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Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent β-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted β-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa. Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent β-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted β-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa. Transgenic mice are routinely used to study the molecular and cellular basis of normal and pathological states in intestinal mucosa (1Sweetser D.A. Hauft S.M. Hoppe P.C. Birkenmeier E.H. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9611-9615Crossref PubMed Scopus (134) Google Scholar, 2Cohn S.M. Simon T.C. Roth K.A. Birkenmeier E.H. Gordon J.I. J. Cell Biol. 1992; 119: 27-44Crossref PubMed Scopus (154) Google Scholar, 3Hermiston M.L. Green R.P. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8866-8870Crossref PubMed Scopus (50) Google Scholar, 4Markowitz A.J. Wu G.D. Birkenmeier E.H. Traber P.G. Am. J. Physiol. 1993; 265: G526-G539PubMed Google Scholar, 5Crossman M.W. Hauft S.M. Gordon J.I. J. Cell Biol. 1994; 126: 1547-1564Crossref PubMed Scopus (86) Google Scholar). The major limitation regarding the targeting of exogenous transgenes in this tissue is that the epithelium of the mouse intestinal mucosa is renewed every 2–5 days (6Cheng H. Leblond C.P. Am. J. Anat. 1974; 141: 461-479Crossref PubMed Scopus (538) Google Scholar, 7Wright N.A. Irwin M. Cell Tiss. Kinet. 1982; 15: 595-609PubMed Google Scholar, 8Gordon J.I. Hermiston M.L. Curr. Opin. Cell Biol. 1994; 6: 795-803Crossref PubMed Scopus (215) Google Scholar). The epithelial cells arise from multipotent stem cells functionally anchored at the base (more precisely in the lower third) of the proliferative compartment of the epithelium, the crypts of Lieberkühn. These crypts display a monoclonal organization because they are each derived from a single progenitor cell (9Ponder B.A. Schmidt G.H. Wilkinson M.M. Wood M.J. Monk M. Reid A. Nature. 1985; 313: 689-691Crossref PubMed Scopus (254) Google Scholar). Descendants of stem cells multiply in the middle portion of each crypt (10Potten C.S. Loeffler M. Development. 1990; 110: 1001-1020Crossref PubMed Google Scholar) and gradually differentiate into four principal cell types. In the small intestine, absorptive enterocytes (constituting >80% of the epithelial cells), mucus-producing goblet cells, and enteroendocrine cells migrate upward from the crypts to the apex of surrounding villi (whose colonic counterparts are hexagonal-shaped cuffs) (11Schmidt G.H. Wilkinson M.M. Ponder B.A. Cell. 1985; 40: 425-429Abstract Full Text PDF PubMed Scopus (144) Google Scholar), where they become apoptotic and are exfoliated into the gut lumen (12Hall P.A. Coates P.J. Ansari B. Hopwood D. J. Cell Sci. 1994; 107: 3569-3577Crossref PubMed Google Scholar). In contrast, antimicrobial peptides secreting Paneth cells migrate to the bottom of the crypts, where they reside for about 20 days (13Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10335-10339Crossref PubMed Scopus (213) Google Scholar). Given the remarkable protective effect of this epithelium, it is not surprising that most previous studies aiming to induce neoplastic transformation in intestinal mucosa of transgenic mice have failed (14Hauft S.M. Kim S.H. Schmidt G.H. Pease S. Rees S. Harris S. Roth K.A. Hansbrough J.R. Cohn S.M. Ahnen D.J. Wright N.A. Goodlad R.A. Gordon J.I. J. Cell Biol. 1992; 117: 825-839Crossref PubMed Scopus (56) Google Scholar,15Kim S.H. Roth K.A. Moser A.R. Gordon J.I. J. Cell Biol. 1993; 123: 877-893Crossref PubMed Scopus (97) Google Scholar). In these reports, the use of promoter sequences that direct oncogenes in nonproliferating enterocytes located in the upper third of crypts produce only minor phenotypic abnormalities without tumorigenic consequences in the gut epithelium, suggesting that the residence time of these villus-associated cells may not be sufficient for the oncogenes to exert their effects. Furthermore this suggests that transgenic mouse models of neoplasia may require an efficient targeting of oncogenes in crypt stem cells or their immediate descendants. Villin is a cytoskeletal protein that is mainly produced in epithelial cells that develop a brush border responsible for absorption as in the digestive apparatus (epithelial cells of the large and small intestines) and in the urogenital tract (epithelial cells of the kidney proximal tubules). Because it is expressed in the proliferative stem cells of the intestinal crypts (16Robine S. Huet C. Moll R. Sahuquillo-Merino C. Coudrier E. Zweibaum A. Louvard D. Proc. Acad. Natl. Sci. U. S. A. 1985; 82: 8488-8492Crossref PubMed Scopus (149) Google Scholar, 17Boller K. Arpin M. Pringault E. Mangeat P. Reggio H. Differentiation. 