Expression of SV-40 T antigen in the small intestinal epithelium of transgenic mice results in proliferative changes in the crypt and reentry of villus-associated enterocytes into the cell cycle but has no apparent effect on cellular differentiation programs and does not cause neoplastic transformation

The mouse intestinal epithelium represents a unique mammalian system for examining the relationship between cell division, commitment, and differentiation. Proliferation and differentiation are rapid, perpetual, and spatially well-organized processes that occur along the crypt-to-villus axis and involve clearly defined cell lineages derived from a common multipotent stem cell located near the base of each crypt. Nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene were used to establish three pedigrees of transgenic mice that expressed SV-40 large T antigen (TAg) in epithelial cells situated in the uppermost portion of small intestinal crypts and in already committed, differentiating enterocytes as they exited these crypts and migrated up the villus. T antigen production was associated with increases in crypt cell proliferation but had no apparent effect on commitment to differentiate along enterocytic, enteroendocrine, or Paneth cell lineages. Single- and multilabel-immunocytochemical studies plus RNA blot hybridization analyses suggested that the differentiation programs of these lineages were similar in transgenic mice and their normal littermates. This included enterocytes which, based on the pattern of [3H]thymidine and 5-bromo-2'-deoxyuridine labeling and proliferating nuclear antigen expression, had reentered the cell cycle during their migration up the villus. The state of cellular differentiation and/or TAg production appeared to affect the nature of the cell cycle; analysis of the ratio of S-phase to M-phase cells (collected by metaphase arrest with vincristine) and of the intensities of labeling of nuclei by [3H]thymidine indicated that the duration of S phase was longer in differentiating, villus-associated enterocytes than in the less well-differentiated crypt epithelial cell population and that there may be a block at the G2/M boundary. Sustained increases in crypt and villus epithelial cell proliferation over a 9-mo period were not associated with the development of gut neoplasms--suggesting that tumorigenesis in the intestine may require that the initiated cell have many of the properties of the gut stem cell including functional anchorage.     

Use of fetal intestinal isografts from normal and transgenic mice to study the programming of positional information along the duodenal-to-colonic axis.

The four principal cellular constituents of the mouse intestinal epithelium are all derived from a multipotent stem cell functionally anchored near the base of its crypts. Differentiation of enterocytes, enteroendocrine, and goblet cells occurs during an orderly upward migration from monoclonal crypts supplied by a single active stem cell to adjacent polyclonal small intestinal villi or to their colonic homologs, the surface epithelial cuffs. Paneth cells differentiate as they descend to the base of crypts. This epithelium undergoes rapid and perpetual renewal yet is able to maintain cephalocaudal (duodenal-to-colonic) differences in the differentiation programs of its four cell types from the time of its initial cytodifferentiation in late fetal life (embryonic (E) days 16-17). Rat liver fatty acid-binding protein/human growth hormone transgenes (Fabpl/hGH) have been used as novel phenotypic markers to describe the biological properties of gut stem cells and the differentiation programs of their enterocytic and enteroendocrine lineages. To determine whether the multipotent stem cell is able to retain a "positional" address in the absence of luminal signals, we prepared isografts from the proximal small intestine or distal small intestine and colon of E15-E16 Fabpl/hGH transgenic mice and their normal littermates and implanted them into the subcutaneous tissues of young, adult male CBY/B6 nude mice. Immunocytochemical and histochemical studies indicate that appropriate position-specific differences in the differentiation programs of each of the four principal cell lineages are present along the cephalocaudal and crypt-to-villus (or crypt-to-epithelial cuff) axes of isografts harvested 4-6 weeks after implantation. This suggests that the gut stem cell can be characterized not only by its multipotency and enormous capacity for self-renewal but also by its ability to be programmed (? imprinted) with positional information. Transgene expression is reduced in a number of enteroendocrine subpopulations in small intestinal and colonic isografts compared to the intact gut. Moreover, the decision to express the Fabpl/hGH transgene appears to be coordinated between adjacent crypts as evidenced by (i) the presence of multicrypt patches of wholly reporter (hGH)-positive or reporter-negative cells in the intact colon and in colonic isografts and (ii) by the presence of coherent bands of reporter-positive or -negative cells that emanate from adjacent monophenotypic crypts and extend to the apical extrusion zone of distal small intestinal villi.(ABSTRACT TRUNCATED AT 400 WORDS)     

ERBB3 Positively Correlates with Intestinal Stem Cell Markers but Marks a Distinct Non Proliferative Cell Population in Colorectal Cancer

Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts.     

Expression of liver fatty acid-binding protein/human growth hormone fusion genes within the enterocyte and enteroendocrine cell populations of fetal transgenic mice.

