The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time

The decay in the number of grains over [3H]-thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDF1 mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC-zone) of 18.8 +/- 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.     

The stem cells of small intestinal crypts: where are they?

Recently, there has been resurgence of interest in the question of small intestinal stem cells, their precise location and numbers in the crypts. In this article, we attempt to re-assess the data, including historical information often omitted in recent studies on the subject. The conclusion we draw is that the evidence supports the concept that active murine small intestinal stem cells in steady state are few in number and are proliferative. There are two evolving, but divergent views on their location (which may be more related to scope of capability and reversibility than to location) several lineage labelling and stem cell self-renewing studies (based on Lgr5 expression) suggest a location intercalated between the Paneth cells (crypt base columnar cells (CBCCs)), or classical cell kinetic, label-retention and radiobiological evidence plus other recent studies, pointing to a location four cell positions luminally from the base of the crypt The latter is supported by recent lineage labelling of Bmi-1-expressing cells and by studies on expression of Wip-1 phosphatase. The situation in the human small intestine remains unclear, but recent mtDNA mutation studies suggest that the stem cells in humans are also located above the Paneth cell zone. There could be a distinct and as yet undiscovered relationship between these observed traits, with stem cell properties both in cells of the crypt base and those at cell position 4.     

Tracking the cell hierarchy in the human intestine using biochemical signatures derived by mid-infrared microspectroscopy

Markers of gastrointestinal (GI) stem cells remain elusive. We employed synchrotron Fourier-transform infrared (FTIR) microspectroscopy to derive mid-infrared (IR) spectra along the length of human GI crypts. Tissue sections (10-μm thick) were floated onto BaF₂ windows and image maps were acquired of small intestine and large bowel crypts in transmission mode with an aperture of ≤  10 μm × 10 μm. Counting upwards in a step-size (≤ 10 μm) fashion from the crypt base, IR spectra were extracted from the image maps and each spectrum corresponding to a particular location was identified. Spectra were analyzed using principal component analysis plus linear discriminant analysis. Compared to putative crypt base columnar/Paneth cells, those assigned as label-retaining cells were chemically more similar to putative large bowel stem cells and, the small intestine transit-amplifying cells were closest to large bowel transit-amplifying cells; interestingly, the base of small intestine crypts was the most chemically-distinct. This study suggests that in the complex cell lineage of human GI crypts, chemical similarities as revealed by FTIR microspectroscopy between regions putatively assigned as stem cell, transit-amplifying and terminally-differentiated facilitates identification of cell function.     

The morphogenesis of the human Paneth cell. An immunocytochemical ultrastructural study

In human duodenal mucosa Paneth cells originate away from the base of crypts and migrate towards the base during maturation. The earliest cells in the Paneth cell lineage could be identified by labelling of lysozyme in the Golgi apparatus. Specific labelling for lysozyme was present in the rough endoplasmic reticulum, Golgi apparatus, condensing vacuoles, granules and many lysosomes of mature Paneth cells. The maturation of the Paneth cell is accompanied by an increase in the content of lysozyme in the secretory granules and with senescence lysozyme diffuses into the cytoplasm.     

The morphogenesis of the human Paneth cell

Summary In human duodenal mucosa Paneth cells originate away from the base of crypts and migrate towards the base during maturation The earliest cells in the Paneth cell lineage could be identified by labelling of lysozyme in the Golgi apparatus. Specific labelling for lysozyme was present in the rough endoplasmic reticulum, Golgi apparatus, condensing vacuoles, granules and many lysosomes of mature Paneth cells. The maturation of the Paneth cell is accompanied by an increase in the content of lysozyme in the secretory granules and with senescence lysozyme diffuses into the cytoplasm.     

Zinc-secreting Paneth cells studied by ZP fluorescence.

We have used a new family of zinc-specific-responsive fluorescent dyes (ZPs) to study the sequestration and secretion of zinc from Paneth cells, which are located in the bases of the crypts of Lieberkühn within the rat small intestine. Vivid ZP fluorescence zinc staining of Paneth cell secretory granules is seen in both cryostat sections and isolated crypts, providing firm evidence for a pool of labile (rapidly exchangeable) zinc within these cells. We further demonstrate that this ionic zinc pool is secreted under physiological conditions. In vivo stimulation of the small intestine by IP injection of the secretagogue pilocarpine results in discrete zinc staining within the lumens of subsequently isolated crypts, concomitant with a decrease in the zinc staining of Paneth cell granules located within the same crypts. In contrast, the secretion of zinc into the lumens of isolated crypts stimulated in vitro with either carbachol or LPS (lipopolysaccharide) is not observed. However, a distinct change in Paneth cell morphology, suggesting attempted secretion, is seen in response to the direct application of cholinergics but not LPS. These findings suggest that zinc is coreleased with other Paneth cell anti-microbials, and that the intact intestine is necessary for secretion into the crypt lumen.     

