Abstract
Background & aims
The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce postmitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor Atoh1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type II immune responses to parasite infections and appear to differentiate independently of Atoh1. We investigated the role of Sox4 in ISC differentiation.Methods
We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre; SOX4 conditional knockout [cKO]) and mice without disruption of Sox4 (control mice). Crypt- and single-cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative polymerase chain reaction, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice) and single-cell RNA-seq analysis were used to identify cells that coexpress Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1 inducible knockout) mice and assessed differentiation.Results
Sox4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly after helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by interleukin 13 in control organoids; SOX4cKO mice had impaired tuft cell hyperplasia and parasite clearance after infection with helminths. In single-cell RNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISC, progenitor, and tuft cell genes; 12.5% of Sox4-expressing cells coexpressed Atoh1 and were enriched for enteroendocrine genes. In organoids, overexpression of Sox4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of Atoh1.Conclusions
We found Sox4 promoted tuft and enteroendocrine cell lineage allocation independently of Atoh1. These results challenge the longstanding model in which Atoh1 is the sole regulator of secretory differentiation in the intestine and are relevant for understanding epithelial responses to parasitic infection.Free full text
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SOX4 Promotes ATOH1-independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates
Associated Data
Abstract
Background & Aims:
The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce post-mitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor ATOH1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type 2 immune responses to parasite infections and appear to differentiate independently of ATOH1. We investigated the role of SOX4 in ISC differentiation
Methods:
We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre;SOX4cKO) and mice without disruption of Sox4 (control mice). Crypt- and single cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative PCR, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 (IL13) and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice), and single-cell RNA-seq analysis, were used to identify cells that co-express Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1iKO) mice and assessed differentiation.
Results:
SOX4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly following helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by IL13 in control organoids; SOX4cKO knockout mice had impaired tuft cell hyperplasia and parasite clearance following infection with helminths. In scRNA-seq analysis, Sox4+/Atoh1− cells were enriched for ISCs, progenitors, and tuft cells; 12.5% of Sox4- expressing cells co-expressed Atoh1 and were enriched for enteroendocrine cells. In organoids, overexpression of SOX4 was sufficient to induce differentiation of tuft and enteroendocrine cells—even in the absence of ATOH1.
Conclusions:
We found SOX4 to promote tuft and enteroendocrine cell lineage allocation independently of ATOH1. These results challenge the long-standing model in which ATOH1 is the only regulator of secretory differentiation in the intestine, and are relevant for understanding epithelial responses to parasitic infection.
Abstract
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Introduction
The intestinal epithelium is essential for both digestive and barrier function and contains a diverse complement of post-mitotic cells which carry out complex physiological functions critical for homeostasis. Functional cell types in the intestine can be broadly subdivided into: (a) absorptive enterocytes and (b) secretory lineages, which include antimicrobial-producing Paneth cells, mucus-producing goblet cells, and hormone-secreting enteroendocrine cells (EECs) 1. Tuft cells, which represent a less well-characterized lineage, initiate immune responses to parasitic infections in the intestine 2–5. With the exception of long-lived Paneth cells, these post-mitotic lineages are subject to the rapid (5–7 day) and continuous turnover of the intestinal epithelium. Therefore, their numbers must be maintained through constant proliferation and differentiation of the intestinal stem cells (ISCs), which reside in a specialized stem cell niche at the base of the intestinal crypts.
Extrinsic signals from the niche induce regulatory programs in ISCs and their immediate progeny, transit-amplifying progenitor cells (TAs), to drive commitment to different cellular lineages. For example, loss of Notch signaling in ISCs and early progenitors is associated with the induction of secretory differentiation through the derepression of Atoh1, which is widely regarded as the master regulator of secretory fate 6, 7. Certain pathological settings can shift lineage allocation in the intestine, as exemplified by goblet and tuft cell hyperplasia in the setting of type II immune responses. While tuft cells are also derived from ISCs, the transcriptional regulatory mechanisms governing their differentiation and hyperplastic response to infection remain less well characterized 3. This is due in part to evidence that tuft cells differentiate independently of Atoh1, but still appear to require downregulation of Notch 6, 8, 9. Recent studies have demonstrated upregulation of tuft cell numbers following Atoh1 deletion in vivo, and systems biology approaches have supported an Atoh1-independent differentiation program for this lineage 10. Together, these data suggest an apparently paradoxical Notch-repressed, Atoh1−independepent mechanism for tuft cell specification.
Sry-box containing (Sox) factors are transcription factors with broad regulatory roles in stem cell maintenance and differentiation in many adult tissues, including the intestinal epithelium 11, 12. Sox9 is known to contribute to Paneth cell differentiation, and previous work from our group has demonstrated that distinct expression levels of Sox9 mark stem and progenitor cell populations from intestinal crypts, including label-retaining cells (LRCs) 13–16. The role of other Sox factors in ISC function and differentiation remain uncharacterized.
Sox4 has been associated with the ISC genomic signature and is correlated with increased invasion/metastasis and decreased survival in colon cancer, but its role in intestinal epithelial homeostasis is unknown 17, 18. In the hematopoietic system, as well as the developing nervous system, pancreas, kidney, and heart, Sox4 is required for both proper cell lineage allocation and maintenance of progenitor pools 19–21. The diverse regulatory potential of Sox4, coupled with its known role in a broad range of tissue-specific progenitor populations, led us to hypothesize that it may function as a regulator of ISC maintenance and differentiation. In this study we establish a role for Sox4 in ISC homeostasis and regulation of tuft cell hyperplasia during parasitic helminth infection, which represents a novel pathway for tuft cell specification with important implications for human responses to parasitic disease.
