Testing the hypothesis that crypt size determines the rate of enterocyte development in neonatal mice

Pieces of mid-jejunum taken from 7-9 day old mice have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis to test the hypothesis that enterocyte development of structure is directly determined by the physical characteristics of the intestinal crypt. Parallel measurements of enterocyte migration rate were carried out using tritiated thymidine to determine the time course of microvillus elongation in neonatal mice. Microvillus length approximately doubled during early enterocyte migration from the crypt base to the lower part of the villus. Enterocyte migration rate was only 0.9 micron/hr at this stage of development, a value considerably less than that found in adult intestine. Results plotting the time dependency of microvillus elongation were fitted by a logistic curve giving a maximal rate for microvillus growth of 0.004 micron/hr. The corresponding estimate of crypt depth was 35 microns. Both these values are considerably less than those found in adult intestine. These results provide strong support for the general hypothesis that some factor associated with the physical length of the crypt, called crypt factor or CF, is directly responsible for controlling the way enterocytes organize subsequent structural differentiation of their surface membranes.     

Galactose increases microvillus development in mouse jejunal enterocytes.

1. Mice fed low carbohydrate and galactose-containing diets have been used to determine both positional and temporal aspects of microvillus development during enterocyte migration from intestinal crypts towards the tips of jejunal villi. 2. The positional dependence of microvillus growth was found to be similar in mice fed low carbohydrate (3.0 kcal/g), galactose-containing lipid substituted (2.9 kcal/g) and galactose-containing agar substituted (5.1 kcal/g) diets. The daily calorific intake by mice fed these diets was about 10.4 kcal/mouse. The maximal microvillus length reached by enterocytes fed galactose was nearly twice that measured in mice fed the low carbohydrate diet. 3. Enterocyte migration rate in mice fed the low carbohydrate and the high calorie galactose-containing diet was twice that measured in mice fed the low calorie galactose-containing diet. These changes were not associated with any noticeable alteration in the size of intestinal crypts. 4. Changes in maximal microvillus length (M) can be predicted from the equation M = 0.0016 CD + 0.073 CD/R, where CD and R refer to crypt depth and enterocyte migration rate respectively, Smith M. W. and Brown D. (1989). Dual control over microvillus elongation during enterocyte development. Comp. Biochem. Physiol. 93A, 623-628. Substituting measured values for CD and R in this equation revealed a specific capacity of galactose to potentiate microvillus development when presented in the form of a high calorie diet. 5. The possibility that galactose, which is poorly metabolized in mice, can increase microvillus expression by interfering specifically with some aspect of carbohydrate metabolism is discussed.     

Comparative aspects of microvillus development in avian and mammalian enterocytes

1. Pieces of chicken mid-jejunum have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis to determine the positional dependence of microvillus elongation. 2. The rate at which enterocytes migrate along the crypt-villus axis has also been measured, by injecting tritiated thymidine into the same animals, to determine the time course of microvillus elongation. 3. In mammals it had been shown previously that the maximal rate of microvillus elongation correlated closely with the physical size of the intestinal crypt (Smith et al., 1984). Comparison of crypt size with maximal rate of microvillus growth in chickens showed this correlation to apply to both animal classes. 4. The maximal microvillus length of a chicken enterocyte was much longer (3.3 microns) and the rate of enterocyte migration much less (4.0 microns/hr) than values reported previously for mammalian intestines. 5. The possibility that some other factor in addition to crypt size is affecting microvillus growth is discussed.     

A comprehensive description of brush border membrane development applying to enterocytes taken from a wide variety of mammalian species

Pieces of small intestine taken from rabbit, rat, mouse, guinea-pig, hamster and pig have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis. Thymidine labelling has also been used to convert measurements of enterocyte position into age of enterocyte. Microvillus lengths showed lower and upper plateaux (a and a + c respectively) with fairly rapid transition from one to the other defined by an exponential coefficient b. The mid-point of elongation (m) usually occurred within 60 micron of the crypt-villus junction. Correlations were found to exist between a and m and between a, m and the depth of the intestinal crypt. Values of b and bc/4, the maximal rate of microvillus elongation, were also found to be correlated with the size of the crypt. None of these parameters were related in any way to the villus height or enterocyte turnover time. The possibility that some factor associated with the physical size of the crypt might be exerting positional and temporal control over the subsequent structural differentiation of enterocytes is discussed.     

