MODE OF GROWTH OF THE JEJUNAL CRYPT CELLS OF THE RAT: AN AUTORADIOGRAPHIC STUDY USING DOUBLE LABELLING WITH 3H- AND 14C-THYMIDINE IN LOWER AND UPPER PARTS OF CRYPTS

The frequency distribution of cells through the mitotic cycle in lower and upper portions of jejunal crypts of the rat was examined by the ³H-¹⁴C-thymidine double labelling technique. Isolated crypts were cut perpendicular to the longitudinal axis so that the percentage of cells in the lower portion varied from 16 to 74 %. The lower and upper portion of the same crypt were squashed separately on one microscope slide and the number of ³H- and ¹⁴C-only labelled cells were scored to determine the flow rate into and out of S for the two portions. The mitotic cycle and its phases of the crypt epithelial cells were also determined. For lower portions of crypts which contained less than 40 % of the total cell number in that crypt the flow rate into S was about 1–7 times that of the flow rate out of S indicating that nearly every mitosis in this region produced two proliferative daughter cells. As the proportion of cells in the lower part of the crypt increased the quotient of the flow rate into S divided by the flow rate out of S decreased, and approached the steady state value of 1 0 in lower portions containing 60–74 % of the cells. For upper portions of crypts which contained less than 40% of the total crypt cells the flow rate into S was about 0 2 times that of the flow rate out of S, indicating that in this region mitoses predominantly produced non-proliferative daughter cells. The results obtained were in good agreement with the model of crypt cell proliferation proposed by Cairnie, Lamerton & Steel (1965b).     

The effects of repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine.

Keratinocyte growth factor (KGF) administered on a daily basis for 3 or more days can result in dramatic changes in tissue architecture, particularly the thickness in oral epithelia, and can afford protection against the cytotoxic effects of radiation on the clonogenic stem cells in the crypts. This protection of intestinal stem cells (increased numbers of surviving crypts) is reflected in an increased survival of animals exposed to a lethal dose of irradiation. The mechanisms underlying these effects are not clear. The present experiments were designed to investigate the nature of any proliferative changes induced in the crypts of the small intestine by protracted exposure to KGF. Tritiated thymidine or bromodeoxyuridine labeling showed statistically significant increases in labeling in the stem cell zone of the crypt, with a concomitant reduction in labeling in the upper regions of the crypt corresponding to the late-dividing transit population. The increase in labeling in the lower regions of the crypt was also observed with Ki-67 staining, but the reduction in the upper regions of the crypt seen with tritiated thymidine was not observed with Ki-67. Metaphase arrest data suggest that the rate of progression through the cell cycle is essentially the same in KGF-treated animals as in controls, but there is a statistically significant increase in the number of mitotic events per crypt. Double labeling studies suggest that, at certain times of the day, there is a greater influx into S phase than efflux. The data overall indicate that KGF induces some complex proliferative changes in the intestinal crypts and are consistent with the hypothesis that the radioprotection may be afforded, at least in part, by a KGF-induced increase in stem cell numbers and/or increases in the number of stem cells in the S phase of the cell cycle. This alteration in the homeostasis of the crypt is compensated for by a foreshortening of the dividing transit lineage.     

Renewal of the epithelium in the descending colon of the mouse. IV. Cell population kinetics of vacuolated-columnar and mucous cells.

