Effects of puromycin, cycloheximide and noradrenaline on cell migration within the crypts and on the villi of the small intestine. A model to explain cell movement in both regions

The normal process of cell migration, occurring as part of the replacement scheme within the small intestinal epithelium, was investigated extensively. The effects of puromycin, cycloheximide and noradrenaline on the movement of tritiated thymidine [( 3H]TdR) prelabelled crypt or villus cells have been studied. These studies have led to the formulation of a model for the mechanism of cell migration, postulating that the crypts and villi behave as separate units, with regard to cell migration, in addition to their distinct structural and functional properties. It is proposed that crypt cell migration is an active process requiring protein synthesis and protein glycosylation, whilst movement of villus epithelial cells is passive, depending on the continued contraction of smooth muscle cells in the lamina propria.     

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse

Summary Suspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron microscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.Supported by a research grant from the Medical Research Council of Canada (J.H.)     

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.     

The influence of various cell kinetic conditions on functional differentiation in the small intestine of the rat. A study of enzymes bound to subcellular organelles

The process of cell maturation and cell ageing of absorptive epithelial cells was investigated in normal rat duodenum. The development of a number of enzymes bound to subcellular organelles was studied by using microchemical analyses on various cell compartments dissected from crypts and villi from freeze-dried cryostat sections. The development of the ultrastructural features of the absorptive epithelium was investigated by electron microscopy of various cell positions along the whole length of the crypt and the base of the villus. The data obtained were related to cell position along the crypt and villus and to cell age during migration from the bottom of the crypt to the tip of the villus.The influence of changes in the life-span of the cells and of increasing proliferative activity was studied by comparing normal rat duodenum with that from germfree rats and rats recovering from low radiation doses (72 hr after 400 R).Our data show that the specific activity of nonspecific esterases mainly localized in the endoplasmic reticulum increases when the cells migrate along the upper half of the crypt and the basal part of the villus. Activity of alkaline phosphatase, measured as a marker for the microvilli, is absent in the crypt, but increases linearly from the base of the villus to the tip. The longer life-span of villus cells in germfree animals does not result in a higher activity of these enzymes than in normal animals. An increased proliferative activity in the crypt, as present 72 hr after X-irradiation, is accompanied by a decreased activity of both enzymes but the pattern of activity during cell migration remains the same. The specific activity of enzymes bound to mitochondria or lysosomes (monoamineoxidase and β-N-acetylglucosaminidase) are not affected by changing crypt cell kinetics.Electrophoretic analyses of isolated cell compartments showed that the increase during normal differentiation or the decrease after X-irradiation of esterase activity is due to changes in overall activity, not to the appearance or disappearance of specific isoenzymes. Electron microscopy showed that in the normal intestine there is a gradual development of ultrastructural features during migration of the cell along the crypt while the most drastic changes in cell structure occur at the moment the cell enters the villus. Contrary to our expectation, the ultrastructural development was not influenced by increased proliferative activity in the crypt 72 hr after irradiation, and hence the decrease in enzyme activity found cannot be related to changes in ultrastructure.     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Acute and Subacute Effects of Thiamine Deficiency On the Morphometry and Cell Kinetics of Jejunal and Ileal Epithelial Cells

Abstract. The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (I_p) and crypt cell cycle time (T_c) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. the jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, I_p and T_c.     

The effect of increased crypt cell proliferation on the activity and subcellular localization of esterases and alkaline phosphatase in the rat small intestine

Synopsis The activity and ultrastructural localization of alkaline phosphatase and esterase has been studied in normal rat intestine and after the increased crypt cell proliferation that occurs during recovery after 400 rad X-irradiation. Alkaline phosphatase activity is not present in crypt cells of normal intestine, but becomes apparent after the cell has migrated on to the villus. The enzyme is localized in the microvilli, along the lateral cell membranes and in dense bodies. Its activity increases 10 to 15-fold from the base to the tip of the villus. Morphometric analysis of the cell structureswhere this enzyme is localized reveals no marked changes in their relative proportions during crypt cell development.The expansion of the proliferative cell compartment along the whole length of the crypt which occurs during recovery after irradiation (72 hr after 400 rad X-irradiation) results in a marked reduction of alkaline phosphatase activity in the lower 10–15 cell positions at the base of the villus. During subsequent migration of these cells, the activity increases with cell age but normal values are not attained. From a morphometric analysis it was found that the ultrastructural development is similar to that in controls. These results suggest that during cell maturation, normal values for alkaline phosphatase activity are only attained after a 10–12 hr period of maturation in a non-proliferative state and only after the cell has migrated on to the functional villus compartment.In normal intestine, esterase activity shows a 3-fold increase from the bottom to the tip of the crypt and a 3 to 4-fold increase during migration up to the middle of the villus. Enzyme activity is localized in the endoplasmic reticulum, the dense bodies and the perinuclear space. Morphometric analyses reveal a 2 to 3-fold increase in the absolute size of these subcellular compartments during crypt cell differentiation and a 2-fold increase at the crypt-villus junction. The relative sizes increase 1.5-fold during crypt cell differentiation and at the time of transition of the cells on to the villus.Increased crypt cell proliferation after irradiation leads to a marked decrease in esterase activity both in crypts and villi. Morphometric analyses of electron micrographs indicate that these changes in activity are not related to any changes in the subcellular structures in which the enzyme is localized. It appears that the normal development of esterase activity depends both on the functional state of the cell and its localization in the crypt or villus.     

