FoxF1 and FoxL1 Link Hedgehog Signaling and the Control of Epithelial Proliferation in the Developing Stomach and Intestine

The hedgehog (Hh) signaling pathway is a key component of cross-talk during vertebrate gut development, involving endodermally secreted Sonic (Shh) and Indian hedgehog (Ihh) proteins that directly signal to adjacent mesoderm. Here we show that the closely linked mesenchymal forkhead transcription factors Foxf1 and Foxl1 are part of this signaling cascade. Analysis of conserved non-coding sequences surrounding Foxf1 and Foxl1 identified seven Gli binding sites, with two sites near Foxl1 being identical among mammalian, bird, fish, and amphibian species. In vitro experiments indicate that Gli2 binds to these Gli sites, several of which are critical for Gli2-mediated activation of a luciferase reporter in 293 cells. In addition, we demonstrate occupancy of one of these elements by Gli proteins in the intestine in vivo using chromatin immunoprecipitation. Furthermore, expression of both Foxf1 and Foxl1 is reduced in the Gli2/Gli3 mutant gut. These results provide compelling evidence that Foxf1 and Foxl1 are mediators of the Hh (endoderm) to mesoderm signaling pathway. The hedgehog (Hh) signaling pathway is a key component of cross-talk during vertebrate gut development, involving endodermally secreted Sonic (Shh) and Indian hedgehog (Ihh) proteins that directly signal to adjacent mesoderm. Here we show that the closely linked mesenchymal forkhead transcription factors Foxf1 and Foxl1 are part of this signaling cascade. Analysis of conserved non-coding sequences surrounding Foxf1 and Foxl1 identified seven Gli binding sites, with two sites near Foxl1 being identical among mammalian, bird, fish, and amphibian species. In vitro experiments indicate that Gli2 binds to these Gli sites, several of which are critical for Gli2-mediated activation of a luciferase reporter in 293 cells. In addition, we demonstrate occupancy of one of these elements by Gli proteins in the intestine in vivo using chromatin immunoprecipitation. Furthermore, expression of both Foxf1 and Foxl1 is reduced in the Gli2/Gli3 mutant gut. These results provide compelling evidence that Foxf1 and Foxl1 are mediators of the Hh (endoderm) to mesoderm signaling pathway. The Hedgehog (Hh) 2The abbreviations used are: Hh, hedgehog; Shh, Sonic hedgehog; Ihh, Indian hedgehog; Hox, Homeobox; E, embryonic day; GI, gastrointestinal; EMSA, electrophoretic mobility shift assay; RT-PCR, reverse transcription-PCR; QPCR, quantitative PCR; ChIP, chromatin immunoprecipitation; CMF, Ca2+ and Mg2+-free; HBSS, Hanks' balanced salt solution; oligo, oligonucleotide.2The abbreviations used are: Hh, hedgehog; Shh, Sonic hedgehog; Ihh, Indian hedgehog; Hox, Homeobox; E, embryonic day; GI, gastrointestinal; EMSA, electrophoretic mobility shift assay; RT-PCR, reverse transcription-PCR; QPCR, quantitative PCR; ChIP, chromatin immunoprecipitation; CMF, Ca2+ and Mg2+-free; HBSS, Hanks' balanced salt solution; oligo, oligonucleotide. signaling pathway is one of the earliest regulatory cascades activated during organogenesis of the gut tube. Shh and Ihh, expressed in the gut endoderm, bind to the receptor Ptch1, which is expressed exclusively in the mesoderm of the vertebrate gut (1Motoyama J. Heng H. Crackower M.A. Takabatake T. Takeshima K. Tsui L.C. Hui C. Mech. Dev. 1998; 78: 81-84Crossref PubMed Scopus (58) Google Scholar, 2Ramalho-Santos M. Melton D.A. McMahon A.P. Development (Camb.). 2000; 127: 2763-2772Crossref PubMed Google Scholar, 3Wang L.C. Nassir F. Liu Z.Y. Ling L. Kuo F. Crowell T. Olson D. Davidson N.O. Burkly L.C. Gastroenterology. 2002; 122: 469-482Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 4Sukegawa A. Narita T. Kameda T. Saitoh K. Nohno T. Iba H. Yasugi S. Fukuda K. Development (Camb.). 