Neural crest fate restriction models. (a) Direct fate restriction (DFR). In the DFR model, highly multipotent NCC (rainbow colored) generates each fate directly (solid arrow), under direct influence of environmental signals (shown in red). Work in mammals suggests that Wnt signaling is important for both melanocyte and sensory neuron fate specification, with differences in signal timing being key [27]. (b) Progressive fate restriction (PFR). In the PFR model, multipotent NCC rapidly generates a series of intermediate progenitors whose options are restricted. In this schematic, for simplicity, they are shown only as the best-characterized bipotent intermediates (two color), but these models also posit higher potency intermediates as well. The partially restricted (here, bipotent) intermediates then generate individual fates under the influence of environmental signals (red). (c) New cyclical fate restriction (CFR) model. In our novel CFR model, NCCs enter a highly multipotent progenitor state (NC-HMP, grey circle), characterized by a highly dynamic transcriptome, which cycles (dashed arrows) through a series of sub-states; note that this state is highly multipotent (rainbow colors), but each sub-state is biased towards an individual fate (broader band of one color); under the influence of environmental signals (red), cells in each sub-state are driven to adopt an individual fate (solid arrow). Note that these simplified schematics focus on only four key fates—melanocyte (black), Schwann cell (green), sensory neuron (yellow) and sympathetic neuron (cyan)—and on key features of each model

Pigment cell development in zebrafish: conventional PFR model. The three zebrafish pigment cell types (xanthophore (orange), melanocyte (black) and iridophore (blue)) are generated from early neural crest cells (NCCs) by a mechanism involving production of intermediate progenitors that are fate-restricted, respectively, to two or three pigment cell options: melanoiridoblasts (blue, black), generating melanocytes and iridophores only, and chromatoblasts, generating all pigment cell fates (black, blue, orange); other progenitors give rise to other derivatives (e.g., neuroglioblasts (cyan, green) to peripheral ganglia). Key molecular markers as revealed by in situ hybridization are indicated in italics for each proposed cell type; note how recent studies have shown that maintenance and upregulation (rather than de novo transcription) of expression of these markers often characterizes the fate specification and differentiation phases, as indicated here by number of ‘+’ symbols after each gene name. In this diagram, the absence of a marker (e.g., the neuronal transcription factors neurog1 and phox2bb) is significant. Key transcription factor genes (yellow) and environmental signaling molecules (red) driving specific fate choices are indicated on the transition arrows. Note that the development of xanthophores has been less well studied. The mechanisms driving the formation of each intermediate progenitor have not been proposed.

Putative molecular mechanism underpinning CFR. Multipotent neural crest-derived highly multipotent progenitors (NC-HMP) cycles repeatedly through individual sub-states (circles) biased to be receptive to environmental signals driving specific fate choices (indicated by expanded color on each sub-state); we have proposed that this might simply reflect elevated levels of expression of receptors for fate-specific environmental signals (e.g., Ltk for ALK-ALs involved in iridophore specification; BMPRs for BMPs in sympathetic neuron specification). Transition between sub-states (curved, dashed arrows) is proposed to be dependent upon activity of key fate-specific transcription factors (e.g., Tfec for iridophore; Mitfa for melanocyte; Phox2bb and/or MASH1 for sympathetic neurons). Hence, when these genes are mutated, the NC-HMP lingers in the previous sub-state for a longer period (e.g., in mitfa mutant, cell lingers in pro-iridophore sub-state), although cells eventually overcome this block using a less favored transition (straight, dashed arrow shown for mitfa mutant). Order of transition is predicted based upon mutant phenotypes (e.g., in mitfa mutant, iridophore numbers are elevated), but note that order for other fates is rather more hypothetical and subject to further experimental analysis). Key fate specification environmental factors (red) for melanocyte (Wnt), iridophore (ALK-Als), glia (NRG and Notch ligands, e.g., Delta), sympathetic neurons (BMPs) are deduced from zebrafish or mammalian studies; such signal for xanthophore not quite so clear, but Csf1 is a candidate. Note that in this simplified scheme we have assumed just one type of neuron; of course, other neuron types (e.g., sensory neurons) are derived from the NC-HMP.

Simple genetic regulatory networks of mutual inhibition and their temporal response to a steadily increasing strength of inhibitory coupling g(t): (a,b) Cyclic repression between three genes (x₁–x₃) generates oscillatory transcription factor (TF) expression levels as shown by the repressilator circuit; (c,d) Two superimposed sets of mutual inhibition between three genes, with equal inhibitory strengths and using OR gate (i.e., repression occurs so long as one of TFs is expressed), allows for non-oscillatory, multipotent state (panel (d), left), oscillatory expression (giving cells that in a snap-shot view appear fate specified, but which retain multipotency (central region of panel (d), with three distinct biased sub-states depicted diagrammatically) and also for equilibrium states in which one TF is expressed at much higher levels than the other two, i.e., differentiation (panel (d), right). (e) If inhibitory strengths between TFs are asymmetric, this creates oscillation between sub-states, but with temporal bias to one, i.e., biased sub-state favoring specific fate; here (in contrast to the other panels) shown for constant coupling strength, i.e., g(t) = const. (f) Where inhibitions between TFs use AND gate (i.e., both required for the repression), oscillatory sub-states and resultant differentiation states combine expression of 2 TFs, consistent with combinatorial fate determination.