Jamie Elifritz Are Bone Morphogenic Proteins the Negative Schwann Cell Factors in Urodelian Limb Regeneration? The Intrigue Continues Division Sciences Department Biology Year 2002 Advisor Ken Klatt

Urodele amphibians have the capability to regenerate lost limbs via the formation of a blastema of progenitor cells through the dedifferentiation of mature cells. The early phase of this process has been suggested to be nerve-dependent. If the peripheral nerve is intact following amputation, the neurons supply a protein, glial growth factor (GGF), which binds to Schwann cells, and leads to normal limb regeneration. If the peripheral nerve is cut following amputation, however, the glial growth factor is not produced by the neurons and the Schwann cells secrete a negative factor that inhibits blastemal cell cycling and regeneration. In the embryonic limb, Bone Morphogenetic Proteins (BMPs) inhibit undifferentiated cell proliferation, thus, we hypothesized that in the regenerating limb, the scarcity of GGF would cause the secretion of BMP-4, and regeneration would be inhibited. We have cloned a 275 base pair portion of a BMP messenger RNA from young Mexican axolotl early bud stage RNA. We proved that a PCR product discovered by Paul Bryson (DU ’00) was BMP and we showed that the timing of RNA extraction correlates with the observation of BMP mRNA production.

BMP has been shown to inhibit FGF, which is a crucial signal for the proliferation of cells within the apical ectodermal cap (AEC) and outgrowth of the bud. Additionally, overexpression of BMP-2 and BMP-4 in embryonic chick limbs  has been found to lead to abnormal morphological limb structures. These findings led us to hypothesize that injecting axolotl limbs with human BMP-4 would either halt or slow the regeneration process and lead to visible morphological deformities of the limb. After six injections of human BMP-4 at high concentration (600ng/mL), the rate of regeneration slowed, and there were malformations of the limb.