1988; 39: 51-57Crossref PubMed Scopus (34) Google Scholar), it is believed to be an early marker for committed intestinal cells. The multiple levels of regulation control villin gene activity during mouse embryogenesis (18Maunoury R. Robine S. Pringault E. Huet C. Guenet J.L. Gaillard J.A. Louvard D. EMBO J. 1988; 7: 3321-3329Crossref PubMed Scopus (65) Google Scholar, 19Maunoury R. Robine S. Pringault E. Leonard N. Gaillard J.A. Louvard D. Development. 1992; 115: 717-728Crossref PubMed Google Scholar, 20Ezzell R.M. Chafel M.M. Matsudaira P.T. Development (. 1989; 106: 407-419PubMed Google Scholar) and account for the strict pattern of tissue-specific expression observed in adults. Moreover, the expression of the villin gene in intestinal epithelial cells is conspicuously maintained in their corresponding carcinomas (21Carboni J.M. Howe C.L. West A.B. Barwick K.W. Mooseker M.S. Morrow J.S. Am. J. Pathol. 1987; 129: 589-600PubMed Google Scholar, 22Moll R. Robine S. Dudouet B. Louvard D. Virchows Arch. B. Cell Pathol. 1987; 54: 155-169Crossref Scopus (88) Google Scholar, 23West A.B. Isaac C.A. Carboni J.M. Morrow J.S. Mooseker M.S. Barwick K.W. Gastroenterology. 1988; 94: 343-352Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 24Bacchi C.E. Gown A.M. Lab. Invest. 1991; 64: 418-424PubMed Google Scholar). The specific expression pattern of villin suggests that it is an appropriate candidate for the characterization of regulatory sequences that could allow targeting of heterologous genes into a selected population of cells in the mouse digestive tract. With this goal in mind, the human villin gene has been isolated and characterized (25Pringault E. Robine S. Louvard D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10811-10815Crossref PubMed Scopus (27) Google Scholar). A 2-kb 1The abbreviations used are: kb, kilobase(s); PCR, polymerase chain reaction; bp, base pair(s).1The abbreviations used are: kb, kilobase(s); PCR, polymerase chain reaction; bp, base pair(s). 5′-flanking region has been found to contain sufficient regulatory elements to promote tissue-specific expression of a reporter gene in intestinal and renal cell lines (26Robine S. Sahuquillo-Merino C. Louvard D. Pringault E. J. Biol. Chem. 1993; 268: 11426-11434Abstract Full Text PDF PubMed Google Scholar). In transgenic mice, this regulatory region is able to drive the expression of the human Ha-ras oncogene in the tissues in which the endogenous gene is actively transcribed. However, low levels of expression were observed that did not trigger malignant tissue appearance into the gut of these animals. 2G. Tremp, personal communication.2G. Tremp, personal communication. These observations led us to further analyze an extended genomic region of the mouse villin gene with the goal of elements and/or and in levels of transgenic expression in the intestinal mucosa. the of tissue-specific expression of the mouse villin gene and transgenic cells, kidney proximal cells, and kidney cells were as (26Robine S. Sahuquillo-Merino C. Louvard D. Pringault E. J. Biol. Chem. 1993; 268: 11426-11434Abstract Full Text PDF PubMed Google Scholar). were of with each of β-galactosidase reporter construct and the control which contains the gene the control of the were and cell were of β-galactosidase and a activity was for in as were at are expressed as that of the without was isolated from mouse with of to to of the villin was with of at for with of was at for in of a and The were in The was with the of the A control was the a to to of the mouse intestinal protein and an R.P. Cohn S.M. Gordon J.I. Cell Biol. 1992; PubMed Scopus Google Scholar). from mice were used of intestine, kidney, and and were as C. M. 1991; PubMed Scopus Google Scholar) with minor were without or with 20 to of for at of was with or The were in were into the with isolated from a a region kb upstream and kb from the of the mouse villin gene Tremp, M. Robine S. A. D. C. Louvard D. Cell. Biol. PubMed Scopus Google Scholar). The construct in the to was of kb kb upstream from the of the mouse villin to the a polymerase chain at the site in a the region of the mouse villin gene to the region The and an have from in the region and in a the region of the mouse villin gene to the The and were derived from the and with and To the from the region was and into the site of the The and to the and from the intron The the transcription