2000; 127: 1971-1980Crossref PubMed Google Scholar). Interaction with the Ptch1 receptor causes derepression of Smo, a transmembrane protein, which prevents post-translational processing of Gli2 and Gli3 by inhibiting phosphorylation and proteasomal degradation (5Wang Y. McMahon A.P. Allen B.L. Curr. Opin. Cell Biol. 2007; 19: 159-165Crossref PubMed Scopus (102) Google Scholar). In the absence of Ptch1-mediated repression of Smo, an N-terminal proteolytic fragment of Gli3, and to a lesser extent Gli2, emerges from the proteasome as a transcriptional repressor, whereas Gli1 appears to be completely degraded. Both Shh and Ihh are expressed in mouse definitive endoderm from E8.5 onward along the entire gut tube, except in the domain of pancreatic bud endoderm (6Apelqvist A. Ahlgren U. Edlund H. Curr. Biol. 1997; 7: 801-804Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). In the intestine, Shh and Ihh are both required for proper specification and development of smooth muscle of the muscularis externa and neurons of the myenteric plexus (2Ramalho-Santos M. Melton D.A. McMahon A.P. Development (Camb.). 2000; 127: 2763-2772Crossref PubMed Google Scholar, 4Sukegawa A. Narita T. Kameda T. Saitoh K. Nohno T. Iba H. Yasugi S. Fukuda K. Development (Camb.). 2000; 127: 1971-1980Crossref PubMed Google Scholar, 7Madison B.B. Braunstein K. Kuizon E. Portman K. Qiao X.T. Gumucio D.L. Development (Camb.). 2005; 132: 279-289Crossref PubMed Scopus (294) Google Scholar). Severe embryonic inhibition of all Hh signals, through overexpression of an Hhip cDNA or via injection of a neutralizing antibody against Hh proteins, revealed that Shh and Ihh are also indispensable for villus development and later critical for patterning the cryptvillus axis and restricting epithelial proliferation via modulation of adjacent mesenchymal cells (3Wang L.C. Nassir F. Liu Z.Y. Ling L. Kuo F. Crowell T. Olson D. Davidson N.O. Burkly L.C. Gastroenterology. 2002; 122: 469-482Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 7Madison B.B. Braunstein K. Kuizon E. Portman K. Qiao X.T. Gumucio D.L. Development (Camb.). 2005; 132: 279-289Crossref PubMed Scopus (294) Google Scholar). Recent computational approaches have attempted to identify Gli target genes. One investigation used a local alignment algorithm termed “enhancer element locator,” which evaluates the spatial order of transcription factor binding sites from two different species (8Hallikas O. Palin K. Sinjushina N. Rautiainen R. Partanen J. Ukkonen E. Taipale J. Cell. 2006; 124: 47-59Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). Gli binding sites near Foxf1, Foxc2, and Foxl1, all located within a 50-kb region of mouse chromosome 8, scored very highly using the enhancer element locator method. In addition, expression of Foxf1 is dependent on Shh in the developing oral cavity, lung, and sclerotome, and both Foxf1 and Foxf2 expression can be extinguished in E12.5 intestine explants when treated with the Smo inhibitor cyclopamine (9Mahlapuu M. Enerback S. Carlsson P. Development (Camb.). 2001; 128: 2397-2406Crossref PubMed Google Scholar, 10Ormestad M. Astorga J. Landgren H. Wang T. Johansson B.R. Miura N. Carlsson P. Development (Camb.). 2006; 133: 833-843Crossref PubMed Scopus (179) Google Scholar). These reports suggest that Foxf1 and Foxf2 could be Hh target genes. Intestinal epithelial phenotypes of Foxf1, Foxf2, and Foxl1 mutants also suggest connections to the Hh signaling pathway. Foxf1+/-, Foxf2-/-, and Foxl1-/- mice all have perturbations in the crypt-villus axis, characterized by increased proliferation and Wnt signaling, reminiscent of epithelial abnormalities caused by Hh signal ablation (3Wang L.C. Nassir F. Liu Z.Y. Ling L. Kuo F. Crowell T. Olson D. Davidson N.O. Burkly L.C. Gastroenterology. 2002; 122: 469-482Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 7Madison B.B. Braunstein K. Kuizon E. Portman K. Qiao X.T. Gumucio D.L. Development (Camb.). 