Recent evolution of the developing human intestine affects metabolic and barrier functions

Abstract

Diet, microbiota, and other exposures make the intestinal epithelium a nexus for evolutionary change; however, little is known about genomic changes associated with adaptation to a distinctly human environment. In this work, we interrogate the evolution of cell types in the developing human intestine by comparing tissue and organoids from humans, chimpanzees, and mice. We find that recent changes in primates are associated with immune barrier function and lipid and xenobiotic metabolism and that human-specific genetic features affect these functions. Enhancer assays, genetic deletion, and in silico mutagenesis resolve evolutionarily important enhancers of lactase (LCT) and insulin-like growth factor binding protein 2 (IGFBP2). Altogether, we identify the developing human intestinal epithelium as a rapidly evolving system and show that great ape organoids provide insight into human biology.

Fig. 1.. Enterocytes of the developing human small intestinal epithelium are rapidly evolving.

A, Schematic describing human proximal small intestine (duodenum) specimens profiled using scRNA-seq (light gray) or scMultiome (dark gray). The data was used to assess nonsynonymous to synonymous substitution (dN/dS) ratios of expressed genes and deepest ancestor of accessible chromatin. B-C, UMAP of developing human intestinal single-cell transcriptome data with each cell colored by annotated cell class and numbered by the cell type (B, See also Fig. S1), and average normalized nonsynonymous to synonymous substitution ratio (dN/dS) in protein coding regions of expressed genes across primates (C). D, Violin plots show the distribution of average normalized dN/dS ratio per cell type. M1-M4, mesenchymal subtype 1–4. EN, enteric neuron. E, Gene ontology enrichment analysis for enterocyte enriched genes with high dN/dS scores. F, Ancestry enrichment plot with x-axis depicting nodes grouped into vertebrate (nodes 12–14), tetrapod (–11), mammal (–7), primate (–4), and y-axis depicting the enrichment of intestine open chromatin regions mapped to their deepest ancestry. Colors depict cell class (top) or intestinal epithelial cell type (bottom). G, Heatmap shows the scaled enrichment of specific gene ontologies which is defined as the difference between the -log10-transformed binomial test Benjamini-Hochberg(BH)-corrected P values of the primate evolutionary group of regulatory regions and the mean of others.

Fig. 2.. Human intestinal organoids recapitulate gene regulatory features underlying intestinal epithelial development.

A, Schematic depicting the protocol to generate transplanted PSC-derived human intestinal organoids (tHIO) and developing small intestine epithelial stem cell derived enteroids, and multiomic comparison of mRNA expression and chromatin accessibility measurements of epithelial cell types of each specimen. WNRIFAG : Wnt3a, Noggin, R-spondin-3, IGF1, FGF2, A-83–01, Gastrin. B, tHIO stained with H&E to highlight tissue organization (top, scale bar, 100 μm) and bright field image of developing enteroids (bottom, scale bar, 200 μm). C, Integrated UMAP representation depicting contribution of each organoid/tissue source of developing human small intestine (left), tHIO (middle) and developing enteroid (right) based on scRNA-seq data with cells colored by cell types. D, Representative scATAC-seq peaks associated with the promoter or nearby regulatory element of the epithelial cell types of the developing enteroid (top), developing human small intestine tissue (middle) and tHIO (bottom). CHGA, SLC15A1 and MUC2 peaks are associated with enteroendocrine cells, enterocytes, and goblet cells, respectively. E, Heatmap showing per-source scaled accessibility of putative cell type-specific cis-regulatory elements (CREs) consistently detected in primary developing intestine tissue, developing enteroid, and tHIOs (ESC, iPSC). Cell type colors follow panel C. F, Schematic illustrating the single-cell STARR-seq approach used to test the enhancer activity of candidate CREs in human intestinal enteroids. Candidate CREs were pooled and co-transfected with a constitutively expressed GFP reporter to enable selection of transfected cells prior to scRNA-seq and normalization of CRE activity. mP, minimal promoter; ORF, open reading frame; pA, polyadenylation sequence; CMV, cytomegalovirus promoter; GFP, green fluorescent protein. G, Heatmaps showing the expression of putative target genes of selected CREs (left), chromatin accessibility of selected CREs (middle), and CRE activity measured with the scSTARR-seq reporter assay (right) in developing enteroids. Two distinct CREs were measured for CYP3A4 and are defined as (up, upstream CRE) and (dn, downstream CRE). BNC2ma denotes the BNC2 intronic CRE with mammalian ancestry (fig. S2B).

Fig. 3.. Chimpanzee intestinal organoids model great ape intestinal development.

