GATA4 and GATA6 regulate intestinal epithelial cytodifferentiation during development.
ABSTRACT: The intestinal epithelium performs vital roles in organ function by absorbing nutrients and providing a protective barrier. The zinc-finger containing transcription factors GATA4 and GATA6 regulate enterocyte gene expression and control regional epithelial cell identity in the adult intestinal epithelium. Although GATA4 and GATA6 are expressed in the developing intestine, loss of either factor alone during the period of epithelial morphogenesis and cytodifferentiation fails to disrupt these processes. Therefore, we tested the hypothesis that GATA4 and GATA6 function redundantly to control these aspects of intestinal development. We used Villin-Cre, which deletes specifically in the intestinal epithelium during the period of villus development and epithelial cytodifferentiation, to generate Gata4Gata6 double conditional knockout embryos. Mice lacking GATA4 and GATA6 in the intestinal epithelium died within 24h of birth. At E18.5, intestinal villus architecture and epithelial cell populations were altered. Enterocytes were lost, and goblet cells were increased. Proliferation was also increased in GATA4-GATA6 deficient intestinal epithelium. Although villus morphology appeared normal at E16.5, the first time at which both Gata4 and Gata6 were efficiently reduced, changes in expression of markers of enterocytes, goblet cells, and proliferative cells were detected. Moreover, goblet cell number was increased at E16.5. Expression of the Notch ligand Dll1 and the Notch target Olfm4 were reduced in mutant tissue indicating decreased Notch signaling. Finally, we found that GATA4 occupies chromatin near the Dll1 transcription start site suggesting direct regulation of Dll1 by GATA4. We demonstrate that GATA4 and GATA6 play an essential role in maintaining proper intestinal epithelial structure and in regulating intestinal epithelial cytodifferentiation. Our data highlight a novel role for GATA factors in fine tuning Notch signaling during intestinal epithelial development to repress goblet cell differentiation.
Project description:Studies of adult mice lacking either GATA4 or GATA6 in the small intestine demonstrate roles for these factors in small intestinal biology. Deletion of Gata4 in the adult mouse intestine revealed an essential role for GATA4 in jejunal function. Deletion of Gata6 in the adult mouse ileum alters epithelial cell types and ileal enterocyte gene expression. The effect of deletion of Gata4 or Gata6 alone during embryonic small intestinal development, however, has not been examined. We recently demonstrated that loss of both factors in double conditional knockout embryos causes severe defects in jejunal development. Therefore, the goal of this study is to provide phenotypic analysis of the small intestine of single Gata4 and Gata6 conditional knockout embryos.Villin-Cre was used to delete Gata4 or Gata6 in the developing intestinal epithelium. Elimination of either GATA4 or GATA6 in the jejunum, where these factors are co-expressed, caused changes in enterocyte and enteroendocrine cell gene expression. Ectopic expression of markers of the ileal-specific bile acid metabolism pathway was induced in GATA4-deficient jejunum but not in GATA6-deficient jejunum. A subtle increase in goblet cells was also identified in jejunum of both mutants. In GATA6-deficient embryonic ileum, villus length was altered, and enterocyte gene expression was perturbed including ectopic expression of the colon marker Car1. Goblet cells were increased, and enteroendocrine cells were decreased.Overall, we show that aspects of the phenotypes observed in the small intestine of adult Gata4 and Gata6 conditional knockout mice emerge during development. The effect of eliminating GATA6 from the developing ileum was greater than that of eliminating either GATA4 or GATA6 from the developing jejunum likely reflecting functional redundancy between these factors in the jejunum. Although GATA4 and GATA6 functions overlap, our data also suggest unique functions for GATA4 and GATA6 within the developing intestine. GATA4 likely operates independently of GATA6 within the jejunum to regulate jejunal versus ileal enterocyte identity and consequently jejunal physiology. GATA6 likely regulates enteroendocrine cell differentiation cell autonomously whereas GATA4 affects this population indirectly.
Project description:Reports have suggested that the two Notch ligands, Dll1 and Dll4, are indispensable to maintain the homeostasis of the intestinal epithelium. However, within the intestinal epithelium, the precise distribution of the cells that express those ligands at the protein level remains largely unknown. Here, we show a series of immunohistochemical analysis through which we successfully identified mice intestinal epithelial cells (IECs) that endogenously express Dll1 or Dll4. Results showed that Dll1-positive (Dll1+ve) IECs reside exclusively within the crypt, whereas Dll4-positive (Dll4+ve) IECs can locate both in the crypt and in the villus of the small intestine. Also in the colon, Dll1+ve IECs resided at the lower part of the crypt, whereas Dll4+ve IECs resided at both upper and lower part of the crypt, including the surface epithelium. Both Dll1+ve and Dll4+ve IECs were ATOH1-positive, but Hes1-negative cells, and located adjacent to Hes1-positive cells within the crypts. A sub-population of both Dll1+ve and Dll4+ve IECs appeared to co-express Muc2, but rarely co-expressed other secretory lineage markers. However, as compared to Dll1+ve IECs, Dll4+ve IECs included larger number of Muc2-postive IECs, suggesting that Dll4 is more preferentially expressed by goblet cells. Also, we identified that Dll4 is expressed in the Paneth cells of the small intestine, whereas Dll1 and Dll4 is expressed in the c-kit-positive IECs of the colon, indicating that Dll1+ve and Dll4+ve IECs may contribute to constitute the intestinal stem cell niche. Compared to the normal colon, analysis of DSS-colitis showed that number of Dll1+ve IECs significantly decrease in the elongated crypts of the inflamed colonic mucosa. In sharp contrast, number of Dll4+ve IECs showed a significant increase in those crypts, which was accompanied by the increase in number of Hes1-positive IECs. Those Dll4+ve IECs were mostly found adjacent to the Hes1-positive IECs, suggesting that Dll4 may act as a major Notch ligand in the crypts of the inflamed colonic mucosa. Our results illustrate distinct expression patterns of Dll1 and Dll4 within the intestinal epithelium, and suggest that these two ligands may have different roles in normal and inflamed mucosa.