2005; 132: 279-289Crossref PubMed Scopus (294) Google Scholar, 11Kaestner K.H. Silberg D.G. Traber P.G. Schutz G. Genes Dev. 1997; 11: 1583-1595Crossref PubMed Scopus (186) Google Scholar, 12Perreault N. Katz J.P. Sackett S.D. Kaestner K.H. J. Biol. Chem. 2001; 276: 43328-43333Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Foxl1-/- mice also have delayed villus morphogenesis, with very stunted villi evident at E16.5 and E18.5, similar to the severe defects in villus development caused by Hh inhibition (7Madison B.B. Braunstein K. Kuizon E. Portman K. Qiao X.T. Gumucio D.L. Development (Camb.). 2005; 132: 279-289Crossref PubMed Scopus (294) Google Scholar, 11Kaestner K.H. Silberg D.G. Traber P.G. Schutz G. Genes Dev. 1997; 11: 1583-1595Crossref PubMed Scopus (186) Google Scholar). The Foxl1 mRNA expression pattern also highly resembles that of the known Hh target genes Ptch1, Gli1, and Bmp4 during intestine development (2Ramalho-Santos M. Melton D.A. McMahon A.P. Development (Camb.). 2000; 127: 2763-2772Crossref PubMed Google Scholar, 13Kaestner K.H. Bleckmann S.C. Monaghan A.P. Schlondorff J. Mincheva A. Lichter P. Schutz G. Development (Camb.). 1996; 122: 1751-1758Crossref PubMed Google Scholar, 14Karlsson L. Lindahl P. Heath J.K. Betsholtz C. Development (Camb.). 2000; 127: 3457-3466Crossref PubMed Google Scholar, 15Fukuda K. Yoshida H. Sato T. Furumoto T.A. Mizutani-Koseki Y. Suzuki Y. Saito Y. Takemori T. Kimura M. Sato H. Taniguchi M. Nishikawa S. Nakayama T. Koseki H. Dev. Biol. 2003; 255: 278-289Crossref PubMed Scopus (19) Google Scholar). Our studies indicate that Foxfl and Foxl1 are direct target genes of the Hh signaling pathway in the developing stomach and intestine. Their expression is dependent on Gli2 and Gli3 and induced by Shh-N recombinant protein. Several highly conserved Gli binding sites appear crucial for Gli-mediated binding and transcriptional activation of the Foxf1 and Foxl1 promoters. These findings provide a critical link toward understanding the molecular mechanisms that control epithelial proliferation and organization of the developing gastrointestinal (GI) tract. EMSA Analysis—Stomach and intestine primordia (E14.5) were isolated from ∼36 CD1 embryos and snap-frozen in liquid N2, and nuclear extracts were prepared as described previously (16Greenbaum L.E. Li W. Cressman D.E. Peng Y. Ciliberto G. Poli V. Taub R. J. Clin. Investig. 1998; 102: 996-1007Crossref PubMed Scopus (229) Google Scholar), with some modifications. Complete (Roche Applied Science) protease inhibitor tablets were used in all buffers, supplemented with 0.1 μm phenylmethylsulfonyl fluoride and 1.0 mm sodium metavanadate. Oligonucleotides (oligos) were labeled by incubating 50 ng of double-stranded DNA with 30 μCi of a 32PdCTP and 2.5 units of Klenow (New England Biolabs) for 30 min at room temperature followed by removal of unincorporated nucleotides with a MicroSpin G-50 column (Amersham Biosciences). Electrophoretic mobility shift assays (EMSAs) were performed as described previously (17Gupta R.K. Gao N. Gorski R.K. White P. Hardy O.T. Rafiq K. Brestelli J.E. Chen G. Stoeckert Jr., C.J. Kaestner K.H. Genes Dev. 2007; 21: 756-769Crossref PubMed Scopus (131) Google Scholar); 3-4 μg of protein extract were incubated with 20 mm Hepes, pH 7.9, with 0.1 mm EDTA, 1 mm MgCl2, 12% glycerol, 60 mm KCl, 0.5 mm dithiothreitol (added fresh), 50 ng/μl sonicated dIdC, and 125 ng/μl bovine serum albumin for 10 min to prebind. Labeled probe (25,000 cpm) was then added and incubated for 30 min at room temperature. For some assays, 1 μg of a polyclonal rabbit antibody to Gli2 (AbCam ab26056) or 1 μg of rabbit IgG (R PubMed Scopus Google Scholar). Luciferase assays were performed as described previously (17Gupta R.K. Gao N. Gorski R.K. White P. Hardy