Parental spleen cells accelerate the development of intestinal brush border structure and function in neonatal mice

The influence of parental spleen cells on the postnatal development of brush border microvillus membrane structure and the ability to transport lysine and alanine has been studied in the mouse jejunum during the second week of postnatal life. Control tissue taken from 7-11 day old mice has an unchanging crypt-villus structure and a low enterocyte migration rate of about 1 micron hr-1. Microvillus elongation in crypt enterocytes takes 6 days to complete under these conditions. Lysine and alanine transport begin 2 days after structural differentiation has ceased. Parental spleen cells injected into 1-2-day-old F1 mice cause crypt cell hyperplasia, villus shortening and a 3-6-fold increase in enterocyte migration rate after a period of 8 days. These effects are associated with large reductions in the time needed to complete microvillus membrane development and first express absorptive function. Lysine and alanine transport begin approximately 6 hr after structural differentiation has ceased under these conditions. Adaptive changes in the development of enterocyte structure and function, induced by injection of parental spleen cells, bear some resemblance to other changes found to occur normally at weaning and in adult animals subjected to controlled changes in diet and environmental temperature. The possibility that common principles govern enterocyte adaptation and that some of these still apply in an intestine undergoing an immune reaction is discussed.     

Effects of genetic selection on growth rate and intestinal structure in the domestic fowl (Gallus domesticus)

1. Pieces of small intestine taken from chickens subjected previously to continuous selection, relaxed selection or no selection for rapid growth were used to estimate villus surface area and microvillus development to determine what effects genetic selection might have on factors controlling intestinal function. 2. Crypt size and the rates at which enterocytes migrated out of crypts were also measured, after injection of tritiated thymidine, to determine the time course of microvillus elongation. 3. Differences in growth rates measured between highly selected, relaxed selected or unselected birds were found to be correlated with parallel changes in villus surface area. Selection for growth did not change the density, dimensions or pattern of development of enterocyte microvilli. Microvilli did, however, produce a maximal 20-fold increase in villus surface area under all conditions. 4. Crypt size and enterocyte migration rates did not vary significantly between tissue taken from unselected and relaxed selected chickens. Tissue taken from highly selected birds had a crypt size and enterocyte migration rate 40% higher than values found for the other two groups of chickens. 5. The possibility that early genetic selection increased growth potential by uncoupling diet-induced changes on crypt hyperplasia from secondary effects on villus structure, and that later selection increased growth potential by increasing appetite, is discussed.     

Galactose effects on enterocyte differentiation in the mouse jejunum

The present work investigates the ability of galactose to affect enterocyte differentiation during normal development in vivo. Energy intake has also been varied to take account of the fact that galactose is poorly metabolized in mice. Brush-border lactase, alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N, alkaline phosphatase and microvillus length were measured as markers of enterocyte differentiation in mice fed diets containing galactose (G diet), corn oil (E diet) or galactose + corn oil (G + E diet). Maintaining mice on a G instead of E diet reduced brush-border lactase activity and enterocyte migration rates; alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N and microvillus length expression increased and alkaline phosphatase activity remained unchanged. Feeding the G + E diet restored enterocyte migration rates, lactase, aminopeptidase N and dipeptidylpeptidase-IV activities to values found in mice fed the E diet. Galactose stimulation of alpha-glucosidase and microvillus length expression was, however, fully maintained in mice fed the G + E diet. Present results show that enterocyte differentiation is affected independently by varying dietary galactose and energy levels; that galactose effects always increase and energy effects usually decrease expression of enterocyte components and that energy stimulation of lactase activity is exceptional.     

A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells

Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.     

