Intestinal epithelial gene expression is conserved in vertebrates

In order to understand the extent of gene expression similarity across vertebrate intestinal epithelia, we compared newly generated gene expression data from IECs isolated from adult human colon, adult zebrafish intestine, adult stickleback intestine, and from data we previously generated from adult mouse colon and ileum [16] (Fig 1A). We find a strong correlation in gene expression between fish and mammalian IECs throughout the dynamic range of the transcriptome (Fig 1B and 1C, S1A Fig). Using principal component analysis (PCA) and hierarchical clustering, we find that the expression of orthologous genes is more similar amongst IECs across species than mouse IECs are to other mouse tissues (Fig 1B–1E, S1 and S2 Figs). Furthermore, unrelated RNA sequencing (RNA-seq) data from whole mouse intestine cluster with data from all vertebrate IECs (Fig 1D and 1E) [29]. These results reveal that gene expression levels in IECs are similar across these 4 vertebrate species and suggest that many aspects of IEC physiology have been conserved since the common ancestor of mammals and fish.

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Fig 1. Transcriptional profiling of intestinal epithelial cells (IECs) from multiple species show conserved expression after 420 million years since a common ancestor.

(A) (Left) A phylogenetic tree showing time since a common ancestor for human (Homo sapiens), mouse (Mus musculus), zebrafish (Danio rerio), and stickleback (Gasterosteus aculeatus) species. (Right) Simplified schematics showing the intestinal tract of all 4 organisms in gray with the region of collected IEC sample colored: green (human colon IECs), orange (mouse ileum IECs), red (mouse colon IECs), blue (zebrafish whole intestine IECs), and purple (stickleback whole intestine IECs). (B) Scatterplot of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values for 1-to-1 orthologs from mouse ileum IEC and zebrafish IEC samples shows a positive correlation coefficient (Pearson r² = .273; Spearman r² = .416). (C) Same as (B) for mouse colon IECs and zebrafish IECs (Pearson r² = .341; Spearman r² = .379). (D) Complete linkage cluster analysis using FPKM values for 1-to-1 orthologs show similarity between mRNA levels for IECs in comparison to other mouse tissues. Scale represents values of Pearson distance. Black data sets marked by asterisks are RNA sequencing (RNA-seq) experiments from non-IEC tissues [29]. (E) Scatterplot of principal component 1 and 2 (PC1 and PC2) using principal component analysis (PCA) of FPKM values for all IEC data sets and other mouse tissues. (F) Top reported Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from Database for Annotation Visualization and Integrated Discovery (DAVID) using IEC signature genes. The enrichment of these gene groups do not clear Bonferroni correction thresholds (S2 Table). (G) Top tissues showing overlap with IEC signature genes from human and mouse Gene Atlas using Enrichr. (H) The University of California Santa Cruz (UCSC) screenshot of RNA-seq levels at cadherin 17 (CDH17) in 4 species’ IECs (top) and other tissues (bottom) [29] shows expression largely restricted to IECs.

https://doi.org/10.1371/journal.pbio.2002054.g001

In our PCA of mRNA expression from IECs and mouse tissues, we found that principal component 1 (PC1) separated mammalian and fish IECs from all other tissues. We identified 470 genes whose expression levels highly correlate with PC1 and exhibit high expression in IECs relative to other tissues, though their expression and function is not necessarily exclusive to IECs (Fig 1D and 1E, S1 Fig, S1 Table, Materials and methods). These IEC signature genes are representative of physiologic functions and cell types in the intestinal epithelium and include genes involved in lipid, carbohydrate, and protein metabolism (Fig 1F and 1G, S2 Table). IEC signature genes, including retinol binding protein 2 (RBP2, with rbp2a assigned as the zebrafish ortholog by Ensembl), fatty acid binding protein 6 (FABP6), and cadherin 17 (CDH17), are amongst the most highly expressed genes in IECs from several species (Fig 1H, S1D Fig). In addition, we identified genes indicative of different IEC subtypes within the intestinal epithelium including RBP2 [30] and FABP6 (enterocyte) [31], peptide YY (PYY; enteroendocrine) [32], and polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6; goblet cells) [33], consistent with our RNA-seq data representing heterogeneous populations of IECs (S1D Fig). Ribosomal protein genes and translation components were also highly represented within this signature, consistent with the intestinal epithelium being one of the most highly-proliferative tissues [33]. Amongst the IEC signature genes, we found TFs known to be involved in development and function in the intestine, including the epithelial-specific E26 transformation-specific (ETS) TF ELF3 [34], HNF4A [18], HNF4G [35], FXR [36], GATA5 [37], and OSR2 [38], suggesting a conserved basal IEC transcriptional network. Several IEC signature TFs have known associations with human Inflammatory Bowel Diseases (IBD), including SMAD7 [39], CEBPG [40], STAT3 [40–42], XBP1 [43], HNF4A [44], ELF3 [41], IRF1 [45], and NFκB components IKBKB, IKBKG, and NFKBIZ [46]. Furthermore, the most common human disorders associated with IEC signature genes were obesity-related traits, IBD, and Type 2 Diabetes (S1 Table). Together these data highlight the utility of mouse, zebrafish, and stickleback in modeling human intestinal development and disease and suggest a basal similarity in the transcriptional mechanisms underlying intestinal epithelial homeostasis in vertebrates.

Evidence for transcriptional conservation and regulation along the length of the intestine

The results described above revealed conserved IEC signatures by comparing RNA levels from intestinal IECs to other tissues. However, we speculated that signatures of intestinal identity might be further resolved by comparing gene expression along the intestine’s anteroposterior axis in zebrafish and mice. Using previously published datasets we identified gene orthologs that showed similar expression patterns along the adult zebrafish intestine (divided into 7 sections of equal length) [15] and along the adult mouse intestine (divided into duodenum, jejunum, ileum, and colon) (Fig 2, S3 Fig, Materials and methods) [47]. Of 493 genes that showed high expression in the anterior of the zebrafish intestine, we found over 70 genes sharing similar high expression in the anterior intestine of mouse [15], including IEC signature genes like Rbp2, Aldob, and Ehhadh (Fig 2A and 2B, S3 Fig). Many of these genes (e.g., Fabp2, Acsl5, Agpat2, Slc27a4, and Dgat2) are critical in lipid metabolism and uptake, which is consistent with lipid absorption and metabolism taking place mostly in the small intestine in mouse and anterior portion of the intestine in zebrafish [48]. However, ordering genes by mouse duodenum expression level reveals that some of these genes have surprisingly high similarities in cross-species expression patterns along the length of the intestine (Fig 2A and 2B, S3C Fig). For example, adenosine deaminase (Ada), which is most highly expressed in duodenum in mouse [49], is most highly expressed in the zebrafish sections 1–2 (Fig 2B, S3C Fig). Similarly, Fabp2 and Enpep, which are expressed most highly in the jejunum and ileum in mouse, are most highly expressed in sections 3–5 in zebrafish intestine (Fig 2C and S3C Fig). This suggests an unappreciated similarity between IEC gene expression along the small intestine in mammals (mouse) and teleosts (zebrafish) and the potential for further examples of subregionalization in the zebrafish intestine that have not been previously described [13–15].

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Fig 2. Identification of genes with conserved regional transcriptional specification along the length of the intestine in zebrafish and mouse.