Proliferation and migration of cells in the vacuolated-columnar and mucous cell lines were studied in the descending colon of adult female mice given a single injection or a continuous infusion of 3H-thymidine and killed at various intervals from one hour to 12 days. This investigation was carried out using one mum-thick Epon sections which were radioautographed after staining with the periodic acid-Schiff technique and iron-hematoxylin. In the normalized crypts with ten equal segments, labeled vacuolated cells at one hour after injection of 3H-thymidine were encountered in the lower four segments and in decreasing numbers in segments 5 through 7. From the percent labeled cells in segments of the crypt, the birth rate and fluxes of cells were computed. Moreover, it was found that a cell in the vacuolated-columnar cell line would undergo three mitotic cycles on the average from its birth at the cryptal base to its extrusion from the surface; of these three cycles, the last one which took place from segment 3 to segment 7 appeared to be a changeover from dividing cells to non-dividing cells, in accordance with the "slow cut-off" model of Cairnie et al. ('65b). Mucous cells located in segments 1 through 6 of the crypt were capable of incorporating 3H-thymidine and thus capable of undergoing mitosis. However, the rate of turnover of mucous cells based on proliferative rate was found to be much lower than the rate of turnover of mucous cells based on the transit time in the non-dividing segments of the crypt. Since there was a concomitant overproduction of cells in the vacuolated cells and newly formed mucous cells in the lower portion of the crypt, it was concluded that some vacuolated cells would give rise to mucous cells. This putative transformation occurred in the lower four segments of the crypt. Mucous cells which were formed by transformation would migrate upward along the cryptal wall and accumulate more mucus in the theca; in doing so, they would undergo two divisions, on the average, before they became non-dividing mucous cells. In ascending the cryptal walls, both vacuolated-columnar cells and mucous cells appeared to migrate at a similar speed; they moved much slower at the base of the crypt and accelerated toward the upper portion of the crypt, but they migrated at a constant speed in the non-dividing segments of the crypt.     

AUTORADIOGRAPHIC CONFIRMATION OF THE MITOTIC DIVISION OF EVERY MOUSE JEJUNAL CRYPT CELL LABELLED WITH 3H-THYMIDINE

The question was investigated of whether for crypt epithelia of the jejunum of the mouse all cells labelled after a single injection of ³H-TdR subsequently divide or whether cells exist in the crypt which synthesize metabolic DNA and, therefore, do not undergo division after labelling.
A double labelling experiment was performed with a first injection of ³H-TdR followed 1 hr later by an injection of ¹⁴C-TdR. Then from double emulsion autoradiographs of isolated squashed crypts the number of ³H-only, ¹⁴C-only and double labelled cells and mitoses were counted.
The double labelling produced a narrow, 1 hr wide sub-population of ³H-only labelled cells. This subpopulation of S cells completed its division before labelled cells were lost from the crypts by migration onto the villi. The results showed that this subpopulation of ³H-only cells completely doubled within 3 hr and then remained constant through 6 hr. From this result it was concluded that every cell labelled after a single injection of ³H-TdR divides.
From the same autoradiographs the flow rate through the end of mitosis was measured. From the flow rate and the mitotic index a mitotic duration of 0·5 hr was determined. The agreement of this measured mitotic time with the value calculated from the labelling index, mitotic index and S duration is also strong evidence that every labelled cell divides.
Both experiments show that the intestinal crypt does not contain cells synthesizing metabolic DNA.     

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.     

CONTROL OF INTESTINAL EPITHELIAL REPLACEMENT: LACK OF EVIDENCE FOR A TISSUE-SPECIFIC BLOOD-BORNE FACTOR

The small intestine of rats was cut across in two places, about 14 and 50% of the length of the small intestine from the pylorus, and continuity was re-established by suturing the proximal and distal ends. The resulting sac of small intestine, averaging 36% of the total length of the small intestine, had its upper end closed off, and its lower end anastomosed, either to the intestine-in-continuity (an ‘intestine-sac’), or to the skin of the abdominal wall (a ‘skin-sac’). On the ninth post-operative day, the cell production rate in squashes of micro-dissected whole crypts of Lieberkühn was measured by mitotic blockade with Colcemid. The rate of cell production in unoperated and sham-operated rats was 30 cells/crypt/hr, throughout the length of the small intestine. In the intestine in continuity, the rate increased to an average of 46 cells/crypt/hr above the anastomosis, and to 54 cells/crypt/hr below it. At the lower end of the ‘intestine-sac’, which drained into the intestine-in-continuity, the rate was 39 cells/crypt/hr, while in the lower end of the sac which drained to skin the rate of cell production was only 16 cells/crypt/hr. This significantly lower cell production rate in intestine which was not in contact with ingesta is taken to be evidence of the importance of local, rather than blood-borne factors in the control of epithelial replacement.     

