Mechanisms of cell diversification

Cell identity and function is determined by the intrinsic wiring of the gene regulatory network that endows progenitors with the competence to respond appropriately to extrinsic cues in a spatiotemporally-dependent manner. One such class of cues, morphogens, instruct cells in their identity by virtue of a concentration gradient, but how this is interpreted at gene regulatory levels to result in sharp and robust boundaries of gene expression is poorly understood. The patterning of the dorsoventral (DV) axis of the developing vertebrate nervous system by Sonic hedgehog (Shh) and its bifunctional transcriptional mediator, Gli, results in the specification of distinct neural subtypes and serves as a model of morphogen function. The identification and functional analysis is described of cis-regulatory modules (CRMs) required for the neural-specific interpretation of morphogen activity by genes that pattern the dorsoventral axis of the CNS and coordinately specify progenitor subtype identity. The results presented are consistent with a model in which morphogen exposure is interpreted via distinct transcriptional mechanisms by genes induced close to the morphogen source as compared to those induced at long-range. In particular, long-range genes directly interpret the Gli repressor (GliR) gradient, resulting in target gene derepression in response to Shh. As a result, expression of long-range targets is critically reliant on additional activators that act in synergy with Gli activators (GliA) as well as direct repressive input from other TFs that restrict expression to the ventral neural tube. By contrast, locally induced Shh target genes directly interpret the balance between GliA and GliR and require input by GliA for their expression. Although synergy with other activators is required for expression, locally induced genes appear to be largely insensitive to mutations of their Gli-binding sites. Evidence is provided that input from other morphogens that pattern the DV axis as well as from Hox proteins that regulate cell identity along the anteroposterior axis is directly integrated into the same set of Shh-regulated CRMs to modulate the relative sizes of progenitor domains along these axes. The high dependence of local targets on the balance of Gli isoforms to regulate their range of expression obviates the need for other direct repressive input, and, consistent with this, genetic and gain-of-function evidence is presented that Pax6 cell-autonomously suppresses expression of local responses by upregulating Gli3 and, hence, GliR. Conversely, the locally induced Shh target, Nkx2.2, is shown to cell-autonomously amplify the Shh response by downregulating Gli3. Extracanonical feedback modulation by Shh-regulated genes offers a mechanism for the phenomenon of cellular memory that is essential to produce qualitative responses to quantitative input, including previous observations that the highest Shh responses are not immediately accessible, but rather depend on ongoing morphogen exposure. Accordingly, whereas Pax6 suppresses floor plate (FP) differentiation, ectopic expression of Nkx2 proteins at early stages promotes FP differentiation in a Shh-dependent manner, whereas misexpression at later stages specifies p3 identity, and it is suggested that the loss of this ability reflects a temporal switch of progenitor competence. Shh signaling is transduced through the primary cilium, which is absolutely required for stabilization of GliA and facilitates GliR formation. The differential sensitivity of local and long-range target genes to perturbed Shh signaling is consistent with the phenotypes of mutants that impact cilia morphology but do not prevent ciliogenesis. Mutants of Rfx4, which regulates ciliogenesis, display a selective reduction of the size of locally regulated domains. Surprisingly, this is due not to a delayed induction of local target genes, but rather to a failure to maintain them as Shh signaling declines. This period is characterized by reactivation and extended co-expression of Olig2 and Pax6 in Nkx2.2-expressing progenitors that do not commit to FP fate. It is suggested that this mixed identity corresponds to a metastable cell state that is acutely sensitive to ongoing fluctuations in morphogen exposure and required to generate sharp domain boundaries. Consistent with impaired Shh signaling, Rfx4 mutants fail to extinguish Gli1 expression at the ventral midline, which is correlated with an extension to the ventral midline of the zone of Olig2/Pax6 reactivation and delayed FP commitment. Evidence is presented that the neural-specific response of morphogen target genes is regulated by Soxb1 proteins, which are sufficient to induce these genes in the developing limb in response to Shh, retinoid, or Bmp morphogen exposure. Moreover, the collocation of Soxb1- and Gli-binding sites constitutes a genomic signature that reliably predicts the neural-specific expression of nearby genes