(A) A heat map comparing gene median-centered z-scores of 1-to-1 orthologs from previously published datasets profiling expression levels along the length of the zebrafish [15] and mouse intestine (Materials and methods, S3 Fig) [47]. Similarly expressed genes are ordered by expression values of mouse (A) duodenum, (B) jejunum, (C) ileum, and (D) colon. Gene names with asterisks are also intestinal epithelial cell (IEC) signature genes as defined in Fig 1. Genes can exist in multiple subfigures.

https://doi.org/10.1371/journal.pbio.2002054.g002

In support of this conserved regional specification, we also found a small group of genes that are expressed highly only in the terminal portion of the zebrafish anterior intestine (section 5 [15]), which occupies a location similar to the mammalian ileum (Fig 2C and S3D Fig). The mammalian ileum is involved in the uptake of bile salts following their use in emulsification of lipids in the anterior small intestine [50]. Two IEC signature genes involved in bile handling, Fabp6 and Slc10a2, show high expression in this narrow region of zebrafish intestine and mouse ileum (Fig 2C and S3D Fig). In addition, the proteases Lgmn, Scpep1, and 3 cathepsins are in this cluster and show similar high expression largely in the mouse ileum, suggesting a regionally conserved utilization of lysosomal-cathepsin—mediated degradation (Fig 2C and S3D Fig) [51]. These observations suggest that the cellular differentiation and physiological programs deployed in this region of the zebrafish intestine are specialized for bile salt recovery, with strong homology to the mammalian ileum. We also found numerous genes expressed more highly in the posterior end of the mouse and zebrafish intestine, suggesting similar physiologic functions in zebrafish distal intestine and mouse colon (Fig 2D, S3E and S3F Fig) [15]. Collectively, these observations suggest that the teleost intestine has the capacity to articulate complex gene differentiation patterns along the length of the intestine that are functionally and spatially analogous to segments in the evolutionarily distant mammalian intestine.

Accessible chromatin profiling of IECs in 4 species identifies putative IEC regulatory regions

To test the hypothesis that conserved regions of accessible chromatin underlie the transcriptional similarities we found in IECs, we profiled accessible chromatin in the same cell preparations that we used to generate our RNA-seq data using Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) from stickleback, zebrafish, and human colon IECs. We also used recently published data from our group that profiled mouse ileum and colon IECs using DNase I hypersensitive sites sequencing (DNase-seq) [16]. Combining IEC accessible chromatin maps with species-transferable regulatory landmarks such as the transcription start site (TSS) and conserved nonexonic elements (CNEs) allowed us to profile and define common regulatory information utilized in all species. Accessible chromatin peaks were frequently enriched at orthologous TSS in IECs, including at IEC signature genes such as ELF3 (Fig 3A, S3 Table and S2 Fig). This accessible chromatin signal is consistent with typical genome-wide distributions, though the relationship with accessibility may not be strictly driven by regulatory regions or transcription that is specific to IECs (Fig 3B and 3C). However, the related IEC PC1 correlation values, transcription levels, and presence of IEC signature genes were both higher on average at the TSS with higher accessible chromatin levels, suggesting that the magnitude or presence of accessible chromatin may be conserved at related regulatory regions in IECs in distantly related species (Fig 3B and 3C, S4A and S4B Fig).

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Fig 3. Accessible chromatin maps in intestinal epithelial cells (IECs) from multiple species reveals common regulatory information without substantial sequence conservation.

(A) Accessible chromatin and RNA sequencing (RNA-seq) data from representative replicates for the IEC signature gene ELF3 for each organism. For each organism, gene models are represented with thick bars (exons) and thin bars (introns and untranslated regions). (B) Accessible chromatin signal at the 1,000 bp surrounding the transcription start site (TSS) of 1-to-1-to-1-to-1 orthologs ordered by zebrafish IEC Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) signal at the base pair coordinate of the gene’s TSS for zebrafish, stickleback, mouse ileum, mouse colon, and human colon (Right). Moving medians (Left) are shown for PC1 correlation value used to identify IEC signature genes (500 gene window, 1 gene step; black), Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of associated genes (250 gene window, 1 gene step; color scheme based on data sets presented in A and throughout), and IEC signature genes are marked by black horizontal bars. (C) Moving median of accessible chromatin signal at TSS of stickleback, mouse ileum, mouse colon, and human ordered by signal at zebrafish TSS (250 gene window, 1 step) highlight the relationship between IEC accessible chromatin data in multiple species. Numerical values can be found in S1 Table. (D) A heatmap of cluster analysis of overlap between accessible chromatin peak calls for IEC samples and additional published accessible chromatin datasets with the region 1,000 bp upstream of the TSS (TSS-1,000 bp) for IEC signature genes (red bars). Overlap between TSS-1,000 bp and conservation metrics are represented with blue bars. The total number of overlaps is represented in parentheses for each conservation metric column, and overlaps are defined as having at least 1 shared base pair. Initials are used to specify IEC data sets: Z; Zebrafish, S; Stickleback, MI; Mouse Ileum, MC; Mouse Colon, HC; Human Colon. (E) Closeup of the heatmap of clustered genes from panel 3D that frequently showed putative conserved accessible chromatin at TSS in IEC samples (light blue and green). Color scheme is shared with 3D. (F) A heatmap highlighting common transcription factor binding sites (TFBS) motif enrichment within the accessible chromatin peaks that fall between the region 10,000 bp upstream of the TSS and 10,000 bp downstream of the transcription termination site (TTS) of IEC signature genes. Motif enrichment includes known transcription factors (TFs) involved in IEC biology which are also often expressed highly in our IEC samples. E26 transformation specific (ETS) factors include motifs for ELF5, EHF, ELF1, GABPA, ETS, Elk1, Fli1, Elk4, ETS1, ERG, SPDEF, and EWS:FLI. (G) The percentage of DNA bases that are either adenosine or thymine (AT%) (20 bp smooth: 20 bp window, 1 bp step) surrounding TSS as ordered by IEC zebrafish FAIRE signal from (B) and IEC mouse Ileum DNase signal (Right) shows nonrandom patterns at TSS that (H) vary on average from species to species. (I) Comparison of AT% (20 bp smooth) at conserved nonexonic elements (CNEs) that by their nature show some similar sequence composition.

https://doi.org/10.1371/journal.pbio.2002054.g003

We compared accessible TSS call overlaps at the TSS for all IEC samples and several cell lines and tissues from mouse and human Encyclopedia of DNA Elements (ENCODE)/Roadmap [52–54] to determine if accessible chromatin status could identify IEC-specific regions. We clustered this information to identify common patterns of accessible chromatin status in all these samples (Fig 3D). While the majority of TSS regions appeared to be constitutively accessible at most genes in most species and tissues represented, a group of IEC signature genes had TSSs that were accessible frequently in IECs but less often in other tissues (Fig 3E). Importantly, genes within this group were also almost always accessible at the TSS in several independent datasets of intestinal tissue from mouse and human (Fig 3E). This group included key genes involved in IEC biology such as HNF4A, HNF4G, RBP2, A1CF, CFTR, and CDH17. Further, we found 3 genes, FABP6, SLC10A2, and TMIGD1, that showed high similarity of mRNA levels along the length of the intestine at the mouse ileum and zebrafish section 5 (Fig 2C) and showed limited accessibility in nonintestinal tissues with accessibility in some of the intestine-related datasets (Fig 3E).

To determine if the regions that show chromatin accessibility largely in IECs also showed conservation at a sequence level, we used multiple metrics that measure sequence conservation from teleost to mammals. This included zebrafish conserved nonexonic elements (zCNEs), a stringent comparison of noncoding regions of 14 species, including human and mouse [55]. We also used UCSC liftOver and zebrafish-to-human and zebrafish-to-mouse multiple alignment format (MAF) blocks to identify potentially DNA-conserved regions. Though metrics at the RNA and chromatin levels suggest substantial similarities between teleost and mammalian intestine and these TSS regulatory regions often show sequence constraint at some level, less than 15% of IEC signature genes had detectable conservation from zebrafish to mouse or human in TSS regions. This hampered our ability to infer which regulatory regions and putative TFBS are actually conserved across these species (Fig 3E) [56]. Additionally, most of these conserved regions were identified by MAF blocks and appeared to have only small regions of highly degenerate sequence conservation from teleosts to mammals that were located immediately upstream of transcribed regions and suggestive of minimal functional conservation. We speculated that common regulatory information could still be shared in these TSS regions because short, modular TFBS could escape sequence conservation metrics [56–58]. We looked for enrichment of TFBS using a library of 303 position weight matrices (PWMs) (primarily derived from human ChIP-seq datasets) within 2 sets of accessible chromatin peaks: (1) those between 10 kb upstream of the TSS to 10 kb downstream of the transcription termination site (TTS) [59] (Fig 3F) and (2) those at the TSS of IEC signature genes (S4C and S4D Fig). Common enrichment of PWMs in accessible chromatin regions at IEC signature genes of different species identified several TF motifs known to regulate IEC expression in mammals including HNF1, HNF4A, GATA4, KLF5, and the often similar ETS factors, including ELF3 and SPDEF (Fig 3F, S4C and S4D Fig) [60–63], that may represent a TF network that control core conserved aspects of IECs in animals.

