Summary
Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals; possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.
Adrenomedullin (AM), a recently discovered hypotensive peptide, is expressed in the endocrine pancreas of different species, as demonstrated by immunocytochemistry. Electron microscopic studies with double immunogold showed colocalization of AM and pancreatic polypeptide. A homogeneous expression of AM receptor was found throughout the islet using in situ hybridization. Six different insulin- producing cell lines have been analyzed by reverse transcription-PCR and showed expression of both AM and its receptor. Two experimental models have been used to study the effects of AM in pancreatic physiology. 1) Analysis of isolated rat islets shows that AM inhibits insulin secretion in a dose-dependent manner. The monoclonal antibody MoAb-G6, which neutralizes AM bioactivity, was able to increase insulin release 5-fold; this effect was reversed by the addition of synthetic AM. 2) Oral glucose tolerance tests showed that iv injection of AM reduces the levels of insulin in the bloodstream with a concomitant increase in circulating glucose. These studies implicate AM as a newly defined factor of the insulin regulatory system that could be involved in disorders such as diabetes and obesity.
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