start site and the the intron was from that of the villin and was into of the construct a of a and a in the The and the were derived from the an and from the region at the site in the were The transgenes were into the of the of the mice in with the transgenes were first of genomic to the of the β-galactosidase gene and to the of the of the transgene intestine, kidney, and were from transgenic mice and for or to was isolated with 20 of were with of at for in transcription with of was at for in a of and of the were in for of at at transgene and and and at the transgene and were a villin and were a for and were a of the was an from the tissues were with for and in a that of at for To the start site of the mouse villin was isolated from and an to the mouse villin of the The of the was of the mouse intestinal protein gene from the R.P. Cohn S.M. Gordon J.I. Cell Biol. 1992; PubMed Scopus Google Scholar). of the a with a a of a of as The of villin was bp, that the start site as was upstream of the of the villin of the genomic kb upstream from the with the of site sequences in the 9-kb genomic and of the transcription start site that the mouse villin gene has transcription start site that is from the a region To the regulatory in the specific control of villin have the 1994; PubMed Scopus Google Scholar) in the mouse villin gene a region kb upstream and kb from the as in The of the mouse villin gene in tissues kidney, and was to and with the appropriate and a of the of were at 5.5 and kb, and corresponding to as at kb from the transcription start upstream from the and at kb from the was observed in isolated from intestine, kidney, and was only in intestinal specific were in isolated from The and of these were with the to the of the not and the of were at and kb and and kb, corresponding to the at kb upstream from the at kb upstream from the at kb upstream from the and at kb upstream from the was observed in isolated from and kidney, was only in intestinal tissue as The and were only in tissue which villin is and were located upstream from the transcription start site in that have not been and that could to an for these these were not for specific were in isolated from and the and kb in were not In four major to were to be in the region from kb to kb in to the transcription start site of the mouse villin gene. These were in to kidney and and tissues in which villin is but they were not found in a tissue that not produce These with the tissue-specific control of villin gene expression and that the regulatory elements these and were only in and are with tissue-specific transcription in the control of villin gene intestinal To the of the the activity and to precisely the villin gene expression in the intestine, were upstream of a LacZ The construct the from the the four to and the intron and were to for the of site and intron and were to but the from to kb and to kb to the transcription start was to but the region from to and were to and for the of intron The was to for the of the transcription start site and the region upstream from this The which not contain a promoter or and a which the were also in each with these were in and cell which and in kidney epithelial cells, in which villin expression is from the villin promoter was β-galactosidase activity in from the cells, and the were expressed as that of the levels of β-galactosidase activity in the cell lines cells, that of cells, the of efficient mechanisms in these cells not low levels of β-galactosidase activity in cells with cells that the transcription start site was necessary for an efficient specific transcription of the reporter gene and that transcription was not in the villin regulatory The construct expressed the β-galactosidase gene at the in cells as with cells suggesting that the four with the first intron are necessary to efficiently promote transcription in cells of intestinal of the the site β-galactosidase expression in cells to about of that of that a major that intestinal activity was this were the region upstream from the transcription start site and was with or without and The of the region or in with sequences upstream from the transcription start site and only from and bp, to a β-galactosidase expression in the intestinal cells with a to only about of that of that the regulatory elements that were sufficient to promote transcription in cells. However, the of β-galactosidase activity the and were in cells and that the of the first in with the of was able to promote transcription in a kidney cell that elements that in kidney transcription are in these To specificity, the villin