Dab1 and reelin participate in a common signal pathway that controls intestinal crypt/villus unit dynamics

This is an Accepted Manuscript of an article published by Wiley-Blackwell in Biology of the Cell, 106 (3), 83 – 96 on March 2014, available at: https://doi.org/10.1111/boc.201300078 . It is deposited under the terms of the Creative Commons Attribution-NonCommercialNoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. Dab1 and reelin participate in a common signal pathway that controls intestinal crypt/villus unit dynamics Mar ´ ıa D. V a ´ zquez-Carreter o, Pablo Gar c ´ ıa-Miranda, Mar ´ ıa L. Calonge, Mar ı ´ a J. Peral 1 and Anunciacio ´ n A. Ilundain Departamento de Fisiologı´a, Facultad de Farmacia, Universidad de Sevilla, Spain Background information. The myofibroblasts placed underneath the epithelium of the rodent small intestine express reelin, and the reelin absence modifies both the morphology and the cell renewal processes of the crypt–villus unit. In the developing central nervous system, the reelin effects are mediated by the disabled-1 (Dab1) protein. The present work explores whether Dab1 mediates the reelin control of the crypt–villus unit dynamics by examining in the mouse small intestine the consequences of the absence of (i) Dab1 (scrambler mutation) on crypt–villus unit cell renewal processes and morphology and (ii) reelin (reeler mutation) on the intestinal expression of Dab1. Results. The effects of the scrambler mutation on the crypt– villus unit renewal processes are remarkably similar to those caused by the lack of reelin. Thus, both mutations significantly reduce epithelial cell proliferation, migration and apoptosis, and the number of Paneth cells; affect the morphology of the villus, and expand the intercellular space of the adherens junctions and desmosomes. The Western blot assays reveal that the Dab1 isoform present in the enterocytes has a molecular weight of ~63 kDa and that in the brain of ~82 kDa. They also reveal that the absence of reelin increases Dab1 abundance in both brain and enterocytes. Conclusions. All together, the current findings link reelin with Dab1 and suggest that Dab1 functions downstream of reelin action on the homeostasis of the crypt–villus unit. Introduction The homeostasis of the epithelium of the small intestine is preserved through the strict regulation of cell proliferation, growth arrest, migration/ differentiation and apoptosis. Epithelial cells originate from multipotent stem/progenitor cells, located near the bottom of each crypt of Lieberku¨ hn, and cell cycle arrests when cell progenitors reach the crypt– villus junction. As the progeny migrate out of the crypt towards the villus tip, they differentiate in absorptive enterocytes, hormone-secreting enteroendocrine cells, opioid-producing brush cells, microfold cells and mucus-producing Goblet cells, and eventually shed into the lumen within less than a week (van der Flier and Clevers, 2009). The antibacterial peptide-secreting Paneth cells also arise from the multipotent crypt stem cells, but they migrate towards the bottom of the crypt, where they survive for around 6–8 weeks before being eliminated by phagocytosis (Porter et al., 2002). Spontaneous apoptosis in the crypts is rare and it may serve to remove defective/injured progeny cells and senescent Paneth cells (Potten, 1997). Epithelial cell turnover and morphogenesis of the small intestine are controlled by cell–cell and cell-underlying basement membrane interactions (Gumbiner, 1996). The nature of cell–basement membrane interactions and their intracellular processing remain largely undefined. Beneath the epithelia are the myofibroblasts, which orchestrate several functions such as the control of epithelial turnover, tissue repair, inflammation and the immune response. They do so by secreting various substances to the extracellular matrix as well as expressing receptors for many of them, allowing information flow to and from the intestinal epithelium and