Mice carrying Villin-Cre
(Madison et al., 2002
) and Itgb1flox
(Raghavan et al., 2000
) transgenes () were crossed to generate villin-Cre
mice. Intestinal epithelial–specific recombination of the loxP sites was confirmed by PCR (), and loss of β1 integrin protein expression was verified by immunodetection ( and Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200602160/DC1
). Consistent with previous results, Cre-mediated recombination was detected by PCR only in genomic DNA isolated from the small intestine and the proximal large intestine, but not from the stomach or kidneys (; Madison et al., 2002
Figure 1. Conditional deletion of Itgb1 in mouse intestinal epithelium. (A and B) Agarose gels of PCR products of genomic DNA extracted from tail snips of mice hetero- or homozygous for Itgb1flox (A) or expressing the villin-Cre transgene (B). (C) Agarose gel of (more ...)
Villin-Cre/Itgb1flox/flox mouse pups were born at the expected frequencies. At embryonic day 18 and birth, the appearance and size of the pups and intestinal organs were similar in all littermates (unpublished data). However, by postnatal day (P) 4, the villin-Cre/Itgb1flox/flox mice were less than half the body weight of their control littermates (villin-Cre/Itgb1flox/+ or Itgb1flox/flox; ) and died between P7 and P14 from severe malnutrition. The early deaths were not due to a lack of feeding, as the stomachs of all the newborn mice were full of colostrum at the time of death (unpublished data).
The intestinal epithelium not only carries out nutrient absorption but also presents a barrier to the environment. Because of previous reports that β1 integrins mediate IEC survival ex vivo (Strater et al., 1996
), the villin-Cre
mice were carefully examined for evidence of mucosal defects resulting from a loss of IECs. Cleaved caspase 3 immunohistochemistry revealed the presence of rare apoptotic IECs in the villin-Cre
mice and control littermates, but the prevalence of apoptosis was similar (<2% of total IECs counted) in both types of mice (). There was no evidence of mucosal defects or inflammation (specifically, crypt abscesses, intraepithelial leukocytes, and loss of crypts) that would have arisen from barrier defects in the intestines of the villin-Cre
mice (). Conditional deletion of Itgb1
in skin disrupted basement membrane formation (Brakebusch et al., 2000
; Raghavan et al., 2000
), but ultrastructural examination revealed no differences in basement membrane structure between the villin-Cre
mice and their control littermates (). These results suggest that other adhesion molecules mediate epithelial adhesion, survival, and basement membrane formation in the intestine.
Figure 2. Lack of increased intestinal apoptosis or basement membrane disruption in villin-Cre/Itgb1flox/flox mice. (A and B) Immunohistochemical detection of cleaved caspase 3 (brown; arrows) positive IECs in small intestine of control (wt) and villin-Cre/Itgb1 (more ...)
The distal small and proximal large intestines of the villin-Cre/Itgb1flox/flox mice were noticeably larger in external diameter compared with their control littermates (). This was found to be due in part to a dramatic expansion of the intestinal stroma (Fig. S1, C and D), muscularis (Fig. S1, E and F), and ECM (Fig. S1, G–N).
The epithelium in the villin-Cre
mice was markedly expanded compared with the control littermates as well (). The intestinal crypts and villi of P14 villin-Cre
mice () were much larger than those of their control littermates (). In addition, the majority of the crypts in the P14 villin-Cre
mice were dysplastic, as indicated by pseudostratified, enlarged and crowded nuclei, and abnormal crypt architecture in both the small () and large intestines (). The crypt expansion, enlargement, and dysplasia were much more pronounced in the cecum, a specialized portion of the large intestine (). Villous enlargement with expansion of the stroma was apparent, and there were multiple polypoid structures in the small intestinal mucosa of the villin-Cre
mice () but not in their control littermates (). The mucosa overlying the polyps was not dysplastic, indicating normal maturation of the villous epithelium. The polyps had the appearance of juvenile type polyps because of the stromal expansion and cystic dilation of the crypts (Desai et al., 1995
Figure 3. Crypt hyperplasia and dysplasia and villous enlargement in villin-Cre/Itgb1flox/flox mice. Hematoxylin-eosin sections of the intestines of P14 control (WT) and villin-Cre/Itgb1flox/flox (KO) littermates. Duodenal (A and B) and ileal (C and D) sections (more ...)
Figure 4. Small intestinal polyps, lipid inclusions, and defective microvilli in villin-Cre/Itgb1flox/flox mice. (A–D) Transverse jejunal sections from P6 control (A) and villin-Cre/Itgb1flox/flox (B–D) littermates show polyps with stromal expansion (more ...)