Conservation metrics rely on relatively long stretches of DNA sequence, so we tried to identify sequence properties that varied between species and might interrupt the maintenance of easily detected conserved sequence. By comparing the percentage of DNA bases that are either adenosine or thymine (AT%) at TSSs ordered by zebrafish FAIRE signal, we found that gross sequence characteristics are substantially different at the TSS of orthologs between species (Fig 3G and 3H). AT% decreases at the TSS from approximately 51% in zebrafish to 46% in stickleback to 35% in mouse and human (Fig 3H). These differences are maintained on average in the area surrounding the TSS (Fig 3G and 3H). This general phenomenon likely has a substantial influence on the maintenance of sequence at these regulatory regions [64,65] and may represent a particular challenge for the identification of conserved regions and regulators. However, the substantial difference in AT% observed at TSS is absent on average at CNEs, which, based on the method that they are identified, are inherently similar in sequence (Fig 3I). This suggests that general sequence utilization differences seen at TSSs in different species on average are important but may not influence all regulatory regions.

Conservation of regulators that define regional intestinal expression from fish to mammals without apparent sequence conservation

The apparent maintenance of TFBS enrichment suggested that our accessible chromatin and RNA-seq data were identifying functionally conserved regions, although they frequently lacked conserved DNA sequence. To test this, we cloned regions upstream of the rbp2a and fabp6 TSS that showed no substantial sequence conservation from fish to mammals, but appeared accessible largely in IECs, and tested them using a functional in vivo zebrafish reporter assay (Materials and methods). Because both RBP2 and FABP6 are IEC signature genes that showed strong regional conservation of expression in zebrafish and mouse (Fig 2), this allowed us to simultaneously test if the conservation of regulatory information was interpreted by zebrafish to specify intestinal regionality in addition to IEC expression generally.

We generated a transgenic reporter construct with the 1.3 kb region upstream of the TSS of zebrafish retinol binding protein gene, rbp2a, cloned upstream of the mouse cFos minimal promoter and green fluorescent protein (GFP) [Tg(rbp2a:GFP)] (Figs 4A and 3E, S5A, S6A Figs and S4 Table). Tg(rbp2a:GFP) was capable of driving high expression in the anterior portion of the intestinal epithelium in larvae (Fig 4A), which was consistent with the known expression patterns of rbp2a [15,66,67], suggesting that sufficient regulatory information to drive regional expression in IECs is contained within this fragment. This expression pattern is distinct from a general control reporter construct in which no additional DNA was cloned upstream of the mouse cFos minimal promoter driving GFP, and no consistent IEC expression is found (S6B–S6I Fig) [68]. Using the same reporter assay, we then tested the region immediately upstream of Rbp2 from mouse Tg(Mmu.Rbp2:GFP) and RBP2 from human Tg(Hsa.RBP2:GFP) and found that both were capable of driving GFP in IECs in the anterior portion of the larval zebrafish intestine (Fig 4A and S5A Fig). We tested if we could identify common, small TFBS that might have escaped detection by conservation metrics in these regions but explain the conserved patterns of IEC expression. TF motif searching within the RBP2 fragments for zebrafish, mouse, and human identified shared strong matches to HNF4A and GATA factors within a few hundred bases of the TSS (Fig 4B and 4C). To test how often HNF4A and GATA motifs occurred generally, we queried a similarly sized area 150 bp upstream of the TSS of all genes. Only .23% (79 genes), .25% (51 genes), and .25% (47 genes) had both HNF4A and GATA4 motifs in zebrafish, mouse, and human, respectively, and no other 1-to-1-to-1 orthologs contained both HNF4A and GATA4 motifs at this location in all 3 species. This suggests that the presence of HNF4A and GATA sites in the RBP2 TSS is conserved among vertebrate genomes and is unlikely to have occurred by chance. Indeed, HNF4A and GATA4 are likely a common vertebrate regulatory cassette as studies in multiple species have identified their importance in small intestinal IEC biology (S3G and S3H Fig) [26,69]. Collectively, these results establish that accessible chromatin maps can help discern conserved motif information and how in vivo reporter assays can be used to test for potential conserved tissue-specific regulatory activity.

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Fig 4. Exogenous regulatory regions identified by intestinal epithelial cell (IEC) accessible chromatin can drive regionally conserved IEC expression in zebrafish larvae.

(A) (Left) Whole-mount stereofluorescence images of 7 dpf zebrafish stable lines expressing a green fluorescent protein (GFP) reporter construct for rbp2a/Rbp2/RBP2 accessible regulatory regions from zebrafish, mouse, and human show high levels of expression in IECs in the anterior portion of the intestine. Scale bar 100 μm. (Right) Representative cross-sections of zebrafish intestine confirming high IEC expression for each corresponding stable line. The zebrafish cross-section was taken in the posterior of the intestinal bulb, and the mouse and human cross-sections were taken within the middle of the intestinal bulb. Scale bar 25 μm. (B) Common motifs detected in the RBP2(a) cloned region for each species colored by Homer motif scores. Motif score scale is shared with Figure 4G. (C) Schematic of common transcription factor binding sites (TFBS) motifs found immediately upstream of the transcription start site (TSS) of RBP2(a) in zebrafish, mouse, and human. (D) (Left blue) Whole-mount stereofluorescence images of 7 dpf zebrafish stable line Tg(fabp6:GFP) show high levels of GFP expression in IECs in the middle of the intestine. (Below blue) Average projection of confocal stacks of whole-mount zebrafish Tg(fabp6:GFP). Scale bar 100 μm. (Right blue) Representative cross-section of zebrafish intestine confirming high IEC expression for Tg(fabp6:GFP). Scale bar 25 μm. (Left orange) Whole-mount stereofluorescence image of 7 dpf zebrafish stable line Tg(Mmu.Fabp6:GFP) shows high levels of GFP expression in IECs in the middle of the intestine. Scale bar 100 μm. (Right orange) Representative cross-section of zebrafish intestine confirming high IEC expression for Tg(Mmu.Fabp6:GFP). Scale bar 25 μm. A smaller conserved region from mouse and 2 inclusive regions from human FABP6 did not drive IEC GFP expression in our zebrafish reporter assay. Throughout, the white dashed line marks the boundary of intestine and IECs. (E) Confocal whole-mount maximum projection image showing lack of overlap between Tg(fabp6:GFP) and the segment 2 marker TgBAC(lamp2-RFP). Scale bar 100 μm. (F) Confocal whole-mount average projection image showing lack of overlap between Tg(fabp6:GFP) and the segment 1 marker Tg(-4.5fabp2:DsRed). Scale bar 100 μm. (G) Common motifs detected in the cloned or corresponding region for each species colored by Homer motif score.

https://doi.org/10.1371/journal.pbio.2002054.g004

Fabp6 regulatory region drives conserved expression in a region of the zebrafish intestine positionally and functionally analogous to the mammalian ileum

We next sought to interrogate the regulatory potential of regions upstream of FABP6, an IEC signature gene expressed primarily in the ileum in mouse and human and whose TSS was accessible in all IECs tested except mouse and human colon (Figs 3E and 4D). The region 258 bp upstream of the zebrafish fabp6 TSS [Tg(fabp6:GFP)] drove a very specific GFP expression domain exclusively in IECs in the middle of the larval zebrafish intestine, consistent with the endogenous pattern of fabp6 mRNA expression (Figs 2C and 4D, S5B and S5G–S5J Fig) [15,47,70]. Unlike rbp2a, fabp6 had a small region upstream of the fabp6 TSS that was conserved to mouse and human. However, this appeared to only correspond to a TATA-box (Fig 4G, S5B and S5C Fig), and cloning of this small region from mouse to test in our zebrafish reporter assay did not drive expression in IECs. However, when we included the entire accessible chromatin region from mouse Tg(Mmu.Fabp6:GFP) (a 503 bp region upstream of mouse Fabp6 TSS), which included the minimal conserved region, we found this larger sequence sufficient to drive an IEC expression pattern that was positionally identical to the corresponding region from zebrafish (Fig 4D). This suggests the regulatory information necessary to drive IEC expression in the putative zebrafish ileum is within the additional region defined by accessible chromatin from mouse and not solely detected by conservation.