Project description:Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors due to their conversion into postmitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SCs), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiologic ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4).Transgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ER(T2)).Notch1 signaling was found to be activated in intestinal SCs. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into postmitotic goblet cells, concomitant with loss of SCs (Olfm4(+), Lgr5(+), and Ascl2(+)). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut.Notch signaling in SCs and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SCs. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice.
Project description:Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE.
Project description:GATA transcription factors regulate proliferation, differentiation, and gene expression in multiple organs. GATA4 is expressed in the proximal 85% of the small intestine and regulates the jejunal-ileal gradient in absorptive enterocyte gene expression. GATA6 is co-expressed with GATA4 but also is expressed in the ileum; its function in the mature small intestine is unknown.We investigated the function of GATA6 in small intestine using adult mice with conditional, inducible deletion of Gata6, or Gata6 and Gata4, specifically in the intestine.In ileum, deletion of Gata6 caused a decrease in crypt cell proliferation and numbers of enteroendocrine and Paneth cells, an increase in numbers of goblet-like cells in crypts, and altered expression of genes specific to absorptive enterocytes. In contrast to ileum, deletion of Gata6 caused an increase in numbers of Paneth cells in jejunum and ileum. Deletion of Gata6 and Gata4 resulted in a jejunal and duodenal phenotype that was nearly identical to that in the ileum after deletion of Gata6 alone, revealing common functions for GATA6 and GATA4.GATA transcription factors are required for crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene expression in the small intestinal epithelium.
Project description:The embryonic small intestinal epithelium is highly proliferative, and although much is known about mechanisms regulating proliferation in the adult intestine, the mechanisms controlling epithelial cell proliferation in the developing intestine are less clear. GATA4, a transcription factor that regulates proliferation in other developing tissues, is first expressed early in the developing gut in midgut endoderm. GATA4 function within midgut endoderm and the early intestinal epithelium has not been investigated.Using Sonic Hedgehog Cre to eliminate GATA4 in the midgut endoderm of mouse embryos, we determined the impact of loss of GATA4 on intestinal development, including epithelial cell proliferation, between E9.5-E18.5.We found that intestinal length and width were decreased in GATA4 mutants compared with controls. GATA4-deficient intestinal epithelium contained fewer cells, and epithelial girth was decreased. We further observed a decreased proportion of proliferating cells at E10.5 and E11.5 in GATA4 mutants. We demonstrated that GATA4 binds to chromatin containing GATA4 consensus binding sites within Cyclin D2 (Ccnd2), Cyclin dependent kinase 6 (Cdk6), and Frizzled 5 (Fzd5). Moreover, Ccnd2, Cdk6, and Fzd5 transcripts were reduced at E11.5 in GATA4 mutant tissue. Villus morphogenesis was delayed, and villus structure was abnormal in GATA4 mutant intestine.Our data identify GATA4 as an essential regulator of early intestinal epithelial cell proliferation. We propose that GATA4 controls proliferation in part by directly regulating transcription of cell cycle mediators. Our data further suggest that GATA4 affects proliferation through transcriptional regulation of Fzd5, perhaps by influencing the response of the epithelium to WNT signaling.
Project description:Notch signalling maintains stem cell regeneration at the mouse intestinal crypt base and balances the absorptive and secretory lineages in the upper crypt and villus. Here we report the role of Fringe family of glycosyltransferases in modulating Notch activity in the two compartments. At the crypt base, RFNG is enriched in the Paneth cells and increases cell surface expression of DLL1 and DLL4. This promotes Notch activity in the neighbouring Lgr5+ stem cells assisting their self-renewal. Expressed by various secretory cells in the upper crypt and villus, LFNG promotes DLL surface expression and suppresses the secretory lineage . Hence, in the intestinal epithelium, Fringes are present in the ligand-presenting 'sender' secretory cells and promote Notch activity in the neighbouring 'receiver' cells. Fringes thereby provide for targeted modulation of Notch activity and thus the cell fate in the stem cell zone, or the upper crypt and villus.
Project description:The foodborne pathogen Listeria monocytogenes (Lm) crosses the intestinal villus epithelium via goblet cells (GCs) upon the interaction of Lm surface protein InlA with its receptor E-cadherin. Here, we show that Lm infection accelerates intestinal villus epithelium renewal while decreasing the number of GCs expressing luminally accessible E-cadherin, thereby locking Lm portal of entry. This novel innate immune response to an enteropathogen is triggered by the infection of Peyer's patch CX3CR1+ cells and the ensuing production of IL-23. It requires STAT3 phosphorylation in epithelial cells in response to IL-22 and IL-11 expressed by lamina propria gp38+ stromal cells. Lm-induced IFN-γ signaling and STAT1 phosphorylation in epithelial cells is also critical for Lm-associated intestinal epithelium response. GC depletion also leads to a decrease in colon mucus barrier thickness, thereby increasing host susceptibility to colitis. This study unveils a novel innate immune response to an enteropathogen, which implicates gp38+ stromal cells and locks intestinal villus invasion, but favors colitis.
Project description:Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine.Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4(-/-)) mice were generated by breeding TLR4(loxp/loxp) mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days.TLR4(-/-) mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells.TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.
Project description:BACKGROUND & AIMS:Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells. METHODS:We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. RESULTS:Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a ?-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased. CONCLUSIONS:cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.