Mowat-Wilson syndrome factor ZEB2 controls early formation of human neural crest through BMP signaling modulation

Abstract

Mowat-Wilson syndrome is caused by mutations in ZEB2, with patients exhibiting characteristics indicative of neural crest (NC) defects. We examined the contribution of ZEB2 to human NC formation using a model based on human embryonic stem cells. We found ZEB2 to be one of the earliest factors expressed in prospective human NC, and knockdown revealed a role for ZEB2 in establishing the NC state while repressing pre-placodal and non-neural ectoderm genes. Examination of ZEB2 N-terminal mutant NC cells demonstrates its requirement for the repression of enhancers in the NC gene network and proper NC cell terminal differentiation into osteoblasts and peripheral neurons and neuroglia. This ZEB2 mutation causes early misexpression of BMP signaling ligands, which can be rescued by the attenuation of BMP. Our findings suggest that ZEB2 regulates early human NC specification by modulating proper BMP signaling and further elaborate the molecular defects underlying Mowat-Wilson syndrome.

Keywords: ATAC-seq; NuRD; RNA-seq; Schwann cells; ZEB2; epigenetics; human embryonic stem cells; neural crest; osteoblasts; peripheral neurons.

Figure 1

Expression of ZEB2 in human neural crest and early chick embryos (A) qRT-PCR analysis of ZEB2 expression during hNC cell induction. Data from 3 independent experiments with SEM. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.001. (B) Whole-mount in situ hybridization revealing Zeb2 expression in the early chick embryo. A representative image is shown. Scale bars, 1 mm.

Figure 2

Knockdown of ZEB2 causes misregulation of NC, non-neural, and pre-placodal ectodermal genes (A) Schematic of siRNA knockdown. (B) Volcano plots representing RNA-seq differentially expressed genes measured between non-targeting control and ZEB2 knockdown at day 3 and day 5. Blue, fold change ≥ 1.5 and adjusted p ≤ 0.05; red, fold change ≤ −1.5 and adjusted p ≤ 0.05; gray, −1.5 < fold change < 1.5 and/or adjusted p > 0.05. (C) Gene expression changes of select gene modules measured using RNA-seq following ZEB2 knockdown. (D) Gene enrichment analysis of hNC day 5 differentially expressed genes.

Figure 3

N-terminal ZEB2 truncation inhibits neural crest cell formation and differentiation (A) Schematic of the ZEB2 N-terminal mutant compared with WT ZEB2. (B) Western blot analysis of ZEB2 protein from day 5 WT and ZEB2 N-mutant hNC cells. (C) Co-immunoprecipitation of ZEB2 and HDAC1 in day 5 hNC cells. Total HDAC1 protein probed from input collected prior to immunoprecipitation. (D) Volcano plots representing RNA-seq differentially expressed genes between WT and ZEB2 N-mutant hNC at day 3 and day 5. Blue, fold change ≥ 1.5 and adjusted p ≤ 0.05; red, fold change ≤ −1.5 and adjusted p ≤ 0.05; gray, −1.5 < fold change < 1.5 and/or adjusted p > 0.05. (E) Osteoblasts differentiated from day 5 WT and ZEB2 N-mutant hNC cells. Calcification was assessed after 30 days of culture using Alizarin red (scale bars, 100 μm) and Von Kossa staining (scale bars, 500 μm). A representative image is shown. (F) S100B and P75NTR immunostaining of day 21 Schwann cells differentiated from WT and ZEB2 N-mutant NC cells. A representative image is shown. Scale bars, 50 μm. (G) ISL1 and PRPH immunostaining of day 10 peripheral neurons differentiated from WT and ZEB2 mutant NC cells. A representative image is shown. Scale bars, 50 μm. (H) qRT-PCR analysis of Schwann cell markers at day 21. Data from 3 independent experiments with SEM. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.001. (I) qRT-PCR analysis of markers of peripheral neurons at day 10. Data from 4 independent experiments with SEM. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.001.

Figure 4

ZEB2 truncation derepresses putative enhancers in the neural crest gene regulatory network (A) ATAC-seq peaks differentially enriched between WT and ZEB2 N-mutant hNC cells. (B) Integrated Genome Viewer tracks displaying ATAC-seq signal associated with select genes. Differentially enriched peaks are marked in red. (C) Heatmaps displaying day 3 (left) and day 5 (right) ATAC-seq signal centered on the differentially enriched peaks at the indicated stage. Heatmaps were k-means clustered with 7 clusters for day 3 and 10 clusters for day 5. (D) De novo motif analysis of genomic sequences (150 bp) centered on the summits of differentially enriched ATAC-seq peaks. Top motifs are shown in the table and full motif output is reported in Table S5.

Figure 5

Inhibition of BMP signaling during neural crest induction rescues the ZEB2 truncation phenotype (A) Gene expression changes of BMP ligands and BMP-responsive genes at day 3 observed in ZEB2 mutant NC RNA-seq. See also Figure 3D. (B) Expression levels of BMP ligands during hNC cell induction. Mean of 2 independent experiments. (C) Schematic of BMP inhibition experiment. Indicated concentrations of DMH1 was added to the hNC media beginning at day 1. (D) qRT-PCR analysis of DLX5, MSX2, and PAX7 under isogenic WT, ZEB2 mutant, and DMH1 rescue conditions at day 3. Data from 3 independent experiments with SEM. p values calculated to ZEB2 mutant: ^∗p ≤ 0.1 and ^∗∗p ≤ 0.05. (E) qRT-PCR analysis of FOXD3, PAX7 (left), and CDH1 (right) at day 5. Data from 3 independent experiments with SEM. p values calculated to ZEB2 mutant: ^∗p ≤ 0.1, ^∗∗p ≤ 0.05, ^∗∗∗p ≤ 0.01, and ^∗∗∗∗p ≤ 0.001.