In order to define the relationship between this fabp6 domain in the context of the canonical 3 segments of the zebrafish intestine [14], we compared the larval expression pattern of Tg(fabp6:GFP) with zebrafish segment 2 marker TgBAC(lamp2-RFP) [71] and intestinal segment 1 marker Tg(-4.5fabp2:DsRed) [72,73]. Strikingly, we found Tg(fabp6:GFP) did not overlap with either, suggesting the intestinal region marked by Tg(fabp6:GFP) is a novel distinct segment of the zebrafish larval intestine (Fig 4E–4F). Similar regions upstream from human FABP6 were negative for driving expression in zebrafish IECs, despite the putative presence of shared IEC-related TFBS like CDX2, RBPJ, and RXR (Fig 4G, S5B Fig and S4 Table). The combined evidence of conserved positional expression of Fabp6 and other ileal genes (Fig 2C and S3D Fig) together with the maintenance of region-specific cis-regulatory information at zebrafish and mouse Fabp6 orthologs (Fig 4D–4G) indicate that an intestinal segment functionally and regionally homologous to mammalian ileum is maintained in zebrafish larvae and likely specified by similar regulators.

Transcriptional domains identified in larval zebrafish intestine are maintained in adults

To determine if the transcriptional patterns and domains we detect in the larval zebrafish intestine also occur in adult stages, we assayed expression patterns in the adult (3+ months) zebrafish intestine using the same stable transgenic lines we queried in larvae. Tg(rbp2a:GFP) showed a similar expression pattern restricted to the anterior intestine as larval zebrafish; however, consistent with adult expression data (Fig 2B), high GFP expression did not extend to the most anterior IECs (Fig 5A–5B). This suggests that additional transcriptional domains or functional differences exist in the most anterior zebrafish intestine. We also found that Tg(fabp6:GFP) had a very similar pattern between larval and adult stages with a relatively small and discreet region of IEC GFP expression immediately after the second bend and between the segment 2 marker TgBAC(lamp2-RFP) (Fig 5F–5G) and the segment 1 marker Tg(-4.5fabp2:DsRed) expression domains (Fig 5H–5I). Collectively, this suggests that regional transcriptional programs in the zebrafish intestine are maintained between larval and adult stages. Further, the extent of functional homology between the zebrafish and mammalian intestine may be greater than previously appreciated (Fig 5J–5K). We propose a working model with at least 5 transcriptional/functional domains in zebrafish, although additional studies are needed to comprehensively resolve these domains, their interplay, boundaries, regulators, as well as the full nature and limitations of the homology between teleost and mammalian regional IEC programs (Fig 5K).

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Fig 5. Transcriptional domains identified in larval zebrafish intestine are maintained in adults.

(A) Stereomicroscopy image of 2 dissected adult Tg(rbp2a:GFP) intestines (top) and 2 wild-type (WT) intestines without green fluorescent protein (GFP) (bottom) shows high GFP expression between the first and second bend of the adult intestine in Tg(rbp2a:GFP). Autofluorescence can be seen in several intestines following the second bend, presumably due to bile or feces (see brightfield microscopy in B). Scale bar 1,000 μm. Confocal cross-section of adult Tg(rbp2a:GFP) intestinal folds with DAPI in blue shows GFP expression in intestinal epithelial cells (IECs) in the inset panel. Scale bar 50 μm. (B) Brightfield microscopy of 2 dissected adult Tg(rbp2a:GFP) intestines (top) and 2 wild-type intestines (bottom). Dissection pins can be seen as long black lines anchoring the intestine in the intestinal bulb. (C) Stereomicroscopy image of brightfield and GFP overlay for the dissected intestine of adult Tg(fabp6:GFP), showing GFP expression in a discreet domain immediately following the second bend. The anus was excluded in this preparation. Scale bar 1,000 μm. (D) Stereomicroscopy image of GFP (top) and brightfield (bottom) for the dissected adult intestine of Tg(fabp6:GFP) opened lengthwise to show internal detail of the discreet domain of GFP expression. Scale bar 1,000 μm. (E) Confocal cross-section of adult Tg(fabp6:GFP) intestinal folds with DAPI in blue shows high GFP expression in IECs. Scale bar 50 μm. (F) Stereomicroscopy close-up of red fluorescent protein (RFP) and brightfield for adult WT and TgBAC(lamp2-RFP) zebrafish lines (top) showing entire intestines with the segment 2 relative position marked with a red horizontal line (middle). Scale bar 2,000 μm. Fluorescence-only image showing the distribution of the lamp2-RFP signal, highest in segment 2 (bottom). The relative orientation of the WT (top) and TgBAC(lamp2-RFP) (bottom) lines is maintained throughout. (G) Confocal cross-section of adult TgBAC(lamp2-RFP) intestinal folds from segment 2 with DAPI in blue shows high lamp2-RFP expression in IECs. Scale bar 50 μm. (H) (Top) Stereomicroscopy image for a dissected intestine of adult Tg(-4.5fabp2:DsRed) showing DsRed expression most highly between the first and second bend with additional expression in the intestinal bulb. The white dotted line references the dissected intestine. A similar Tg(fabp2:RFP) result has previously been reported [15]. Scale bar 1,000 μm. Overlay between DsRed and brightfield is shown in the inset in the upper right. (I) Confocal cross-section of adult Tg(-4.5fabp2:DsRed) intestinal folds with DAPI in blue shows high DsRed expression in IECs. Scale bar 50 μm. (J) Schematic of dissected adult intestine showing major anatomical features overlaid with proposed transcriptional/functional domains shown in K. (K) Linear schematic representation of proposed transcriptional and functional domains of zebrafish intestine with potential regional gene markers and previously defined regional annotation. Boundaries should not necessarily be considered discrete, and domains may vary or overlap for different genes. Markers refer to proposed region-defining transcriptional markers determined using transgenic lines from this study (*) or from Wang et al. (^)[15]. Additional markers can be inferred from Fig 2 and S3 Fig, including markers for the most posterior region of the zebrafish intestine.

https://doi.org/10.1371/journal.pbio.2002054.g005

Accessible conserved zCNEs in IECs are rarely IEC-specific and are likely open in most species and tissues

In addition to TSS regions, we also specifically queried a published dataset of 54,533 zCNEs, of which 11,792 are also conserved to mouse and human, for accessibility in IECs [55]. Ordering zCNEs by the FAIRE-seq signal from zebrafish identified that the neighboring genes of the most accessible CNEs were also highly expressed in other-species IEC samples (Fig 6A). There was also a surprising overlap in the magnitude of accessibility at these conserved sites between species (Fig 6A, 6B and 6F). However, 48 of the 77 CNEs that were accessible in all zebrafish, mouse ileum and colon, and human colon IEC datasets were also accessible in at least 82% (14/17) of additional nonintestinal ENCODE and Human Roadmap datasets (Fig 6B–6D) [52–54]. This suggests that these regions are not specifically responsible for IEC expression. However, these frequently accessible conserved sites could represent a particular pan-vertebrate primitive transcriptional networks as zCNEs are commonly found at TSSs and near developmental and TF genes (Fig 6B–6E) [55].

