The intestinal epithelium renews every three to four days in mice through the differentiation of transit amplifying cells emerging from the crypts. Multiple transcription factors are involved in this process, including Math1, Ngn3, Isl-1, Pax4 and Pax6, NeuroD, Nkx2.2 and Insm-1. Here, we propose that Foxa1 and Foxa2 are additional transcription factors important for proper differentiation of goblet and enteroendocrine cells. Goblet cells function to produce mucus to protect the gut, and alteration in mucin expression has been implicated in intestinal inflammation and carcinogenesis [39
]. We observed decreased numbers of goblet cells throughout the intestinal tract of Foxa1/a2
mutants also had significantly reduced Muc2 expression in the colon and rectum at age of 8 weeks, while the entire mucin pattern was perturbed (). Taken together, Foxa1 and Foxa2 are important factors for goblet cell differentiation, and possibly the maintenance of intestinal health.
The endocrine cells of the gastrointestinal tract form the largest endocrine system of the body. Hormones produced by enteroendocrine cells control various functions such as glucose metabolism, exocrine pancreatic secretion, the growth and repair of the intestinal epithelium, and the motility of the intestinal wall [35
]. Several transcription factors such as Isl-1, Pax6, Pax4, Beta2, Nkx2.2 and Insm-1 are not only important for the expression of gut hormones but also involved in the differentiation of these enteroendocrine cells. The present study shows that Foxa1 and Foxa2 control the differentiation of enteroendocrine L- and D-cells via regulating expression of several important transcription factors including Isl-1 and Pax6 (). The current model of the differentiation of the secretory cell lineages in the mammalian intestine is summarized in .
The principal hormones expressed in L-cells, Glp-1 and Glp-2, were absent from the intestine of Foxa1/a2
mutants (). Transcription of the preproglucagon gene is controlled by numerous transcription factors, including Isl-1, Pax6, and Foxa1 or Foxa2 [17
]. Preproglucagon promoter activity is regulated through G1, G2, G3, and G4 elements. Both Foxa1 and Foxa2 can interact with similar affinities to either the G1 or G2 element of preproglucagon promoter in vitro
]. Recently, an additional conserved Foxa binding site located close to the transcription start site of the preproglucagon promoter has been identified and shown to bind preferentially to Foxa1 in a glucagon-producing pancreatic islet α-cell line [43
]. Moreover, Foxa1
null mice have markedly reduced levels of plasma glucagon [17
]. Consistent with prior work in the pancreas, our study shows that deletion of both Foxa1 and Foxa2 ablates preproglucagon transcripts in the small intestine ().
Pax6 is a critical regulator of pancreatic α-cell differentiation during development [42
]. It is a transcription factor of preproglucagon gene and inhibition of Pax6 led to decreased endogenous glucagon expression in pancreatic α-cell lines [44
]. Mice expressing a dominant-negative Pax6 mutant have reduced preproglucagon mRNA levels and lack Glp-1 and Glp-2 positive cells in the small and large intestine [37
]. Pax6 activates the preproglucagon promoter through G1 and G3 elements [45
]. In vitro
, Pax6 binds to an overlapping site on G1 element with a greater affinity as Foxa1 or Foxa2 [34
]. Binding of Foxa1 and Foxa2 impairs Pax6-mediated transactivation of the preproglucagon promoter via G1 and G3 elements in co-transfection assays, suggesting an antagonism between Pax6 and the Foxa factors in the regulation of this gene [34
]. We show here that intestine-specific deletion of Foxa1
causes decreased Pax6, Glp-1 and Glp-2 expression ( and ). Thus, our data indicate that in small intestine Foxa1 and Foxa2 control Pax6 transcription, and Pax6 acts downstream of and in concert with Foxa1 and Foxa2 to regulate preproglucagon gene transcription in a feed-forward loop.
Isl-1 is another important transcription factor of preproglucagon gene transcription, and Isl-1
null mice exhibit defective islet development and α-cell formation [36
]. In early embryogenesis, Isl-1 can be detected in the foregut, implicating its possible role in the gut development [47
]. Our data showed that Isl-1
expression is significantly reduced in mice with intestine specific deletion of Foxa1
(), suggesting that Foxa1 and Foxa2 may regulate Isl-1
gene transcription and Isl-1 lie downstream of Foxa1/a2
-mediated enteroendocrine lineage. Reduction of Isl-1
expression could contribute to the significant decrease of preproglucagon gene expression in Foxa1/a2
mutants are smaller and have lower bone mineral content, less lean muscle and body fat than age-matched controls. These phenotypes can be explained, in part, by reduced action of Glp-2, PYY and somatostatin. Glp-2 functions as a key regulator of mucosal integrity, permeability, and nutrient absorption (reviewed in [48
]). While we did not observe decreased epithelial cell proliferation in the small intestine of Foxa1/a2
mutants, the lack of Glp-2 may affect nutrient absorption and thus body weight. PYY is important for water and electrolyte absorption [49
], and indeed we observed increased water loss via the feces of Foxa1/a2
mutants (data not shown). Somatostatin controls gastric emptying, reduces smooth muscle contraction and blood flow within the intestine, and suppresses the release of several gastrointestinal hormones including gastrin, cholecystokinin, secretin, VIP, GIP, Glp-1 and Glp-2 ([50
], reviewed in [51
mutants have relatively normal intestinal gastrin, cholecystokinin, secretin, VIP, and GIP expression as shown by immunohistochemical analysis and qRT-PCR (data not shown), but we cannot exclude that altered somatostatin levels might affect plasma levels for these hormones.
Since Glp-1 is important for glucose metabolism, intestine-specific deletion of Foxa1
may have an impact on glucose homeostasis. Glp-1 stimulates insulin and inhibits glucagon secretion from the endocrine pancreas, thus contributing to postprandial glucose uptake (reviewed in [53
]). It also inhibits gastrointestinal motility, regulates appetite and food intake (reviewed in [53
]). In the pancreas, Glp-1 stimulates β-cell proliferation and neogenesis, and prevents β-cell apoptosis [55
]. Surprisingly, the resting glucose level was normal in Foxa1/a2
mutants (data not shown). One possibility for this finding is that deletion by the Villin-Cre transgene is not complete in the proximal duodenum, and that the remaining L-cells present there produce enough Glp-1 to control glucose homeostasis. Another possibility for the normal glucose homeostasis in our model is the fact that GIP expression is normal in Foxa1/a2
mutants, and both GLP-1 and GIP are insulinotropic peptides. GIP-receptor deficient mice have higher blood glucose levels with impaired initial insulin response in the oral glucose test [59
]. GLP-1 receptor null mice had mild glucose intolerance and impaired insulin secretion after intraperitoneal glucose injection [60
]. Interestingly, GLP-1 receptor null mice had increased plasma GIP levels, suggesting a compensatory relationship between the two signaling mechanisms [61
]. It might be interesting to test the metabolic response of Foxa1/a2
mutants to a high fat diet, which is often used to uncover metabolic defects, in the future.
In conclusion, we demonstrate that intestine-specific deletion of Foxa1 and Foxa2 alters the differentiation of goblet and enteroendocrine L- and D-cells. In goblet cells, Foxa1 binds to the Muc2 promoter preferentially over Foxa2. In L- and D-cells, Foxa1 and Foxa2 control expression of Glp-1/2, somatostatin and PYY both directly and through feed-forward regulation of Isl-1 and Pax6.