Formed stool was found in the large intestines of the villin-Cre
mice but was absent in the large intestines of their control littermates, suggesting diarrhea (Wang et al., 2002
). The intestinal contents of the villin-Cre
mice stained positively for large fat droplets (unpublished data), which was not observed in the control littermates and indicated the presence of steatorrhea and fat malabsorption in the former mice. Fat is absorbed by enterocytes along the length of the small intestine, and examination of intestinal epithelium from villin-Cre
mice revealed large lipid inclusions within the villous enterocytes that were not present in their control littermates (). Total serum lipid levels were significantly reduced in the villin-Cre
mice compared with their control littermates (unpublished data), confirming the presence of fat malabsorption.
Because the villin-Cre
mice appeared to die from severe malnutrition between P7 and P14, the absorptive lineage of IECs (enterocytic) was examined. Expression of the enterocytic marker sodium hydrogen exchanger 3 (NHE3) was detected in the vast majority of the IECs of the villi of the villin-Cre
mice and their control littermates (), demonstrating the abundance of enterocytes in both mice. However, ultrastructural examination of the small intestinal epithelium of villin-Cre
mice by electron microscopy revealed a severely defective microvillus brush border on the apical surfaces of the villous enterocytes (). Microvilli greatly increase the surface area of the intestine for nutrient absorption, are essential for proper nutrition, and express nutrient transporters and digestive enzymes (Davidson et al., 1978
). The intestinal microvilli were diminished in size and poorly formed in the villin-Cre
mice compared with their control littermates (), indicating defective enterocyte differentiation (Smith et al., 1986
). Other IEC lineages were examined as well. The Paneth cell marker, Defensin/Cryptdin5, which was properly restricted to Paneth cells in control mice, was markedly increased in expression by the IECs along the entire crypt–villous axis of the villin-Cre
mice (Fig. S2, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200602160/DC1
). However, electron microscopy revealed that the villous IECs of the villin-Cre
mice expressed microvilli and lacked the secretory granules characteristic of true Paneth cells (unpublished data). The secretory goblet cell lineage was preserved in the villin-Cre
mice and their control littermates (Fig. S2, C and D) as well. Thus, proper cell fate determination occurred in the absence of β1 integrin expression.
The enlarged and dysplastic crypts in the villin-Cre
mice suggested that the aberrant epithelial proliferation could be responsible for the defective enterocytic differentiation. In the control mice, IECs with nuclear immunostaining for the cell cycle progression marker Ki-67 were confined to the crypt bases () as expected. However, the number of IECs with nuclear Ki-67 was greatly increased in the crypts of the villin-Cre
mice () compared with their control littermates. The villin-Cre
mice also demonstrated ectopic foci of Ki-67–positive IEC nuclei in the villi (), which were absent in the control littermates (). Immunodetection of Musashi-1, a putative intestinal stem cell marker (Potten et al., 2003
) revealed an expansion of intestinal stem cells in the crypts of the villin-Cre
mice compared with their control littermates (). Musashi-1 was not detected in the villi of the villin-Cre
mice, suggesting that the ectopic foci of proliferating IECs were not stem cells mislocalized to the villi and were instead properly retained in the bottoms of the crypts. Thus, β1 integrin expression is not necessary for proper intestinal epithelial stem cell localization to the crypt bases.
Figure 5. Intestinal hyperproliferation in the villin-Cre/Itgb1flox/flox mice. (A–D) Ki-67 immunofluorescence (red; nuclei are shown in blue) reveals localization to IEC nuclei in the crypt bases (small arrows) in the jejunum of P6 control mice (A). Increased (more ...)
During the first two postnatal weeks, crypt development in the mouse intestine occurs. Through the use of chimeric mice, it was previously shown that the nascent crypts are initially polyclonal and become monoclonal by P14, suggesting that stem cell selection occurs and yields a single pluripotent progenitor cell in each mature crypt (Wong et al., 2002
). Genetic deletion of Tcf-4 in mice resulted in early postnatal lethality because of a complete lack of IEC proliferation in the nascent crypts, which led to intestinal failure shortly after birth (Korinek et al., 1998
), demonstrating the essential role of Tcf-4 in intestinal stem cell proliferation and maintenance. Although the expression of nuclear Tcf-4 was limited to a few IECs in the bases of the nascent crypts in P6 control mice (), it was expressed by many more IECs in the nascent crypts and even the villi of P6 villin-Cre
mice (). Immunoblotting of nuclear lysates of the IECs confirmed the greater nuclear Tcf-4 protein expression in IECs of the villin-Cre
mice compared with IECs of their control littermates (). In addition, quantitative RT-PCR showed a significantly greater expression of Tcf-4 mRNA in the IECs from the villin-Cre
mice compared with IECs from their control littermates (). Thus, β1 integrin deletion in the intestinal epithelium causes increased and mislocalized expression of Tcf-4 along the crypt–villous axis.