the extracellular matrix (Andoh et al., 2007; Mifflin et al., 2011). We reported that the myofibroblasts placed underneath the epithelium of the rodent small intestine express reelin (Garc´ıa-Miranda et al., 2010) and that reelin absence (reeler mutation) modifies both the morphology and the cell renewal processes of the crypt–villus unit (Garc´ıa-Miranda et al., 2012, 2013). Within the intestinal mucosa, reelin expression is restricted to the myofibroblasts, but both epithelial cells and myofibroblasts express the reelin effector protein disabled-1 (Dab1) (Garc´ıa-Miranda et al., 2010). In the developing central nervous system, the binding of reelin to its receptors results in tyrosine phosphorylation of Dab1 and in activation of multiple downstream signalling pathway/s, resulting in cytoskeleton remodelling and precise neuronal positioning (Howell et al., 1999). Whereas the information on the cell signalling cascades initiated by the reelin/Dab1 signalling system in brain is profuse, the studies on the role of reelin/Dab1 in non-neural tissues are scarce. The purpose of the current work was to examine whether Dab1 mediates the observed reelin effects on the crypt–villus unit dynamics. To achieve this, we have examined in mice small intestine the consequences of (i) Dab1 gene deficiency (scrambler mice) on the morphology and on epithelial cell proliferation, migration, differentiation and apoptosis and (ii) the absence of reelin (reeler mice) on Dab1 expression in the enterocytes. We have used scrambler mice because they exhibit a phenotype indistinguishable from that of the reeler mice (Sweet et al., 1996; Sheldon et al., 1997) even though the scrambler mice produce about 5% of the normal level of Dab1 protein (Sheldon et al., 1997). A preliminary report of some of these results was published as an abstract (Va´zquez-Carretero et al., 2012). Results Weights of the body and small intestine Dab1-deficient mice (scrambler) are used in this study to elucidate whether Dab1 mediates the reelin-induced effects on the crypt–villus unit homeosta sis previously reported (Garcia-Miranda et al., 2013). The study was started evaluating body and intestinal weights, as well as intestinal length, of 15 and 60 day-old scrambler and control mice. The data summarised in Figure 1 reveal that the body weight of the scrambler mice is similar to that of the control littermates during the suckling period, but smaller in the 60 day-old mice. The scrambler mutation also decreases both the weight and length of the small intestine at the two ages tested. Intestinal morphology In order to determine whether the scrambler mutation affects the morphology of the epithelium of the small intestine, the height and width of the villi and the depth and diameter of the crypts were measured in the jejunum and ileum of 15 and 60 day-old control and scrambler mice. The results summarised in Table 1 show that the scrambler mutation significantly affects the morphology of the villi but not that of the crypts. As compared with control mice, the scrambler villi are shorter, mainly in the jejunum, at the two ages tested, and thinner only in the 60 day-old mice. As the absence of Dab1 reduces the villus length and similarly in the absence of reelin, the cell renewal processes of the crypt–villus unit are modified (Garc´ıa-Miranda et al., 2012, 2013) we decided to examine the effects of the scrambler mutation on epithelial cells to determine whether this mutation also affects epithelial cell proliferation, migration, differentiation and apoptosis. Cell proliferation and migration rates in the epithelium of the small intestine of control and scrambler mice To assess the effects of the scrambler mutation on epithelial cell proliferation and migration rates, the incorporation of BrdU into DNA was measured in the jejunum and ileum of 15 and 60 day-old control and scrambler mice as described in the Materials and Methods section. Cell proliferation was determined by detecting BrdU-marked