The aneurogenic limb identifies developmental cell interactions underlying vertebrate limb regeneration

Abstract

The removal of the neural tube in salamander embryos allows the development of nerve-free aneurogenic limbs. Limb regeneration is normally nerve-dependent, but the aneurogenic limb regenerates without nerves and becomes nerve-dependent after innervation. The molecular basis for these tissue interactions is unclear. Anterior Gradient (AG) protein, previously shown to rescue regeneration of denervated limbs and to act as a growth factor for cultured limb blastemal cells, is expressed throughout the larval limb epidermis and is down-regulated by innervation. In an aneurogenic limb, the level of AG protein remains high in the epidermis throughout development and regeneration, but decreases after innervation following transplantation to a normal host. Aneurogenic epidermis also shows a fivefold difference in secretory gland cells, which express AG protein. The persistently high expression of AG in the epithelial cells of an aneurogenic limb ensures that regeneration is independent of the nerve. These findings provide an explanation for this classical problem, and identify regulation of the epidermal niche by innervation as a distinctive developmental mechanism that initiates the nerve dependence of limb regeneration. The absence of this regulation during anuran limb development might suggest that it evolved in relation to limb regeneration.

AGR2 Gene Function Requires a Unique Endoplasmic Reticulum Localization Motif

“…AREG and CDX2 expression thus provided an opportunity to evaluate the protein domains necessary for AGR2 function. Published studies to date have reported an extracellular role for AGR2 as a secreted protein as well as an intracellular role as a protein chaperone or stress response factor (7,10,14,16,32,33). Among the factors favoring an intracellular role based in the ER is the presence of a carboxyl-terminal motif, KTEL, that is conserved from Xenopus to humans.…”

“…The AGR2 N terminus contains a sequence motif characteristic of signal peptides, which results in protein targeting to the secretory pathway of the cell. Indeed, several studies have proposed that AGR2 secretion from the cell is necessary for its action (7,10,14,16). In addition, yeast two-hybrid screens identified AGR2 binding proteins that naturally occur on the cell surface (9,22).…”

“…AGR2 was first described in Xenopus laevis where its expression is responsible for the development of a glandular organ called the cement gland (7,8). A significant role in tissue regeneration was established for AGR2 in salamanders where it functions in nerve-dependent limb regeneration (9,10). AGR2 is also expressed by secretory cells in the normal murine intestine (11).…”

Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration

“…These limbs can be transplanted to the flank of a normal larva, where they can regenerate in the absence of nerve supply (33). This lack of nerve dependence is made possible by the substitution of nerve-derived factors from ectodermal appendages, including epidermis and glands (33).…”

A brief history of the study of nerve dependent regeneration

“…One candidate factor is anterior gradient, which is expressed in the developing aneurogenic limb but decreased after grafting and innervation. 35 A series of grafting and in vitro studies in the latter half of the 20th century implicated proliferating blastemal cells as a major target of the nerves. In 1977, Liversage & Globus showed that blastemas cultured in vitro proliferated only if they remained innervated by spinal cord implants, 36 while Goldhamer et al found that implantation of dorsal root ganglia into the blastemas of denervated limbs rescued cell cycling, although it did not increase cell cycling in fully-innervated blastemas.…”