Results
SOX4 is expressed at distinct levels in the ISC zone
To characterize Sox4 expression, we first used in situ hybridization to detect Sox4 mRNA. Consistent with previous reports, Sox4 is restricted to the base of the intestinal crypts in the stem cell zone (Figure 1A) 18. Since the cellular resolution of in situ hybridization is low, we leveraged a previously characterized Sox9EGFP reporter mouse to examine the expression of Sox4 in different crypt cell types. Distinct Sox9EGFP levels differentially mark active ISCs (Sox9low), secretory progenitors/reserve ISCs (Sox9high), TA progenitors (Sox9sublow), Paneth cells (Sox9EGFP-neg:CD24+), and non-Paneth post-mitotic cells (Sox9EGFP-neg:CD24neg) 13, 14, 22, 23. Sox9EGFP populations were isolated by fluorescence-activated cell sorting (FACS) and analyzed by RT-qPCR. Sox4 was significantly enriched in two populations: Sox9low, which is consistent with active ISCs, and Sox9high, which is consistent with secretory progenitors (Figure 1B) 23, 24.
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(A) In situ hybridization localizes Sox4 mRNA to the base of small intestinal crypts. (B) FACS isolation of Sox9EGFP populations demonstrates enrichment of Sox4 in active ISCs (Sox9low) and secretory progenitors/reserve ISCs (Sox9high) (different letters indicate statistically significant differences, p < 0.05). (C) SOX4 protein is expressed in the ISC and supra-Paneth cell zones at variable levels, with (D) SOX4low cells primarily localized to the CBC position (1–3) and SOX4high cells occurring mainly in the supra-Paneth cell zone (4+). (E/E’) SOX4high and Sox9high cells do not co-localize; arrows indicate SOX4hi cells and arrowheads indicate Sox9high cells (n=3 mice, 100 SOX4high cells per mouse, scale bar represents 20um). (F) SOX4high cells are not LRCs by 28d continuous EdU labeling followed by 10d washout; white arrow indicates SOX4high cell and yellow arrow indicates LRC (scale bar represents 20um; note: EdU detection kit reacts with Paneth cell granules). (G) A small portion of SOX4high cells co-localize with the tuft cell marker DCLK1 and (H) a majority of SOX4high cells are positive for proliferative marker KI-67.
Next, we examined SOX4 protein expression by immunofluorescence. SOX4 protein was expressed in a more restricted pattern relative to mRNA. Like Sox9, we observed distinct expression levels of SOX4, which we characterized as “low” and “high” (Figure 1C). SOX4low cells were associated with the crypt base columnar (CBC), active ISC population, in cell positions +1–3 counting from the base of the crypt (Figure 1C, D). Interestingly, SOX4high cells were predominantly found in the supra-Paneth cell position, which is commonly associated with early secretory progenitors, at cell positions +4–7 (Figure 1C, D) 25. Unlike SOX9, which is expressed in villus-based EECs/tuft cells, we did not detect any Sox4 mRNA or protein outside of the crypts 13.
Since Sox9high cells are known to represent EEC-like secretory progenitors, we asked if the SOX4high population overlapped with Sox9high cells. However, we found no co-localization between high levels of SOX4 protein and high levels of Sox9 mRNA, as determined by Sox9EGFP, which is an accurate surrogate of endogenous Sox9 mRNA and protein expression. (n = 3 mice, 100 SOX4+ cells per mouse) (Figure 1E, E’) 13, 26. Of note, the Sox9EGFP transgene is selectively silenced in Paneth cells, and SOX4 expression was never observed in the Paneth cell position. Sox9high-expressing cells are consistent with a subset of label-retaining reserve ISCs (LRCs) located in the supra-Paneth cell position. However, not all LRCs are Sox9high23. We reasoned that the supra-Paneth cell location of SOX4high cells suggests that they may represent the non-Sox9high subset of LRCs. To examine this, we counted the number of SOX4high cells co-localizing with LRCs, as determined by 28 days of continuous labeling with 5-ethynyl-2-deoxyuridine (EdU), followed by 10 days of washout 23, 25. Surprisingly, we found that no SOX4high cells were LRCs (n = 3 mice, 100 SOX4+ cells per mouse) (Figure 1F). We observed rare co-localization between DCLK1, a tuft cell/reserve ISC biomarker also associated with the +4 position, and SOX4 (5.0% ± 3.0%; Figure 1G). Finally, we addressed the possibility that SOX4high cells may represent a quiescent population by co-staining with KI67, which marks all proliferating cells. 90.6 ± 3.2% of SOX4high cells were found to be proliferative (Figure 1H). Together, these data suggest that SOX4high cells represent ISCs or progenitors, and are distinct from LRC Sox9high secretory precursors. Co-localization with DCLK1 suggests that a subset of SOX4high cells may represent tuft cell or non-LRC secretory precursors.
Loss of Sox4 results in increased proliferation and decreased ISC function
Complete deletion of Sox4 results in embryonic lethality 27. To enable examination of Sox4 function in adult intestinal epithelium, we crossed previously generated Sox4fl/fl mice to mice expressing Cre recombinase driven by the villin promoter (vilCre), which is constitutively expressed in the intestinal epithelium beginning at E13.5 28, 29. To examine Sox4 expression, we isolated CD44− and CD44+ populations from CD326 (EPCAM)+ control and Sox4cKO adult intestinal epithelium by FACS and confirmed CD44 enrichment by qPCR (Supplementary Figure 1A–C). CD44 marks crypt cells as well as EECs in the villi (Supplementary Figure 1A). As expected, control animals exhibited significant enrichment of Sox4 in the CD44+ population, consistent with restricted expression of Sox4 in the intestinal crypts (Supplementary Figure 2A). Sox4cKO animals did not express detectable levels of Sox4 mRNA or protein (Supplementary Figure 2A, B).
To assay the role of Sox4 in ISC/TA proliferation, we examined KI67 and EdU incorporation, which was administered as a single, 2hr pulse. Sox4cKO crypts exhibited significantly higher percentages of total proliferating cells as well as cells in S-phase (Figure 2A, B). This increase in proliferation was reflected by increased crypt length in Sox4cKO intestines (Supplementary Figure 2C). In organoid cultures, Sox4cKO crypts grew at the same rate as control crypts, suggesting that hyperproliferation in the absence of Sox4 may be masked by exogenous growth factors required for organoid culture (Figure 2C).