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Fig 6. Intestinal epithelial cell (IEC)-specific accessible chromatin at conserved noncoding elements identifies IEC regulatory elements.

(A) Conserved nonexonic elements (CNEs) ordered by zebrafish IEC Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) signal at zebrafish conserved nonexonic elements (zCNE) center. (Left) Moving median of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) levels of CNEs nearest gene feature (250 gene window, 1 step; color scheme based on data sets presented in Fig 1A and throughout), showing the relationship between accessible chromatin level and transcription at these conserved regions. (Right) Heat maps of accessible chromatin signals at the 1,000 bp, surrounding the CNE center, highlighting the similarity of signal in IECs at CNEs in all 4 species. Gray represents no mappable signal or failure to liftover to the stickleback genome, as the stickleback genome was not included in the CNE set [55]. (B) Peak overlap of IEC accessible chromatin datasets and human and mouse ENCODE/Roadmap data sets with CNEs in each respective species. Overlap is defined as having at least 1 shared base pair. (C) Cluster analysis of CNEs with at least 1 IEC accessible peak overlap from any of the IEC datasets. (D) Subset of CNEs containing peak overlap from each of the zebrafish, mouse ileum, mouse colon, and human colon IEC datasets, as well as substantial overlap with additional mouse and human data from mostly unrelated tissues. (E) The entire group of CNEs that have overlap with zebrafish intestine, mouse ileum, mouse colon, and human colon but few other data sets, identify potentially highly conserved IEC-specific regulatory elements. (F) Scatterplot of accessible chromatin signal at the center of CNEs for zebrafish IECs and mouse ileum IECs, which shows a positive conserved relationship between accessible chromatin signal at these sites. (G) Accessible chromatin data from the EGR1 locus highlighting the commonly accessible CNE_11264 in multiple species. (H) Whole-mount stereofluorescence of stable transgenic line harboring egr1 Tg(zCNE_11264:GFP) showing highest green fluorescent protein (GFP) expression in IECs in the mid intestine. (Bottom) Close-up view of zebrafish egr1 Tg(zCNE_11264:GFP) intestine. Scale bar 100 μm. (Right) Whole-mount confocal images confirming egr1 Tg(zCNE_11264:GFP) GFP expression in IECs. Scale bar 25 μm. (I) The same as H for stable line Egr1 Tg(mzCNE_11264:GFP). (J) The same as H for stable line EGR1 Tg(hzCNE_11264:GFP). (K) The distribution of CA/T-rich-G (CArG)/MCM1, AGAMOUS, DEFICIENS, and SRF (MADS box) and E26 transformation specific (ETS) motifs in human, mouse, zebrafish, and stickleback CNE_11264 show distinct motif distributions despite sequence conservation. Bronze bar represents conserved region. Dashed bar for human hzCNE_11264 represents the apparently conserved region that is adjacent to the hzCNE_11264 boundary marked with solid bronze bar. (L) Heatmap showing motif number (left) and highest motif score (right) for common motifs detected in CNE_11264. (M) Similarity relationship of CArG boxes detected in CNE_11264 shows the diversity of transcription factor binding sites (TFBS) despite overall sequence conservation. Numbering of CArG box sites is arbitrary.

https://doi.org/10.1371/journal.pbio.2002054.g006

Some of these conserved regions that are accessible in most tissues were near genes known to have roles in IEC biology, like Egr1 [74], Nr1d1 [75], and Jun [75], and we did not want to exclude that these regions could still be important in IEC expression. Cloning constitutively accessible regions from nr1d1 and jun were negative for IEC expression. When we cloned the egr1-neighboring CNE regions from zebrafish (zCNE_11264), mouse (mzCNE_11264), and human (hzCNE_11264) and tested them separately using our reporter assay, unsurprisingly, multiple tissues showed GFP expression. However, we observed a distinct differential pattern across the intestine, with GFP expressing most highly in IECs in the mid intestine in zebrafish reporter lines representing all 3 species (Fig 6G–6J). TF motif searching identified multiple ETS and CA/T-rich-G (CArG)/MCM1, AGAMOUS, DEFICIENS, and SRF(MADS) box sites in all 3 species in CNE_11264, which are often immediately adjacent (Fig 6K), consistent with a serum response element that has been characterized at human EGR1 [76–78].

We noticed the size of the conserved region and spacing and number of CArG and ETS binding sites of zCNE_11264 (458 bp) and mzCNE_11264 (352 bp) were greater than hzCNE_11264 (120 bp) (Fig 6G–6L). Searching for local CArG and ETS binding sites identified a neighboring cluster of additional CArG and ETS binding sites, approximately 200 bp outside of the conserved hzCNE_11264 region (Fig 6K). hzCNE_11264 was capable of driving similar IEC expression without these additional putative redundant conserved binding sites (Fig 6G, 6J and 6K). However, this highlights the imperfect nature of defining conserved regions across distantly related species and suggests that accessible chromatin maps combined with searching for common neighboring motif language seeds may identify additional sequence conservation information (S7J–S7L Fig). This same combination of ETS and CArG TFBS was found immediately upstream of Egr1 in the stickleback genome, although zCNEs are not specifically annotated in stickleback (Fig 6K). Identifying these discrete nonoverlapping ETS and CArG binding sites at CNE_11264 in multiple species suggests that multiple ETS and CArG sites have functional relevance for this regulatory region (Fig 6K). However, this also demonstrates the complexity of identifying conservation that often relies on flexible, redundant regulatory logic (Fig 6K and 6L). Similarly, the diversity of detected CArG boxes also show how degenerate TF sequences can deviate while potentially maintaining a similar functional output (Fig 6M) [79].

Accessible chromatin can identify conserved tissue-specific regulatory elements at broadly expressed genes

In an attempt to find zCNEs and upstream regulators that act primarily in IECs, we identified a group of 15 zCNEs that were not accessible in at least 9 out of 17 non-IEC tissues but were always accessible in IECs in zebrafish, mouse, and human (Fig 6E). Motif analysis of these IEC-accessible CNEs revealed common putative IEC-related TFBS in zebrafish, mouse, and humans such as GATA, HNF4A, HOX, CDX2, HNF6, HNF1, and TEAD (S7A–S7I and S8 Figs).

One of these zCNEs neighbors, the gene hairy and enhancer of split-1/hairy-related 6 (HES1/her6) (zCNE_44665), showed remarkable accessible chromatin specificity within intestinal datasets (Fig 6E; S9A and S9B Fig). Hes1 is a transcriptional repressor known to play diverse roles in many tissues including embryogenesis and neural and T-cell development [80–82]. Importantly, it also plays critical roles in the differentiation of IEC subtypes from intestinal stem cell progenitors and in mouse is expressed exclusively in the intestinal crypt [17,83]. While crypts are not present in the zebrafish intestine, and IEC progenitor or stem cells in fish have only been recently characterized [11], hes1/her6 has been found to be expressed in a distinct subset of IECs in an analogous compartment at the base of zebrafish intestinal folds [84]. It is not fully known what genomic regions regulate Hes1 IEC expression and if these regions control aspects of Hes1’s transcriptional response to microbes [85] or in intestinal cancer [86].

We were curious if our IEC chromatin data did discriminate important conserved regulatory regions that drove expression in IECs (Fig 7A). In our reporter assay zCNE_44665, a region approximately 3,600 bp upstream of the hes1/ her6 TSS in zebrafish, revealed strong IEC expression and expression in other tissues including liver (Fig 7B–7D, S10 Fig). Whole-mount and cross-sections of 7 dpf fish identified that high GFP expression was within a subset of IECs, often at the base of slight invaginations of this cell layer (Fig 7B–7E, S10A Fig). These invaginations are ultimately analogous to the base of epithelial folds (rugae) seen in older fish and the intestinal crypt in mammals, although, at 7 dpf, substantial folds are not articulated (Fig 7D and 7E) [12]. This Tg(zCNE_44665:GFP) population did not overlap with cells that were positive with the enterocyte marker Tg(-4.5fabp2:DsRed) (Fig 7F and S10G Fig) [73], the enteroendocrine marker Tg(neurod1:TagRFP) (Fig 7G and S10H Fig) [87], or cells with characteristic goblet cell morphology. This suggests the hes1-neighboring conserved region zCNE_44665 drives GFP in an undercharacterized population of IECs that is distinct from these known differentiated IEC subtypes in zebrafish.