cells 90 min after the intraperitoneal injection of the marker. The results are given in Figure 2 and show that, in both types of mice and at the two ages tested, BrdU is only observed in nuclei of the crypt cells. The quantification of the ± ± ± marked nuclei reveals that the cell proliferation rate is greater in the 60 dayold mice than in the suckling mice in both control and scrambler mice. The mutation significantly reduces the epithelial cell proliferation rate at the two ages and intestinal regions examined by 26 2%. To evaluate the cell migration rate, intestinal BrdU-marked cells were detected 32 h after injection of the marker and the results are shown in Figure 3. In both types of mice, the cell migration rate along the villi is greater in the 60 day-old mice than in the suckling mice and it is significantly reduced by the mutation in all the experimental conditions tested by 34 ± 4%. Cell apoptosis in the epithelium of the small intestine of control and scrambler mice Cell apoptosis was evaluated by immunological detection of cleaved Caspase-3 in the jejunum and ileum of 15 and 60 day-old control and scrambler mice. The anticleaved Caspase-3 antibody detects on a Western blot a band of 17 kDa that is indicative of cell apoptosis (see Figure 4A). The immunohistochemistry assay reveals that in both control and scrambler mice the apoptotic cells are mainly observed at the villus (Figure 4C). In control mice, both the density of the 17-kDa band (Figure 4A) and the number of apoptotic cells (Figure 4B) are higher in the 60 day-old than in the 15 day-old mice. The scrambler mutation decreases the density of the band (42 5% decrease) and the number of apoptotic cells (59 4% decrease) in all the experimental conditions tested. Cell differentiation in the epithelium of the small intestine of control and scrambler mice Epithelial cell differentiation was evaluated by measuring the number of Goblet and Paneth cells in the jejunum and ileum of 15 and 60 day-old control and scrambler mice. Goblet cells were identified by the periodic acidShiff (PAS) ± staining system, as described in Materials and Methods section, and Figure 5 shows that the number of PAS positive cells increases with the age in both types of mice. The scrambler mutation decreases the number of Goblet cells, mainly in the 15 day-old jejunum. No significant differences are observed in the 60 day-old jejunum. Paneth cells were quantified by immuno-detection of lysozyme and the results are summarised in Figure 6. They reveal that age increases the number of Paneth cells in both control and scrambler mice and that the mutation reduces their number at the two ages and intestinal regions examined by 29,3%. Electron microscopy studies The results discussed so far reveal that the scrambler mutation reduces epithelial cell proliferation, migration, differentiation and apoptosis. Because intercellular junctions control these processes (Gumbiner, 1996; Herve´, 2009) and the reeler mutation affects those junctions in the intestinal epithelium (Garc´ıaMiranda et al., 2013), the effects of the scrambler mutation on the cell-to-cell junctions were examined at the electron microscopy. In the intestinal epithelium, the intercellular junctions are located on the apical side of the lateral cell membrane and form the apical junctional complex that comprises tight junctions (TJ), adherens junctions (AJ) and desmosomes. The microphotographs of Figure 7 reveal that the TJ appear normal, but the intercellular space of both the AJ and desmosomes is significantly wider in the scrambler than in the control mice. Immunolocalisation of E-cadherin and β -catenin in the epithelium of the small intestine of control and scrambler mice Since the electron microscopy studies revealed that the scrambler mice present AJ with wider intercel lular space than the control mice, the cell location of Ecadherin in the epithelium of the control and scrambler small intestine was detected by immunohistochemistry (Figure 8). βcatenin associates with the cytosolic domain of the E-cadherin and regulates several cell processes. As its cell membrane location in part depends on the amount of Ecadherin present in the cell membrane, β-catenin localisation was also in vestigated by immunostaining in both types of mice (Figure 8). The specific signal produced by the anti-Ecadherin antibody is seen at the lateral membrane of the epithelial cells in both control and scrambler mice. β-Catenin-specific staining is also concentrated at the lateral cell membrane. No significant differences between the two types of mice are observed, indicating that the mutation does not modify the cell location of either protein. Immunoreactive signal was not seen in the absence of the primary antibodies (data not shown). Dab1 protein in enterocytes isolated from control, reeler and scrambler mice The last set of experiments was designed to (i) determine the Dab1 isoform expressed in mice enterocytes,( ii) compare the intestinal isoform with that expressed in brain and (iii) determine whether reelin modifies the intestinal expression of Dab1. This was done by Western blot assays using an anti-Dab1 antibody raised against the C terminus of Dab1 that recognises all Dab1 isoforms. The specificity of the antibody was verified using brain and enterocytes isolated from the scrambler mice, which should show drastic reduction in Dab1 expression on a Western blot. The results are given in Figure 9A. The bands detected by the antiDab1 antibody that is absent in the scrambler tissues are a polypeptide band at ~63 kDa in the enterocytes and a polypeptide of ~82 kDa in the brain. To test whether reelin modifies the expression of Dab1 its abundance was measured in enterocytes isolated from control and reeler mice. Figure 9A shows that the reeler mutation increases Dab1 abundance by a factor of approximately 2 relative to control in both enterocytes and brain. The Western blot does not provide evidence on the subcellular location of Dab1, which was investigated by immunocytochemistry. Figure 9B reveals that the specific signal produced by the anti-Dab1 antibody is seen throughout the cytosol, and in some cells the signal is stronger at the terminal web domain. The specific labelling was absent from the enterocytes isolated from scrambler mice. Discussion The reelin-signalling system and its role in tissues other than the brain is poorly understood. We reported that in the rodent small intestine reelin is released by the myofibroblasts placed underneath the epithelium and involved in the homeostasis of the crypt–villus unit (Garc´ıaMiranda et al., 2010, 2012, 2013). The current work extends the knowledge of the reelin-signalling system by revealing that in the small intestine Dab1 also might transmit the reelin signal to cytosolic signalling pathway/s, which ultimately might affect the cell renewal processes of the crypt–villus unit. As compared with the brain, mouse enterocytes express significant amounts of Dab1 and a different Dab1 isoform: the enterocyte isoform is ~63 kDa and that in the brain ~82 kDa. Both the size of the bands and the differences between brain and enterocytes are consistent with the high diversity observed in Dab1 expression. Dab1 polypeptide bands ranging from 36 to 120 kDa have been identified in mouse embryonic brain, the 80-kDa Dab1 being the predominant form (Howell et al., 1997). It has also been reported that the Dab1 forms resulting from alternative splicing are species specific, have different tissue expression profiles and sometimes are expressed in the same tissue at the same stage of development or in different subpopulations of cells depending on the stage of development (Bar et al., 2003; Katyal and Godbout, 2004; Costagli et al., 2006; Gao et al., 2010; Long et al., 2011; Gao et al., 2012). The physiological meaning of the differing presence of a Dab1 isoform in the small intestine from that in brain is unclear at present. The molecular weight of the