CVD: the intestinal crypt/villus in situ hybridization database

The intestinal crypt/villus in situ hybridization database (CVD) query interface is a web-based tool to search for genes with similar relative expression patterns along the crypt/villus axis of the mammalian intestine. The CVD is an online database holding information for relative gene expression patterns in the mammalian intestine and is based on the scoring of in situ hybridization experiments reported in the literature. CVD contains expression data for 88 different genes collected from 156 different in situ hybridization profiles. The web-based query interface allows execution of both single gene queries and pattern searches. The query results provide links to the most relevant public gene databases. AVAILABILITY: http://pc113.imbg.ku.dk/ps/     

Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na(+) bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na(+)/H(+) exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunner's glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl(-)-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl(-) secretion by goblet cells and Cl(-) and HCO(3)(-) secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.     

Cell migration pathway in the intestinal epithelium: an in situ marker system using mouse aggregation chimeras

The cell migration pathway in the intestinal epithelium of DDK in equilibrium C57BL/6JLac mouse chimeras is demonstrated using Dolichos biflorus agglutinin-peroxidase as strain-specific marker. Cell sheets of one genotype extend in relatively straight lines from crypt to villus apex. Narrow sheets are mostly interrupted in the distal two-thirds of duodenal but not ileal villi, suggesting that in the duodenum cell loss occurs below the apical extrusion zone. These differences between duodenum and ileum correspond to differences in villus shape. The pattern of cell migration in Peyer's patch epithelium is consistent with that of the duodenum. In chimeric colon, sharply demarcated territories of crypts with a narrow cuff of surface epithelium represent the counterpart of the villus/crypt unit of the small intestine.     

Murine intestinal organoids resemble intestinal epithelium in their microRNA profiles

Intestinal organoids were established as an ex vivo model of the intestinal epithelium. We investigated whether organoids resemble the intestinal epithelium in their microRNA (miRNA) profiles. Total RNA samples were obtained from crypt and villus fractions in murine intestine and from cultured organoids. Microarray analysis showed that organoids largely resembled intestinal epithelial cells in their miRNA profiles. In silico prediction followed by qRT-PCR suggested that six genes are regulated by corresponding miRNAs along the crypt-villus axis, suggesting miRNA regulation of epithelial cell renewal in the intestine. However, such expression patterns of miRNAs and their target mRNAs were not reproduced during organoids maturation. This might be due to lack of luminal factors and endocrine, nervous, and immune systems in organoids and different cell populations between in vivo epithelium and organoids. Nevertheless, we propose that intestinal organoids provide a useful in vitro model to investigate miRNA expression in intestinal epithelial cells.     

Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB

In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. We show here that beta-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3(-/-) mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. We conclude that in the intestinal epithelium beta-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system.     

Existence of serotonin and neuropeptides-immunoreactive endocrine cells in the small and large intestines of the mole-rats (Spalax leucodon)

The present study was conducted to clarify the regional distribution and relative frequency of endocrine cells secreting serotonin, substance P (SP), cholecystokinin-8 (CCK-8), vasoactive intestinal polypeptide (VIP) and neurotensin in the small and large intestine of the mole-rats (Spalax leucodon), by specific immunohistochemical methods. In the small and large intestine of mole-rats (Spalax leucodon), serotonin, SP and VIP were identified with various frequencies, but CCK-8 and neurotensin were not observed. Most of the IR cells in the small and large intestine were located in the intestinal crypt and epithelium however, they were more frequency in the intestinal crypt. Serotonin-IR cells were detected throughout the whole intestinal tract, predominantly in the duodenum and colon. SP-IR cells were demonstrated throughout the whole intestinal tract except for the ileum and rectum with highest frequencies in the cecum. VIP-IR cells were found in all parts of the small intestine except for the large intestine.In conclusion, the general distribution patterns and relative frequency of intestinal endocrine cells of the mole-rats (Spalax leucodon) was similar to those of some rodent species. However, some species-dependent unique distributions and frequencies characteristics of endocrine cells were also observed in the present study.     

Differentiation-dependent expression and localization of the class B type I scavenger receptor in intestine