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(A) Sox4cKO intestines exhibit increased total proliferation by KI67 staining, as well as (B) increased numbers of cells in S-phase, as indicated by EdU (scale bar represents 100um). (C) Whole crypts isolated from Sox4cKO intestines form organoids that undergo similar growth rates to control crypts (scale bar represents 100um). (D) Numbers of OLFM4+ ISCs remain unchanged in Sox4cKO crypts, while (E) Lgr5 is downregulated in CD44+ populations from Sox4cKO intestines. Functional stemness, as assayed by organoid forming ability, is significantly decreased in CD44+ populations isolated from Sox4cKO intestines (scale bar represents 25um; asterisks indicate significance; p < 0.05).
Due to its known role in maintenance of stem/progenitor pools in other tissues, we sought to determine if Sox4 regulates the size or function of the ISC pool. OLFM4, which is an established marker of ISCs, was detected by immunofluorescence to assess ISC numbers, which were unchanged between control and Sox4cKO intestines (Figure 2D) 30. Next, we FACS-isolated CD44− and CD44+ cells from control and Sox4cKO mice and conducted single cell organoid forming assays to test ISC function 31. As expected, CD44− cells failed to form organoids regardless of Sox4 status (Figure 2E). CD44+ cells from Sox4cKO mice produced approximately 50% fewer organoids than controls, demonstrating a significant loss of stemness (Figure 2E).
To determine if this functional deficit was coincident with an increase in apoptosis in Sox4cKO samples, we examined cleaved-caspase-3 expression in the crypts of control and Sox4cKO intestines, as well as the number of apoptotic cells in CD44− and CD44+ populations of cells prepared for FACS. In both cases, we found no significant difference in apoptosis between control and Sox4cKO samples (Supplementary Figure 3A, B). However, CD44+ cells from Sox4cKO mice expressed significantly lower levels of Lgr5, consistent with decreased stem cell function (Figure 2F). Consistent with immunofluorescence results, Olfm4 mRNA levels were unchanged between control and Sox4cKO samples (Figure 2F). Together, these data indicate that Sox4 does not regulate ISC numbers or apoptosis, but is required for normal ISC function and Lgr5 expression in the intestinal crypt.
Sox4 regulates secretory lineage allocation
We next sought to determine if Sox4 regulates differentiation and intestinal lineage allocation. Goblet cells, detected by expression of MUC2, were found at similar numbers between Sox4cKO and control intestines (Figure 3A). CHGA+ EECs were found in significantly reduced numbers in the villi of Sox4cKO mice (Figure 3B). Numbers of DCLK1+ tuft cells were significantly reduced in crypt and villus compartments of Sox4cKO intestines (Figure 3C). Paneth cells were increased in number in Sox4cKO intestines (Figure 3D), and located at higher positions in Sox4cKO crypts, but determined to be non-proliferative by co-staining with KI67 (Supplementary Figure 4A and 4B). Staining patterns for sucrose isomaltase (SIS), a brush-border enzyme associated with absorptive enterocytes, were unchanged between control and Sox4cKO samples (Supplementary Figure 5A). Additionally, villus height was unchanged, and no change was observed for Hes1, which specifies absorptive enterocyte fate (Supplementary Figure 2C and Supplementary Figure 5B).
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(A) MUC2+ goblet cells remain unchanged in Sox4cKO intestines, while (B) CHGA+ EECs are found at decreased numbers in Sox4cKO villi, and (C) DCLK1+ tuft cells are decreased in both crypt and villus compartments of Sox4cKO intestines (scale bar represents 100um). (D) Paneth cell numbers are increased in Sox4cKO crypts (scale bar represents 50um). (E) Assessment of EEC differentiation by immunofluorescence against markers associated with specific EEC subtypes reveals that Sox4 is required for proper specification of a subset of EEC lineages. Conversely, numbers of cells positive for serotonin (5-HT) and Substance P (SUB-P) are unaffected in Sox4cKO intestines. (asterisks indicate significance; p < 0.05). (F) The top 20 most significantly downregulated, GO-identified terms for biological processes in RNA-seq of Sox4cKO vs. control CD44+ populations are consistent with EEC function.
In order to examine gene regulatory changes in Sox4cKO samples in an unbiased manner, we subjected FACS isolated CD44- and CD44+ populations to RNA-seq. Consistent with histological observations, decreased expression of genes associated with EEC subpopulations, including pro-EEC transcription factors Isl1, Nkx2.2, and Pax6, was the predominant change observed in Sox4cKO CD44+ populations (Supplementary Figure 1D and Supplementary Table 1) 32, 33. There were no significant gene expression changes in the CD44- populations consistent with the restricted expression pattern of SOX4 in CD44+ intestinal crypts and CD44+ status of EECs (Supplementary Figure 1D). Gene set enrichment analysis (GSEA) revealed a decreased gene expression signature consistent with pancreatic beta cells, another well-characterized endocrine population (Supplementary Figure 1E and Supplementary Table 2). MAPK signaling, which was recently shown to inhibit EEC and tuft lineages, was enriched, as was IL-6 JAK/STAT signaling, which is associated with increased epithelial proliferation and may reflect increased Paneth cell numbers through Paneth cell-specific expression of IL-6R (Supplementary Figure 1E) 34–36. Collectively, transcriptomic data support the phenotypic changes in lineage allocation observed in Sox4cKO mice, and indicate that these changes occur in CD44+ cells.
Since the EEC lineage is comprised of a diverse complement of subpopulations, we re-examined Sox4cKO intestines for the presence of hormones produced by specific EEC subtypes. Compared to the modest reduction in CHGA+ cell numbers, we found substantial and significant decreases in EEC subpopulations expressing GIP, CCK, SST, GCG, PYY, and GLP-2 (Figure 3E). Interestingly, numbers of 5-HT and SUBP-expressing populations remained unchanged (Figure 3E). Gene ontology (GO) analysis of significantly downregulated genes in Sox4cKO CD44+ cells was consistent with a loss of EEC populations, and yielded terms for biological processes involving hormone regulation, signaling, and nutrient homeostasis (Figure 3F).