intestinal isoform is close to that of the “Dab1 early (Dab1-E) isoform” expressed in the progenitor cells of the human/chicken retina and chicken embryos gut (Gao et al., 2010; Katyal et al., 2011). A “late (Dab1-L) isoform” (commonly referred to as Dab1) has been isolated from differen tiating retinal cells (Gao et al., 2010). The Dab1-E is missing two tyrosine ± The results are given as the number of positive lysozyme cells per crypt. Electron microscopy assays Segments of small intestine were fixed in 4% glutaralde hyde/0.1 mol/l sodium cacodylate, pH 7.4 at 4°C for 3 h. After three rinses in cacodylate-buffered solution, the tissues were postfixed in 1% OsO4 in 0.1 M phosphate buffer at 4°C for 1 h and washed in cacodylate-buffered solution containing 7.5% sucrose. The segments were then dehydrated in a graduated series of acetone (30%, 50% and 70%), stained with 2% uranyl acetate and embedded in Spurr’s epoxy resin. Ultrathin sections were examined under a Philips CM-10 transmission electron microscope equipped with an Olympus Veleta. The photographs were processed with iTEM software and ImageJ program version 1.46 (National Institutes for Health, http://rsb.info.nih.gov/ij/index.html). Statistical analysis Data are presented as mean SEM. The number of animals is indicated in the legends. In Figures 7 and 9 comparisons between different experimental groups were evaluated by the two-tailed Student’s t test. One-way ANOVA followed by the Newman– Keuls’ test was used for multiple comparisons (GraphPad Prism program). Differences were set to be significant for P < 0.05. Author contribution M.J.P. and A.A.I. conceived and designed the experiments. M.D.V-C, P.G-M. and M.L.C. performed the experiments. M.J.P., M.L.C. and A.A.I. analysed and interpreted the data. A.A.I. carried out the discussion and wrote the paper. Acknowledgements W e thank Dr. O. Pintado (Centro de Producci o ´ n y E xperimentaci o ´ n Animal, U niversidad de Sevilla), Dr. L. Collinson (Cancer Research Institute of London), Dr. F. Romero and Dr. N. Wright’s labora tory (Cancer Research Institute of London) for their technical advice and R. Caracuel for technical support The antilysozyme and anti-β-catenin antibod ies were kindly gifted by Dr. N. Wright. Electronmicroscopy images were obtained in the Centro de Investigaci o ´ n, T ecnolog ´ ıa e I nnovaci o ´ n, U niversidad de Sevilla. Funding This work was supported by a grant from the Junta de Andalucia (CTS 884) and by a fellowship from the Spanish Ministerio de Educaci o ´ n y Ciencia (AP200704201) to M.D. Vazquez-Carretero. Conflict of interest statement The authors have declared no conflict of interest. References Andoh, A., Bamba, S., Brittan, M., Fujiyama, Y. and Wright, N.A. (2007) Role of intestinal subepithelial myofibroblasts in inflammation and regenerative response in the gut. Pharmacol. Ther. 114, 94–106 Arnaud, L., Ballif, B.A. and Cooper, J.A (2003). Regulation of protein tyrosine kinase signaling by substrate degradation during brain development. Mol. Cell Biol. 23, 9293-9302 Bar, I., Tissir, F., Lambert de Rouvroit, C., De Backer, O. and Goffinet, A.M. (2003) The gene encoding disabled-1 (DAB1), the intracellular adaptor of the Reelin pathway, reveals unusual complexity in human and mouse. J. Biol. Chem. 278, 5802–5812 Bento-Abreu, A., Velasco, A., Polo-Hernandez, E., Lillo, C., Kozyraki, R., Tabernero, A. and Medina, J.M. (2009) Albumin endocytosis via megalin in astrocytes is caveolaand Dab-1 dependent and is required for the synthesis of the neurotrophic factor oleic acid. J. Neurochem. 