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Fig 7. Hes1 conserved nonexonic element (CNE)-driven expression overlaps with Notch signaling and marks a specific population of intestinal epithelial cells (IECs).

(A) Accessible chromatin at HES1 loci. (B) Whole-mount stereofluorescence of stable transgenic line 7 dpf zebrafish harboring hes1 Tg(zCNE_44665:GFP) showing green fluorescent protein (GFP) expression in IECs. Scale bar 100 μm. (C) Same as B, a closeup showing high GFP+ IECs in a population of cells. (D) Same as B, a confocal whole-mount z-stack maximum projection of zebrafish intestine showing high GFP+ in a population of IECs. (E) A confocal image of intestinal cross-section of 7 dpf hes1 Tg(zCNE_44665:GFP) showing GFP expression in IECs that appear at the base of slight invaginations (white arrows). Scale bar 25 μm. (F) A confocal intestinal cross-section of 7 dpf zebrafish shows lack of overlap between hes1 Tg(zCNE_44665:GFP) and enterocyte marker Tg(-4.5fabp2:DsRed). GFP+ IECs lacking Discosoma sp. red fluorescent protein (DsRed) are marked with a white arrow. Scale bar 25 μm. (G) Lack of overlap between hes1 Tg(zCNE_44665:GFP) and the enteroendocrine marker Tg(neurod1:TagRFP) [87]. A reduction in background GFP can be seen at red fluorescent protein (RFP)+ cells (white arrow). Scale bar 25 μm. H) A confocal cross-section image shows overlap between hes1 Tg(zCNE_44665:GFP) and Tg(EPV.Tp1-Ocu.Hbb2:hmgb1-mCherry) in 7 dpf zebrafish. Individual channels are shown in inset with DAPI in blue. Scale bar 25 μm. I) A confocal image of cross-section of 8-week-old zebrafish intestinal folds in hes1 Tg(zCNE_44665:GFP) and Tg(EPV.Tp1-Ocu.Hbb2:hmgb1-mCherry). (J) A stereoscopic image showing the overlap of whole-mount human HES1 Tg(hzCNE_44665:GFP) and Tg(EPV.Tp1-Ocu.Hbb2:hmgb1-mCherry) in a subset of IECs in 7 dpf zebrafish. Scale bar 100 μm. (K) A confocal cross-section of 7 dpf zebrafish showing the overlap of human HES1 Tg(hzCNE_44665:GFP) and Tg(EPV.Tp1-Ocu.Hbb2:hmgb1-mCherry). Scale bar 25 μm. (L) Heatmap of common TFBS motifs in CNE_44665 in zebrafish, mouse, and human. (M) A schematic of site-directed mutagenesis on Hepatocyte nuclear factor 1 (HNF1) (top) and Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) (bottom) putative binding sites from zCNE_44665. (N) A confocal whole-mount image showing the overlap between hes1 Tg(zCNE_44665 ΔHNF1 sdm:GFP) and Tg(EPV.Tp1-Ocu.Hbb2:hmgb1-mCherry). Scale bar 25 μm. O) A confocal whole-mount image showing the overlap between hes1 Tg(zCNE_44665 ΔRBPJ sdm:GFP) and Tg(EPV.Tp1-Ocu.Hbb2:hmgb1-mCherry). Scale bar 25 μm.

https://doi.org/10.1371/journal.pbio.2002054.g007

We scanned the sequence of the orthologous CNEs from zebrafish, mouse, and human and found TFBS for hepatocyte nuclear factor 1 (HNF1), hypoxia inducible factor 2 alpha (HIF2A), and recombination signal binding protein for immunoglobulin kappa J region (RBPJ) in all 3 CNEs (Fig 7L; S9A–S9C Fig). RBPJ is known to regulate HES1 expression in the presence of Notch signaling [88], resulting in the alteration of the proportion of secretory and absorptive IEC lineages [17]. Therefore, we tested if this Tg(zCNE_44665:GFP) population was positive for Notch signaling. Crosses of Tg(zCNE_44665:GFP) with Tg(EPV.Tp1-Ocu.Hbb2:hmgb1-mCherry), which uses a viral-derived promoter with Notch-responsive RBPJ binding sites [89], revealed substantial overlap between mCherry+ and GFP+ cells (Fig 7H and 7I, S10 Fig, S1 Movie). The mCherry+ IECs were always GFP+, and only 64.6% of GFP+ cells were mCherry+, which could be due in part to the relatively slow maturation of mCherry protein. This overlap suggests Notch signaling is important in regulating this element upstream of HES1/her6 (Fig 7H and 7I, S10 Fig). A cross-section of dissected intestine from 8-week-old fish revealed a more sophisticated expression pattern in relationship to the now articulated intestinal folds (Fig 7I). GFP+ cells included cells at the base and typically in the bottom half of the intestinal folds and included cells positive for Notch signaling. Interestingly, these cells frequently had more apical nuclei than most IECs, whose nuclei typically are located basally within the epithelium (Fig 7I and S10E Fig).

To determine if a similar regulatory capacity was conserved to mammals, we then tested the hzCNE_44665 from human (approximately 10 kb from the TSS of HES1). This human region also drove intestinal expression that was similarly limited to a subset of IECs that often overlapped with Notch-positive cells (30.5% GFP+/mCherry+, 33.6% GFP+ only, 35.7% mCherry+ only) (Fig 7J and 7K). To determine if common putative TFBSs found in CNE_44665 were regulating expression in IECs, we generated a zebrafish line with the HNF1 binding site abolished through mutation in the zCNE_44665 GFP reporter construct Tg(zCNE_44665 ΔHNF1:GFP) (Fig 7L and 7M). We still identified GFP expression in a subset of cells that was coincident with Notch signaling in IECs from stable fish lines containing this construct (Fig 7N). However, when we abolished the RBPJ binding site, we found that high GFP expression was completely lost in IECs, including the subset of cells that overlapped with Notch-positive cells (Fig 7O). This suggests a conserved RBPJ binding site is necessary for expression in IECs and may contribute to common HES1 regulation in fish and mammals in progenitor IEC populations (Fig 7O). Collectively, our chromatin data analysis is capable of distinguishing IEC regulatory regions and putative causative TFBS from complex regulatory landscapes that may regulate a broadly expressed gene’s transcription in IECs.

Transcriptional similarities are detectable in IECs separated by 420 million years of evolution

Conserved regulatory elements that are stable over millions of years are likely to have analogous functions in their respective genomes and could coordinate conserved tissue-specific transcriptional patterns. However, identifying and functionally annotating these regions at individual loci or across a single genome is problematic, tedious, and does not accurately predict or robustly identify conserved function. As demonstrated above, our strategy of combining tissue-specific transcription and accessible chromatin datasets with conservation and TFBS prediction inferred from DNA sequences from multiple species identified putative conserved regulatory and functional information in IECs that could not have been identified by any one data set alone.

Tissues and cell types like IECs are defined by complex patterns of gene expression. Despite specializations in each animal species, IECs serve core inherent functions such as absorption and metabolism of dietary nutrients and xenobiotics and as a barrier to microbes residing within the intestinal lumen. As evidence for this common conserved function, we identified 470 orthologous genes expressed highly in IECs with relative tissue-specificity across 420 million years of vertebrate evolution. Functional conservation was maintained across a broad range of IEC biology, including genes involved in IEC subtypes, lipid transport and metabolism, and a response to microbes and inflammation. We highlight commonly expressed TFs because they may underlie the expression of conserved networks that are associated with IEC function, identity, and regionalization. YBX1, HNF4A, ELF3, XBP1, ID3, HMGB2, IRF1, STAT3, GATA5, and OSR2, amongst other TFs, appear to be more highly expressed in IECs than other tissues. Importantly, many of these highly expressed TFs also show enrichment for their cognate TFBS in accessible chromatin surrounding IEC signature genes in multiple species including the ETS factors ELF3 and ELF4, HNF4A, GATA5, and STAT3.

