Characterization of the developing small intestine in the absence of either GATA4 or GATA6
© Walker et al.; licensee BioMed Central. 2014
Received: 18 August 2014
Accepted: 28 November 2014
Published: 11 December 2014
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.
The zinc-finger DNA binding transcription factors GATA4 and GATA6 are expressed in the small intestinal epithelium throughout development and adulthood [1–7]. GATA4, unlike GATA6, is present in a restricted pattern in the adult small intestine; it is expressed in the proximal small intestine (duodenum and jejunum) but absent from the distal small intestine (ileum) [1, 4, 8]. Global Gata4 or Gata6 knockout causes early embryonic lethality necessitating conditional knockout (cKO) strategies to analyze their roles in organogenesis [5, 9–11]. Several studies performed by our laboratory and others to eliminate GATA4 or GATA6 specifically in the intestinal epithelium have uncovered roles for these factors in small intestinal biology [1, 2, 4]. Fat and cholesterol absorption are disrupted in adult mice lacking GATA4 in the jejunal epithelium . Moreover, expression of many jejunal-specific transcripts is lost and expression of many ileal-specific transcripts is induced in GATA4-deficient jejunum demonstrating a role for GATA4 in regulating jejunal versus ileal intestinal identity [1, 4]. Although constitutive Villin-Cre has been used to delete Gata4 in the small intestine during development , GATA4-deficient embryonic intestine was not examined.
Unlike Gata4 cKO adult mice, elimination of Gata6 from the adult jejunal epithelium using tamoxifen-inducible Villin-Cre does not decrease expression of jejunal enterocyte markers or induce expression of ileal enterocyte markers suggesting that jejunal identity is maintained in its absence . Increased Paneth cells with atypical granules are reported in GATA6-deficient jejunum . In contrast, loss of Gata6 from the adult ileum, a tissue lacking GATA4, results in shortened villi, reduced proliferative, enteroendocrine, and Paneth cells, and increased crypt goblet cells . Changes in ileal enterocyte gene expression also occur; small intestinal enterocyte marker expression is decreased, and colonocyte marker expression is induced suggesting that GATA6 plays a role in regulating intestinal identity in the distal small intestine . These studies did not induce Gata6 deletion during embryonic development precluding analysis of embryonic intestine.
We recently demonstrated that simultaneous deletion of both Gata4 and Gata6 within the developing intestinal epithelium using constitutive Villin-Cre severely disrupts jejunal development causing Gata4-Gata6 double cKO mice to die within a day of birth . Intestinal epithelial architecture is altered in the absence of both GATA4 and GATA6 with the jejunum of Gata4-Gata6 double cKO embryos containing short, blunted villi. Furthermore, differentiated epithelial cell populations are skewed in Gata4-Gata6 double cKOs. Enterocytes are decreased and goblet and proliferative cells are increased in mutant jejunum. The effect of deletion of Gata4 or Gata6 alone during embryonic development of the small intestine, however, has not been examined. Therefore, the goal of this study is to provide phenotypic analysis of intestinal development in single Gata4 and Gata6 cKO embryos derived using constitutive Villin-Cre. We examined the jejunum of Gata4 Villin-Cre and Gata6 Villin-Cre cKO embryos and the ileum of Gata6 Villin-Cre cKO embryos at E18.5. We found that jejunum lacking either GATA4 or GATA6 was largely normal. Changes in enterocyte gene expression reflecting a transition from jejunal to ileal identity were identified only in GATA4 mutant jejunum. Analysis of GATA6 mutant ileum revealed a phenotype similar to that observed when GATA6 is eliminated from the adult ileal epithelium. We observed shortened villi and subtle changes in epithelial cell populations including altered enterocyte gene expression, decreased enteroendocrine cells, and increased goblet and proliferative cells.
Results and discussion
Because the enterocyte gene expression profile of GATA4 mutant adult jejunum shifts from that of the jejunum toward that of the ileum , we performed qRT-PCR using epithelial cells from control and GATA4-deficient jejunum to compare expression of sets of jejunal-enriched transcripts (Apoa4, Apoc2, Apoc3, Fabp1, Lct, Slc2a2, Slc2a5, and Slc5a11), ileal-enriched transcripts (Slc10a2, Fabp6, Fgf15, and Cldn8), and transcripts expressed at similar levels in jejunum and ileum (Fabp2, Abcg5, and Abcg8). The majority of jejunal-enriched transcripts examined (5/8) were decreased in GATA4 mutant epithelium compared with control (Figure 4D). Moreover, expression of all ileal-enriched transcripts examined (4/4) was increased in GATA4 mutant epithelium compared with control (Figure 4E). Notably, ectopic expression of bile acid metabolism markers (Slc10a2, Fabp6, and Fgf15) was induced. None of the transcripts expressed ubiquitously in jejunum and ileum were changed in GATA4 mutant epithelium (Figure 4D). Taken together, these data suggest that gene expression changes in GATA4 mutant jejunal epithelium reflect a shift in jejunal enterocyte identity toward that of the ileum rather than a change in enterocyte cell number.