Use of a mouse chimaeric model to study cell migration patterns in the small intestinal epithelium

The pattern of cells migration in the small intestinal epithelia of a RIII/ro----C57BL/6J mouse aggregation chimaera is demonstrated using Dolichos biflorus agglutinin-peroxidase (DBA) conjugate as a strain-specific marker. Using serial tangential sections of heterogeneously stained villi and plotting the distribution of labelled/unlabelled cells with a drawing tube, and by three-dimensional reconstruction with the aid of computer graphics, we show the migration pathway to be in tight cohorts of similar DBA-peroxidase staining type, which move upwards in straight lines. There is little cell mixing either on the villus or along the crypt-villus junctions. Our observations also show for the first time that a single crypt can feed cells to more than one villus. They also suggest that either cell loss is not confined to the villus tips but can take place from the villus sides, or that there is marked asynchrony of cell production between crypts.     

Cell Migration Velocities In the Crypts of the Small Intestine After Cytotoxic Insult Are Not Dependent On Mitotic Activity

Abstract. The role of mitotic activity in the normal process of intestinal epithelial cell migration was investigated. the movement of [³H]TdR-labelled cells in the crypt-villus column was used to study migration both in the crypts and on the villi. Radiation alone or in conjunction with other cytotoxic agents (hydroxyurea, cyclophosphamide and isopropyl-methane sulphonate) was used to eliminate cell division activity and to decrease crypt cellularity. This was done in order to determine the role of 'mitotic pressure' in driving cell migration.
It has been clearly demonstrated in this study that cell migration, both within the crypts and on the villi, can take place in the complete absence of mitotic activity and after a drastic decrease in crypt cellularity. These results add to the continually mounting evidence against the idea that the 'pressure' generated by mitoses within the crypt or indeed in other epithelial regions is responsible for propelling epithelial cells. the data also demonstrate that the migration mechanisms are resistant to cytotoxic exposure.     

Relative acylglycerol acyltransferase activities in homogenates of enzymically dispersed rat jejunal villus and crypt cells

The relative acyltransferase activities were compared in homogenates of rat jejunal villus and crypt cells isolated by differential scraping and hyaluronidase dispersion. The contributions of the monoacylglycerol and phosphatidic acid pathways to the higher acylglycerol and phospholipid biosynthesis were assessed using 2-oleoyl-sn-[3H] glycerol and [I-14C] palmitic acid as tracers. The stereochemical course of the diacylglycerol biosynthesis was determined by stereospecific analysis. Using 2-oleoyl-sn-glycerol as a tracer, the villus cells exhibited for times higher diacylglycerol and 19 times higher triacylglycerol biosynthesis than crypt cells on an equivalent protein basis. Furthermore, while the villus cell homogenates yielded a preponderance (75%) of the 1, 2-diacyl-sn-glycerols, the crypt cell homogenates formed essentially racemic proportions of 1, 2- and 2,3-diacyl-sn-glycerols. Both villus and crypt cell homogenates exhibited comparable acyl acceptor and acyl donor concentration dependence and the same cofactor requirements. It is unlikely that these acyltransferase activities in the crypt cell preparation are due to contamination with the villus cells, because then more comparable proportions of the enantiomeric diacylglycerols and triacylglycerols would have been anticipated. It is concluded that the crypt cells possess intrinsic monoacylglycerol and to a much lesser extent diacylglycerol acyltransferase activities, which are acquired prior to the development of a distinct brush border and which probably do not require dietary stimulus for induction.     

EFFECT OF VILLUS LENGTH ON CELL PROLIFERATION AND MIGRATION IN SMALL INTESTINAL EPITHELIUM

For the interpretation of data supporting the hypothesis of a feedback regulation of proliferative activity in intestinal crypts by the functional villus cell compartment the life span and migration rate of epithelial cells on villi of experimentally reduced length should be known. Autoradiographic studies and scintillation counting of isolated villi at different time intervals after ³H-thymidine labelling were carried out 36, 48 and 60 hr intervals after X-irradiation. The results showed that the life span of epithelial cells in rat small intestine (36–48 hr) is independent of the villus length. In villi of reduced length the migration rate of the epithelial cells was found to be decreased compared with controls. Changes in the migration rate in turn seem to be dependent on the production of epithelial cells in the crypt. Comparative studies on the recovery of crypt and villus epithelium after various doses (300 and 700 R) of X-radiation support the hypothesis that increased proliferative activity in the crypt cell compartment is related to a reduction of the number of functional villus cells below a critical villus length. The importance of these findings in the interpretation of data on (micro) biochemical analyses of certain cell differentiation characteristics during increased proliferative activity is discussed.     

