Control of normal and malignant pigment cell development
Final Report Abstract
Our long-term goal is to understand the genetic control of the pathway from a normal to a malignant pigment cell in melanomagenesis. In this project we followed two approaches. Firstly, making use of the well-established genetics of melanoma formation in the Xiphophoms hybrid system, we attempted to moleculariy clone and functionally analyze loci involved in pigmentation and melanomagenesis. Such loci have been mapped to the vicinity of the oncogene xmrk on the platyfish sex chromosomes. An existing BAC collection from the X and Y chromosome was refined and assembled into contigs spanning the pigmentation and melanoma region on the X and Y. Sequencing has identified so far more than 60 predicted genes. One, the GTP cyclohydrolase I, represents a reasonable candidate for the red/yellow pigmentation pattern (and derived pigment cell tumor) locus. The encoded enzyme is the key protein for red/yellow pigment production and initiates the pteridine pathway. We found a perfect correlation in gene expression with red/yellow pigmentation pattems. Functional analyses of the protein in vitro and transgenic experiments have to be performed in the future to substantiate the role ofthis gene for pigment pattern formation. Interestingly up to 10 copies of a melanocortin receptor subtype (mc4r) gene were identified in each BAC contig. Expression in skin and melanoma, as well as the localization of some copies to a segment, where also the macromelanophore locus (Mdl) was mapped, proposes that these genes are candidates for Mdl. Functional analysis, using the prototypic Mc1r as reference revealed that at least some mc4r copies encode functional receptors, while others appear to act as dominant negative versions. Further analyses with genetically modified medaka are in progress. As a second approach we analyzed the development of stem cells to melanocytes in the nonneoplastic situation as a basis for understanding the melanoma stem cell / derived melanoma cell transition, reasoning that there are common mechanisms of development and disease. We could show that the transcription factor Mitf can act as a master regulator of physiological pigment cell differentiation. Transfection of a Mitf expression vector into medaka embryonic stem cells and spermatogonial stem cells initiated the full differentiation program towards functional melanophores without the need for further factors or manipulations. Monitoring expression of pigment cell differentiation genes and genes involved in melanomagenesis revealed that surprisingly the differentiating embryonal and adult stem cells pass through stages, which are in the developmental pathway before the stage where Mitf is normally active, e.g. a neural crestlike stage. The protocol for producing melanocytes and melanophores from embryonal and adult stem cells was also successful for two other master regulators, namely cbfa1 and mash1, which led to the induced differentiation of neuronal and osteoblast-like cells in vitro. Again, we observed a recapitulation of the temporal expression pattern of marker genes and obviously passage through differentiation stages prior to those where the respective master regulator are active.
Publications
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(2003) Mitf expression is sufficient to direct differentiation of medaka blastula derived stem cells to melanocytes. Development 130; 6545-53
J. Bejar, Y. Hong and M. Schartl
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(2006) Molecular analysis of the sex-determining region of the platyfish Xiphophorus maculatus. Zebrafish 3: 295- 305
Schultheis C , Q. Zhou, A. Froschauer, I. Nanda, Y. Selz, C. Schmidt, S. Matschl, M. Wenning, A.-M. Veith, M. Naciri, R. Hanel, I. Braasch, A. Dettai, A. Böhne, C. Ozouf-Costaz, B. Segurens, A. Couloux, S. Bernard-Samain, M. Schmid, M. Schartl, J.-N. Volff
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(2007) Evolution of melanocortin receptors in teleost fish: the melanocortin type 1 receptor. Gene 401: 114-122
Selz Y., I. Braasch, C. Hoffmann, C. Schmidt, C. Schultheis, M. Schartl, J.-N. Volff
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(2007) Evolution of pigment synthesis pathways by gene and genome duplication in fish. BMC Evolutionary Biology 7: 74
Braasch I., M. Schartl, J.-N. Volff
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(2008) Identification of new gene candidates on the sex chromosomes of the platyfish Xiphophoms maculatus. Cybium 32:69-71.
Böhne A., C. Schultheis, Q. Zhou, A. Froschauer, C. Schmidt, Y. Selz, I. Braasch, C. Ozouf- Costaz, A. Dettai, B. Segurens, A. Couloux, S. Bernard-Samain, S. Chilmonczyk, A. Gannouni, K. Madani, F. Brunet, D. Galiana-Arnoux, Manfred Schartl, J.-N. Volff