β1 integrins are well known to mediate cell proliferation through extracellular signal–regulated kinase (ERK) activation (for review see Giancotti, 1997
). Examination of intestinal epithelial lysates from P6 villin-Cre
mice and their control littermates failed to show differences in ERK activation that could explain the large differences in IEC proliferation (). This result suggested that other proliferative signaling pathways mediated the hyperproliferation observed in the intestinal epithelium of the villin-Cre
The phenotypic changes observed in the villin-Cre
mice (crypt hyperplasia, defective enterocyte differentiation, severe malnutrition, lipid inclusions, juvenile-like polyps, ectopic intestinal epithelial proliferation, and stromal expansion) were similar to those described in mice with defective Hedgehog signaling (Ramalho-Santos et al., 2000
; Wang et al., 2002
; Madison et al., 2005
). Thus, Hedgehog expression was examined.
Immunodetection and quantitative RT-PCR demonstrated large reductions of Shh and Ihh in the IECs of P6 villin-Cre
mice compared with their control littermates ( and ). In a previous study, neonatal mice with defective intestinal Hh signaling displayed the most severe changes in the distal ileum and cecum, the latter of which was enlarged (Wang et al., 2002
). The crypt hyperplasia in the villin-Cre
mice was most dramatic in the distal small intestine, proximal large intestine, and cecum, which were enlarged in diameter as well ().
Figure 6. Loss of Hedgehog expression in villin-Cre/Itgb1flox/flox mice. (A and B) Immunofluorescent detection of Shh (red) shows crypt (small arrows) and villous (large arrows) small intestine epithelial expression in control (Itgb1flox/flox) mice (A) but not (more ...)
To determine if defective Hh signaling in the intestine could be a cause of aberrant expression of Tcf-4, neonatal mice were randomized to vehicle or the Hh inhibitor cyclopamine treatment for 7 d. Treatment with cyclopamine resulted in increased and mislocalized expression of Tcf-4 along the entire crypt–villous axis, suggesting that the dysregulation of Tcf-4 expression in the villin-Cre/Itgb1flox/flox mice may be due to diminished Hh expression and signaling (Fig. S2, E and F).
To further investigate the regulation of Hh expression by β1 integrins, studies on IECs were performed in vitro. Caco-2 cells differentiate into enterocyte-like and electrically resistant monolayers when postconfluent (Damstrup et al., 1999
). As Caco-2 cells differentiated in culture, β1 integrin as well as Shh and Ihh expression increased (). Overexpression of β1 integrin in subconfluent Caco-2 cells in the presence of fibronectin caused increases in Ihh and Shh protein expression above constitutive levels (). β1 integrin overexpression in a rat intestinal epithelial (RIE) cell line with relatively low levels of Shh expression also increased Shh expression (). These results show that intestinal epithelial Hh expression requires an intact β1 integrin signaling pathway.
Figure 7. β1 integrins regulate Hedgehog and HNF-3β (Foxa2) expression. (A) Western blots of β1 integrin, Ihh, and Shh from cell lysates from Caco-2 cells that were allowed to differentiate in culture. (B) Western blots of V5 (C-terminal (more ...)
Because regional Shh
expression in the central nervous system is dependent on HNF-3β (Foxa2
; Epstein et al., 1999
) and the Shh
promoter contains consensus binding sites for HNF-3β (Chang et al., 1997
; Kitazawa et al., 1998
; Epstein et al., 1999
; Odom et al., 2004
), the role of HNF-3β in mediating β1 integrin–induced Shh expression was examined. HNF-3β protein levels were greatly decreased in nuclear lysates of IECs from P6 villin-Cre
mice compared with their control littermates (). Furthermore, HNF-3β mRNA levels were reduced approximately eightfold in villin- Cre
mice compared with their control littermates (). These results suggest a dependence of HNF-3β expression on β1 integrin expression and signaling. Transfection of subconfluent Caco-2 cells () with full-length human FOXA2
induced SHH expression.
Because β1 integrins can modulate cell proliferation through activation of MAPK and PI3-kinase signaling, confluent RIE cells, which express relatively low constitutive levels of HNF-3β, were cultured in the presence of fibronectin and in the presence or absence of inhibitors of MEK-1 (PD98059) and PI3-kinase (LY294002) to determine how β1 integrins might regulate HNF-3β expression. Although MEK-1 inhibition failed to change HNF-3β expression (unpublished data), PI3-kinase inhibition caused HNF-3β expression to decrease (). When RIE cells with low levels of β1 integrin expression were transiently transfected with a full-length human β1 integrin construct, HNF-3β expression increased (). Furthermore, the PI3-kinase inhibitor LY294002 abrogated this β1 integrin–induced increase in HNF-3β expression (). These studies show that β1 integrin–PI3-kinase signaling stimulates HNF-3β expression, which in turn increases Shh transcription in IECs.