Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning

Abstract

In the vertebrate neural tube, regional Sonic hedgehog (Shh) signaling invokes a time- and concentration-dependent induction of six different cell populations mediated through Gli transcriptional regulators. Elsewhere in the embryo, Shh/Gli responses invoke different tissue-appropriate regulatory programs. A genome-scale analysis of DNA binding by Gli1 and Sox2, a pan-neural determinant, identified a set of shared regulatory regions associated with key factors central to cell fate determination and neural tube patterning. Functional analysis in transgenic mice validates core enhancers for each of these factors and demonstrates the dual requirement for Gli1 and Sox2 inputs for neural enhancer activity. Furthermore, through an unbiased determination of Gli-binding site preferences and analysis of binding site variants in the developing mammalian CNS, we demonstrate that differential Gli-binding affinity underlies threshold-level activator responses to Shh input. In summary, our results highlight Sox2 input as a context-specific determinant of the neural-specific Shh response and differential Gli-binding site affinity as an important cis-regulatory property critical for interpreting Shh morphogen action in the mammalian neural tube.

Figure 1.

Genome-wide detection of Gli1 binding in neural progenitors. (A) Schematic of neural tube development at the spinal cord level. (B) Overview of directed differentiation of ESCs into neural progenitors. (C) Large-scale clustering of GBRs within 10-Mb domains comparing observed (red) distribution versus random expectation (blue). (D) Summary of de novo motifs enriched in GBRs focusing on peak regions (CisGenome) or short sequence motifs close to the peak center (DREME). (E) Histogram analysis highlighting the Gli motif centering in peak regions. (F) Neural tube marker analysis along the rostral–caudal axis at E8.5. Note that Sox2 expression precedes the emergence of Shh-dependent cell types marked by Nkx6.1. Bar, 50 μm.

Figure 2.

Intersection and genomic analysis of Gli1 and Sox2 binding in neural progenitors. (A, top) Venn diagram for intersection of GBRs and Sox2-binding regions in neural progenitors. (Bottom) Heat map representation of genes that are differentially expressed in response to Shh pathway stimulation and also associated with Gli1- and Sox2-cobinding regions. Class II targets are marked by an asterisk (red). (B) Normalized expression values from the microarray are shown as box plots. (C) Region-based analysis comparing changes in H3K4me2 and H3K27ac levels with and without Shh pathway activation for regions bound by Gli1, Sox2, or both Gli1 and Sox2. Class II target regions are denoted by a black bar.

Figure 3.

Gli1 and Sox2 binding at class II target genes. (A–E) Gli1 (purple) and Sox2 (red) ChIP-seq signals are shown. Blue underline denotes the Gli1-binding signal that passed the peak detection threshold, with relative distances to TSSs shown below.

Figure 4.

Functional characterization of Gli1/Sox2-cobound CRMs. Transient transgenic analysis of class II regulatory regions performed at the forelimb level in E10.5 embryos. Numbers in the top right corner of each panel show expressing embryos/total transgenic embryos. (A) Nkx6.2(+54kb). Note Nkx6.2 expression flanked by Dbx1 and Nkx6.1 (Briscoe et al. 2000). (B,C,I,J) Analysis of Nkx6.1(+140kb and +540kb) Gli1-bound regions. (D,K) Characterization of Gli input to Olig2(−33kb). (E,L) Sox-binding sites are required for Nkx2.2(−2kb) enhancer activity. (F,G,H) Foxa2(+6kb, +160kb, and +170kb) enhancer activity. Bar, 50 μm.

Figure 5.

Determination and functional characterization of intrinsic Gli-binding affinity. (A) Hierarchical clustering of bound probes in PBM data for Gli1–3. (B) Gli1–3-binding specificity motifs deduced from PBMs. (C) Reporter constructs with associated point mutations to the GBS are highlighted in red. (D–G) Transient transgenic reporter analysis performed at the forelimb level of E10.5 embryos. (D,E) Nkx2.2(−2kb) element with a high-affinity Gli site displays strong reporter activity compared with a low-affinity variant. (F,G) Foxa2(+10kb) element with a wild-type Gli site shows weak expression restricted to the most ventral cell population. The high-affinity variant shows a significant increase in GFP and extends to the dorsal limit of Nkx2.2. Bar, 50 μm.

Figure 6.

Critical role for Gli-binding affinity in patterning the ventral neural tube. (A) Schematic illustration of DNA constructs used in transgenic assay. (B–G) Transgenic embryos were analyzed at E10.5 at the forelimb level. (B,D,F) Expressing Foxa2 under the wild-type +10-kb element does not alter neural tube patterning. (C,E,G) Modifying the wild-type GBS in the Foxa2(+10kb) (Foxa2^low) to a high-affinity variant (Foxa2^high) increases reporter activity, alters floor plate morphology, and perturbs ventral patterning. (D,E) Increased levels of Foxa2 expression result in an expanded Shh domain. (F,G) Ventral neural patterning defects observed in Foxa2^high embryos include a reduced number of Nkx2.2⁺ cells and the emergence of Foxa2/Olig2 double-positive cells. Bar, 50 μm.

Figure 7.

Shh signaling dynamics in developing neural tube. (A) Temporal progression of Shh signaling activity within the neural tube analyzed by smRNA FISH profiling of Hh pathway components. Position along the dorso–ventral (DV) axis is shown as relative position in each embryo (percentage to the entire length along the dorso–ventral axis). (B–G) Heat map representation of transcript densities indicating down-regulation of Ptch1 within the floor plate region followed by activation of Shh. Ventral (V) to dorsal (D), from left to right.