A stochastic branching model with formation of subunits applied to the growth of intestinal crypts

The intestinal epithelium is one of the most rapidly regenerating tissues in mammals. Cell production takes place in the intestinal crypts which contain about 250 cells. Only a minority of 1-60 proliferating cells are able to maintain a crypt over a long period of time. However, so far attempts to identify these stem cells were unsuccessful. Therefore, little is known about their cellular growth and selfmaintenance properties. On the other hand, the crypts appear to exhibit a life cycle which starts by fission of existing crypts and ends by fission or extinction. Data on these processes have recently become available. Here, we demonstrate how these data on the life cycle of the macroscopic crypt structure can be used to derive a quantitative model of the microscopic process of stem cell growth. The model assumptions are: (1) stem cells undergo a time independent supracritical Markovian branching process (Galton-Watson process); (2) a crypt divides if the number of stem cells exceeds a given threshold and the stem cells are distributed to both daughter crypts according to binomial statistics; (3) the size of the crypt is proportional to the stem cell number. This model combining two different stochastic branching processes describes a new class of processes whose stationary stability and asymptotic behavior are examined. This model should be applicable to various growth processes with formation of subunits (e.g. population growth with formation of colonies in biology, ecology and sociology). Comparison with crypt data shows that intestinal stem cells have a probability of over 0.8 of dividing asymmetrically and that the threshold number should be 8 or larger.     

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.     

The response of the intestinal epithelium in B10.A mice to infection with Trichinella spiralis

Previous studies on intestinal trichinosis have dealt mainly with areas other than the intestinal epithelium. Since the epithelium is now known to be the parasite's habitat, its response to infection is important. Infection with Trichinella spiralis in immunologically slow-responding B10.A mice was associated with crypt hyperplasia and villus atrophy. With similar infection levels in both primary and challenge infections, there was no difference in the maximal degree of atrophy or hyperplasia between the 2 groups. However, challenged mice underwent these mucosal changes in about half the time. Expulsion of worms always occurred during regeneration of the intestinal epithelium suggesting that the host's defense mechanism of altering the kinetics of the epithelium was not the prime factor causing expulsion. Pulse labelling of enterocytes with [3H] thymidine showed that there was no significant increase in the relative size of the proliferation zone. This indicates that the crypt cell output was not altered by this parasite. Atrophy of the villus was analysed with respect to its 3-dimensional shape. There was a decrease in both height and width of the villus but not thickness. Thus, there is a real decrease in the size of the enterocyte population per villus. Histochemical staining of the enterocyte brush border by an alkaline phosphatase method showed that (1) hyperplastic crypts have an enlarged maturation zone and (2) the villus epithelium is composed entirely of mature cells. The distribution of the nematode population was compared to these changes in the intestine. Trichinella spiralis showed a marked anteriad (distal to proximal) migration prior to expulsion. Thus, utilizing a novel approach to study intestinal trichinosis, the response of the mucosal epithelium has been characterized.     

Mosaic analysis of small intestinal development using the<Emphasis Type="Italic"> spf</Emphasis><Superscript><Emphasis Type="Italic">ash</Emphasis></Superscript>-heterozygous female mouse

Mosaic analysis using the spf(ash)-heterozygous female mouse was performed to clarify the cell lineage and cell behavior during small intestinal development with special attention given to the villus and crypt formation. The spf(ash) mutation, located on the X-chromosome, causes ornithine transcarbamylase (OTC) deficiency, which leads to mosaic expression of this enzyme in the small intestine of the heterozygous female mouse. In the small intestine in heterozygous fetuses, very small patches, which were aggregates of OTC-positive cells or negative cells, with no definite orientation to the villus structures were observed. In the neonatal small intestine, the intervillus region (the presumptive crypts) was polyclonal, and the majority of crypts were comprised exclusively cells of either genotype in 2-week-old small intestine. These results suggest that extensive migration and cell mixing of small intestinal epithelial cells, which have no definite correlation with the villus formation, occur in fetal stages of development, and that the crypt morphogenesis commences after birth independently of the monoclonality of the epithelial cells. Our data with the mosaic mice also reconfirmed the monoclonality of the adult small intestinal crypts demonstrated in mouse aggregation chimeras.     

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.     

Variation in crypt size and its influence on the analysis of epithelial cell proliferation in the intestinal crypt.

The standard model of epithelial cell renewal in the intestine proposes a gradual transition between the region of the crypt containing actively proliferating cells and that containing solely terminally differentiating cells (Cairnie, Lamerton and Steel, 1965 a, b). The experimental justification for this conclusion was the gradual decrease towards the crypt top of the measured labeling and mitotic indices. Recently, however, we have proposed that intestinal crypts normally undergo a replicative cycle so that at any time in any region of the intestine, crypts will be found to have a wide range of sizes. We show here that if this intrinsic size variation is taken into account, then a sharp transition between the proliferative and nonproliferative compartments of individual intestinal crypts is consistent with the labeling and mitotic index distributions of mouse and rat jejunal crypts. Thus there is no need to invoke the region of gradual transition from proliferating to nonproliferating cells as is done in the standard model. The position of this sharp transition is estimated for both the mouse and rat. Experiments to further test our model are suggested and the significance of the results discussed.     

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.     

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.     

Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium

Caudal-related homeobox (Cdx) proteins play an important role in development and differentiation of the intestinal epithelium. Using cDNA differential display, we identified clusterin as a prominently induced gene in a Cdx2-regulated cellular model of intestinal differentiation. Transfection experiments and DNA-protein interaction assays showed that clusterin is an immediate downstream target gene for Cdx proteins. The distribution of clusterin protein in the intestine was assessed during development and in the adult epithelium using immunohistochemistry. In the adult mouse epithelium, clusterin protein was localized in both crypt and villus compartments but not in interstitial cells of the intestinal mucosa. Together, these data suggest that clusterin is a direct target gene for Cdx homeobox proteins, and the pattern of clusterin protein expression suggests that it is associated with the differentiated state in the intestinal epithelium.     

