Genetic pathways that promote cell-survival during development are poorly defined, yet represent potential targets for anti-cancer drugs. The genes that promote survival of melanocytes, in particular those that operate independently of the well-characterized anti-apoptotic pathway headed by the receptor tyrosine kinase, C-kit, are largely unknown. Through forward and reverse genetic studies in zebrafish, we have identified two genes that regulate the number of embryonic melanocytes independently of C-kit. One is Touchtone (Tct), which appears to prevent necrotic cell death in melanocytes, but whose molecular identity is unknown. The other is transcription factor AP-2alpha, which has long been known to be expressed in neural crest, but whose function there has remained obscure because of redundancy. Interestingly we have evidence that Tct and AP-2 are also required within neural crest-derived Rohon-Beard sensory neurons (RBs). Our overall hypothesis is that Tct and AP-2 regulate survival of neural crest derivatives, and that they may do so as part of a single regulatory pathway.
In Aim 1, to gain insight into the possibly novel Tct pathway, we propose to identify the Tct gene by positional cloning and identify new alleles.
In Aim 2, to learn the function of AP-2-type activity in neural crest and melanocytes, we propose temporal and spatial modulation of AP-2-type activity with cell-type-specific promoters.
In Aim 3, to determine the function of Tct and AP-2 in RBs, we propose ultrastructural analysis of RBs in the Tct mutant, and temporal and spatial modulation of AP-2-type activity with RB-specific promoters. Finally we propose to test for possible interaction of Tct and AP-2 type activity with double mutant experiments. These experiments exploit the advantages of the zebrafish model to dissect poorly understood neural crest regulatory pathways. These pathways are potential entry points for therapies against malignant melanoma and other neural crest diseases.
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