Complex regulatory mechanisms that specify regional function are conserved along the length of the zebrafish and mouse intestine

Our understanding of intestinal evolution has been hindered by a lack of information about the degree to which anteroposterior segments in extant vertebrate species are ancestral or derived traits. We found patterns of conserved expression along the length of the zebrafish and mouse intestine, suggesting that conserved discrete transcriptional regulatory programs may specify homologous duodenal, jejunal, ileal, and colonic segments along the zebrafish intestine [15]. We found striking evidence for transcriptional regulation underlying this conserved similarity, as the genomic region immediately upstream of the highly expressed IEC signature gene FABP6, in both zebrafish and mouse, was capable of driving GFP expression coincident between the zebrafish intestinal segment 1 and segment 2 domains [12–15]. We infer that this discrete segment specified by the expression of FABP6 and other markers functions as the conserved homologous zebrafish ileum and that the zebrafish intestine is more completely defined as at least 5 distinct segments with further evidence that transcriptional domains similar to the duodenum, jejunum, and colon exist (Figs 2 and 5). Collectively, these results indicate that the transcriptional underpinnings of the well-characterized segmental program present in the mammalian intestine are ancestral to the last common ancestor with bony fishes and that the utility of the zebrafish as a model for human intestinal biology is even greater than previously appreciated.

Accessible chromatin maps annotate regulatory regions in IECs in the absence of sequence conservation

Our initial analysis to utilize the presumed regulatory information at the TSS of IEC signature genes identified a number of regions with largely IEC-specific accessible chromatin status. However, we were unable to identify a substantial number of highly sequence-conserved regions at these TSSs. To circumvent the apparent lack of conserved regulatory information despite clear transcriptional similarities, we applied strategies through which regulatory information could be inferred without directly using traditional sequence conservation metrics. Searching for significantly enriched TF motifs found in accessible chromatin regions surrounding genes expressed in IECs in multiple species allowed us to identify common presumptive TF motifs that are used in the regulation of these genes, including HNF1, HNF4A, GATA, and ETS factors [60,62,63]. In addition, we looked for common predicted TFBS in TSS regions that appeared to have accessible chromatin in IECs but no strong sequence conservation. Accordingly, despite the lack of sequence conservation, the RBP2(a) promoter regions from zebrafish, mouse, and human are capable of driving highly similar expression in the IECs of zebrafish. This conserved expression is presumably largely due to common HNF4A and GATA motifs in zebrafish, mouse, and human that escape detection by commonly used sequence conservation metrics. Recently, a microbially responsive element in the zebrafish angptl4 gene was shown to contain an element with HNF4A and GATA motifs that were involved in driving expression that is essentially identical to the expression pattern from our rbp2a fragment [68]. Fabp2 also shows the same regional expression as rbp2a and angptl4 in the intestine and has binding sites for HNF4A and GATA that are shared to mammals [72]. Furthermore, the TFs HNF4A and GATA, and FXR, that have putative binding sites in the regulatory regions from Rbp2 and Fabp6, respectively, show intestinal expression patterns that appear to explain much of the regional IEC expression of these genes (S3G and S3H Fig). This suggests these common combinations [18] are conserved in regulating genes in the IECS of anterior intestines from teleosts to mammals. Intriguingly, HNF4A and FXR were also recently shown to mediate IEC responses to microbiota [26,90], indicating complex relationships between tissue-specific and microbially-responsive transcriptional programs in the intestinal epithelium.

IEC-specific CNE at hes1/her6 drives expression in Notch-positive cells

We were able to identify a small number of highly conserved noncoding elements with apparent conserved IEC-specific chromatin accessibility, representing excellent candidates to understand conserved regulatory mechanisms that drive IEC expression (Fig 6 and S7 Fig). Our strategy highlights the utility of these data in identifying tissue-discriminating regulatory regions at genes that lack clear tissue-specific transcription and may be selectively regulated by discrete regulatory regions in different tissues. Similar strategies using available mammalian accessible chromatin datasets could annotate the remaining CNEs with putative functions. We focused on a CNE upstream of HES1 because of its exceptional accessible chromatin specificity in the intestine (Fig 6E, S9A and S9B Fig) and the known importance of HES1 in IEC biology [91]. We identified a necessary RBPJ binding site within the hes1 CNE that drove expression in IECs coincident with Notch signaling. Notch signaling likely plays a complex function at the hes1 locus, as amongst the 6 hes1/her6 neighboring zCNEs, 4 contain conserved RBPJ binding sites in their zebrafish, mouse, and human CNE counterpart (zCNE_44665, 44657, 44661, 44663), 1 CNE (zCNE_44658) contains an RBPJ site in zebrafish and human, and only 1 additional CNE (zCNE_44660) contains no RBPJ sites [92,93]. The only HES1/her6-neighboring zCNE containing a predicted binding site for HNF1 was zCNE 44665; however, loss of the HNF1 binding site was not sufficient to ablate expression in Notch-positive IECs, suggesting that other mechanisms confer the specific accessibility and expression in IECs. Due to the existence of other RBPJ sites at this locus, it seems unlikely that Notch signaling strictly underlies the IEC specificity, although tissue-specific expression of Notch ligands may also contribute to IEC expression [94].

Hes1 is expressed exclusively in the crypt of the mammalian intestine, including in stem cells and transit-amplifying progenitors coincident with Notch signaling [83,94,95]. Similarly, we see the zebrafish and human HES1 CNE_44665 is capable of driving expression in IECs at the base of and bottom half of intestinal folds using our zebrafish GFP reporter assay, also coincident with Notch signaling [84]. It will be interesting to determine if Hes1 and the CNE_44665-marked cells play an analogous progenitor role in fish as well as mammals. Though we focused on the sequenced conserved region that also showed accessible chromatin specificity, a larger region outside of this conserved region showed intestinal accessible chromatin specificity in human and mouse (S9A and S9B Fig). Interestingly, a GFI1B binding site detected in the zebrafish zCNE, but absent in the mouse and human zCNE, was detected within the adjacent region that was accessible specifically in mouse and human intestinal datasets. GFI1B is a repressor TF that helps specify the IEC-subtype tuft cell [96]. This larger regulatory region may facilitate conserved regulation of HES1 expression and restrict Hes1 expression in tuft cells, even though this GFI1B binding site is not found in a region that is conserved in sequence from zebrafish to human (S9A–S9C Fig) [96]. These results provide an important context for further exploring how conserved DNA regulatory regions and multiple TFs function cooperatively to regulate expression of the HES1 gene in IECs and other distinct tissues.

Towards a complete understanding of IEC specificity

While we framed this study around the idea that particular regulatory regions and transcripts could have specific functionality in IEC cell types, an interesting premise is that important regulatory regions and transcripts may be exclusive to multiple distinct organs or cell types, and these circuits could be conserved across species. Of course, even IECs are heterogeneous, so a complete understanding of IEC-specific programs will require higher resolution maps of IECs and multiple additional tissues [33]. Our examples of Fabp6 and Rbp2 do indeed seem to show high GFP expression that is largely limited to IECs (Fig 2). However, HNF4A and GATA also function in the liver [97] where rbp2a is expressed [66]; therefore, the definition of specificity and understanding of insulation of transcriptional regulation in different cell types requires further study. Though we believe our approach successfully identified genes and regulatory regions that are important in IECs, we did notice that the TSS at many IEC signature genes also showed accessibility in kidney and liver tissue (Fig 3E), suggesting an important overlapping utilization of the regulatory regions and gene functions in these other tissues. We do not want to minimize the importance of the concept that many regulatory regions are used in multiple cell types and should perhaps holistically be thought to function, be selected upon, and be conserved in an interorgan/cell-type regulatory network.