Use of A Mouse Chimaeric Model to Study Cell Migration Patterns In the Small Intestinal Epithelium

The pattern of cells migration in the small intestinal epithelia of a RIII/?ro C57BL/6J mouse aggregation chimaera is demonstrated using Dolichos biflorus agglutinin-peroxidase (DBA) conjugate as a strain-specific marker. Using serial tangential sections of heterogeneously stained villi and plotting the distribution of labelled/unlabelled cells with a drawing tube, and by three-dimensional reconstruction with the aid of computer graphics, we show the migration pathway to be in tight cohorts of similar DBA-peroxidase staining type, which move upwards in straight lines. There is little cell mixing either on the villus or along the crypt-villus junctions. Our observations also show for the first time that a single crypt can feed cells to more than one villus. They also suggest that either cell loss is not confined to the villus tips but can take place from the villus sides, or that there is marked asynchrony of cell production between crypts.     

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.     

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.     

Distribution of Ca-ATPase, ATP-dependent Ca-transport, calmodulin and vitamin D-dependent Ca-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca²⁺-ATPase activity and ATP-dependent Ca²⁺-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na⁺ + K⁺)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca²⁺-transport in fraction II (8.2 ± 0.3 nmol Ca²⁺/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca²⁺-transport activity (0.9 ± 0.1 nmol Ca²⁺/min per mg protein). The distribution of high-affinity Ca²⁺-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca²⁺-transport. The activity of Na⁺/Ca²⁺ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca²⁺-transport system, the Na⁺/Ca²⁺ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

(Na---K)-stimulated adenosinetriphosphatase in isolated intestinal villus tip and crypt cells

The migration of intestinal epithelial cells from the crypt area to the villus tip is associated with progressive differentiation of these cells. The distribution of (Na⁺---K⁺) stimulated adenosinetriphosphatase ((Na⁺---K⁺)-ATPase; EC 3.6.1.3) along the intestinal villus may have functional as well as developmental implications. To define this distribution, rat jejunal and ileal segments were incubated in vitro with a citrate solution that dissociates epithelial cells sequentially from villus tip to crypt area. ATPase activity in cell collections from villus tips and crypt areas were compared. The specific activity of (Na⁺---K⁺)-ATPase was higher in the villus tip than in the crypt cells of both jejunum and ileum. Crypt cell (Na⁺---K⁺)-ATPase activity in the jejunum and ileum were similar. Thus, (Na⁺---K⁺)-ATPase activity of villus tip cells in the jejunum was greater than in the ileum. There was no difference in villus tip and crypt cell Mg²⁺-ATPase activity in either jejunum or ileum. The steep gradient for (Na⁺---K⁺)-ATPase along the intestinal villus may signify an improtant difference in Na⁺ transport between the villus tip and crypt area. The higher level of (Na⁺---K⁺)-ATPase activity in the jejunal villi is consistent with the more important role of the jejunum in Na⁺ and substrate-linked Na⁺ transport.     

Conversion of somatostatin-28 to somatostatin-14 during maturation of epithelial cells in the porcine jejunum

Fractions of isolated epithelial cells were harvested from a segment of porcine jejunum by ten successive incubations with a chelating buffer. The cell fractions showed a progressive decrease in the activity of the brush-border enzymes, alkaline phosphatase and sucrase, with increasing incubation number but a progressive increase in the ability to incorporate labelled thymidine into DNA. Fractions enriched in cells from the crypt region (fractions 9 and 10) contained higher concentrations per mg protein of somatostatin-like immunoreactivity (1.8-fold), glucagon-like immunoreactivity (5.3-fold) and serotonin (3.0-fold) than fractions enriched in cells from the villus tip (fractions 1 and 2). Analysis of extracts of the fractions by gel filtration/radioimmunoassay showed that somatostatin-28 represented the predominant molecular form of somatostatin-like immunoreactivity in all cell fractions but the relative proportion of somatostatin-14 (and related metabolites) to somatostatin-28 was significantly higher (P less than 0.05) in fractions enriched in villus cells (fraction 1 and 2) than in fractions enriched in crypt cells (fractions 5-10). This result suggests that metabolism of somatostatin-28 to somatostatin-14 takes place during migration of the D cell from the crypt base to the villus tip. Heterogeneity in the somatostatin-14 region of the chromatograms indicates that the peptide may be further metabolized by the action of aminopeptidases.