Since Notch repression is required for EEC specification, we asked if Sox4 is activated following downregulation of Notch. To do so, we treated wild type organoids with the gamma secretase inhibitor DAPT. Since Sox4 has been reported to be regulated by Wnt in colon cancer cell lines and inhibited by Bmp in hair follicle progenitors, we also treated organoids with WNT3A and Noggin, a Bmp antagonist 37, 38. While Sox4 levels and cell numbers did not respond to Wnt activation or Bmp inhibition, we found that Notch inhibition drove a 2-fold increase in Sox4 expression that coincided with decreased expression of Ascl2, and ISC marker, and increased expression of Ngn3, an early EEC transcription factor (Supplementary Figure 6). Together, these data demonstrate that Sox4 is required for proper secretory lineage allocation. Furthermore, Sox4 is upregulated following Notch inhibition, consistent with early secretory differentiation responses, and required for proper specification of EEC subpopulations.
Sox4 contributes to tuft cell differentiation and hyperplasia
Since a subset of SOX4high cells were found to co-localize with DCLK1 and DCLK1+ tuft cells were decreased in Sox4cKO intestines, we sought to examine the regulatory role of Sox4 in tuft cell differentiation more closely. IL-13 is known to induce tuft cell differentiation, and is produced by Group 2 innate lymphoid cells (ILC2) that are recruited to the intestinal epithelium in response to tuft cell secreted IL-25. This mechanism produces an extrinsic feed-forward loop that signals through unknown intrinsic response elements in ISCs and progenitors to drive tuft cell hyperplasia following parasitic helminth infection 2, 5. We treated wild type intestinal organoids with IL-13 to determine its effect on Sox4. As previously reported, IL-13 induced expression of tuft cell-specific Dclk1 (Supplementary Figure 7A). Compellingly, IL-13 also induced a dose-dependent upregulation of Sox4, driving an approximately 3-fold increase at the optimal dose (Supplementary Figure 7A). Levels of Sox4 upregulation correlated closely with Dclk1 induction. As expected, goblet cell Muc2 was the only non-tuft lineage specific gene also upregulated in response to IL-13 (Supplementary Figure 7B) 2. To determine if Sox4 is required for proper Dclk1 upregulation in response to IL-13, we next treated Sox4cKO and control organoids with IL-13. While control organoids exhibited robust induction of Dclk1 and the tuft cell transcription factor Pou2f3, Sox4cKO organoids failed to significantly upregulate these genes (Figure 4A).
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(A) Tuft cell markers Dclk1 and Pou2f3 are upregulated in control, but not Sox4cKO organoids, in response to treatment with recombinant IL-13 (different letters indicate statistically significant differences, p < 0.05). (B) 7 days post-infection with N. brasiliensis, control intestinal crypts exhibit a significant expansion of SOX4high cells (scale bar represents 20um). (C) Control mice demonstrate expected tuft cell hyperplasia at 7dpi, where Sox4cKO intestines have significantly fewer tuft cells as quantified by DCLK1, COX1, and COX2 (ND = no data; COX1+ cells are not found in intestinal crypts. Scale bar represents 100um). (D) Sox4cKO mice also fail to fully clear adult worms by 7dpi (asterisks indicate significance, p < 0.05, n=3 mice per group).
Next, we asked if Sox4-driven tuft cell responses were physiologically necessary for clearance of helminth infection. Sox4cKO and control animals were infected with N. brasiliensis, a helminth known to induce tuft cell-mediated type II immune reactions 2, 4, 5. Intestines were examined 7 days post-infection (d.p.i.), at the peak of tuft cell hyperplasia, when most worms are typically cleared from wild type mice 5. We first examined SOX4+ cells in control mice. Helminth infection drove a 4-fold increase in the number of SOX4+ cells per crypt (Figure 4B). As expected, DCLK1+ cell numbers were largely upregulated in control animals (Figure 4C). In contrast, Sox4cKO intestines exhibited an attenuated increase in tuft cell numbers (Figure 4C). To determine if impaired tuft cell hyperplasia in Sox4cKO animals affected worm burden at 7d.p.i., we quantified the number of adult worms present in the intestines of infected animals. In order to match worm clearance data with tuft cell hyperplasia in the same mouse, duodenal and ileal tissues were harvested to establish worm counts and jejunal tissues were used for histological studies, discussed above. Control animals exhibited a near-total clearance of adult worms, while Sox4cKO animals bore a significantly higher worm burden at 7d.p.i. (Figure 4D). These data implicate Sox4 as a regulator of tuft cell differentiation at physiological baseline, with an important role in tuft cell hyperplasia during helminth infection.
Sox4− and Atoh1-expressing cells form overlapping and distinct populations
Early tuft cell specification remains a paradox in intestinal epithelial differentiation. Atoh1 is accepted as the master regulator of early secretory fate in the intestine, and is de-repressed following loss of Notch signaling 39. While tuft cells fail to differentiate properly in the presence of constitutively active Notch, multiple lines of evidence demonstrate that a majority of tuft cells are not derived from Atoh1-expressing progenitors 3, 6. These data suggest the existence of Notch-repressed, Atoh1-independent tuft cell regulatory pathways that remain uncharacterized. Since our data demonstrate that Sox4 is upregulated following Notch inhibition and is involved in tuft cell differentiation, we hypothesized that it might be expressed in an Atoh1-independent manner. We examined SOX4 expression in Atoh1GFP intestines, and found that 55.3 ± 6.1% of SOX4high cells were Atoh1-negative, with the remainder co-localizing with Atoh1-GFP (Figure 5A).
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(A) Co-detection of SOX4 and Atoh1, using a transgenic Atoh1GFP allele, reveals that 55.3 ± 6.1% of SOX4+ cells co-express Atoh1 (yellow arrowhead indicates SOX4+/Atoh1+ cell; white arrowhead indicates SOX4+/Atoh1− cell. Scale bar represents 25um). (B) scRNA-seq 40 identifies unique cell populations in the intestinal epithelium (C) Classifying cells based on Sox4 and Atoh1 expression status (Sox4+:Atoh1-, Sox4-:Atoh1+, and Sox4+:Atoh1+) reveals that Sox4+ populations contain the highest proportion of tuft cells, EECs, and ISCs, consistent with observed phenotypes in vivo and in vitro.