111, 49–60 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 Bock, H.H., Jossin, Y., May, P., Bergner, O. and Herz, J. (2004) Apolipoprotein E receptors are required for reelin-induced proteasomal degradation of the neuronal adaptor protein Disabled-1. J. Biol. Chem. 279, 33471–33479 Bryja, V., Grad, l.D., Schambony, A., Arenas, E. and Schulte, G. (2007) Beta-arrestin is a necessary component of Wnt/beta-catenin signaling in vitro and in vivo. Proc. Natl. Acad. Sci. USA. 104, 6690–6695 Costagli, A., Felice, B., Guffanti, A., Wilson, S.W. and Mione, M. (2006) Identification of alternatively spliced dab1 isoforms in zebrafish. Dev. Genes Evol. 216, 291–299 D’Arcangelo, G., Miao, G.G. and Curran, T. (1996) Detection of the reelin breakpoint in reeler mice. Brain Res. Mol. Brain Res. 39, 234–236 D’Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I. and Curran, T. (1995) A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 374, 719–723 Gao, Z., Monckton, E.A., Glubrecht, D.D., Logan, C. and Godbout, R. (2010) The early isoform of disabled-1 functions independently of Reelin-mediated tyrosine phosphorylation in chick retina. Mol. Cell Biol. 30, 4339–4353 Gao, Z., Poon, H.Y., Li, L., Li, X., Palmesino, E., Glubrecht, D.D., Colwill, K., Dutta, I., Kania, A., Pawson, T. and Godbout, R. (2012) Splice-mediated motif switching regulates disabled-1 phosphorylation and SH2 domain interactions. Mol. Cell Biol. 32, 2794–2808 Garcıa-Miranda, P., Peral, M.J. and Ilundain, A.A. (2010) Rat small intestine expresses the reelin-disabled-1 signalling pathway. Exp. Physiol. 95, 498–507 Garcıa-Miranda, P., Vazquez-Carretero, M.D., Gutierrez, G., Peral, M.J., and Ilunda´ in, A.A. (2012) Lack of reelin modifies the gene expression in the small intestine of mice. J. Physiol. Biochem. 68, 205–218 Garcıa-Miranda, P., Vazquez-Carretero, M.D., Sesma, P., Peral, M.J. and Ilundain, A. A. (2013) Reelin is involved in the crypt-villus unit homeostasis. Tissue Eng. 19, 188–198 Gumbiner, B.M. (1996). Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84, 345–357 Herve , J.C. (2009). The apical junctional complexes, roles, and dysfunctions. Biochim. Biophys. Acta 1788, 753–754 Honda, T. and Nakajima, K. (2006) Mouse Disabled1 (DAB1) is a nucleocytoplasmic shuttling protein. J. Biol. Chem. 281,38951–38965 Howell, B.W., Gertler, F.B. and Cooper, J.A. (1997) Mouse disabled (mDab1): a Src binding protein implicated in neuronal development. EMBO J. 16, 121– 132 Howell, B.W., Herrick, T.M. and Cooper, J.A. (1999) Reelin-induced tyrosine phosphorylation of Disabled 1 during neuronal positioning. Genes Dev. 13, 643– 648 Katyal, S., Glubrecht, D.D., Li, L., Gao, Z. and Godbout, R. (2011) Disabled-1 alternative splicing in human fetal retina and neural tumors. PLoS One 6, e28579 Katyal, S. and Godbout, R. (2004) Alternative splicing modulates Disabled-1 (Dab1) function in the developing chick retina. EMBO J. 23, 1878–1888 Khialeeva, E., Lane, T.F. and Carpenter, E.M. (2011) Disruption of reelin signaling alters mammary gland morphogenesis. Development 138, 767–776 Lau, C., Lytle, C., Straus, D.S. and DeFea, K.A. (2011) Apical and basolateral pools of proteinase-activated receptor-2 direct distinct signaling events in the intestinal epithelium. Am. J. Physiol. Cell Physiol. 300, C113–C123 Long, H., Bock, H.H., Lei, T., Chai, X., Yuan, J., Herz, J., Frotscher, M. and Yang, Z. (2011) Identification of alternatively spliced Dab1 and Fyn isoforms in pig. BMC Neurosci. 12, 17–31 Martın-Lopez, E., Blanchart, A., De Carlos, J.A. and Lopez-Mascaraque, L. (2011) Dab1 (Disable homolog-1) reelin adaptor protein is overexpressed in the olfactory bulb at early postnatal stages. PLoS One 6, e26673 Massalini, S., Pellegatta, S., Pisati, F., Finocchiaro, G., Farace, M.G. and Ciafre, S.A. (2009) Reelin affects chain-migration and differentiation of neural precursor cells. Mol. Cell Neurosci. 42, 341–349 Mifflin, R.C., Pinchuk, I.V., Saada, J.I. and Powell, D.W. (2011)Intestinal myofibroblasts: targets for stem cell therapy. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G684-G696 Peral, M.J., Garcıa-Delgado, M., Calonge, M.L., Dura´ n, J.M., De La Horra, M.C., Wallimann, T., Speer, O. and Ilundain, A.A. (2002) Human, rat and chicken small intestinal Na+–Cl–-creatine transporter: functional, molecular characterization and localization.J. Physiol. 