A, Schematic showing the generation of chimpanzee intestinal organoids (CIOs) from pluripotent stem cells. Organoids mature after transplantation into the mouse kidney capsule. B, Brightfield image showing epithelial and mesenchymal CIO domains, scale bar, 100 μm. C, Immunofluorescence (IF) of CIOs showing epithelial (ECAD) part of the organoid with enterocytes (DPP4) polarized apically and smooth muscle cells (SM22) covering the outermost basal layer of the emerging mesenchyme, scale bar, 60 μm. D, Intestine epithelial cell (EPCAM) transcription factor expression (SOX9 and CDX2) in the in vitro zoomed-in images, scale bar, 20 μm, nuclei (DAPI). E, CIOs 12.5 weeks after transplantation (tCIOs) (top left), scale bar, 5 mm, H&E staining of a tCIO (top right), scale bar, 100 μm. IF staining of tCIO for epithelial cell type markers CDX2 for intestinal epithelium, MKI67 for proliferative cells, DPP4 for enterocytes, MUC2 for goblet cells, DEFA5 for Paneth cells and CHGA for enteroendocrine cells co-stained with the epithelial marker (ECAD, blue) and DAPI (gray), scale bars, 100 μm (bottom). F, UMAP embedding of tCIO epithelial cells based on scRNA-seq colored by cell types. scATAC-seq data was projected to the UMAP. G, Representative feature plots depicting gene expression (top) and chromatin accessibility (bottom) for enterocytes (APOA4), stem cells (LGR5), and enteroendocrine cells (CHGA). H, Top 20 cell type-enriched marker genes, and I, Top 50 cell type enriched accessible chromatin regions of tCIO epithelial cell types. J, Normalized intestinal epithelial cell type chromatin accessibility of the promoter or nearby regulatory element of tCIOs: BEST4+ cells (BEST4/MEIS1), enteroendocrine cells (CHGA), enterocytes (APOA4), goblet cells (MUC2), and stem cells (OLFM4) markers.

Fig. 4.. Conservation and divergence between human and chimpanzee intestinal epithelial gene regulation.

A, Comparing single-cell sequencing data between human, chimpanzee, and mouse developing intestinal epithelium can illuminate similarities and differences between species. B, Developing human, chimpanzee and mouse epithelium integrated UMAP with cells colored according to cell type (left) or source (right). EC: Enterochromaffin cells. C, Barplots showing the number of differentially expressed genes (DEGs, left) and accessible chromatin regions (DARs, right) between human and chimpanzee for each intestinal epithelial cell type. D-E, Epithelial cell type average profiles of human-chimp differential and conserved gene expression (D) and chromatin accessibility (E) features. F, Heatmap indicates top 10 human-chimp differentially expressed enterocyte marker genes and nearby differentially accessible chromatin regions. G, Distinct human-specific pseudo temporal expression patterns along stem cell (SC) to enterocyte (Ent) trajectory (co-expression gene module index with number of genes in parenthesis) whereby each line depicting average expression patterns of each gene module in each species and shaded area depicting one standard deviation in expression patterns with corresponding KEGG pathway enrichment analysis (right). H, Expression profile of SLC5A12 along the stem cell to enterocyte differentiation trajectory (left) and the accessibility profile in enterocytes and stem cells of different sources of differentially accessible chromatin regions (right). Light grey color box highlights an SLC5A12 intronic region, chr11:26717719–26718218.

Fig. 5.. Small intestinal epithelial regulatory regions harbor human selection signatures that impact diverse functions.