O.T. Rafiq K. Brestelli J.E. Chen G. Stoeckert Jr., C.J. Kaestner K.H. Genes Dev. 2007; 21: 756-769Crossref PubMed Scopus (131) Google Scholar). Gli sites within the and within were to of the (Stratagene). of the Gli within the Foxf1 was performed by PCR using the PfuUltra J.K. PubMed Scopus Google Scholar). was isolated using the The was used for cDNA from ng of of genomic DNA in 0.5 of was added to reverse to the of were incubated for min at 10 min at and then on the was performed in with 0.5 of cDNA on a were to of was prepared from the intestine of embryos at of as described previously L. Brestelli J. Kaestner K.H. Cell. Biol. 2005; PubMed Scopus Google Scholar). was with a polyclonal antibody antibody of the the fragment was using and as described DNA and DNA was to that of the of the protein which is expressed in the and a target of Gli of Gli2 and Gli3 mice were by mice were obtained from The were on a were isolated from and as described previously PubMed Scopus Google Scholar, R. D.L. Crackower M.A. J. Heng Tsui L.C. Hui C. Development (Camb.). 1997; 124: PubMed Google Scholar). and intestine primordia from E12.5 embryos were and in in with was then using the was performed as with and intestine primordia (E14.5) were isolated from CD1 embryos and into Ca2+ and were incubated 10 min at room temperature in 10 mm and then to the endoderm and to an was all endoderm was as a was in was then incubated at for 30 with in 1 of a of with was added and then at for min at was with of and then of a in 0.5 of of bovine serum albumin or mouse recombinant Shh-N Systems) was added to a concentration of an of was using and quantitative was performed as described of Hh target genes is by Gli factors binding to the Cell. Biol. PubMed Scopus Google Scholar, H. S. T. S. S. Davidson McMahon A.P. Development (Camb.). 2007; PubMed Scopus Google Scholar). identify conserved sequences within a region surrounding Foxf1 and Foxl1, we and species using the M. L. C. 2000; PubMed Scopus Google Scholar, L. A. PubMed Scopus Google Scholar), which this we two sites near Foxf1 and sites near Foxl1 that or the Gli binding One Gli near Foxf1 was within a conserved region which also two forkhead sites and one similar to the of transcription known to be in and smooth muscle development B.L. Olson Cell Dev. Biol. 1998; PubMed Scopus Google The two Gli sites to Foxl1 were within a region of the that the region of Foxl1 Gli binding sites within to forkhead binding sites, with of seven Gli sites located within of a conserved forkhead The Gli Foxl1, at was also adjacent to a highly conserved the Gli binding of these conserved we performed on sites with the to the Gli Gli sites near Foxl1 at and with to the of the Foxl1 mRNA at which was with an a Gli from the Ptch1 M. P. M. R. PubMed Scopus Google and an antibody against Gli2 reduced the of the signal of the and a of Gli2 binding and The in assays performed with the Gli to Foxf1 Gli except that the of the was and the binding of these Gli sites, with were performed and These that sequences at and the Gli near Foxf1 at These experiments also revealed that the two closely and the in this was to with the could binding of a factor to the highly conserved adjacent to the Gli we the Gli sites within Foxf1 and Foxl1 fragments for to a luciferase reporter in cells. Gli sites were and was in the of a Gli2 protein that an N-terminal of the domain or E. Wang A. M. M. 2005; PubMed Scopus Google Scholar). The Foxf1 very reporter in 293 cells was when with a of the Gli reduced the of The conserved non-coding region near Foxf1, to the Gli near Foxf1, was for on reporter in the of the Foxf1 Foxf1 and when the Gli within was reporter was be that a mouse with a cDNA by a Foxf1 the region the expression pattern of Foxf1 in the developing and gut Y. S. D.E. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of a Foxl1 we a of reporter of the Gli sites at and were of and to bind Gli2 in The Gli at caused of whereas of the of both sites to that these two sites provide the of the Gli2 of the Foxl1 in this a was in the that some Gli2 at the and Gli sites, in which all sites except are or in which all sites are completely by all Gli sites, the appears to have of Gli binding and key in in this the element the of the Foxf1 was a on Foxl1 regulatory the of this of the Gli within the of These suggest that as a enhancer for the Foxl1 Gli proteins of the binding sites identified in we performed chromatin assays with an antibody to Gli1 on chromatin isolated from intestine in the Gli binding in the of the Foxf1 located at with to the transcriptional of Foxl1, is highly in the DNA when with binding to this by Gli1 in this is the of Gli1 binding to in vivo in the using chromatin immunoprecipitation. The in vitro and in vivo described indicate that Gli transcription factors directly the Foxf1 and Foxl1 that of Gli2 Gli3 of Foxf1 and Foxl1 mRNA in Gli1 is expressed in the absence of Gli2 and Gli3 and for D. Dev. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). we Foxf1 and Foxl1 mRNA in E12.5 Gli2 and Gli3 stomach and intestine primordia and In the stomach of Foxl1 expression is whereas Foxf1 expression is The stomach of embryos a in expression of Foxf1 and a of Foxl1 of Gli3 that to expression of Foxf1 in the this in the intestine, Foxf1 and Foxl1 expression was when both of Gli2 or Gli3 are Foxl1 mRNA were reduced in embryos In of Foxf1 and Foxl1 expression of these genes appears dependent on of both Gli2 and Gli3 in the developing intestine in the of Foxf1 and mRNA was to a of caused by Gli2 Gli3 of the mRNA for an expressed in mesenchymal cells expression of Gli1 was reduced in embryos we activation of the Hh pathway was to the expression of these genes in a mesoderm from embryos was isolated and treated with a recombinant mouse N-terminal fragment of Shh and for of Foxf1 and Foxl1 Foxl1 was induced in both stomach and intestine whereas Foxf1 was induced in the intestine In this the Hh signaling pathway is as by of the Gli2/Gli3 target Gli1 these demonstrate that Foxf1 and Foxl1 are dependent on Gli2 and Gli3 and induced Hh pathway activation in the mesoderm of developing stomach and intestine. In this evidence that Foxf1 and Foxl1 are target genes of the Hh signaling pathway via Gli transcription a absence of Gli factors completely Foxf1 and Foxl1 expression in the developing this is The expression of Foxf1 and Foxl1 in gut is to and transcription factors on promoters. as in conserved sites, forkhead sites, and binding sites are located in these and could provide on Foxf1 and Foxl1 In the developing expression of Foxf1 in the mesoderm and is in and embryos in the and J. Carlsson P. Development (Camb.). 2007; PubMed Scopus Google Scholar), that some of Foxf1 expression in of Gli in embryos expression of Foxf1 and Foxl1 in the stomach and intestine, an of Foxf1 and of Foxl1 in the intestine could for a of the defects Hh inhibition in the developing gut. In to Foxf1 and Foxl1, are to be Hh target genes activated via Gli transcription factors in the developing gut mesoderm. These target genes in One of this the factor signaling in which of identified factor target genes of the phenotypes of the and mutants J. P. 2007; PubMed Scopus Google Scholar). of Foxl1 results in a of the gut severe that caused by inhibition of the Hh signal (7Madison B.B. Braunstein K. Kuizon E. Portman K. Qiao X.T. Gumucio D.L. Development (Camb.). 