To examine Sox4 and Atoh1 populations more thoroughly, we reanalyzed previously published single cell RNA-seq (scRNA-seq) data from primary mouse intestinal epithelial cells 40. Cells were visualized by tSNE dimensionality reduction and classified into previously characterized phenotypic populations (Figure 5B) 40. Next, we queried the distribution of cell types within each of three populations of interest: (1) Sox4+:Atoh1− (n = 617 cells), (2) Sox4+:Atoh1+ (n = 88 cells), and (3) Sox4-:Atoh1+ (n = 162 cells). Within the Sox4+ population, 12.5% of cells were also Atoh1+. Next, we determined the cellular composition of each of our populations of interest. The Sox4+:Atoh1− population consisted primarily of ISCs and progenitor cells (88%), but also contained the highest proportion of tuft cells relative to other populations (Figure 5C). The Sox4+:Atoh1+ population was a mix of ISCs and secretory cell types, and contained the highest proportion of EECs relative to other populations (Figure 5C). As expected, the Sox4-:Atoh1+ population was comprised mostly of secretory cells, with the lowest proportion of ISCs and progenitor cells and the highest proportion of goblet and Paneth cells relative to other populations (Figure 5C). To ask the inverse question, we next binned scRNA-seq data by cell type and queried the percent distribution of Sox4 and Atoh1 within defined intestinal epithelial cell subpopulations. This analysis indicates that 46.1% of tuft cells are Sox4+:Atoh1− and 48.0% are negative for both Sox4 and Atoh1, while Sox4-:Atoh1+ and Sox4+:Atoh1+ populations represent 2.9% of total tuft cell numbers, each (Supplementary Figure 8). These data demonstrate that Sox4 and Atoh1 form distinct and overlapping populations, and that Sox4 is most strongly correlated with ISCs, progenitors, and tuft cells when expressed exclusively of Atoh1 and more strongly correlated with EECs when co-expressed with Atoh1.
Sox4 drives tuft cell differentiation independently of Atoh1
We next sought to determine if Sox4 is sufficient to drive secretory EEC and tuft cell differentiation. To this end, we generated stable transgenic organoids carrying a cumate-inducible Sox4 overexpression vector (Sox4OE). Gain of function was validated by immunofluorescent detection of SOX4 in empty vector control and induced Sox4OE organoids (Supplementary Figure 9). To validate organoid models for assessment of Sox4-dependent differentiation phenotypes, we included organoids isolated from Sox4cKO mice in our analyses. As expected, Sox4cKO organoids exhibited very low numbers of DCLK1+ tuft cells and CHGA+ EECs (Figure 6A, B). In contrast, overexpression of Sox4 resulted in a significant increase in numbers of both tuft cells and EECs, relative to empty vector controls (Fig 6A, B).
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(A) Tuft cells, as identified by DCLK1, are rare in control organoids, absent in Sox4cKO organoids, and upregulated in Sox4OE organoids. (B) Similar results are observed for CHGA+ EECs, indicating that Sox4 is sufficient to increase tuft and EEC differentiation (scale bars represent 50um; different letters indicate statistically significant differences, p < 0.05, n=25 organoids per group). (C) 4-OHT treatment downregulates Atoh1 in control and Sox4OE organoids, resulting in (D) loss of Muc2 expression, which demonstrates loss of Atoh1-driven transcription. (E) Loss of Atoh1 in organoids also results in downregulation of Dclk1, which is rescued to normal levels in Sox4OE organoids, regardless of Atoh1 expression. (F) Chga is not affected by Atoh1 deletion in organoids, but is upregulated by Sox4OE in the absence of Atoh1 (different letters indicate statistically significant differences, p < 0.05, n=3 different wells of organoids per group). Cell counts of (G) DCLK1+ tuft cells and (H) CHGA+ EECs demonstrate correlation between changes in gene expression and lineage specification in Atoh1iKO and Sox4OE organoids. Notably, CHGA+ cells numbers are increased in Sox4OE organoids both with and without Atoh1 expression (scale bars represent 50um; different letters indicate statistically significant differences, p < 0.05, n=25 organoids per group).
Since scRNA-seq demonstrated differential distribution of Sox4 and Atoh1 across progenitors and secretory cell types, we hypothesized that Sox4 may function independently of Atoh1 in secretory differentiation. To test this, we generated Sox4OE organoids derived from Atoh1fl/fl:vilCreER mice. Atoh1 recombination was induced by treating organoids with 1uM 4-OHT once a day for three days and collecting for analysis 24hr following final treatment with 4-OHT. Analysis of Atoh1 expression confirmed the effectiveness of this strategy in generating Atoh1iKO organoids (Figure 6C). To assess whether residual ATOH1 protein was exerting continued effects in Atoh1iKO organoids, we next analyzed Muc2 expression, which is associated with Atoh1-dependent goblet cell differentiation. We found that Muc2 levels were also significantly downregulated in Atoh1iKO organoids, suggesting no residual ATOH1 function at the time of organoid analysis (Figure 6D).
Next, we examined the effect of Atoh1 and Sox4 on tuft and EEC differentiation. Surprisingly, Dclk1 was significantly downregulated following loss of Atoh1, in direct conflict with reported in vivo data demonstrating an increase in tuft cells in Atoh1-null intestines (Figure 6E) 10. However, treatment of Atoh1iKO organoid cultures with IL13 restored Dclk1 expression to control levels, in contrast to the loss of IL13 responsiveness in Sox4cKO organoids (Supplementary Figure 10). Overexpression of Sox4 was also sufficient to restore Dclk1 expression levels in the absence of Atoh1 (Figure 6E). Unexpectedly, Chga expression levels remained unchanged following loss of Atoh1, and were upregulated by Sox4 in Atoh1iKO organoids (Figure 6F). Notably, Sox4OE failed to rescue or upregulate Muc2 expression levels, suggesting specificity to tuft and EEC lineages (Figure 6D). To refine these observations, we conducted whole mount staining on Atoh1iKO and Sox4OE organoids and directly quantified tuft and EEC numbers in whole organoids. Quantification by immunofluorescence recapitulated gene expression results, demonstrating upregulation of DCLK1+ tuft cells and CHGA+ EECs in Sox4OE organoids regardless of Atoh1 expression (Figure 6G and H). Together, these data demonstrate that Sox4 is sufficient to drive an increase in tuft and EEC differentiation and can do so independently of Atoh1.