The intestinal epithelium establishes and maintains a precise spatial organization despite its continuous and rapid renewal. We have used transgenic mice containing liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to begin to define the molecular mechanisms which are responsible for appropriate regional and cell-specific expression of genes in the gut. Multilabel immunocytochemical methods were employed to characterize the patterns of expression of two transgenes in the enteroendocrine and enterocytic populations of late gestation fetal mice at the time of initial cytodifferentiation of the gastrointestinal epithelium (fetal days 16-19). Surveys of the enteroendocrine cell population using a panel of antibodies directed against 11 neuroendocrine products revealed that these cells are scarce prior to fetal day 17, show a progressive increase in number through day 19, and while the relative proportion of subpopulations (defined by their principal peptide product) are somewhat different than in adults, their geographic distribution along the duodenal to colonic and intervillus(crypt) to villus axes are very similar to that encountered in adult (2-5 month old) mice. Immunoreactive L-FABP is first detectable at fetal day 17 and at this time of first appearance shows an adult pattern of regional enterocytic expression: i.e. it is present in cells overlying nascent villi but not those in the intervillus zone, it is highest in proximal small bowel, declines distally, and is absent from colonocytes. Colocalization studies indicate that L-FABP is not present in enteroendocrine cells during fetal life. Mapping studies indicate that nucleotides -596 to +21 of the rat L-FABP gene are sufficient to reproduce an appropriate temporal, cellular, and regional pattern of reporter (hGH) expression in fetal transgenic mice (with the exception that a subset(s) of enteroendocrine cells, typically containing immunoreactive gastric inhibitory peptide, support transgene but not L-FABP expression). This is in marked contrast to adult transgenic mice where inappropriate hGH accumulation occurs in crypt-associated epithelial cells, in colonocytes, and in many enteroendocrine populations. These studies indicate the importance of considering developmental stage when interpreting the results of any mapping study of cis-acting elements that regulate cell-specific and regional expression of genes in the perpetually renewing intestinal epithelium. Moreover, they also raise the possibility of using transgenes to define fundamental temporal changes in the gut's epithelial cell populations.     

Structure and histochemistry of the normal intestine of the fowl

The fine structures of the development and maturation of intestinal chief cells from the duodenal crypts of the fowl has been described in detail. Three areas within the crypt can be recognised: 1) the crypt base where cell division is frequent but cell development is minimal; (2) the mid-crypt region where rapid growth and development of the cells takes place; and (3) the zone of differentiation where final developments and functional maturation occurs. The structure of the fowl's intestinal crypt is very similar to that described for the mammals. The results are discussed in relation to previous histochemical studies.     

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.     

Development of the pattern of cell renewal in the crypt-villus unit of chimaeric mouse small intestine

We have previously shown that the epithelium of each adult intestinal crypt in chimaeric mice is derived from a single progenitor cell. Whether the crypts are monoclonal from the outset-that is, are formed by the proliferation of a single cell-or whether their formation is initiated by several cells was not known. Here we report that many crypts contain cells of both chimaeric genotypes in the neonatal period indicating a polyclonal origin at this stage of morphogenesis. The cellular organization of the early neonatal crypt is therefore different from that of the adult crypt, which includes a zone of 'anchored' stem cells above the crypt base. Within 2 weeks, however, the crypt progenitor cell and its descendants displace all other cells from the crypt and the crypt attains monoclonality. The distribution of enterocytes on chimaeric villi in the neonate shows a mottled pattern of mosaicism which is progressively replaced by coherent sheets of cells from the crypts, and within two weeks the orderly adult clonal pattern is established.     

Chimeric mice reveal clonal development of pancreatic acini, but not islets

Intestinal crypt stem cells establish clonal descendants. To determine whether the pancreas is patterned by a similar process, we used embryonic stem (ES) cell chimeric mice, in which male ES cells were injected into female blastocysts. Fluorescence in situ hybridization for the Y chromosome (Y-FISH) revealed clonal patterning of ES-derived cells in the adult mouse small intestine and pancreas. Intestinal crypts were entirely male or entirely female. Villi contained columns of male or female epithelial cells, consistent with upward migration of cells from the crypts which surround them. Within the exocrine pancreas, acini were entirely male or entirely female, consistent with patterning from a single stem/progenitor cell. Pancreatic islets contained a mixture of male and female cells, consistent with patterning from multiple progenitors. Male-female chimeric mice demonstrate that the adult mouse exocrine pancreatic acinus is patterned from a single stem/progenitor cell, while the endocrine pancreas arises from multiple progenitors.     

The distribution of endocrine cells along the mouse small intestine

The topographical distribution and incidence of endocrine cells in the crypt and villus epithelium and along the length of the mouse intestine was studied. Cells containing somatostatin and bombesin like reactivity were stained by immunocytochemical techniques using polyclonal antiserum. Most of the somatostatin cells were found in the duodenum, jejunum and ileum, and these cells were generally more frequent on the villus compared to the crypts. This may indicate that the somatostatin cells develop late in the endocrine cell lineage. Bombesin like cells were rare in occurence, and were only present in measureable numbers in the ileum, where they were observed in the crypt and villi. The application of ELISA assays to determine the specificity of the antisera for these peptides is also discussed.     

