Enhancer, transcriptional, and cell fate plasticity precedes intestinal determination during endoderm development

Abstract

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.

Keywords: chromatin plasticity; developmental competence; developmental plasticity; fate determination; homeodomain transcription factors; lineage commitment; tissue specification.

Figure 1.

Transcriptional and chromatin landscapes in the developing and neonatal gut. (A) Gut epithelia with distinctive morphologies arise from contiguous endodermal regions. (Eso) Esophagus; (FS) forestomach; (HS) hindstomach; (Int) small intestine. (B) Principal component analysis (PCA) of Eso/FS (red), HS (purple), and Int (blue) transcriptomes at embryonic day 12 (E12), E14, and E16. n = 2 per group. The largest sources of variation reflect the emergence of tissue-specific mRNA profiles after E14. Photomicrographs of intestinal endoderm show pseudostratified epithelium at E12, early villus formation at E14, and mature villus structures at E16. (C) Differential chromatin access in postnatal day 1 (P1) endoderm from different regions of the tubal gut. Each cluster of ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing) summits ±1.5 kb represents candidate enhancers selectively accessible in one region or common to the FS and HS (Overlap). In adult intestinal villi, the enhancers identified in P1 intestine carry H3K4me1, H3K27ac, and RNAPII, but these marks are largely missing from enhancers detected in P1 foregut tissues. Integrated Genome Viewer (IGV) tracks show ATAC-seq and RNA sequencing (RNA-seq) data at representative loci from Eso/FS and Int clusters. Numbers represent signal scales, and shaded boxes highlight region-specific enhancers. (D) Gene set enrichment analysis (GSEA) of genes located within 50 kb of sites selectively accessible in the Eso/FS or Int (derived using binding and expression target analysis [BETA]) (Wang et al. 2013), plotted with respect to differential mRNA expression in the E16 Eso/FS and Int. (NES) Normalized enrichment score.

Figure 2.

Extensive FG enhancer marking and activity in early Int endoderm. (A) ATAC-seq signals at region-specific P1 enhancers (from Fig. 1C) at successive stages in each regional endoderm: Eso/FS and HS (FG) and Int (midgut). FG endoderm lacked open chromatin at Int-specific enhancers at all times, whereas early Int endoderm showed extensive open chromatin at FG enhancers and loss of this chromatin access by P1. (B) H3K27ac ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) signals in E12, E14, and P60 Int endoderm (Kazakevych et al. 2017) at region-specific P1 enhancers. Early open chromatin at FG-specific enhancers is associated with H3K27ac, and both features are later lost. (C) Distributions of RNA levels among 824 Eso/FS-enriched genes (E16) (from Supplemental Fig. S1D) showing comparable global expression in the Eso/FS, HS, and Int at E12, before expression becomes selectively enriched in the Eso/FS. Dots in each violin plot represent median mRNA counts, and the significance of differences between groups was determined by the Mann-Whitney U-test. (n.s.) Not significant; (***) P < 0.01; (****) P < 0.0001. The table shows normalized RNA read counts for representative region-specific genes, illustrating FG-specific gene activity in early Int endoderm. (D) A representative Eso/FS locus, Sox15, carries open chromatin and H3K27ac in early (E11, E12, and E14) Int endoderm, later losing both features. Early, but not late, H3K27ac is also shown at a representative HS locus: Gkn3. (E) Aggregate intestinal ATAC-seq (blue; left) and H3K27ac ChIP-seq (brown; right) signals in the “Overlap,” HS, and Int enhancer clusters at different times. Chromatin access and H3K27ac at HS and “Overlap” enhancers decline during development, while both features arise at Int-specific enhancers.

Figure 3.

Shh^Cre-mediated Cdx2 deletion results in anterior homeotic transformation. (A) The highest enriched sequences within region-specific enhancer clusters represent canonical TF motifs. (B) Trajectories of Eso/FS- and Int-specific TF transcripts in E12, E14, and E16 Eso/FS (left) and Int (right) endoderm. Log₂ transformed RPKM values, plotted over time, show increases and decreases in the different regions. (C) Intestinal regions (from the duodenum to the ileum) stained with PAS or with ATP4B or TRP63 antibody in E18 wild-type and Shh^Cre;Cdx2^Fl/Fl embryos. In the mutant duodenum and jejunum, gastric foveolar cells (recognized by a mauve apical rim; black arrows) extensively replace intestinal goblet cells (recognized by a purple cytoplasmic blob; black arrowheads). These contrasting features of wild-type and mutant jejunal villi are highlighted in the dashed boxes and magnified at the right. In addition, ATP4B⁺ gastric parietal cells appear in the mutant jejunal (white arrowheads) but not duodenal crypts. The mutant ileum loses crypt–villus structure, including PAS⁺ goblet cells, and is lined instead by TRP63⁺ stratified squamous mucosa (red arrows). Bars, 25 µm.