Dissecting regulatory roles for Sox4 in intestinal stemness and lineage allocation
To further dissect the roles of Sox4 and Atoh1 in intestinal epithelial cell fate, we analyzed gene expression for known regulators of ISCs and differentiation in our Sox4OE:Atoh1iKO organoids. First, we examined expression of ISC markers Lgr5, which is downregulated in Sox4cKO intestines in vivo, and Ascl2 and Tcf4, which activate a master ISC regulatory program together with beta catenin 41. While loss of Atoh1 results in significantly decreased Lgr5 expression, Sox4 is insufficient to upregulate Lgr5 and cannot rescue Lgr5 expression levels in the absence of Atoh1 (Figure 7A). Atoh1iKO organoids exhibit decreased Ascl2 expression which is not rescued by Sox4OE and Tcf4 is unaffected by loss of Atoh1 or Sox4OE. Collectively, these data indicate that Sox4 alone is insufficient to upregulate ISC core genes.
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(A) ISC regulatory genes Lgr5 and Ascl2 are downregulated in Atoh1iKO and unaffected by Sox4 overexpression, while Tcf4 is unresponsive to Atoh1iKO or Sox4OE. (B) Tuft cell-associated transcription factors are differentially responsive to Atoh1 and Sox4. Pou2f3 requires Atoh1 for normal expression levels and is not regulated by Sox4OE. Stat6 is unresponsive to Atoh1iKO, but is mildly repressed by Sox4OE in an Atoh1-dependent manner. (C) Canonical IEC differentiation-associated transcription factors exhibit different requirements for Atoh1 and Sox4. Hes1, which drives absorptive differentiation, is unaffected by Atoh1 and Sox4 expression. Spdef and Gfi1 are significantly downregulated in Atoh1iKO organoids and unaffected by Sox4OE. Foxa1 and Foxa2 trend toward downregulation, but are not significantly affected by loss of Atoh1, and are also unresponsive to Sox4OE. (D) Early EEC transcription factors Ngn3 and Neurod1 do not significantly change in Atoh1iKO or Sox4OE organoids, while late EEC transcription factors Isl1 and Pax6 are significantly repressed by Sox4OE independently of Atoh1 expression. EEC hormones exhibit differential responses, all independently of Atoh1 expression, with Sox4OE resulting in (E) Gip upregulation and (F) Cck, Gcg, and Nts downregulation, and (G) having no significant effect on Tph1 and Tac1 (different letters indicate statistically significant differences, p < 0.05, n=3 different wells of organoids per group).
Next, we examined Sox4 and Atoh1 regulation of tuft cell-associated transcription factors Gfi1b, Pou2f3, and Stat6 2–4. Gfi1b was not detected in any samples (data not shown). We found that Pou2f3 is significantly downregulated in Atoh1iKO organoids (Figure 7B). Interestingly, while Sox4 is required for Pou2f3 upregulation in response to IL13 treatment (Figure 5A), Sox4OE organoids did not exhibit increased Pou2f3 expression, demonstrating that Sox4 is necessary but not sufficient for Pou2f3 induction (Figure 7B). Stat6, which is activated by tuft cell-associated cytokines IL-4 and IL-13, was weakly inhibited in the combined presence of normal levels of Atoh1 and Sox4OE, but is otherwise unaffected by loss of Atoh1 or Sox4OE (Figure 7B).
As EEC-related genes comprise the majority of transcriptomic changes in Sox4cKO intestines, we sought to examine canonical secretory differentiation pathways in the setting of our Sox4OE:Atoh1iKO organoid model. As expected, goblet and Paneth cell-associated transcription factors Spdef and Gfi1 were found to be highly dependent on Atoh1 expression, exhibiting near total loss of expression in the setting of Atoh1iKO (Figure 7C). Additionally, neither gene was responsive to Sox4OE, regardless of Atoh1 expression. Foxa1 and Foxa2, which are implicated in goblet and EEC differentiation, trended toward reduction following Atoh1iKO and were unresponsive to Sox4OE (Figure 7C) 42. Hes1 was unaffected by either Sox4 or Atoh1 (Figure 7C). To assess EEC-specific transcriptional changes, we examined Ngn3, Neurod1, Isl1, Pax6, and Nkx2–2. All but Nkx2–2 were expressed in Sox4OE:Atoh1iKO organoids, as assessed by RT-qPCR. While Ngn3 trended toward reduction following loss of Atoh1, this effect was not significant (Figure 7D). Neurod1, Isl1, and Pax6 were also unaffected by loss of Atoh1 (Figure 7D). Surprisingly, while Sox4OE had no effect on Ngn3 or Neurod1 expression, it significantly repressed Isl1 and Pax6 regardless of Atoh1 expression and in contrast to observations made in Sox4cKO intestines (Figure 7D). To extend these observations, we next examined genes associated with terminally-differentiated EEC function. While none of the genes examined were significantly affected by loss of Atoh1, they could be categorized by differential responsiveness to Sox4OE. Gip, which was downregulated following loss of Sox4 in vivo, was significantly upregulated by Sox4OE, regardless of Atoh1 expression and consistent with the effect of Sox4 on Chga (Figure 7E). Unexpectedly, Cck, Gcg, and Nts, which were all strongly downregulated in Sox4cKO intestines, were also significantly inhibited in the presence of Sox4OE (Figure 7F). Tph1 and Tac1, which were not affected by loss of Sox4 in vivo, were also unaffected by Sox4OE in vitro (Figure 7G). Together, these data suggest that transcriptional functions of Sox4 in EEC regulation are largely independent of Atoh1 and are exerted differentially at a gene-by-gene level, with portions of EEC genes being activated, repressed, or unaffected by Sox4. Collectively, the repression of late EEC transcription factors Isl1 and Pax6, as well as a subset of EEC hormones, may suggest that Sox4 exerts its effect on the EEC lineage by maintaining a progenitor state and preventing premature differentiation.
