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

Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these compo- nents form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signal- ing, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unex- pected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic pheno- type. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the bal- ance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernu- merary DRG sensory neurons in a wild-type background, and can rescue the sensory neu- ron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10 (baz1) protein\u2019s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lat- eral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates

A Systematic Survey of Expression and Function of Zebrafish <em>frizzled</em> Genes

<div><p>Wnt signaling is crucial for the regulation of numerous processes in development. Consistent with this, the gene families for both the ligands (Wnts) and receptors (Frizzleds) are very large. Surprisingly, while we have a reasonable understanding of the Wnt ligands likely to mediate specific Wnt-dependent processes, the corresponding receptors usually remain to be elucidated. Taking advantage of the zebrafish model's excellent genomic and genetic properties, we undertook a comprehensive analysis of the expression patterns of <em>frizzled</em> (<em>fzd</em>) genes in zebrafish. To explore their functions, we focused on testing their requirement in several developmental events known to be regulated by Wnt signaling, convergent extension movements of gastrulation, neural crest induction, and melanocyte specification. We found fourteen distinct <em>fzd</em> genes in the zebrafish genome. Systematic analysis of their expression patterns between 1-somite and 30 hours post-fertilization revealed complex, dynamic and overlapping expression patterns. This analysis demonstrated that only <em>fzd3a, fzd9b,</em> and <em>fzd10</em> are expressed in the dorsal neural tube at stages corresponding to the timing of melanocyte specification. Surprisingly, however, morpholino knockdown of these, alone or in combination, gave no indication of reduction of melanocytes, suggesting the important involvement of untested <em>fzds</em> or another type of Wnt receptor in this process. Likewise, we found only <em>fzd7b</em> and <em>fzd10</em> expressed at the border of the neural plate at stages appropriate for neural crest induction. However, neural crest markers were not reduced by knockdown of these receptors. Instead, these morpholino knockdown studies showed that <em>fzd7a</em> and <em>fzd7b</em> work co-operatively to regulate convergent extension movement during gastrulation. Furthermore, we show that the two <em>fzd7</em> genes function together with <em>fzd10</em> to regulate epiboly movements and mesoderm differentiation.</p> </div

<i>fzd</i> gene knockdown has no effect on NCC induction, but causes CE defects.

<p>Embryos were injected with standard control-MO alone (A, B, C), or a mixture of <i>fzd7a, fzd7b</i>, and <i>fzd10</i>-M (D, E, F) or <i>fzd3a, fzd7a, fzd7b, fzd9b</i> and <i>fzd10</i>-MO (G, H, I), respectively. The amounts of each morpholino were as indicated. Embryos were examined by in situ hybridization analysis at 1-somite stage with three early NCC markers, <i>foxd3</i> (A, D, G), <i>pax3a</i> (B, E, H) and <i>sox10</i> (C, F, I). All are dorsal views with anterior oriented to the top. Scale bar: 100 µm.</p

Sequence of morpholinos.

<p>Sequence of morpholinos.</p

Expression of <i>fzd7a</i>, <i>fzd7b</i> and <i>fzd10</i> at gastrula stage.

<p>A, B) <i>fzd7a</i> expression. C, D) <i>fzd7b</i> expression. E–H) <i>fzd10</i> expression. Left column shows expression at 6 hpf and right column shows expression at 80% epiboly stage as indicated. All are left side views with anterior to the top. Embryos in panel E and G, and embryos in panel F and H are the same ones. We focused on medial plane along the animal-vegetal axis for embryos in panel A–F. Embryos in panel G and H alone were focused on lateral surface to show signal in marginal area. Arrowheads in panels A, G, H indicate the marginal expression, and arrows in B and D show the lack of expression (see text). Scale bar: 100 µm.</p

Melanisation occurs in <i>sox10</i> mutant heterozygous embryos injected with <i>fzd3a, 9b, 10</i>-MO at 1 dpf.

<p>Notes.</p><p>50% of embryos examined for each category should be <i>sox10</i> mutant heterozygotes. Numbers in parenthesis are of significantly melanised embryos. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054833#pone-0054833-t003" target="_blank">Table 3</a> for definition of categories.</p