The strategies and methods we used here were able to detect diverse types of conserved transcriptional and regulatory information in fish and mammalian IECs; however, important limitations apply. Specifically, with our strategy, some pathways utilized broadly in many tissues, genes expressed in a small number of cells, or lowly expressed genes may be difficult to characterize for their conservation across species in the intestine and other tissues. For example, though the Wnt pathway functions in a broad range of tissues [98], it has been established as functioning in a similar manner in zebrafish and mammalian intestine. The Wnt coactivator TCF4 is tied to microbially regulated epithelial cell proliferation in zebrafish [99]. Remarkably, deletion of the Wnt pathway inhibitor, Apc, results in intestinal tumors in both zebrafish [100] and mammals [101]. However, only a small number of Wnt-related genes, including FZD5, were identified here as IEC signature genes, although individual ligands or pathway components may signal the use of broader pathways in IECs. As a result, the lack of highlighting a conserved gene or pathway cannot be considered as the lack of conservation, generally. Furthermore, the ability to find conserved function in a particular cell type, gene family, or pathway may depend highly on the proposed definition of conservation and be complicated by each genome’s unique history.

The influence of orthology and teleost genome duplication on understanding conserved gene function and regulation

Teleosts underwent a genome duplication approximately 340 million years ago [102]. Approximately 20% of these duplicated gene pairs are maintained in the extant zebrafish and other teleost species, although the same duplicated genes are not always maintained in each genome [102–104]. Retained duplicated genes can undergo, amongst other fates, neo- and subfunctionalization specific to a lineage, spurring adaptation and potentially driving speciation [105]. This duplication can complicate comparative genomics and the parsing of function across vertebrate species with limited transcriptional profiling, but it also provides a rich platform for understanding gene evolution, function, and regulatory regions [106]. To create a focused strategy using genes that likely had maintained function and therefore detectable transcription and regulatory signal across species, our RNA analysis was largely limited to 1-to-1 orthologs across the 4 species we assayed, although our CNE analysis was essentially independent of orthology. We suspect that the diversity of types of regulatory information aren’t unique to genes with specific types of orthology across species, although novel regulation of gene function may more frequently arise during sub- and neofunctionalization of duplicated paralogs [105]. In addition, certain gene groups such as TFs are less likely to be lost following duplication in the zebrafish genome [102], suggesting that additional analysis may be required to uncover and integrate the functions and regulatory information that are contained within groups of genes that do not show 1-to-1 orthology.

Traditional conservation metrics are not sufficient to identify all conserved regulatory information

We found instances at Rbp2, Fabp6, Egr1, and Hes1 in which traditional sequence conservation metrics were not sufficient to fully identify common putative regulatory information from fish to mammals even when it is located approximately the same distance relative to the gene body in all species [57,107]. In all instances, accessible chromatin data provided additional context to identify conserved regulatory information. Importantly, because TFBS can be modular, conserved transcriptional regulation can occur with almost no sequence conservation signal being detectable across species. This property is largely due to the inherent nature of TFs because plasticity in the number, arrangement, and affinity of TFBS can result in nearly identical transcriptional responsiveness and output even in the absence of long stretches of sequence conservation. To more accurately identify conserved regulatory information, metrics are needed that incorporate short degenerate TF motifs or identify highly conserved short motifs between closely related species that are then identified as a conserved block in distantly related groups of similar species in syntenic regions. This information could be further anchored by accessible chromatin [108,109]. This may only partially circumvent the somewhat inherent statistical problem of short sequences arising by chance.

The interrogation of regulatory DNA sequences using assays such as GFP reporters can determine how complex orthologous regulatory sequences are interpreted in vivo. However, these assays are not necessarily sufficient to determine functional conservation across species. For example, orthologous TFs may show an altered TF binding motif preference in each species, although the regulation by this common factor is itself otherwise conserved. Alternatively, functional TFBS from one species may be invalidated by other neighboring sequences in cross-species assays, although the same TF is successfully regulating the same gene in each species. This issue is highlighted by the difference in AT% usage at the TSS of the 4 species used in this study that presumably is partially distinct from traditional TF binding content (Fig 3G). This property may partially be driven by more general sequence characteristics that require even less stringency in conserving specific long blocks of DNA like CpG methylation [64], nucleosome positioning, and overall dynamics of nucleosome accessibility [110], binding competition dynamics [111], or TSS selection [65] that may vary from organism to organism. Detecting sequences and principles that account for these confounding factors will need to be accomplished and compensated for before a complete understanding of the similarity and differences between analogous organs and transcriptional programs can be realized. Tissue-specific transspecies high-throughput enhancer activity assays [112] may be required to sufficiently sample and test transcription rules and conservation for specificity across species. These will need to be further combined with a similar deep understanding of posttranscriptional control and epigenetic mechanisms to develop a more complete picture of signals encoded in DNA and if they are commonly or divergently utilized.

Prospectus

Our experimental strategy utilized IECs from healthy adult animals representing 4 vertebrate lineages to reveal conserved mechanisms underlying tissue- and region-specific IEC transcriptional regulation. These results provide an important frame of reference for future efforts to uncover similar mechanisms in IEC subtypes or in the context of other developmental stages, disease states, or environmental exposures. Many of the genes and upstream TFs broadly implicated here as conserved features of vertebrate IECs have already been implicated in human diseases (S1 Table) and in the intestinal response to microbiota [26,90], prompting further studies into the mechanistic relationships between transcriptional regulatory networks governing IEC identity, environmental sensitivity, and disease pathogenesis. We believe that using the strategy of simultaneously leveraging genome-wide data sets from multiple species can identify key ancient aspects of biology more quickly than studying any species alone.

Ethics statement

Zebrafish studies were approved by the Institutional Animal Care and Use Committees of Duke University (protocol A165-13-06) and University of North Carolina at Chapel Hill (protocol 12–058.0). Stickleback studies were approved by the Institutional Animal Care and Use Committee of Stanford University (protocol 13834). Stickleback were collected under California Scientific Collecting Permit #3260. Studies involving human tissues were performed under University of North Carolina at Chapel Hill IRB approval numbers 10–0355 and 14–2445.

Intestinal epithelial preparation and genomic assays for each vertebrate

Mouse

Mouse tissue dissection and IEC extraction protocols and all initially processed data were previously described (GSE57919) [16].

Zebrafish

Stickleback

RNA levels and downstream analysis

Accessible chromatin and downstream analysis

Conservation metrics

Cloning and generation of reporter constructs

Putative regulatory elements were amplified from genomic DNA with primers containing FseI and AscI restriction site overhangs and then cloned, maintaining orientation relative to their native TSS, into the p5E-FSE-ASC entry plasmid (381) (http://tol2kit.genetics.utah.edu/; Tol2kit v1.2) [125]. Putative clones were confirmed by PCR and sequencing to ensure an exact match to genomic sequence from the corresponding species. Four-way LR reactions were performed using the LR Clonase II Plus Kit (12538120, Invitrogen) combining p5E-FSE-ASC modified with a putative regulatory element, pME cFos (S4 Table), p3E EGFP (366), and pDestTol2pA2 (394) using the provided protocol. This generated a single plasmid recombined with a putative regulatory region upstream of a minimal mouse cFos promoter-driving eGFP and flanked by Tol2 transposon insertion sites, which was confirmed to contain the putative regulatory region by PCR. Site-directed mutagenesis constructs were generated by randomly changing key bases over 10 bp in TFBS, detected by Homer within the zebrafish hes1/her6-neighboring zCNE_44665. Homer could no longer detect the targeted TFBS using the mutated genomic sequence in the context of the otherwise original zCNE_44665 sequence. For each site-directed mutation, complementary primers containing the 10 bp mutated region flanked by wildtype 20 bp sequences on either side were used in circular PCR with the original zCNE_44665 containing 381 plasmid as template followed by DpnI treatment to digest methylated plasmid. Site-directed mutagenesis was confirmed by sequencing.