Discussion
Here we show that genetic ablation of Sox4 in the intestinal epithelium results in increased numbers of proliferating cells and Paneth cells, as well as a reduction in EEC and tuft cell lineages. Our study supports Sox4 mediated regulation of stem cells in other organ systems by demonstrating that Sox4 regulates ISC function and differentiation toward EECs and tuft cells in the intestinal epithelium. Additionally, we demonstrate that Sox4 has an essential role in pushing lineage allocation toward tuft cell differentiation in response to helminth infection, implying an important role for Sox4 in host responses to pathogens.
Our group and others have reported Sox4 expression in Lgr5+ CBCs, but a role for Sox4 in ISCs has yet to be established 18, 22. We demonstrate that the numbers of ISCs quantified by OLFM4 immunostaining are unchanged between Sox4cKO and control samples. These data suggest that the associated increase in proliferating cells in Sox4cKO crypts is likely due to increased progenitor numbers and not more robust ISC activity. Rather, in vitro assay of ISC function demonstrated impaired organoid-forming efficiency of single ISCs isolated from Sox4cKO mice. As this dysfunction is not associated with a change in the number of apoptotic cells between control and Sox4cKO mice, it is likely that decreased organoid-forming efficiency is caused by loss of stemness and premature differentiation in Sox4 deficient ISCs. In this regard, Sox4cKO mice demonstrate reduced Lgr5 mRNA. Lgr5 is highly expressed in ISCs and serves as a receptor for R-Spondin ligands that potentiate Wnt-signaling, which is in turn required to maintain stemness 43. Interestingly, our gain-of-function experiments in organoids indicate that Sox4 alone is insufficient to upregulate Lgr5 and suggest that Sox4 contributes to proper Lgr5 expression through a co-regulatory manner involving other transcription factors or non-cell autonomous mechanisms.
In addition to loss of ISC function, Sox4cKO intestines also demonstrate decreased EEC and tuft cell numbers, implying that Sox4 plays a key role in the specification of these secretory lineages. In vivo, RNA-seq analysis demonstrates that Sox4 regulates the expression of the EEC transcription factors, including Isl1, Pax6, and Nkx2–2. Isl1 deletion in the intestine is known to lead to loss of EECs subtypes expressing GIP, CCK, SST, GCG, PYY, and GLP-2 33. We demonstrate that loss of Sox4 results in a marked reduction of Isl1 expression with a concomitant reduction in the numbers of the same EEC subtypes. An unexpected result of our studies is the observation that Sox4 overexpression is sufficient to drive an increase in CHGA+ cell numbers in the absence of Atoh1. This effect does not appear to be the result of residual ATOH1 activity or non-specific Sox4 regulation of all secretory differentiation, as goblet cell-associated Muc2 expression remains severely downregulated in Atoh1iKO organoids at the timepoint examined, regardless of Sox4 expression status. These data raise interesting questions regarding the role of Atoh1 as a master regulator of secretory differentiation. To our knowledge, no other studies to date have expressed transcriptional regulators of secretory differentiation that are normally expressed upstream of Atoh1 in the absence of Atoh1. Further studies in this area would be necessary to determine if other transcription factors are capable of rescuing secretory differentiation in Atoh1iKO models.
Paradoxically, Sox4OE in organoid cultures results in the downregulation of many of the same genes that are downregulated in Sox4cKO in vivo, including Isl1, Pax6, Cck, and Gcg. Collectively, our in vivo and in vitro results indicate that Sox4 is required for proper EEC specification through established EEC transcriptional regulators Isl1, Pax6, and Nkx2–2 and suggest that Sox4 must be downregulated in order for EEC differentiation to proceed along these pathways. As CHGA is expressed in both mature EECs and cycling EEC progenitors, the Atoh1-independent increase in CHGA+ cells in Sox4OE organoids may reflect an expansion of progenitors as opposed to mature EECs. Further, Sox4OE does not regulate Ngn3 or Neurod1, suggesting that Sox4 can promote an EEC progenitor state without upregulation of established EEC transcription factors, though the requirement of Ngn3 and Neurod1 in Sox4-mediated CHGA+ cell expansion remains unknown. In this way, Sox4 may “prime” ISC and progenitor pools for differentiation while simultaneously inhibiting premature cell fate decisions. This role would be consistent with the crypt-restricted expression of Sox4, as well as Sox4 knockout phenotypes in other tissues, including the developing heart, skeletal muscle, nervous, renal, and hematopoietic systems, which exhibit reduced progenitor and post-mitotic cell numbers due to precocious differentiation 19, 20, 27, 44–46.
Relative to EEC differentiation, the mechanisms that give rise to the tuft cell lineage are less well understood due to the relatively new characterization of tuft cells as a distinct lineage. The role of intestinal tuft cells was recently described by a handful of independent studies, all of which demonstrated tuft cell hyperplasia following infection with parasitic helminths. Following infection, tuft cells initiate a type II immune response through ILC-2 cells 2, 4, 5. ILC-2s secrete IL-13, which induces tuft cell hyperplasia 2, 4, 5.Pou2f3 and Stat6 are known to be essential for tuft cell differentiation, but Pou2f3 is also expressed by mature, villus-based tuft cells and Stat6 is activated by type II responses and involved in tuft and goblet cell differentiation 2, 4. Other transcription factors involved in homeostatic tuft cell specification and hyperplasia remain unknown, and tuft cell progenitors have no distinct biomarkers 2.
Though SOX4high cells are relatively rare in homeostasis, we observed a striking increase in SOX4high cell numbers coincident with peak tuft cell hyperplasia following parasitic infection, suggesting that SOX4high cells may represent a tuft cell progenitor population. We show that Sox4cKO epithelium demonstrates attenuated tuft cell hyperplasia, both in response to recombinant IL-13 in vitro and helminth infection in vivo, and that Sox4cKO mice are functionally impaired at clearing worms by 7d.p.i. Notably, IL13-mediated activation of Pou2f3 is also impaired in Sox4cKO organoids, suggesting that Sox4 acts upstream of this established tuft cell transcription factor. Since there is no reporter mouse model for Sox4, we leveraged existing scRNA-seq data to further interrogate the identity of Sox4+ cells. In agreement with histological data, we find that Sox4 is enriched in ISC, progenitor, and secretory populations. Classifying Sox4+ cells by Atoh1 expression reveals a strong correlation between Sox4+:Atoh1− cells and tuft cells and between Sox4+:Atoh1+ cells and EECs, supporting dual roles for Sox4 in tuft and EEC differentiation. Functionally, this role is demonstrated by the ability of ectopic Sox4 expression to induce increased tuft and EEC numbers in organoids.