2005; 132: 279-289Crossref PubMed Scopus (294) Google Scholar, 11Kaestner K.H. Silberg D.G. Traber P.G. Schutz G. Genes Dev. 1997; 11: 1583-1595Crossref PubMed Scopus (186) Google Scholar). Foxf1 and Foxl1 on the Hh signal also different is that both Foxf1 and Foxl1 appear to Shh-N in stomach mesoderm and dependent on Gli2 and Gli3 in the developing stomach when with similar experiments with could a of Gli2 and Gli3 expression in the the to which the Hh pathway is activated on transcription factors expressed in the was used to the the Hh signal and Foxf1 and Foxl1 in this Luciferase assays of Foxf1 and Foxl1 were the could be used to is compelling that Shh of Foxf1 and Foxl1 in closely the by in a of Foxl1 in a in and a of Foxf1 in a in in and suggest that these fragments in cells an for Gli-mediated activation of these expressed forkhead genes. was in the of Shh, Foxl1 was In when we expression in the intestine of Foxf1 was the whereas in the Foxl1 is to Gli2/Gli3 we these is that during the with Shh is a in the could be to signaling expression in the or of the that both Foxf1 and Foxl1 are by hedgehog signaling and Gli the in vivo binding of Gli factors to the Foxf1 and Foxl1 we from embryonic gut for Gli1, which to were to Gli binding to the of the Foxf1 in the in vivo of the Hh of a target be necessary to all Gli binding sites within regulatory be a and for linked genes as Foxl1 or Foxf1, which have Gli binding sites that could the expression of The evidence of Gli sites, binding by EMSA, to Gli2 in luciferase assays, occupancy of target sites by Gli in on Gli2 and Gli3, and Shh in that Foxf1 and Foxl1 are target genes of the Hh pathway. Foxf1 and Foxl1 appear to be in by the signaling pathway Hh and are both expressed in the developing mesoderm of the is compelling to and to Foxf1 and Foxl1 are or Foxl1 and Foxf2 mutants both reduced expression of Bmp4 in the gut M. Astorga J. Landgren H. Wang T. Johansson B.R. Miura N. Carlsson P. Development (Camb.). 2006; 133: 833-843Crossref PubMed Scopus (179) Google Scholar, 11Kaestner K.H. Silberg D.G. Traber P.G. Schutz G. Genes Dev. 1997; 11: 1583-1595Crossref PubMed Scopus (186) Google Scholar), and indicate that Foxf1 mutant embryos have a severe of Bmp4 expression in the and mesoderm J. Carlsson P. Development (Camb.). 2007; PubMed Scopus Google Scholar). Foxf1 is in Bmp4 expression in the developing intestine, then the of Foxf1 and Foxl1 could be in through a known of epithelial proliferation A.P. H. M. J. S. H. PubMed Scopus Google Scholar, L.E. H. Dev. 2006; PubMed Scopus Google Scholar, J. O. J. Y. Li L. PubMed Scopus Google Scholar), via direct on the Wnt signaling pathway Foxf1 was described as a critical in and the was identified as a direct target D. E. S. L. V. Cell. Biol. 2007; PubMed Scopus Google Scholar). Foxf1 in the gut could defects in during villus could the of mesenchymal cells along the crypt-villus axis, and in epithelial are known target genes of Foxf1 and Foxl1, and described that are of the Wnt signaling except that Foxl1 results in expression of the N. Katz J.P. Sackett S.D. Kaestner K.H. J. Biol. Chem. 2001; 276: 43328-43333Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). of the Foxl1 in vivo DNA binding of Foxl1 target genes and the of Foxf1 and Foxl1 bind to the elements and or identical target genes. in Foxl1 or Foxf1 mesenchymal signaling, in development be by to genomic of a mutant is by the of at two and Foxf1 and of the regulatory in the developing evidence Foxf1 and Foxl1 at a in the signaling organogenesis of the genes are in the of gut development, the of genes can have on the of the gastrointestinal of the Wnt signaling pathway the causes of and in very of epithelial proliferation Wnt signaling have One Foxl1, is a of the mouse as a of from Wnt signaling, through the of secreted proteins N. Sackett S.D. Katz J.P. Kaestner K.H. Genes Dev. 2005; 19: PubMed Scopus Google Scholar). Foxl1 with Foxf1 could provide indispensable control of proliferation and of the mesenchymal of epithelial proliferation are in mice and factors with could an of to this the of and for and for the of and the of the for in and with