The current study used the human Caco-2 cell line and mouse intestine to explore the topology of expression of the class B type I scavenger receptor (SR-BI) in intestinal cells. Results showed that intestinal cells expressed only the SR-BI isoform with little or no expression of the SR-BII variant. The expression of SR-BI in Caco-2 cells is differentiation dependent, with little or no expression in preconfluent undifferentiated cells. Analysis of Caco-2 cells cultured in Transwell porous membranes revealed the presence of SR-BI on both the apical and basolateral cell surface. Immunoblot analysis of mouse intestinal cell extracts demonstrated a gradation of SR-BI expression along the gastrocolic axis of the intestine, with the highest level of expression in the proximal intestine and decreasing to minimal expression levels in the distal intestine. Immunofluorescence studies with SR-BI-specific antibodies also confirmed this expression pattern. Importantly, the immunofluorescence studies also revealed that SR-BI immunoreactivity was most intense in the apical membrane of the brush border in the duodenum. The crypt cells did not show any reactivity with SR-BI antibodies. The localization of SR-BI in the jejunum was found to be different from that observed in the duodenum. SR-BI was present on both apical and basolateral surfaces of the jejunum villus. Localization of SR-BI in the ileum was also different, with little SR-BI detectable on either apical or basolateral membranes.Taken together, these results suggest that SR-BI has the potential to serve several functions in the intestine. The localization of SR-BI on the apical surface of the proximal intestine is consistent with the hypothesis of its possible role in dietary cholesterol absorption, whereas SR-BI present on the basolateral surface of the distal intestine suggests its possible involvement in intestinal lipoprotein uptake.     

Influence of high-calorie (cafeteria) diets on the population of Paneth cells in the small intestine of the rat

A high-calorie (cafeteria) diet is known to cause changes in the intestinal morphology and functioning that seem to be related to calorie overfeeding. Among the cell lineages found in the small intestine epithelium, the Paneth cell (PC) population is known to be influenced by factors related mainly to the intestinal microbiota. The role of PCs in the intestinal cell concert remains unclear, because experimental evidence suggests PC involvement in local processes other than protection against pathogens. Participation of PC in digestive mechanisms has been proposed on this basis. We have analyzed the effect of high-carbohydrate (HC) and high-fat (HF) cafeteria diets on the PC population in the small intestine of the adult rat. For 8 weeks, both HC and HF diets caused a gain in body weight, but whereas the HC-fed rats showed reduced counts of intestinal crypts per 5-mum section, the HF-fed group showed the opposite. In control rats, the number of crypts per section showed a slight tendency to decrease along the duodenum - ileum axis, whereas the number of PCs per crypt was increased towards the ileum. As a result, the number of PCs per section (calculated from these data) remained constant along the three segments of the intestine. The hypercaloric diets did not modify the general tendencies seen in the crypt and PC counts, but reduced the number of PCs per section in the duodenum by 50%. HC-fed, but not HF-fed, rats showed a similar reduction in jejunum also. These changes do not correlate particularly with any of the predictable effects of diet composition, so that a multifactorial control of PC density is proposed.     

Na-glutamine co-transporters B(0)AT1 in villus and SN2 in crypts are differentially altered in chronically inflamed rabbit intestine

Glutamine is a major nutrient utilized by the intestinal epithelium and is primarily assimilated via Na-glutamine co-transport (NGcT) on the brush border membrane (BBM) of enterocytes. Recently we reported that B(0)AT1 (SLC6A19) mediates glutamine absorption in villus while SN2 (SLC38A5) does the same in crypt cells. However, how B(0)AT1 and SN2 are affected during intestinal inflammation is unknown. In the present study it was shown that during chronic enteritis NGcT was inhibited in villus cells, however, it was stimulated in crypt cells. Our studies also demonstrated that the mechanism of inhibition of NGcT during chronic enteritis was secondary to a reduction in the number of B(0)AT1 co-transporters in the villus cell BBM without a change in the affinity of the co-transporter. In contrast, stimulation of NGcT in crypt cells was secondary to an increase in the affinity of SN2 for glutamine without an alteration in the number of co-transporters. Thus, glutamine assimilation which occurs via distinct transporters in crypt and villus cells is altered in the chronically inflamed intestine.     

Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection

The intestinal epithelium undergoes a marked adaptive response following loss of functional small bowel surface area characterized by increased crypt cell proliferation and increased enterocyte migration from crypt to villus tip, resulting in villus hyperplasia and enhanced nutrient absorption. Hedgehog (Hh) signaling plays a critical role in regulating epithelial-mesenchymal interactions during morphogenesis of the embryonic intestine. Our previous studies showed that blocking Hh signaling in neonatal mice results in increased small intestinal epithelial crypt cell proliferation and altered enterocyte fat absorption and morphology. Hh family members are also expressed in the adult intestine, but their role in the mature small bowel is unclear. With the use of a model of intestinal adaptation following partial small bowel resection, the role of Hh signaling in the adult gut was examined by determining the effects of blocking Hh signaling on the regenerative response following loss of functional surface area. Hh-inactivating monoclonal antibodies or control antibodies were administered to mice that sustained a 50% intestinal resection. mRNA analyses of the preoperative ileum by quantitative real-time PCR revealed that Indian hedgehog was the most abundant Hh family member. The Hh receptor Patched was more abundant than Patched 2. Analyses of downstream targets of Hh signaling demonstrated that Gli3 was twofold more abundant than Gli1 and Gli2 and that bone morphogenetic protein (BMP)2 was most highly expressed compared with BMP1, -4, and -7. Following intestinal resection, the expression of Hh, Patched, Gli, and most BMP genes was markedly downregulated in the remnant ileum, and, in anti-Hh antibody-treated mice, expression of Patched 2 and Gli 1 was further suppressed. In Hh antibody-treated mice following resection, the enterocyte migration rate from crypt to villus tip was increased, and by 2 wk postoperation, apoptosis was increased in the adaptive gut. However, crypt cell proliferation, villus height, and crypt depth were not augmented. These data indicate that Hh signaling plays a role in adult gut epithelial homeostasis by regulating epithelial cell migration from crypt to villus tip and by enhancing apoptosis.     

