Modulation of specific intestinal epithelial progenitors by enteric neurons.

The proglucagon-derived peptide glucagon-like peptide 2 (GLP-2), a product of a subset of gut epithelial cells, is pursued clinically for its ability to stimulate gut epithelial growth and repair. Here we show that although specific epithelial progenitors respond to GLP-2 administration, the epithelium does not express the GLP-2 receptor. Rather, enteric neurons express the receptor, respond to GLP-2, and transmit a signal (which can be blocked by the voltage-gated sodium channel inhibitor tetrodotoxin) back to the epithelium. Thus the nervous system is a key component of a feedback loop regulating epithelial growth and repair.

Colossal crypts bordering colon adenomas in Apc(Min) mice express full-length Apc.

Enlarged but nondysplastic crypts are frequently observed at the margins of colon tumors, forming what has been called a transitional epithelium. It is now thought that this is a reactive state and not a preneoplastic condition as previously suggested. We have used the mouse familial adenomatous polyposis model, ApcMin, to study these abnormal adenoma-associated crypts. We report that these nondysplastic crypts are enormous (as much as 10 times normal length) and branch more frequently than normal crypts. They express wild-type Apc protein and display the wild-type Apc allele. We conclude that the colossal crypts at adenoma margins have normal Apc gene function, consistent with the suggestion that their phenotype is a reactive state. The cause remains an open question, but the dramatic epithelial response hints at the presence of potent epithelial trophic factors in the vicinity of colon tumors.

Clonal analysis of mouse intestinal epithelial progenitors.

BACKGROUND & AIMS: Little is known about the cell lineages leading from stem cells to the various terminally differentiated cell types of the intestinal epithelium. In particular, the existence and characterization of intermediate progenitor types remain open issues, which are discussed in this study. METHODS: Chemical mutagenesis was used to genetically mark random intestinal epithelial cells by somatic mutation of the Dlb-1 locus. Intact epithelium was isolated at various times thereafter, and the composition, size, and location of mutant clones were scored. RESULTS & CONCLUSIONS: Analysis of clone dynamics showed short-lived (days) progenitors (C1, M1, and Mix) yielding one or two cell types and long-lived (months) mucous cell progenitors (M0), columnar cell progenitors (C0), and pluripotential stem cells (S) capable of giving rise to all epithelial cell types. Furthermore, study of clonal dispersion, during crypt branching morphogenesis or cell migration, shows that mutant progenitors usually partition into only one of the two daughter crypts and that cells are often widely dispersed in spite of the extensive intercellular junctions in the epithelium.

APC mutation and the crypt cycle in murine and human intestine.

Dysplastic colon adenomas are thought to arise from growth of clones of APC -/- colonic epithelial cells. Isolated clusters of dysplastic crypts are often observed in patients with familial adenomatous polyposis. These patients have genotype APC +/-, and the clusters of dysplastic crypts (called microadenoma or aberrant crypt foci) are thought to represent an early stage in the expansion of a mutant clone of APC -/- cells. It is thought that the growth of these clusters of mutant crypts results from crypt replication through a process similar to what occurs in the normal crypt cycle. We measured the relative replication rate of mutant crypts by analyzing the size of clusters of mutant crypts in APC +/- individuals and found that mutant APC -/- crypts replicate more rapidly than do normal APC +/- (i.e., nonneoplastic) crypts. In contrast, the replication rate of mutant crypts in Apc +/- mice is not significantly different from that of normal crypts, thus supporting previous findings that aberrant crypt foci do not contribute significantly to the colon adenoma population in adult Apc +/- mice. Intriguingly, we found an effect of Apc heterozygosity on the frequency of branching crypts in young mice.

Clonality of dysplastic epithelium in colorectal adenomas from familial adenomatous polyposis patients.

Loss of function of both alleles of the APC gene results in colorectal adenoma formation in familial adenomatous polyposis, an autosomal dominant genetic disorder leading to multiple colorectal tumors at an early age. Previous molecular studies indicate that the mutant cells forming dysplastic epithelium within adenomas are clonally derived. We show, using immunostaining and molecular techniques, that the dysplastic epithelium of adenomas actually contains a mixture of cells derived from both mutant and normal stem cells. Well-differentiated mucous cells in the dysplastic epithelium, one of the indicators of severity of dysplasia, were derived from normal stem cells. Carcinomas in these patients, in contrast, contained only mutant cells, indicating that the subsequent mutations incurred by these cells have led to their isolation from the normal population.

TCFL4: a gene at 17q21.1 encoding a putative basic helix-loop-helix leucine-zipper transcription factor.