Lineage specification of tuft cells from the ISC/progenitor pool remains a significant question in the field, as tuft cells are capable of being produced independently of Atoh1, but are inhibited by constitutive Notch signaling 3, 6. Further, it was recently shown that inducible knockout of Atoh1 in vivo leads to increased tuft cell numbers, implying that Atoh1 may suppress tuft cell differentiation 10. Surprisingly, our in vitro analyses of Atoh1iKO organoids demonstrate the opposite effect. That is, loss of Atoh1 in vitro leads to decreased tuft cell numbers. However, in contrast to Sox4cKO organoids, which fail to upregulate Dclk1 in response to exogenous IL13, Atoh1iKO organoids retain IL13-responsiveness in terms of Dclk1 expression. These data suggest that tuft cells may be specified by multiple signaling events and pathways, but that IL13-mediated tuft cell hyperplasia is Sox4-dependent and Atoh1-independent. In agreement, our gain-of-function studies reveal that, although Atoh1iKO organoids exhibit reduced tuft cell numbers relative to non-recombined controls, Atoh1 is dispensable for Sox4-driven tuft cell differentiation.
The rarity of SOX4+ cells in homeostasis and diverse array of ISC, EEC, and tuft cell phenotypes in Sox4cKO mice presents a significant challenge for identifying molecular mechanisms for Sox4 in the intestine. A common theme in our results is that Sox4 appears to be required for IEC function and responses, such as proper Lgr5 expression or Pou2f3 upregulation in response to IL-13, without direct transcriptional regulation of these targets. One interpretation is that Sox4 may act in concert with a number of co-regulatory factors to exert broad phenotypic effects, being required but insufficient to drive observed phenotypes, such as ISC function. Similar observations suggesting broad, co-factor dependent regulatory roles for Sox4 have been demonstrated in other tissues, with mechanistic work in pancreatic cancer showing that Sox4 can drive either enhanced proliferation and tumorigenesis or apoptosis, dependent on co-expression of Klf5 47. Identification of direct transcriptional targets of SOX4 as well as potential cofactors is further hindered by a lack of antibodies appropriate for immunoprecipitation of native SOX4 protein. Future studies utilizing unique approaches will be required to dissect the seemingly complex Sox4 regulatory network.
At the cellular level, SOX4low cells overlap largely with ISCs, while SOX4high cells appear to represent a cycling secretory progenitor population with EEC and tuft cell potential (see Graphical Abstract). The existence of Atoh1-independent lineage specification of tuft cells and EECs challenges the long-standing dogma of Atoh1 as the master transcriptional regulator of secretory differentiation in the intestine. Our findings demonstrate that Sox4 is upregulated following downregulation of Notch or in response to IL13, and drives tuft or EEC specification in a manner that can proceed independently of Atoh1. These findings implicate Sox4 as an important regulator of host responses parasitic infection and shed further light on epithelial components of Type II immune responses.
Supplementary Material
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Acknowledgments:
The authors would like to thank Drs. Rashmi Chandra and Roger Liddle (Duke University, Durham, NC) for providing antibodies against CCK and PYY; Dr. Veronique Lefebvre for providing Sox4fl mice; Brian Golitz and Noah Sciaky for assistance with Illumina sequencing and analysis; Dr. Pablo Ariel and the UNC Microscopy Services Laboratory for assistance with confocal microscopy; Alexei Kouminov for technical support. RNA-seq data associated with this manuscript have been deposited in NCBI’s Gene Expression Omnibus and are available under GEO accession number GSE90795. This work was funded by the National Institutes of Health, R01 DK091427 (Magness), the Center for Gastrointestinal Biology and Disease P30 DK034987 (Magness), National Institutes of Health, R01 AI119004 (Reinhardt), and National Institutes of Health, R01 CA1428260, R01 DK092306 (Shroyer). A.D.G. was partially supported by the UNC GI Division Basic Science Training Grant, T32 DK007737–20 (Sartor), National Institutes of Health, K01 DK111709 (Gracz), and a Research Scholar Award from the American Gastroenterological Association (Gracz). Y-H.L. was partially supported by National Institutes of Healthy, F99 CA212433.
Footnotes
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Funding
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American Gastroenterological Association (8)
Grant ID: R01 CA1428260
Grant ID: R01 DK091427
Grant ID: K01 DK111709
Grant ID: F99 CA212433
Grant ID: R01 AI119004
Grant ID: T32 DK007737-20
Grant ID: R01 DK092306
Grant ID: P30 DK034987
Center for Gastrointestinal Biology and Disease, School of Medicine, University of North Carolina at Chapel Hill (8)
Grant ID: R01 DK092306
Grant ID: P30 DK034987
Grant ID: R01 AI119004
Grant ID: F99 CA212433
Grant ID: K01 DK111709
Grant ID: R01 CA1428260
Grant ID: T32 DK007737-20
Grant ID: R01 DK091427
NCI NIH HHS (2)
Grant ID: F99 CA212433
Grant ID: R01 CA142826
NIAID NIH HHS (2)
Grant ID: R01 AI119004
Grant ID: T32 AI052077
NIDDK NIH HHS (6)
Grant ID: T32 DK007737
Grant ID: K01 DK111709
Grant ID: R01 DK091427
Grant ID: P30 DK034987
Grant ID: R01 DK092306
Grant ID: P30 DK056338
National Institutes of Health (8)
Grant ID: T32 DK007737-20
Grant ID: F99 CA212433
Grant ID: R01 DK091427
Grant ID: R01 DK092306
Grant ID: R01 CA1428260
Grant ID: P30 DK034987
Grant ID: R01 AI119004
Grant ID: K01 DK111709
University of North Carolina (8)
Grant ID: R01 DK091427
Grant ID: R01 DK092306
Grant ID: F99 CA212433
Grant ID: R01 AI119004
Grant ID: T32 DK007737-20
Grant ID: K01 DK111709
Grant ID: P30 DK034987
Grant ID: R01 CA1428260