Figure 4.

Time-sensitive activation of FG-specific transcriptional programs in the absence of CDX2. (A) Schema of Cdx2 deletions mediated by either Shh^Cre (approximately E10) or treatment of Villin-Cre^ER-T2 dams with tamoxifen at E13 or E15. Embryos deleted before E14 were assessed at E16, and those deleted at E15 were assessed at E18. CDX2 immunostains verify protein loss. (B) Unsupervised hierarchical clustering (Pearson coefficients) for the 2604 genes expressed differently (greater than fourfold, q < 0.05) in E16 wild-type tissues (Supplemental Fig. S1D) and E16 Cdx2^−/− Int after deletion at approximately E10 or E13. Transcriptomes in early, but not late, Cdx2-deleted Int endoderm resemble those in FG epithelia. (C) mRNA differences in Cdx2^−/− Int after deletion at approximately E10 (n = 3), E13, or E15 or in adult duodenum and ileum (n = 2 each). Clusters are those with differential expression in wild-type E16 EPCAM⁺ Eso/FS, HS, and Int cells (from Supplemental Fig. S1D). For each FG cluster, colored violin plots represent the distributions of mRNA levels (colors represent the region, and white dots represent the median) in wild-type tissues, and gray violins show the distributions after Cdx2 deletion (knockout) at different times. Violin plots for Int genes are shown in Supplemental Figure S3B. The Mann-Whitney U-test was applied to determine the significance between Cdx2 knockout at different times. (n.s.) Not significant; (*) P < 0.05; (****) P < 0.0001. (D–F) IGV tracks of RNA-seq data showing native (wild-type FS, HS, and Int) and ectopic FG gene expression in Cdx2^−/− intestines after deletion at E10, E13, or E15. Numbers represent the scale for normalized read counts.

Figure 5.

CDX2 binding in developing Int endoderm and the consequences of late depletion. (A) PAS staining of intestinal regions after Cdx2 deletion at E13 or E15. Wild-type and mutant Ints were assessed at E18. Goblet cells (black arrowheads) were intact, and no region showed gastric foveolar differentiation (no cells were observed with apical rim staining). Bar, 25 μm. (B) Normalized ChIP-seq data for CDX2 at different developmental stages. Binding at enhancers from any regional cluster is minimal at E13; thereafter, it increases progressively and selectively at Int enhancers. (C) Lack of CDX2 binding at P1 FG enhancers (red- and gold-shaded areas). The blue-shaded box demarcates a CDX2-bound intestinal enhancer. Numbers represent the scales of ChIP or ATAC signals. (D) E13-specific CDX2-bound sites overlap minimally with FG-specific enhancer regions, implying the absence of direct CDX2 control over FG genes.

Figure 6.

Persistence of open chromatin at FG enhancers after early, but not late, CDX2 loss. (A) Mice were treated to induce Cdx2 deletion according to the schema in Figure 4A, and isolated Int epithelium was assessed for accessible chromatin by ATAC. “Overlap” and HS enhancers, which are ordinarily open in wild-type embryos until E14 (Fig. 2A), remained accessible after Cdx2 deletion at approximately E10 or E13 but were closed after Cdx2 deletion at E15 or in adult mice. Intestinal enhancers were significantly compromised after early, and less compromised after late, Cdx2 deletion. The IGV track shows a representative locus: Foxp4. Shaded boxes denote Eso/FS- and HS-specific enhancers showing open chromatin after early Cdx2 deletion. (B) The fractions of genes up-regulated in E10 Cdx2^−/− intestines (gray) located 25 or 50 kb away from “overlapping” or HS enhancers where ATAC signals persist in E10 Cdx2^−/− intestines. (C) Model for the temporal relation of enhancer accessibility to tissue plasticity in wild-type endoderm (top), as inferred from Cdx2^−/− intestines (bottom). IGV tracks show original ATAC-seq peaks at representative FG- and Int-specific enhancers located far from the respective TSSs. (D) Model depicting midgut developmental potential as a function of temporally regulated waves of enhancer accessibility and region-specific mRNA expression. Low-level expression of FG genes is initially pervasive, while FG enhancers are accessible throughout the gut tube. Regional determination occurs after E14, when enhancer signatures become consolidated, gene expression domains become restricted, and regional anatomy approaches adult forms.