior gradient protein (AG) are expressed by the AEC in vivo (Christensen, Weinstein, & Tassava, 2001, 2002; Han, An, & Kim, 2001; Kumar & Brockes, 2007). Blastema cells express the bek (FGFR2) receptor for Fgfs (Poulin & Chiu, 1995; Poulin, Patrie, Botelho, Tassava, & Chiu, 1993) and the AG receptor Prod1 (Kumar & Brockes, 2007).…”

“…Fgf1, Fgf2, Fgf8, and the anterior gradient protein (AG) are expressed by the AEC in vivo (Christensen, Weinstein, & Tassava, 2001, 2002; Han, An, & Kim, 2001; Kumar & Brockes, 2007). Blastema cells express the bek (FGFR2) receptor for Fgfs (Poulin & Chiu, 1995; Poulin, Patrie, Botelho, Tassava, & Chiu, 1993) and the AG receptor Prod1 (Kumar & Brockes, 2007). Fgf1 elevated the mitotic index of cultured blastema cells (Albert & Boilly, 1988; Albert, Boilly, Courty, & Barritault, 1987; Boilly, Cavanaugh, Hondermarck, Bryant, & Bradshaw, 1991), and Fgf2 elevated the mitotic index of blastema cells in amputated limbs covered by full‐thickness skin (Chew & Cameron, 1983).…”

“…Fgf1 elevated the mitotic index of cultured blastema cells (Albert & Boilly, 1988; Albert, Boilly, Courty, & Barritault, 1987; Boilly, Cavanaugh, Hondermarck, Bryant, & Bradshaw, 1991), and Fgf2 elevated the mitotic index of blastema cells in amputated limbs covered by full‐thickness skin (Chew & Cameron, 1983). The only AEC candidate factors so far reported to be downregulated by denervation and to substitute for the nerve in supporting the regeneration of denervated limbs to digit stages are Fgf2 (Mullen, Bryant, Torok, Blumberg, & Gardiner, 1996) and AG (Kumar & Brockes, 2007). Fgf2 was administered in beads only to late stage blastemas.…”

“…Conditioned medium of Cos7 cells transfected with the AG gene stimulates bromodeoxyuridine (BrdU) incorporation into cultured blastema cells. This incorporation is blocked by antibodies to Prod1, suggesting that AG acts directly on blastema cells through Prod1 to stimulate their proliferation (Kumar & Brockes, 2007). Finally, AG is expressed by the AEC of regenerating aneurogenic limbs (Kumar, Delgado, Gates, Neville, Forge, & Brockes, 2011).…”

Looking proximally and distally: 100 years of limb regeneration and beyond

“…The crucial experiment leading to this idea was the demonstration that a single protein, the anterior gradient protein (AGP), can substitute for the nerves in regenerating newt limbs (Kumar et al, 2007). AGP is a ligand for the blastema cell surface protein Prod1, a member of the Ly6 family of three-finger proteins anchored to the cell surface by a glycosylphosphatidyl inositol (GPI) linkage (Morais da Silva et al, 2002;Brockes and Kumar, 2008;Garza-Garcia et al, 2009).…”