Endothelin receptor B and neural crest derived melanocyte development

Neural Crest cells (NCC) constitute a unique embryonic cell population that arises between the prospective epidermis and the dorsal aspect of the neural tube of vertebrates. NCC migrate ventromedially and dorsolaterally throughout the developing embryo giving rise to the peripheral nervous system constituents and melanocytes that ultimately reside in the skin and hair follicles respectively. Mice and humans with mutations in the Endothelin receptor b (Ednrb) gene manifest strikingly similar phenotypes characterized by hypopigmentation, hearing loss and megacolon these are due to absence of melanocytes in the skin and inner ear and lack of enteric ganglia in the distal part of the gut, respectively. Piebald lethal mice and humans with Hirschsprung's disease or Waardenburg syndrome carry different mutations in the Ednrb gene. The major goals of this project were to determine whether the action of Ednrb in NCC is required prior to commitment of these cells to the melanocytic lineage and to investigate its potential participation in the actual process of commitment. In order to achieve these goals transgenic mice that express Ednrb under two different regulatory elements were created. The first, Dct-Ednrb, expresses Ednrb under the control of the DOPAchrome tautomerase (Dct) promoter to direct expression to already committed melanocyte precursors. The second, Nes-Ednrb, expresses Ednrb under the regulation of the human nestin gene second enhancer to direct expression to pre-migratory NCC. Crosses of the Dct-Ednrb mouse with piebald lethal showed that the transgene was capable of rescuing the hypopigmentation phenotype of the later. This result indicates that the action of Ednrb after NCC commit to the melanocytic lineage is sufficient for normal melanocyte development. The Dct-Ednrb was further crossed with two other hypopigmentation mutants that carry mutations in the transcription factors Sox10 and Pax3. The transgene rescued the phenotype of the Sox10 mutant only. This suggests that Ednrb interacts with Sox10 but not with Pax3 during melanocyte development. The Nes-Ednrb mice developed a hypopigmentation phenotype that was augmented when crossed with piebald lethal or lethal spotting (mutation in Edn3, the ligand for Ednrb) mice but was rescued by over expression of Edn3. These results suggest that alterations in Ednrb expression early in development affect melanocyte development. This study provides novel information necessary to better understand the early embryonic development of NCC, clarifies specific interactions between different melanogenic genes and, could eventually help in the implementation of therapies for human pigmentary genetic disorders. ^

Melanocytes in the developing and adult atrioventricular valves of the murine heart

The Neural Crest (NC) is a multipotential group of cells that arises from the dorsal aspect of the neural tube early in development. It is well established that a group of NC cells named Cardiac Neural Crest (CNC) migrates to the heart and plays a critical role in the remodeling of the aortic arch arteries and septation of the outflow tract. In this study, using the mouse mutant Pax3sp/sp that has CNC deficits I have identified a putative novel role for the CNC in regulating apoptosis in the atrioventricular (AV) endocardial cushion. The AV endocardial cushion undergoes remodeling to give rise to the cardiac AV valves. Using a transgenic mouse that carries the LacZ reporter gene under the control of the Dopachrome tautomerase promoter (Dct-LacZ), I found that another NC derived population, melanocyte precursors, also contribute to the AV endocardial cushion and developing AV valves. The analysis of Dct-LacZ embryos at different stages showed that NC cells already committed to the melanocytic fate migrate to the heart along the same initial pathway taken by those that will populate the skin. Hypopigmented mice carrying mutations in the Kit and Endothelin receptor b genes, that are critical for the proper development of skin melanocytes, do not have cardiac melanocytes indicating that cardiac and skin melanocyte precursors share the same initial signaling requirements. The analysis of murine adult hearts showed that melanocytes are mostly found in the atrial sides of the tricuspid and mitral valve leaflets. The distribution of melanocytes in the AV valves corresponds exactly to areas of high Versican B expression, a proteoglycan essential for the process of AV valve remodeling. To evaluate a potential role for melanocytes in the AV valves, a nanoindentation analysis of the tricuspid valves of wild type, hypopigmented and hyperpigmented mice was performed. The storage modulus, a measure of stiffness, for the leaflets obtained from hyperpigmented mice was considerably higher (10.5GPa) than that for the leaflets from wild type (7.5GPa) and hypopigmented animals (between 3.5 and 5.5 GPa) suggesting that melanocytes may contribute to the mechanical properties of the AV valves.