STUDIES ON SMALL INTESTINAL CRYPT EPITHELIUM : I. The Fine Structure of the Crypt Epithelium of the Proximal Small Intestine of Fasting Humans

Small intestinal crypt epithelium obtained from normal fasting humans by peroral biopsy of the mucosa was studied with the electron microscope. Paneth cells were identified at the base of the crypts by their elaborate highly organized endoplasmic reticulum, large secretory granules, and small lysosome-like dense bodies within the cytoplasm. Undifferentiated cells were characterized by smaller cytoplasmic membrane-bounded granules which were presumed to be secretory in nature, a less elaborate endoplasmic reticulum, many unattached ribosomes and, in some cells, the presence of glycogen. Some undifferentiated cells at the base of the crypts contained lobulated nuclei and striking paranuclear accumulations of mitochondria. Membrane-bounded cytoplasmic fragments, probably originating from undifferentiated and Paneth cells, were frequently apparent within crypt lumina. Of the goblet cells, some were seen actively secreting mucus. In these, apical mucus appeared to exude into the crypt lumen between gaps in the microvilli. The membrane formerly surrounding the apical mucus appeared to fuse with and become part of the plasma membrane of the cell, suggesting a merocrine secretory mechanism. Enterochromaffin cells were identified by their location between the basal regions of other crypt cells and by their unique intracytoplasmic granules.     

Cell population kinetics in the epithelium of the colon of the male rat.

The present study was undertaken in order to try to define some of the kinetic parameters in the colonic mucosa of normal Wistar rats. Preliminary observations showed considerable morphological differences in the mucosa from site to site along the length of the colon. In particular the height of the crypts (measured in cells) was variable. In addition labelling index studies demonstrated dramatic variations in the distribution of labelling along the length of the crypts from site to site in the bowel. A single site in the descending colon was selected for more detailed study using a stathmokinetic agent, vincristine, and the continous labelling technique with tritiated thymidine. The results of these investigations suggest that there exists at the base of the crypt a subpopulation of cells cycling more slowly than the cells in the rest of the proliferative compartment. Growth fraction appears to fall with rising cell positions within the crypt.     

The influence of 400 r x-irradiation on the number and the localization 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 Lieberkuhn 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.     

Effects of AlF4 – and ATP on intracellular calcium dynamics of crypt epithelial cells in mouse small intestine

Previous digital imaging analysis of intracellular calcium ion ([Ca2+]i) dynamics in the crypt of the small intestine showed little response by most columnar cells to cholinergic and adrenergic agonists. The objective of the present study was to demonstrate whether G-protein activators and other transmitters elicit [Ca2+]i changes in crypt cells. We used digital imaging to analyze spatiotemporal dynamics of [Ca2+]i in Fura-2/AM-loaded isolated crypts of mouse duodenum and ileum. A1F4- increased [Ca2+]i in crypt columnar cells. In many cases, we observed [Ca2+]i oscillations, which were synchronized throughout the entire crypt. The oscillations were blocked by octanol. ATP, but not adenosine, caused a [Ca2+]i increase in middle crypt-regions of the duodenum and upper regions of the ileum, and the [Ca2+]i wave propagated towards the crypt bottom. The ATP-induced [Ca2+]i increase was prevented by pretreatment with thapsigargin or suramin, but not by La3+ or an extracellular Ca(2+)-free environment. Neither dopamine, 5-hydroxytryptamine (5-HT), histamine, vasoactive intestinal peptide, substance P. cholera toxin, nor guanylin had significant effects. The [Ca2+]i dynamics of Paneth cells were independent of the AlF4(-)-induced synchronous oscillations of columnar cells and of the ATP-induced [Ca2+]i wave. In conclusion, crypt columnar cells have [Ca2+]i-dependent intracellular signaling mechanisms that are linked with G proteins, and by which the cells communicate with each other. ATP elicited [Ca2+]i mobilization from columnar cells via P2 receptors, although some regional differences were noted between the duodenum and ileum.     

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.     

The spatial organization of the hierarchical proliferative cells of the crypts of the small intestine into clusters of 'synchronized' cells

Abstract. A statistical analysis of the distribution of [³H]TdR-labelled cells in longitudinal and transverse sections of crypts from the ileum of the mouse, indicated that there was a strong tendency for labelled or unlabelled cells to be associated in short vertical runs and lateral clumps, suggesting the presence of clusters of labelled cells on the sides of the crypts. A model is discussed for the cellular spatial organization of the crypt that proposes a vertical alignment of the cells within branches of the proliferative cell lineage. The model would predict vertical alignment of partially synchronized cells as well as some lateral clumping.
In the present studies mitoses were not observed at higher levels in the crypt than labelled (S phase) cells. This observation would be predicted by the non-random spatial organization suggested by the model.
The model would also make certain predictions concerning cell migration. These are discussed in relation to cell migration studies which include evidence that migration continues in the absence of mitotic activity.     

Modelling the spatio-temporal cell dynamics reveals novel insights on cell differentiation and proliferation in the small intestinal crypt

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4-5 days with 10-12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days.     

Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine

We have previously shown that Hes1 is expressed both in putative epithelial stem cells just above Paneth cells and in the crypt base columnar cells between Paneth cells, while Hes1 is completely absent in Paneth cells. This study was undertaken to clarify the role of Hes1 in Paneth cell differentiation, using Hes1-knockout (KO) newborn (P0) mice. Electron microscopy revealed premature appearance of distinct cells containing cytoplasmic granules in the intervillous region in Hes1-KO P0 mice, whereas those cells were absent in wild-type (WT) P0 mice. In Hes1-KO P0 mice, the gene expressions of cryptdins, exclusively present in Paneth cells, were all enhanced compared with WT P0 mice. Immunohistochemistry demonstrated increased number of both lysozyme-positive and cryptdin-4-positive cells in the small intestinal epithelium of Hes1-KO P0 mice as compared to WT P0 mice. Thus, Hes1 appears to have an inhibitory role in Paneth cell differentiation in the small intestine.     

Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.     

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.     

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 Cairnieet 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 (Totafurnoet 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.     

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.     

Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine.

Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A.