TCFL4 (transcription factor like 4) is the HGMW-approved symbol for the gene of a widely expressed putative basic helix-loop-helix leucine-zipper (bHLH-zip) transcription factor which is located 3' to HSD17B1 (17-beta-hydroxysteroid dehydrogenase gene) at 17q21.1, centromeric to the BRCA1 (a gene implicated in familial breast cancer) locus. We report the human gene structure and the murine cDNA sequence of two variants, about 1.5 and 2.2 kb in size. The deduced protein is highly conserved between mouse and man.

Spontaneous intestinal carcinomas and skin neoplasms in Msh2-deficient mice.

Hereditary nonpolyposis colorectal cancer is associated with defects in DNA mismatch repair. Here, we characterize tumor susceptibility of the recently described Msh2-deficient mouse model. Within the first year of observation, all homozygous mice succumbed to disease, with lymphomas observed in at least 80% of the cases. The majority (70%) of animals 6 months or older developed intestinal neoplasms associated with APC inactivation. Microsatellite instability was more common in carcinomas than in adenomas, but uncommon in normal tissues. Some animals (7%) developed a variety of skin neoplasms analogous to the Muir-Torre syndrome. Msh2-/- mice implicate a direct role for mismatch repair in several neoplasms with striking phenotypic similarities to humans.

MSH2 deficiency contributes to accelerated APC-mediated intestinal tumorigenesis.

Accelerated intestinal tumorigenesis is probable in hereditary nonpolyposis colorectal cancer, a condition associated with germ line DNA mismatch repair (MMR) gene defects, and is believed to be caused by rapid accumulation of replication errors in critical genes, such as the APC (adenomatous polyposis coli) tumor suppressor gene. To study the potential contribution of MMR genes to accelerated intestinal tumorigenesis, we crossed the Min mouse, heterozygous for a germ line mutation of Apc, with an MMR gene (Msh2)-deficient mouse. MSH2 deficiency resulted in the development of many colonic aberrant crypt foci, as well as reduced survival of the mice, secondary to both a greater number and more rapidly developing adenomas. The mechanism of inactivation of the wild-type Apc allele depended on MSH2 status. In the presence of functional MSH2, all tumors demonstrated loss of heterozygosity. In contrast, whereas all adenomas were APC negative by immunostaining, only 5 of 34 adenomas from Apc+/-/Msh2-/- mice demonstrated loss of heterozygosity of the wild-type Apc allele, suggesting that somatic Apc mutations are responsible for the additional tumors. These findings provide evidence for the important role of MMR genes in accelerated intestinal tumorigenesis, thus supporting more aggressive surveillance strategies to prevent colorectal cancer in hereditary nonpolyposis colorectal cancer.

CRP-ductin: a gene expressed in intestinal crypts and in pancreatic and hepatic ducts.

BACKGROUND: A subtraction screen isolated CRP-ductin (CRP), a gene expressed in intestinal crypts. METHODS: DNA sequencing, in situ hybridization, immunostaining, Western and Northern blotting were used to characterize murine CRP. RESULTS: CRP is restricted to the intestine and its associated glands. In the small intestine, CRP mRNA is expressed in crypt cells at all stages of differentiation from the stem cells to the terminally differentiating cells of the crypt top, but not in the mature cells of the villus. In the colon, CRP mRNA is most heavily expressed in the mid-crypt. Expression is also seen in the pancreas and pancreatic ducts, and in the epithelium lining larger hepatic ducts, but not in the liver parenchyma or stomach. CRP protein is localized to the lumenal aspect of crypt cells in the small intestine. In the colon, the protein is seen in the lumenal aspect of surface epithelial cells. CRP protein is similarly found in the lumenal aspect of epithelial cells lining the pancreatic duct system and the larger hepatic ducts. Two cDNA variants, CRP-alpha and CRP-beta, were cloned from mouse jejunal epithelium. Their 3'-sequence differs in an 82-bp domain unique to CRP-beta. CONCLUSIONS: The CRP-alpha sequence predicts a protein with a short cytoplasmic region, a transmembrane domain, and a large extracellular region composed of many repeats (8 scavenger receptor domains, 5 CUB-domains, 1 ZP-domain, and 6 copies of a previously unreported domain which we call the CRP-domain). The structure of the CRP protein suggests a role in ligand interaction; possible functions are discussed.

Expansion of mutant stem cell populations in the human colon.