In contrast to GATA4-deficient jejunum, loss of GATA6 in adult mouse jejunum fails to alter enterocyte gene expression patterns . We performed qRT-PCR for the same set of transcripts examined in GATA4 mutants using jejunal epithelial cells from control and GATA6-deficient jejunum. In general, gene expression profiles were conserved in jejunal epithelium lacking GATA6. However, we did find that expression of two jejunal-enriched transcripts, Lct and Slc5a11, and one ileal-enriched transcript, Cldn8, was altered in GATA6 mutant epithelium (Figures 4D, E). These data suggest that loss of GATA6 during intestinal development subtly alters the jejunal enterocyte gene expression profile. Unlike GATA4-deficient jejunum, bile acid metabolism markers were not induced in jejunum lacking GATA6 suggesting that GATA4 uniquely regulates these genes in the developing jejunum.
To assess the enterocyte population in GATA6 mutant ileum, we stained for brush border AP activity. We detected robust AP activity in control ileum whereas AP activity was very low or undetectable in GATA6-deficient ileum (Figure 6C). This difference may reflect a decrease in enterocytes in mutant ileum or a shift in the identity of ileal enterocytes toward that of colonocytes as AP activity is normally lower in the colon compared with the small intestine . A similar shift toward colon gene expression was observed when GATA6 is eliminated from the adult ileum . If AP activity changes reflect decreased enterocyte number, we expected to see decreased enterocyte marker expression and no induction of colon markers. If AP activity reflects an identity change, we expected to see reduced enterocyte marker expression and induced colon marker expression. We observed the latter. Expression of the small intestine markers Fabp2 and Apoa4 was lost and expression of the colonic marker Car1 was induced in GATA6 mutants (Figure 6D). Moreover, expression of the intestinal alkaline phosphatase gene (Alpi) was lower in mutants compared with controls (Additional file 3). Taken together, these data suggest that enterocyte cell identity is shifted toward the colon in GATA6-deficient ileum.
Finally, we examined the intervillus region in control and GATA6 mutant ileum by quantifying SOX9+ cell number (Figure 7C). We found a 1.3-fold increase in SOX9+ cells in GATA6-deficient ileum compared with control ileum suggesting that loss of GATA6 during ileal development causes an expansion of the intervillus region. As the intervillus region contains the proliferative progenitor population, we determined the number of proliferative cells in control and GATA6 mutant ileum by quantifying KI67+ cells (Figure 7D). As predicted by SOX9 staining, we found a slight increase (1.2-fold) in KI67+ cells in GATA6-deficient ileum compared with control ileum. It is somewhat unexpected to find an increase in proliferative cells, yet fewer epithelial cells, in GATA6 mutant ileum. We examined cell death with cleaved caspase 3 IHC and observed only a few positive cells per section in both control and mutant ileum suggesting that increased cell death is not the reason for this disparity (Additional file 4). One alternative explanation is that increased proliferation occurs as a secondary consequence to compensate for epithelial cell loss and decreased villus length. We observed a similar effect when E-cadherin is deleted from the developing small intestine . On the other hand, if this result reflects a direct effect of GATA6 loss, it would suggest that proliferation in the developing intestine is sensitive to total GATA level as we found no change in proliferation in either GATA4 or GATA6 mutant jejunum. When both GATA4 and GATA6 are deleted in the developing jejunum, however, proliferation is increased . Finally, elimination of GATA6 from the adult ileum results in fewer proliferative cells in mature crypts . This suggests that the requirement for GATA6 with respect to proliferation varies over time in the ileum.