545, 133–144 Porter, E.M., Bevins, C.L., Ghosh, D. and Ganz, T. (2002) The multifaceted Paneth cell. Cell. Mol. Life Sci. 59, 156–170 Potten, C.S. (1997) Epithelial cell growth and differentiation. II. Intestinal apoptosis. Am. J. Physiol. 273, 253–257 Rice, D.S., Sheldon, M., D’Arcangelo, G., Nakajima, K., Goldowitz, D. and Curran, T. (1998) Disabled-1 acts downstream of Reelin in a signaling pathway that controls laminar organization in the mammalian brain. Development 125, 3719–3729 Sheldon, M., Rice, D.S., D’Arcangelo, G., Yoneshima, H., Nakajima, K., Mikoshiba, K., Howell, B.W., Cooper, J.A., Goldowitz, D. and Curran, T. (1997) Scrambler and yotari: disrupt the disabled gene and produce a reeler-like phenotype in mice. Nature 389, 730–733 Sweet, H.O., Bronson, R.T., Johnson, K.R., Cook, S.A. and Davisson, M.T. (1996) Scrambler, a new neurological mutation of the mouse with abnormalities of neuronal migration. Mamm. Genome. 7, 798–802 Usman, N., Tarabykin, V. and Gruss, P. (2000) The novel PCR-based technique of genotyping applied to identification of scrambler mutation in mice. Brain Res. Brain Res. Protoc. 5, 243–247 van der Flier, L.G. and Clevers, H. (2009) Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241– 260 Vazquez-Carretero, M.D., Garcıa-Miranda, P., Calonge, M.L., Calvo, E., Lopez, J.A., Romero, F., Ilundain, A.A. and Peral, M.J. (2012) Disabled-1 protein in the intestine. Genes Nutr. 6, S75 Yeung, T.M., Chia, L.A., Kosinski, C.M. and Kuo, C.J. (2011) Regulation of selfrenewal and differentiation by the intestinal stem cell niche. Cell. Mol. Life Sci. 68, 2513–2523 Figure 1 Body weight and small intestinal weight and length of control and scrambler mice Data are presented as the means ± SEM of five different animals per age. One-way ANOVA showed an effect of age and mutation on body and small intestine weights (P < 0.001). Newman–Keuls’ test **P < 0.001 and *P < 0.05 scrambler versus control mice, aP < 0.01 versus 15 day-old mice. Figure 2 Cell proliferation rate in the intestinal epithelium of control and scrambler mice The number of BrdU-labelled cells was determined with a monoclonal anti-BrdU antibody (1:300 dilution) in 30 well-oriented longitudinally crypts per mouse by light microscopy. Ten micrometre cryosections were used. (A) Representative sections of intestinal crypts. Scale bar = 25 and 10 µm in the inset. (B) Means ± SEM of the number of BrdUlabelled cells per crypt. The number of animals used per age was three scrambler and three control mice. One-way ANOVA showed an effect of mutation and age on cell proliferation (P < 0.001). Newman– Keuls’ test: *P < 0.001 scrambler versus control mice, aP < 0.001 versus 15 day-old mice. Table 1 Morphometric parameters of small intestinal mucosa of scrambler and control mice Villi Crypts Height Width Depth Diameter 323* ± 11 162* ± 6 442*a ± 9 198*a ± 4 73* ± 2 Means ± SEM of intestinal mucosa measurements (in µm). Five scrambler and five control mice per age were used. One-way ANOVA Mice Jejunum Ileum Jejunum Ileum Jejunum Ileum Jejunum Ileum Age 15 days Control: Scrambler: 396 ± 9 199 ± 4 70 ± 1 58 ± 1 64 ± 2 54 ± 2 40 ± 1 36 ± 1 33 ± 1 Age 60 days Control: Scrambler: 483 a ± 12 210 a ± 3 80 a ± 2 69 a ± 3 77 a ± 2 72 a ± 1 46 a ± 1 43 a ± 1 43 a ± 1 Figure 3 Cell migration rate in the intestinal epithelium of control and scrambler mice The distance between the foremost BrdU-labelled cells and the base of the villi was measured and used to determine the cell migration rate. Ten micrometre cryosections were used. (A) Representative sections of mice intestine. Lines indicate the starting and the front point of labelled cells used to evaluate the enterocyte migration rate. Scale bar = 50 µm. (B) Means ± SEM of the distance (in µm) migrated per hour. The number of animals used per age was three scrambler and three control mice. One-way ANOVA showed an effect of both, mutation and age on cell migration rate (P < 0.001). Newman–Keuls’ test: *P < 0.001 scrambler versus control mice, aP < 0.001 versus 15 day-old mice.