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

OF THE DISSERTATION MELANOCYTES IN THE DEVELOPING AND ADULT ATRIOVENTRICULAR VALVES OF THE MURINE HEART by Flavia Cameiro Brito Florida International University, 2008 Miami, Florida Professor Lidia Kos, Major Professor The Neural Crest (NC) is a multipotential group o f cells that arises from the dorsal aspect o f 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 o f the aortic arch arteries and septation o f 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 o f 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 o f skin melanocytes, do not have cardiac melanocytes indicating that cardiac and skin melanocyte precursors share the same initial signaling requirements. The analysis o f murine adult hearts showed that melanocytes are mostly found in the atrial sides o f the tricuspid and mitral valve leaflets. The distribution o f melanocytes in the AV valves corresponds exactly to areas o f high Versican B expression, a proteoglycan essential for the process o f AV valve remodeling. To evaluate a potential role for melanocytes in the AV valves, a nanoindentation analysis o f the tricuspid valves o f wild type, hypopigmented and hyperpigmented mice was performed. The storage modulus, a measure o f 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 o f the AV valves. TABLE OF CONTENTS CHAPTER PAGE I. RESEARCH OVERVIEW. 1 1. Neural Crest Cells.2 2. Melanocytes. 3 3. Cardiac Neural Crest Cells. 8 4. Heart Development.11 5. Research questions. 16 5.1 Do cardiac neural crest cells contribute to the proper development of the mammalian heart? 17 5.2 When do NC derived melanocytes precursors reach the heart and what is migratory pathway they take to reach i t ? 18 5.3 Do NC derived melanocytes affect the ECM environment and mechanical properties o f the AV valves? 18 References. 19 II. THE CONTRIBUTION OF NEURAL CREST DERIVED CELLS TO THE DEVELOPING HEART. 27 1. Cardiac Neural Crest Cells. 28 2. The fates o f Cardiac Neural Crest Cells.28 3. Cardiac Neural Crest Cells Markers. 30 4. Alternative Roles for Cardiac Neural Crest Cells. 36 4.1 In the Myocardium. 36 4.2 In the Conduction System and AV Valves.37 4.3 As Stem Cells.41 References.43 III. TIMELINE AND DISTRIBUTION OF MELANOCYTE PRECURSORS IN THE MOUSE HEART.50 1. Abstract.51 2. Introduction . 52 3. Results.54 3.1 NC cells reach the heart already committed to the melanocytic fate.54 3.2 Cardiac melanocytes depend on Ednrb and Kit signaling.59 3.3 Cardiac melanocytes are only found in four-chambered hearts 60 4. Discussion. 61 5. Methods.65 5.1 M ice. 65 5.2 Semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). 66 5.3 l Dopa, P-galactosidase staining and in situ hybridization.66 References. 68