A, Schematic to annotate each putative regulatory region with information on cell type specificity, functional ontology, evolutionary ancestry, evolutionary constraint and selection, functional species comparison, and hominid variation. B, Epithelial marker region classification based on overlap with features, 1: Constrained, 2: GWAS variant, 3: Differentially accessible (DA) between human and chimp, 4: Single nucleotide change specific to modern humans (SNC), 5: human accelerated region (HAR), 6: Positive selection signature (PS). SNC, HAR and PS are collectively termed as human selection signatures. C, Willow plot showing ontology hierarchy of biological processes (nodes) overrepresented in developing intestinal epithelium enriched regulatory regions harboring constrained and human selection signatures. Nodes are colored and labeled based on ontology hierarchy, edges represent ontology term connections in the ontology hierarchy. Ontology terms are labeled to explain the graph, and also highlight significantly enriched processes (BH-corrected binomial test P < 0.05). D, Willow plot colored based on term enrichment of human selection signatures. E, Scatter plot of developing intestine epithelium enriched accessible regions scored by the epithelial accessibility specificity (Tau, x-axis) and by the composite analysis score for genomic signatures (y-axis, See Methods). F, Accessibility profiles of loci with human selection signature in enterocytes. G, Schematic showing enhancer assay to evaluate the activity of LCT enhancer (chr2:135850827–135851326) in human intestinal enteroids (top). Boxplot of per-cell STARR-seq activity (log2 scale) in enterocytes of the putative LCT enhancer carrying either the C variant (−13910C, ancestral, lactose intolerant) or the T variant (−13910T, SNP, lactase persistence) in developing human enteroids (bottom). *, Mann-Whitney U Test P = 1.652e−06. H, Nucleotide importance scores of the LCT enhancer for enterocytes (ChromBPNet derived) (top), overlap with genomic signatures (middle) and in silico saturation mutagenesis showing predicted effect of SNPs on chromatin accessibility (bottom). Predicted TF-binding sites are shown by boxes of the plot on the top, for repressors (ZBTB7A) and activators (GATA4, HNF4, AP1). SNP under positive selection (PS) for lactase persistence (ancestral C, selected T) is highlighted and computational conversion from C to T is predicted to break the ZBTB7A motif and increase chromatin accessibility. I, Schematic of two guide CRISPR/Cas9 targeting of LCT enhancer (chr2:135850727–135851426) for deletion in developing enteroids (top left). Quantitative real time PCR results for LCT mRNA detection in wild type and enhancer knockout (n = 3 biological replicates) (top right). RNA levels were normalized to TBP and expressed as fold over wild type average. Error bars denote SEM. *, t-test P = 0.0037. Representative images of wild type and MCM6/LCT putative enhancer knockout enteroids after 7 days in differentiation media (bottom). J, Boxplot of per-cell STARR-seq activity (log2 scale) in stem cells and enterocytes of the putative PDX1 enhancer carrying either the human or chimp variants in developing human enteroids (top). *, Mann-Whitney U Test P = 1.5e−12. Quantitative real time PCR results for PDX1 mRNA detection in wild type and enhancer knockout (n = 4 biological replicates) (bottom). RNA levels were normalized to TBP and expressed as fold over wild type average. Error bars denote SEM. *, t-test P = 0.0068.

Fig. 6.. Functional evaluation of two human accelerated regions at the IGFBP2 locus.

A, Genome tracks of chromatin accessibility in developing human (Dev. H.) and transplanted human (tHIO) or chimp (tCIO) intestinal organoids across the IGFBP2 locus for enterocytes and stem cells. The functionally tested intronic (IGFBP2i) and distal (IGFBP2d) putative enhancers are highlighted (gray bars (left) and zoom in panels (right)). B, Expression profile of IGFBP2 along the stem cell to enterocyte differentiation trajectory. C, Representative images of RNAscope in situ hybridization for IGFBP2 in transplanted human (tHIO) or chimp (tCIO) intestinal organoids. Boxes indicate crypt areas displayed in the zoom in panels on the right side of each sample. Epithelial marker (ECAD, blue) and DAPI (gray), scale bar, 100 μm. D, Quantification of IGFBP2 mRNA in tHIO (n=21) and tCIO (n=20) crypts. *, unpaired t-test, P = 0.0123. E, Nucleotide importance scores of the putative IGFBP2 distal enhancer (IGFBP2d) for enterocytes (ChromBPNet derived) (top), overlap with human -chimp single nucleotide changes (SNCs) (middle) and in silico saturation mutagenesis showing predicted effect of SNCs on chromatin accessibility (bottom). Predicted TF-binding sites are shown by boxes of the plot on the top for transcriptional activators (AP1, HNF4, CDX2). SNCs within a Human Accelerated Region (HAR) in the CDX2 motif are highlighted and computational conversion to the chimp variants is predicted to negatively impact the CDX2 motif and decrease chromatin accessibility. F, Quantitative real time PCR results for IGFBP2 mRNA detection in wild type and enteroids carrying CRISPR/Cas9 deletion of IGFBP2 putative intronic (IGFBP2i, left) and distal (IGFBP2d, right) enhancers. RNA levels were normalized to TBP and expressed as fold change over wild type average. Error bars denote SEM. IGFBP2i *, t-test P = 0.006. IGFBP2d *, t-test P =0.0063. G, Boxplot of per-cell STARR-seq activity (log2 scale) in stem cells and enterocytes of the putative IGFBP2 distal enhancer carrying either the human or chimp variants in developing human enteroids. *, Mann-Whitney U Test P < 2.2e−16.