The distribution of endocrine cells along the mouse small intestine. Bombesin and somatostatin producing cells

The topographical distribution and incidence of endocrine cells in the crypt and villus epithelium and along the length of the mouse intestine was studied. Cells containing somatostatin and bombesin like reactivity were stained by immunocytochemical techniques using polyclonal antiserum. Most of the somatostatin cells were found in the duodenum, jejunum and ileum, and these cells were generally more frequent on the villus compared to the crypts. This may indicate that the somatostatin cells develop late in the endocrine cell lineage. Bombesin like cells were rare in occurrence, and were only present in measureable numbers in the ileum, where they were observed in the crypt and villi. The application of ELISA assays to determine the specificity of the antisera for these peptides is also discussed.     

Neurogenin 3 and the enteroendocrine cell lineage in the adult mouse small intestinal epithelium

It is thought that small intestinal epithelial stem cell progeny, via Notch signaling, yield a Hes1-expressing columnar lineage progenitor and an Atoh1 (also known as Math1)-expressing common progenitor for all granulocytic lineages including enteroendocrine cells, one of the body's largest populations of endocrine cells. Because Neurogenin 3 (Neurog3) null mice lack enteroendocrine cells, Neurog3-expressing progenitors derived from the common granulocytic progenitor are thought to produce the enteroendocrine lineage, although more recent work indicates that Neurog3+ progenitors also contribute to non-enteroendocrine lineages. We aimed to test this model and better characterize the progenitors leading from the stem cells to the enteroendocrine lineage. We investigated clones derived from enteroendocrine precursors and found no evidence of a common granulocytic progenitor that routinely yields all granulocytic lineages. Rather, enteroendocrine cells are derived from a short-lived bipotential progenitor whose offspring, probably via Notch signaling, yield a Neurog3+ cell committed to the enteroendocrine lineage and a progenitor committed to the columnar lineage. The Neurog3+ cell population is heterogeneous; only about 1/3 are slowly cycling progenitors, the rest are postmitotic cells in early stages of enteroendocrine differentiation. No evidence was found that Neurog3+ cells contribute to non-enteroendocrine lineages. Revised lineage models for the small intestinal epithelium are introduced.     

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.     

THE INFLUENCE OF 400 R X-IRRADIATION ON THE NUMBER and THE OCALIZATION OF MATURE and IMMATURE GOBLET CELLS and PANETH CELLS IN INTESTINAL CRYPT and VILLUS

The influence of 400 R X-irradiation on the localization and the number of mature and immature goblet cells and Paneth cells in rat duodenal epithelium has been studied. At short times after irradiation, when the total proliferative activity in the crypts of Lieberkiihn is reduced, the proportion of mature and immature goblet cells of the total number of crypt cells was increased; also an absolute increase in the number of goblet cells in the crypts was found. The immature goblet cells were localized in the lower half of the crypt as in control animals, whereas the number of the mature cells increased over the whole crypt length. When the proliferative activity of the crypt cells increases again from 12 to 48 hr after irradiation the number of both types of goblet cells decreases. Between 48 and 72 hr, when the whole crypt is involved in proliferation, a second increase of both types of goblet cells was found. However, the localization of the immature goblet cells is no longer restricted to the lower half of the crypt but they also appear at the higher cell positions. On the villus no immature goblet cells were found and the changes in the numbers of mature goblet cells do reflect the changes induced by irradiation in the goblet cell population in the crypt. The absolute number and localization of Paneth cells did not change under the experimental conditions. The findings are discussed in relation to cell proliferation and differentiation processes in intestinal crypts.     

Quantitative characteristics of the endocrine cells of the duodenal glands of Carnivora

In the duodenal glands of the Carnivora investigated endocrine elements have been revealed, a part of them is presented as serotonin-producing EC-cells. Endocrine cells are situated in terminal parts and in glandular ducts, among them elements of open and close types are distinguished. Distribution of these cells in the glandular lobules is subjected to the distal gradient regularity, specific for the gastrointestinal tract mucosal membrane. Amount of endocrinocytes in the glands is much less than in the gut crypts. There is no correlation between distribution of the endocrine cells in the glands and in the crypts. The results of unifactor analysis of variance demonstrate a slight effect of the taxonomic position of the species on the number of endocrine cells in the duodenal glands. The proper endocrine apparatus of the duodenal glands is supposed to produce a local regulatory influence on the secretory activity of exogenic glandulocytes, as well as ensure humoral connections of the duodenal glands with other parts of the gastrointestinal tract.