Development of the bovine ileal mucosa

The development of the bovine ileal mucosa was studied with particular reference to maturation during the fetal and neonatal period. In this region, by 4-5 months of fetal development, vacuolation of the epithelial cells had occurred on the villi, and the goblet and absorptive cells in the crypts were present. By 6-9 months, the villi were longer and more numerous than in the previous stages. At the same time, the vacuolated cells could be seen predominantly on the upper half of each villus. The absorptive cells and goblet cells were more distinct in the crypt and lower half of each villus. Moreover, the goblet cells showed differences in mucin, while in the submucosa the lymphoid follicles were seen to have enlarged to become a prominent feature of the Peyer's patches at this stage. At birth, in suckled animals, the ileal cells on the lower area of each villus and in the crypt appeared more like mature cells. In contrast, there were numerous inclusion bodies in epithelial cells on the upper half of each villus. They appeared in the apical portion of the cytoplasm as vacuoles with stainable or dense contents. By 1 week, however, epithelial cells no longer contained inclusion bodies, and absorptive and goblet cell populations had begun to emerge from the crypts. These histological results suggest that the bovine ileal mucosa has two distinct turning points during its development in the fetus and the neonate. Initially all the mucosal structures are present in fetuses at 6-7 months of gestation, and then the vacuolated cells covering the ileal villi are replaced by mature, nonpinocytosing epithelium which emerges from the crypts on or before the 7th day after birth (ileal closure).     

Scoring mitotic activity in longitudinal sections of crypts of the small intestine

Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. The correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. The results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestine before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor fD for the mitotic index of 0.59 is obtained; (6) the correction factor fT derived from the shape and position of the mitotic figures measured in 3 microns longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor fmod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is fD; for mouse small intestine it is 0.59.     

Neural stimulation of mitotic activity in the crypts of lieberkühn in rat jejunum.

The influence of nerve stimulation and sham-stimulation on the mitotic rate in epithelial cells lining the crypts of Lieberkühn in the jejunum of anaesthetized rats was studied. Administration of the anaesthetic and opening the abdominal cavity was without significant effect on the crypt cell mitotic rate. However, externalizing a loop of jejunum and applying sham-stimuli to its mesenteric nerves resulted in a significant decrease in the crypt cell mitotic rate in that loop. Application of electrical stimuli to the mesenteric nerves of another externalized jejunal loop resulted in a significant increase in the mitotic rate in the crypt cells of that segment. Similar acceleration of crypt cell proliferation by electrical stimuli applied to mesenteric nerves was also seen in chemically sympathectomized rats.     

NEURAL STIMULATION OF MITOTIC ACTIVITY IN THE CRYPTS OF LIEBERKÜHN IN RAT JEJUNUM

The influence of nerve stimulation and sham-stimulation on the mitotic rate in epithelial cells lining the crypts of Lieberkühn in the jejunum of anaesthetized rats was studied. Administration of the anaesthetic and opening the abdominal cavity was without significant effect on the crypt cell mitotic rate. However, externalizing a loop of jejunum and applying sham-stimuli to its mesenteric nerves resulted in a significant decrease in the crypt cell mitotic rate in that loop. Application of electrical stimuli to the mesenteric nerves of another externalized jejunal loop resulted in a significant increase in the mitotic rate in the crypt cells of that segment. Similar acceleration of crypt cell proliferation by electrical stimuli applied to mesenteric nerves was also seen in chemically sympathectomized rats.     

Scoring Mitotic Activity In Longitudinal Sections of Crypts of the Small Intestine

Abstract. Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. the correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. the results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestime before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor f_d for the mitotic index of 0.59 is obtained; (6) the correction factor f_T derived from the shape and position of the mitotic figures measured in 3 μm longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor f_mod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is f_D; for mouse small intestine it is 0.59.     

Patterns of Proliferative Activity in the Colonic Crypt Determine Crypt Stability and Rates of Somatic Evolution

Epithelial cells in the colon are arranged in cylindrical structures called crypts in which cellular proliferation and migration are tightly regulated. We hypothesized that the proliferation patterns of cells may determine the stability of crypts as well as the rates of somatic evolution towards colorectal tumorigenesis. Here, we propose a linear process model of colonic epithelial cells that explicitly takes into account the proliferation kinetics of cells as a function of cell position within the crypt. Our results indicate that proliferation kinetics has significant influence on the speed of cell movement, kinetics of mutation propagation, and sensitivity of the system to selective effects of mutated cells. We found that, of all proliferation curves tested, those with mitotic activities concentrated near the stem cell, including the actual proliferation kinetics determined in in vivo labeling experiments, have a greater ability of delaying the rate of mutation accumulation in colonic stem cells compared to hypothetical proliferation curves with mitotic activities focused near the top of the crypt column. Our model can be used to investigate the dynamics of proliferation and mutation accumulation in spatially arranged tissues.     

Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick.

Epithelial cells were isolated from rat and chick small intestine by techniques which separated subpopulations of differentiating villus and upper crypt cells from each other and from populations of mitotically dividing lower crypt cells. Incorporation of precursors into epithelial-cell DNA, cytoplasmic rRNA and cytoplasmic poly(A)-containing RNA occurred in the lower crypt cells in vivo when precursor was supplied from the vascular system of the intestine. Incorporation of precursor into 28S and 18S rRNA continued in the upper crypt cells, but occurred to only a very slight extent (if at all) in villus cells, whereas incorporation into poly(A)-containing RNA continued (at a diminishing rate) as the differentiating cells migrated along the villi. When precursor was supplied from the intestinal lumen, its incorporation into DNA and into rRNA of crypt cells was not very different from that observed with the other mode of precursor administration, but incorporation into villus-cell poly(A)-containing RNA then occurred at essentially the same rate in all intestinal epithelial cells in vivo. Cytoplasmic poly(A)-containing RNA appeared to turn over in rat crypt cells with a half-life not exceeding 24 h; crypt-cell rRNA showed no turnover and no evidence could be found for the presence of 'metabolic DNA'.     

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.     

PROLIFERATION KINETICS IN EPITHELIUM OF GUINEA-PIG COLON

Autoradiographs of crypts of guinea-pig colon labelled with ³H-thymidine were quantitatively analysed. The analysis involved: (a) comparison of proliferation parameters in crypts situated in two well-defined regions of colonic folds, i.e. in the top and the basal parts of the fold, and (b) comparison of proliferation parameters in the lower and in the upper parts of an individual crypt. The results provide evidence that: (1) Crypts situated in the top region of folds are significantly longer than those situated in the basal region. (2) The duration of cell cycle and its phases is similar in the crypts of the two regions examined. (3) The rate of ³H-thymidine uptake is higher in the middle than in the initial and final parts of the S phase, and its pattern is the same for the crypts of the two regions of the fold as well as for the parts of the individual crypt. (4) The proliferating pool size is larger in crypts situated in the top than in the basal region of the fold. (5) The proliferating pool size is larger in the lower parts of crypts from each region than in the upper parts. Control of cellular proliferation in the crypt epithelium is considered to operate separately at the level of the individual crypt and at the level of a group of crypts situated in a given region of the colonic wall.     

The distribution of endocrine cells along the mouse intestine: a quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30-60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the bottom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3-4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

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.     

Element concentration changes in mitotically active and postmitotic enterocytes. An x-ray microanalysis study

Unfixed freeze-dried and uncoated tissue sections of the mouse duodenum were suspended across a hole in a carbon planchet and analyzed in a scanning electron microscope fitted with energy-dispersive x-ray analytical equipment. Computer analysis of the x-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and late anaphase-early telophase chromatin regions in the cryptal and villus enterocytes. Elemental concentrations (mmol/kg dry wt) were measured for Na, Mg, P, S, Cl, K, and Ca. None of the elements were compartmentalized preferentially in either the nucleus or the cytoplasm of interphase enterocytes of crypts or in postmitotic enterocytes of villi. In contrast, Ca, S, and Cl are detectable in significantly higher concentrations in mitotic chromatin of dividing enterocytes of the crypt as compared to surrounding mitotic cytoplasm, but Na, Mg, and P are in lower concentrations in the mitotic chromatin as compared to mitotic cytoplasm. Interphase enterocytes of crypts have higher concentrations of Mg, P, and K, and lower concentrations of Na than do postmitotic enterocytes of villi.     

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.     

The distribution of endocrine cells along the mouse intestine: A quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30–60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the botom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3–4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.