In general, it is presumed that colonic epithelial stem cells are the principal cell type at risk of incurring the series of somatic mutations leading to carcinoma, since all other epithelial cell types are short-lived. Mutant stem cell clonal expansion increases the risk for subsequent mutations and is therefore a potentially important step in carcinogenesis. The stem cells reside in colonic crypts, simple tubular foldings of the epithelium, and thus counting crypts provides an indirect means to determine stem cell numbers. The normal crypt population is known to expand through a process of crypt replication and this is thought to result in a corresponding expansion of the epithelial stem cell population. A simple mathematical model of the population dynamics of normal and mutant crypts (crypts containing mutant stem cells) is developed and used to estimate a lower bound on the relative rate of expansion of the mutant stem cell population. The model predicts that if mutant and normal crypt populations expand at the same rate, and if the mutation rate is small relative to the rate of growth, then the fraction of clusters of mutant crypts composed of only a single mutant crypt should steadily decrease with age towards one-half. Aberrant crypts are easily recognizable lesions in human colon which have frequently been shown to contain cells with K-ras and occasionally APC gene mutations. Application of the model to recent counts of aberrant crypt cluster sizes indicate that the aberrant crypt population, and the contained mutant stem cell population, is expanding substantially faster than normal.

Patterns of gene expression along the crypt-villus axis in mouse jejunal epithelium.

BACKGROUND: There is considerable interest in gene expression along the crypt-villus axis of the small intestinal epithelium, particularly in the identification of genes expressed in intestinal crypts. METHODS: In an attempt to identify crypt-expressed genes, single-stranded cDNA made from normal mouse jejunal epithelium was used in subtractive hybridization against single-stranded cDNA from epithelium from which crypt cells were depleted by 2,000 rads of gamma irradiation. Partial DNA sequence and in situ hybridization of 72 resulting clones were determined. RESULTS: The sequence of 45 clones matched previously published genes. Gene expression patterns fell into three categories: expression throughout the crypt-villus axis, expression restricted to the villus, and expression restricted to the crypt. Clones in the first two categories could be further divided into three subgroups: those with uniform expression, those with an increasing gradient of expression, and those with a decreasing gradient of expression along the crypt-villus axis. Twenty two clones showed a stronger expression in crypt and lower villus cells, four of these were differentially localized to the crypt. Two of the crypt localized clones were uniformly expressed throughout the crypt, expression of one was stronger in the lower crypt, and expression of the remaining clone was enhanced Paneth cells. We report the full-length cDNA sequence of the Paneth-cell-enhanced clone. CONCLUSIONS: The screen isolated crypt-expressed genes that may prove useful tools in the study of crypt biology. In a companion report, we characterize one of the crypt clones.

Morphogenesis and mechanical instability of a prestressed tissue.

We consider the issue of whether purely mechanical properties of biological systems can, in principle, play a significant role in morphogenesis. As a simple example, we model a spherically arranged epithelium that is symmetrically prestressed under the action of cytoskeletal elements. A three-dimensional exact bifurcation analysis indicates the existence of a critical radius beyond which, for a physiologically attainable prestress, the spherical organoid is mechanically unstable and will buckle. We conclude that the purely mechanical aspects of biological tissues may indeed play a role in morphogenesis.

The crypt cycle and the asymptotic dynamics of the proportion of differently sized mutant crypt clones in the mouse intestine.

The dynamics of clones of intestinal epithelial crypts populated by mutant stem cells offers the hope of new and independent evidence of continued crypt replication throughout adult life, an important prediction of recent models of intestinal stem cell biology. It is shown here that the experimentally most tractable measurement--scoring of the fraction of groups of mutant crypts found as isolated singletons, pairs, or clusters of > or = 3 mutant crypts--should tend to an asymptotic distribution as animals age. In particular, if the rate of mutation is low relative to the rate of crypt production then 1/2, 1/6 and 1/3 of groups of mutant crypts should be found as singletons, pairs, and larger clusters, respectively, as animals reach old age. Such a result is not immediately obvious, and if obtained from an experiment could easily be misinterpreted as implying that the crypt cycle must slow radically as animals age.

Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs.

The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon. It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene. Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3'-end of the 16S rRNA and the P-site, we conclude that there is a unique optimal aligned SD-AUG spacing in the absence of other complicating issues.

The influence of adenine-rich motifs in the 3' portion of the ribosome binding site on human IFN-gamma gene expression in Escherichia coli.