were in kidney cells, which not these cells only levels of β-galactosidase activity with activity not that the villin regulatory sequences were to promote efficient transcription in villin cells and that the expression of the reporter gene in and cells is these regulatory these from of cells that the mouse villin genomic from to kb, an efficient expression of the β-galactosidase reporter gene in cells, this is the site or the region upstream from the site is of the entire first intron to the to promote and of the entire first intron in with site is with a of in transcription in cells. Because the to region of the mouse villin the activity in the of this region to drive expression of the β-galactosidase reporter gene in transgenic that the construct as a transgene were The mice were for reporter gene expression in tissues the and were analyzed. The only the of that from genomic of an of a from the mouse villin promoter upstream of the site and the from the β-galactosidase gene or the villin gene. each reporter gene expression was in the tissues in which villin were not the mice, the reporter gene transcription was along the of the gut and the expression of the villin gene In the kidney, the transgene was only in of was in from tissues in which the reporter gene expression could not be the of from these To the cellular of transgene expression the of small intestine, and kidney were and for β-galactosidase were of β-galactosidase expression not four of transgenic mice, a pattern of expression in small and was be to because animals. The expression was to the of the epithelial cells, as because the β-galactosidase gene contains a The was a in the villi cells with the crypt cells, of small and epithelium, thus that the to region of the mouse villin gene is able to recapitulate precisely the cellular pattern of along the crypt-villus of the villin gene K. Arpin M. Pringault E. Mangeat P. Reggio H. Differentiation. 1988; 39: 51-57Crossref PubMed Scopus (34) Google Scholar). A of cells of the crypt and was suggesting the expression of the transgene in the stem cells (10Potten C.S. Loeffler M. Development. 1990; 110: 1001-1020Crossref PubMed Google Scholar). is that the of the β-galactosidase was to that of intestinal from which a β-galactosidase gene at the villin S. Louvard D. Am. J. Physiol. Google Scholar), that the to region of the mouse villin gene was able to promote intestinal transcription as efficiently as the mouse villin gene In the kidney of the mouse in which the transgene was the was only observed in the epithelial cells of the proximal tubules where the villin gene is expressed not The were able to the transgene to their with a pattern of β-galactosidase expression not In to direct an efficient expression of the reporter gene in the intestinal epithelium with regulatory and were used to transgenic mice, because these display efficient levels of β-galactosidase activity in cells. The of the transgene β-galactosidase was observed in of the four lines of transgenic mice These lines expressed the reporter gene only in the small the immature and differentiated epithelial cells along the crypt-villus and lines failed to the transgene in the tissues particularly is the of expression in the and the kidney not These that the regulatory region upstream from the transcription start site of the mouse villin gene is necessary and sufficient to expression in small of transgenic mice, the first intron of the mouse villin gene is for and kidney expression in transgenic the and transgenic mice, transgene expression was observed in tissues small intestine, and Thus, the cis-acting elements of the villin gene for intestinal and/or expression of in transgenic mice are not located only the region upstream from the transcription start as observed in the epithelial cells. In this that cis-acting sequences located a 9-kb region to kb from the start site of of the mouse villin gene are sufficient to direct tissue-specific and expression of the β-galactosidase reporter gene in transgenic mice, with the endogenous gene R. Robine S. Pringault E. Leonard N. Gaillard J.A. Louvard D. Development. 1992; 115: 717-728Crossref PubMed Google Scholar). gene expression is in the intestinal and restricted to epithelial cells along the crypt-villus of small and In these regulatory elements can a of β-galactosidase gene expression from the crypts of to the of villi that precisely the the villin gene K. Arpin M. Pringault E. Mangeat P. Reggio H. Differentiation. 