Intestinal villus structure contributes to even shedding of epithelial cells

In the intestinal epithelium, proliferated epithelial cells ascend the crypts and villi and shed at the villus tips into the gut lumen. In this study, we theoretically investigate the roles of the villi on cell turnover. We present a stochastic model that focuses on the duration over which cells migrate the shortest paths between the crypt orifices and the villus tips, where shedding cells are randomly chosen from among those older than the shortest-path cell migration times. By extending the length of the shortest path to delay cell shedding, the finger-like shape of the villus would tightly regulate shedding-cell ages compared with flat surfaces and shorter projections; the villus allows epithelial cells to shed at around the same age, which limits them from shedding early or staying in the epithelium for long periods. Computational simulations of cell dynamics agreed well with the predictions. We also examine various mechanical conditions of cells and confirm that coordinated collective cell migration supports the predictions. These results suggest the important roles of the villi in homeostatic maintenance of the small intestine, and we discuss the applicability of our approach to other tissues with collective cell movement.     

Circadian Variation In Migration Velocity In Small Intestinal Epithelium

Abstract. The variation in migration rates of cells within the small intestinal epithelium was studied over a 24-hr period at 3-hr intervals (migration of cells was studied independently for the crypts and the villi using the changing distributions of [³H]TdR labelled cells as an indicator of cell migration).
Clear changes in the rates of cell movement were observed during a 24-hr period for both crypt and villus epithelium. the rates of cell migration in these two compartments did not correlate well with the exception of samples taken at 18.00 hours. At this time of day there appeared to be no cell movement at all in either crypts or villi. There was not a good correlation between the migration velocity throughout the day and the changes in the number of mitoses.
It is proposed that mitotic rates do not directly govern migration rates but that the converse may be true. Further, the lack of correlation between crypt and villus migration rates at any time of day suggest that the mechanisms controlling all movement in these two regions of small intestinal epithelium may be different.     

Circadian variation in migration velocity in small intestinal epithelium

The variation in migration rates of cells within the small intestinal epithelium was studied over a 24-hr period at 3-hr intervals (migration of cells was studied independently for the crypts and the villi using the changing distributions of [3H]TdR labelled cells as an indicator of cell migration). Clear changes in the rates of cell movement were observed during a 24-hr period for both crypt and villus epithelium. The rates of cell migration in these two compartments did not correlate well with the exception of samples taken at 18.00 hours. At this time of day there appeared to be no cell movement at all in either crypts or villi. There was not a good correlation between the migration velocity throughout the day and the changes in the number of mitoses. It is proposed that mitotic rates do not directly govern migration rates but that the converse may be true. Further, the lack of correlation between crypt and villus migration rates at any time of day suggest that the mechanisms controlling all movement in these two regions of small intestinal epithelium may be different.     

小肠干细胞的命运调控

小肠上皮具有快速更新的能力,是研究成体干细胞的理想系统.小肠上皮由绒毛和隐窝两部分组成,而位于小肠隐窝底部的小肠干细胞是其持续更新的源泉.近年来,以Lgr5为代表的小肠干细胞标记物的发现、Lgr5+小肠干细胞的分离培养和多种转基因小鼠模型的出现,极大地促进了对小肠干细胞自我更新和分化调控的研究,使得人们可以更加深入地认识小肠干细胞命运决定的分子机制.本文简要综述了近年来人们对Wnt,BMP,Notch和EGF等信号如何在小肠干细胞命运调控中发挥作用的认识.     

Regulation of intestinal stem cell fate specification

The remarkable ability of rapid self-renewal makes the intestinal epithelium an ideal model for the study of adult stem cells. The intestinal epithelium is organized into villus and crypt, and a group of intestinal stem cells located at the base of crypt are responsible for this constant self-renewal throughout the life. Identification of the intestinal stem cell marker Lgr5, isolation and in vitro culture of Lgr5+ intestinal stem cells and the use of transgenic mouse models have significantly facilitated the studies of intestinal stem cell homeostasis and differentiation, therefore greatly expanding our knowledge of the regulatory mechanisms underlying the intestinal stem cell fate determination. In this review, we summarize the current understanding of how signals of Wnt, BMP, Notch and EGF in the stem cell niche modulate the intestinal stem cell fate.     

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.     

Mitotic orientation in three dimensions determined from multiple projections.

The three-dimensional orientation of mitoses in mouse small intestinal crypts of Lieberkuhn was determined from multiple projections of the mitotic figures in whole mounts of isolated intestinal crypts. We found evidence of a significant orientational bias for mitoses whose daughter cells would be added along the long axis of the crypt, and thus conform to the maintenance of the cylindrical shape of the intestinal crypt. However, we also observed many mitoses whose progeny must be rearranged if the simple cylindrical shape of the intestinal crypt is to be maintained. Our results indicate that the ultimate behavior of progeny cells and hence of local tissue form may not strictly depend on the orientation of mitosis. The methods presented may also be used in the study of mitotic orientation in other tissues.     

A test of the stochastic theory of stem cell differentiation.

Stochastic theories of stem cell renewal are shown to predict turnover of intestinal crypts. While I found ample evidence of production of new crypts from direct in vivo studies in adult mice, I failed to find evidence of crypt loss. Thus, it would appear that the simple stochastic models may not provide an adequate theory of control of intestinal stem cell function.