This study investigated the impact of loss of either GATA4 or GATA6 within the intestinal epithelium during development. We found that intestinal development was generally normal in GATA4 and GATA6 mutants. Phenotypes observed in embryonic GATA4 or GATA6 mutant small intestine paralleled those identified in adult small intestine lacking either GATA4 or GATA6. The effect of loss of GATA6 in the developing ileum was greater than the effect of loss of either GATA4 or GATA6 in the developing jejunum. As the jejunum co-expresses GATA4 and GATA6 whereas the ileum expresses only GATA6, we conclude that overlapping GATA4 and GATA6 function in the jejunum lessens the effect of loss of either factor alone in this tissue. Although our data demonstrate redundancy between GATA4 and GATA6 function within the developing jejunum, examination of single knockouts suggests that GATA4 likely functions independently of GATA6 in the jejunum to regulate jejunal versus ileal enterocyte identity. Moreover, GATA6 likely plays a direct role in regulating enteroendocrine cell differentiation whereas GATA4 affects enteroendocrine cell development indirectly.
CD-1 mice (Charles River Laboratories, Wilmington, MA) were used to determine expression of GATA4 in embryonic small intestine. The Rosa26 conditional reporter strain Gt(ROSA)26Sor tm1Sor (Rosa26R) was used to determine Cre activity . Gata4 tm1.1Sad (Gata4 loxP ), Gata4 tmo1Eno (Gata4 − ), Gata6 tm2.1Sad (Gata6 loxP ), Gata6 tm2.2Sad (Gata6 − ), and Tg(Vil-cre)997Gum (Villin-Cre) mice were used to generate Gata4 cKO (Gata4 loxP/- Villin-Cre) and Gata6 cKO (Gata6 loxP/- Villin-Cre) embryos [10, 13–15]. Control genotypes were either Gata4 loxP/+ or Gata6 loxP/+ . Embryos were obtained by timed mating with noon of the day of a vaginal plug considered as embryonic day (E) 0.5. Genotypes were determined by PCR using primers previously described [1, 12]. The Medical College of Wisconsin’s Animal Care Committee approved all animal procedures.
Intestinal epithelial cell isolation
Small intestine (jejunum or ileum) was isolated from E18.5 embryos, cut longitudinally, and incubated in cell dissociation buffer (BD Biosciences, San Jose, CA) for at least 6 hours at 4°C with shaking [20, 21]. Mesenchymal tissue was removed, and epithelial cells were used to obtain total RNA for qRT-PCR experiments. Jejunal samples excluded epithelial cells from the distal 1–2 cm of the embryonic small intestine. Ileal samples contained epithelial cells from the distal 0.5-1.0 cm of the embryonic small intestine.
Reverse transcription polymerase chain reaction
DNase treated RNA was used to generate cDNA from isolated epithelial cells as previously described [19, 22]. Additional file 5 contains a table listing the TaqMan assays identifiers used for qRT-PCR (Life Technologies, Carlsbad, CA). Gapdh was used as a normalization control. Expression units for each target represent (2-ΔCq)*1000. Multiplying by a factor of 1000 adjusted most expression unit values to >1. For all assays, gene expression was measured at least three times using cDNA generated from 3 control (Gata4 loxP/+ , jejunal assays; Gata6 loxP/+ , ileal assays) and 3–4 experimental (Gata4 loxP/- Villin-Cre, jejunal assays; Gata6 loxP/- Villin-Cre jejunal and ileal assays) E18.5 embryos. P-values were determined by a two-sample Student’s t test. Error bars represent standard error of the mean (SEM).
Histochemistry and immunohistochemistry
Histochemistry and immunohistochemistry were performed using previously described methods [19, 22]. Jejunal tissue was harvested from the midpoint of the small intestine. The distal 0.5- 1.0 cm of the embryonic small intestine was considered ileum for these assays. To assay β-galactosidase activity in tissue, embryonic intestine was dissected and fixed in 4% paraformaldehyde for 15 minutes at room temperature. Intestines were washed and incubated overnight in X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) stain as previously described . Antibodies used for IHC are listed in Additional file 6.
We thank Benjamin Bondow for technical assistance, Dr. Vivian Lee (Medical College of Wisconsin, Milwaukee, WI) for providing SOX9 antibody, Dr. Xiang-Xi (Mike) Xu (Miller School of Medicine, University of Miami, Miami, FL) for providing GATA6 antibody, and Dr. Jeffrey Molkentin (Cincinnati Children’s Hospital Medical Center) for providing Gata4 null mice. We also thank Drs. Stephen Duncan and Allison Ebert (Medical College of Wisconsin, Milwaukee, WI) for helpful discussions and input into the manuscript.
Funding for this project was provided by grants from the US National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (DK087873), the American Gastroenterological Association Foundation Research Scholar Award, and Advancing a Healthier Wisconsin to MAB.
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