1988; 39: 51-57Crossref PubMed Scopus (34) Google Scholar). transgene and endogenous gene expression were also as a with the of β-galactosidase activity in intestinal from transgenic mice and mice in which the reporter gene has been at the villin S. Louvard D. Am. J. Physiol. Google Scholar). In the kidney, for only of mouse reporter gene expression was restricted to epithelial cells of the proximal tubules the villin expression pattern in this suggests that mechanisms gene expression to and kidney tissues are in the to region of the mouse villin gene and that those to kidney may be sensitive to effects. Indeed it is that the transgene expression is the site of and can be regulatory in the C. D. J. P.J. Transgenic 1994; PubMed Scopus Google Scholar). The construct that the first intron of 5.5 kb but that 3.5 kb to the start site of transcription of the mouse villin in of the β-galactosidase the in expression of the reporter gene only into the epithelial cells along the crypt-villus of the small intestine. The of the reporter gene expression in the kidney be to as the to the be to the of regulatory elements of the intron as the site only the first and to the start site of in with the of the first in of the β-galactosidase failed to drive and expression of suggesting that the site upstream from the an in reporter gene expression into the epithelial cells of the small intestine. Thus, and regulatory elements in the mouse villin gene may direct transgene expression along the of the the regulatory elements for transgene expression in the small be in the region the upstream from the transcription start those necessary for the colonic expression be in the first intron the The of regulatory sequences of the mouse villin gene to direct expression of the reporter gene in the of transgenic mice also be of to the of the entire first In the are of those of the gene J.L. D.A. Cell. Biol. 1992; PubMed Scopus Google Scholar) and the gene C. A. C. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), in which elements located in the first intron are for transgene expression in because they may contain cis-acting tissue-specific elements and/or elements in of the the for transcription and be to the of the first region of the mouse villin gene. To the in the of the mouse villin gene regulatory elements to promote transcription of the reporter gene in cell transgenic may that the regulation of gene expression in the intestinal epithelium as cells differentiate and migrate along the crypt-villus axis. the that these cells with cells and with the the M.L. Gordon J.I. J. Cell Biol. 129: PubMed Scopus Google Scholar). Thus, an as the cell used in is to recapitulate the and of this epithelium and the of in models to a for specific regulatory sequences Gordon J.I. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, Simon T.C. Gordon J.I. J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies in transgenic mice to regulatory elements responsible for intestinal expression have been cis-acting sequences of genes expressed in of small A.J. Wu G.D. Birkenmeier E.H. Traber P.G. Am. J. Physiol. 1993; 265: G526-G539PubMed Google Scholar, 5Crossman M.W. Hauft S.M. Gordon J.I. J. Cell Biol. 1994; 126: 1547-1564Crossref PubMed Scopus (86) Google Scholar, Simon T.C. Gordon J.I. J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T.C. A. P. Gordon J.I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In of these in the crypts in with extended expression in the in an Thus, to the 9-kb regulatory region of the mouse villin gene the only characterized cis-acting sequences that allow the expression of a heterologous gene in small and epithelial cells of transgenic mice with the tissue-specific and pattern of expression with that of the endogenous gene In the mice lines that drive a transgenic expression restricted to the intestinal mucosa could be of those that not display expression into the kidney because of the effects. The to target genes of in transgenic mice the pattern of expression and particularly in the crypt stem cells to the of genes in mouse models of human has been and B. Cell. 1990; Full Text PDF PubMed Scopus Google could for be efficiently targeting the oncogenes or genes to the villin regulatory use could in the of cell lines derived from the digestive tract targeting a to the of intestinal cells, as used in S. S. H. D. M. S. D. S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: PubMed Scopus Google Scholar, D. 1988; PubMed Scopus Google Scholar, N. R. J. R. Robine S. Pringault E. P. A. J. Cell Sci. 1993; PubMed Google Scholar). and for and the and of for are also to and of the for the in the of transgenic are to for the and for
Pinto et al. (Mon,) studied this question.