An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development

The mechanisms generating stably differentiated cell-types from multipotent precursors are key to understanding normal development and have implications for treatment of cancer and the therapeutic use of stem cells. Pigment cells are a major derivative of neural crest stem cells and a key model cell-type for our understanding of the genetics of cell differentiation. Several factors driving melanocyte fate specification have been identified, including the transcription factor and master regulator of melanocyte development, Mitf, and Wnt signalling and the multipotency and fate specification factor, Sox10, which drive mitf expression. While these factors together drive multipotent neural crest cells to become specified melanoblasts, the mechanisms stabilising melanocyte differentiation remain unclear. Furthermore, there is controversy over whether Sox10 has an ongoing role in melanocyte differentiation. Here we use zebrafish to explore in vivo the gene regulatory network (GRN) underlying melanocyte specification and differentiation. We use an iterative process of mathematical modelling and experimental observation to explore methodically the core melanocyte GRN we have defined. We show that Sox10 is not required for ongoing differentiation and expression is downregulated in differentiating cells, in response to Mitfa and Hdac1. Unexpectedly, we find that Sox10 represses Mitf-dependent expression of melanocyte differentiation genes. Our systems biology approach allowed us to predict two novel features of the melanocyte GRN, which we then validate experimentally. Specifically, we show that maintenance of mitfa expression is Mitfa-dependent, and identify Sox9b as providing an Mitfa-independent input to melanocyte differentiation. Our data supports our previous suggestion that Sox10 only functions transiently in regulation of mitfa and cannot be responsible for long-term maintenance of mitfa expression; indeed, Sox10 is likely to slow melanocyte differentiation in the zebrafish embryo. More generally, this novel approach to understanding melanocyte differentiation provides a basis for systematic modelling of differentiation in this and other cell-types. Author Summary: In a multicellular organism, one genome is used to make numerous different cell-types. This must require the activity of all these genes to be configured into multiple distinct and stable active states, each corresponding to one of the different cell-types characteristic of a tissue. The stable active states of differentiated cell-types contrast with the different, and transient, states characteristic of multipotent stem cells. We know little of the key features of these states that regulate the switch of a stem cell to stable differentiation. Here we examine this issue in the melanocyte, a genetically well-characterised cell-type, using a combination of dynamic mathematical modelling and experimental manipulation. In humans, disruption of the melanocyte state results in congenital and degenerative pigmentary diseases, whereas their destabilisation is likely to be an important factor in initiating melanoma. Our work predicts, validates, and identifies several novel features to the gene regulatory network of the zebrafish melanocyte, including one stabilising the differentiated state. Our study demonstrates the utility of this systems biology approach to understanding the genetic basis for differentiated cell states.