The ribosome binding site (RBS) of prokaryotic mRNA is divided into 5' and 3' portions by the translation initiation codon. Although it is well known that the presence of an appropriate RBS containing only the 5' portion is sufficient to direct the initiation of protein synthesis, the 3' portion appears to play a significant role in modulating the initiation process as well. Here we examine the influence of adenine-rich motifs frequently found in the 3' portion of highly expressed prokaryotic mRNAs. Two synthetic DNA fragments, GAGAAAAAAATC (corresponding to the first 12 nucleotides following the initiation codon of the chloramphenicol acetyltransferase gene), and AAAAAAATTAA were used to modify the beginning of the coding region of the human immune interferon-gamma (IFN-gamma) gene. The level of the protein synthesis in Escherichia coli directed by plasmids containing these constructs was quantitated. We found that placing either adenine-rich motif in the 3' portion of the RBS strongly enhanced gene expression, probably through an effect on translation initiation. We have also compared the protein expression levels of these gene constructs containing different series of 5'-RBSs with varying precistronic lengths and Shine-Dalgarno sequence lengths. The results suggest a positive functional role for the 3' adenine-rich motif. A possible mechanism for these effects is discussed.

Simple stochastic theory of stem cell differentiation is not simultaneously consistent with crypt extinction probability and the expansion of mutated clones.

It is shown that a simple Galton-Watson branching process model of the stem-cell pool in intestinal crypts is not simultaneously consistent with observations of the dynamics of replacement of normal by mutant crypt stem cells and other observations which put limits on the probability of crypt loss through extinction of all stem cells. This is because the limits on extinction probability forces the probability that a stem-cell division yields only non-stem cells to be small, but such a small probability makes it unlikely that a mutant stem-cell clone would expand while the normal stem-cell clones decay (it is more likely that both normal and mutant clones persist). It is suggested that the fundamental assumptions of independence and constancy of stem-cell behavior are flawed.

Estimation of the extent of the cutoff region from the spatial distribution of labelling and mitotic indices of intestinal crypts of a fixed length.

Current understanding of the pattern of proliferation within intestinal crypts involves the notion of a cutoff region introduced by Cairnie et al. (Exp. Cell. Res. 39, 539-553, 1965b). (Cells produced above the cutoff are non-cycling, whereas cells produced below the cutoff are cycling.) They contrasted the predicted distribution of proliferation in the extreme cases of a cutoff of width 0 (a sharp cutoff) with one eight cells wide (a slow cutoff) and concluded that the data were better explained by the latter. We have shown that crypt size variation artificially broadens the apparent distribution of proliferating cells in the crypt (Totafurno et al., Biophys. J. 54, 845-858, 1988). Here we show that the measurement and analysis of crypts of a specified height reduces this artifact. This work introduces the use of distance from the crypt base (in microns) to specify the location of cells within the crypt as an improvement over the cell position ordering traditionally used in the determination of the distribution of proliferating cells. We also show how to explicitly correct for several artifacts in the measurement of the labelling index. We conclude that cell proliferation within the crypt is more localized than previously realized; in fact, a cutoff as slow as eight cells wide is rejected.

Whole population cell kinetics of jejunal and colonic epithelium in lactating dams.

Previous studies make it likely that the response of the intestinal epithelium as a whole to lactation is different from that observed in the crypt population alone. We confirm this difference by whole population cell kinetics measurements of jejunal and colonic epithelium in mice that have been suckling pups for various lengths of time. We found that the fraction of cells in S phase in jejunal epithelium was significantly increased after only 1 week of lactation, maintained this elevated level after 2 weeks of lactation, but returned to normal during the third week of lactation. The cell number density in jejunum was also significantly higher after 2 and 3 weeks of lactation before returning to normal by 4 weeks. In the colonic epithelium no changes were found in the distribution of cells in G1, S, and G2 + M phases. However, a significant increase in cell number density was observed after 2 weeks of lactation, followed by a sharp decrease to a level significantly below that of normal mice after 3 and 4 weeks of lactation. We conclude that the observed significant increase in the fraction of S phase in jejunal epithelium of lactating mice is probably due to a smaller relative expansion of the villus population when compared with the expansion of the crypt population. Our data also indicate that a number of cell kinetic parameters in the intestinal epithelium of lactating mice are changing throughout the period of lactation. Thus the intestinal epithelium is probably not in a steady state during lactation.

Comparison of mesenteric with antimesenteric crypt and villus populations in the mouse jejunal epithelium.

Variation in the size and composition of crypts and villi along the length of the intestinal tract is well known. Here we investigate possible variation around the circumference of the intestine. This is a concern because most studies have ignored potential circumferential variation and its implications for experimental design in cell kinetic studies. We compared the crypt and villus populations of the mesenteric half with those of the antimesenteric half of proximal mouse jejunum. The branching crypt index and crypt and villus dimensions were measured. We found no evidence of differences in the branching crypt index, in the mean crypt and villus size, nor in the distribution of crypt and villus sizes between these two populations.