c-Myc is required for the formation of intestinal crypts but dispensable for homeostasis of the adult intestinal epithelium

In self-renewing tissues such as the skin epidermis and the bone marrow, Myc proteins control differentiation of stem cells and proliferation of progenitor cell types. In the epithelium of the small intestine, we show that c-Myc and N-Myc are expressed in a differential manner. Whereas c-Myc is expressed in the proliferating transient-amplifying compartment of the crypts, N-Myc is restricted to the differentiated villus epithelium and a single cell located near the crypt base. c-Myc has been implicated as a critical target of the canonical Wnt pathway, which is essential for formation and maintenance of the intestinal mucosa. To genetically assess the role of c-Myc during development and homeostasis of the mammalian intestine we induced deletion of the c-myc(flox) allele in the villi and intestinal stem cell-bearing crypts of juvenile and adult mice, via tamoxifen-induced activation of the CreER(T2) recombinase, driven by the villin promoter. Absence of c-Myc activity in the juvenile mucosa at the onset of crypt morphogenesis leads to a failure to form normal numbers of crypts in the small intestine. However, all mice recover from this insult to form and maintain a normal epithelium in the absence of c-Myc activity and without apparent compensation by N-Myc or L-Myc. This study provides genetic and molecular evidence that proliferation and expansion of progenitors necessary to maintain the adult intestinal epithelium can unexpectedly occur in a Myc-independent manner.     

Distribution of 3-hydroxy-3-methylglutaryl-CoA reductase in isolated villus and crypt cells of chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), the major rate-limiting enzyme of cholesterogenesis, was studied in epithelial cells isolated in a villus to crypt gradient from chick duodenum, jejunum and ileum, in order to resolve the apparent controversy that exists on the anatomical localization of sterol synthesis in the intestine. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villus cells, and thymidine kinase, specific to the crypt cells. No relative difference in stability was observed, as shown by the equal distribution of acid phosphatase. Cells were 90-95 per cent viable. The highest specific activity of reductase was located in the microsomal fraction (41 per cent of the total). The mitochondria had lower specific activity (8 per cent of the total). The distribution of reductase activity in epithelial cells of the villus-crypt axis was also studied. The specific activity in each cell fraction from chick duodenum was clearly lower than that in jejunum and ileum. The jejunal and ileal crypt regions showed lower specific activity than the villus cells. About 70 per cent of total reductase activity was found in cells from the upper and the mid villus fraction in each intestinal segment.     

Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats

Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.     

Expression of functionally distinct variants of the beta(4)A integrin subunit in relation to the differentiation state in human intestinal cells.

Integrins are important mediators of cell-laminin interactions. In the small intestinal epithelium, which consists of spatially separated proliferative and differentiated cell populations located, respectively, in the crypt and on the villus, laminins and laminin-binding integrins are differentially expressed along the crypt-villus axis. One exception to this is the integrin alpha(6)beta(4), which is thought to be ubiquitously expressed by intestinal cells. However, in this study, a re-evaluation of the beta(4) subunit expression with different antibodies revealed that two forms of beta(4) exist in the human intestinal epithelium. Furthermore, we show that differentiated enterocytes express a full-length 205-kDa beta(4)A subunit, whereas undifferentiated crypt cells express a novel beta(4)A subunit that does not contain the COOH-terminal segment of the cytoplasmic domain (beta(4)A(ctd-)). This new form was not found to arise from alternative beta(4) mRNA splicing. Moreover, we found that these two beta(4)A forms can associate into alpha(6)beta(4)A complexes; however, the beta(4)A(ctd-) integrin expressed by the undifferentiated crypt cells is not functional for adhesion to laminin-5. Hence, these studies identify a novel alpha(6)beta(4)A(ctd-) integrin expressed in undifferentiated intestinal crypt cells that is functionally distinct.     

Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development

Background

Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations.

Methods

VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration.

Results

Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2.

Conclusions

Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future cell therapies for intestinal dysfunction or disease.