Effects of microflora on the dimensions of enterocyte microvilli in the rat

The length and diameter of enterocyte microvilli at mid-villus position were measured on electron-micrographs. The duodenum, jejunum and ileum of axenic (germfree) and holoxenic (conventional) inbred rats fed the same diet have been studied. The microvilli were significantly shorter in all these intestinal regions when the microflora was present. The decrease in microvillus length (due to the presence of microflora), expressed as a percentage of the length in axenic rat, was 5% in the duodenum, 9% in the jejunum and 18% in the ileum. This was not true of microvillus diameter where only the values found for the ileum of axenic rat were significantly lower, the others not differing among themselves. In conclusion, the presence of a total microflora led to a decrease in the length of enterocyte microvilli. These results have been discussed in relation with studies on renewal and enzyme activity in the intestinal epithelium.     

IGF-I augments resection-induced mucosal hyperplasia by altering enterocyte kinetics

Our objective was to determine if exogenous insulin-like growth factor-I (IGF-I) augments the adaptive growth response to mid small bowel resection in association with changes in enterocyte kinetics. We determined structural adaptation and concomitant changes in enterocyte proliferation, apoptosis, and migration of the jejunum in growing, parenterally fed rats after mid small bowel resection or small bowel transection, and treatment with IGF-I or vehicle. IGF-I treatment in resected rats significantly increased jejunal mucosal mass by 20% and mucosal concentrations of protein and DNA by 36 and 33%, respectively, above the response to resection alone. The enhancement of resection-induced adaptive growth and cellularity by IGF-I reflected an increase in enterocyte proliferation, an expansion of the proliferative compartment in the crypt, and no further decrease in enterocyte apoptosis or increase in enterocyte migration beyond the effects of resection. The ability of IGF-I to augment the mucosal hyperplasia stimulated by the endogenous response to resection substantiates the role of IGF-I as an intestinal mitogen that promotes tissue regeneration.     

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.     

Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection

The intestinal epithelium undergoes a marked adaptive response following loss of functional small bowel surface area characterized by increased crypt cell proliferation and increased enterocyte migration from crypt to villus tip, resulting in villus hyperplasia and enhanced nutrient absorption. Hedgehog (Hh) signaling plays a critical role in regulating epithelial-mesenchymal interactions during morphogenesis of the embryonic intestine. Our previous studies showed that blocking Hh signaling in neonatal mice results in increased small intestinal epithelial crypt cell proliferation and altered enterocyte fat absorption and morphology. Hh family members are also expressed in the adult intestine, but their role in the mature small bowel is unclear. With the use of a model of intestinal adaptation following partial small bowel resection, the role of Hh signaling in the adult gut was examined by determining the effects of blocking Hh signaling on the regenerative response following loss of functional surface area. Hh-inactivating monoclonal antibodies or control antibodies were administered to mice that sustained a 50% intestinal resection. mRNA analyses of the preoperative ileum by quantitative real-time PCR revealed that Indian hedgehog was the most abundant Hh family member. The Hh receptor Patched was more abundant than Patched 2. Analyses of downstream targets of Hh signaling demonstrated that Gli3 was twofold more abundant than Gli1 and Gli2 and that bone morphogenetic protein (BMP)2 was most highly expressed compared with BMP1, -4, and -7. Following intestinal resection, the expression of Hh, Patched, Gli, and most BMP genes was markedly downregulated in the remnant ileum, and, in anti-Hh antibody-treated mice, expression of Patched 2 and Gli 1 was further suppressed. In Hh antibody-treated mice following resection, the enterocyte migration rate from crypt to villus tip was increased, and by 2 wk postoperation, apoptosis was increased in the adaptive gut. However, crypt cell proliferation, villus height, and crypt depth were not augmented. These data indicate that Hh signaling plays a role in adult gut epithelial homeostasis by regulating epithelial cell migration from crypt to villus tip and by enhancing apoptosis.     

Vitamin A deficiency inhibits intestinal adaptation by modulating apoptosis,proliferation, and enterocyte migration

In a prior study, vitamin A-deficient rats subjected to submassive small bowel resections did not mount a normal intestinal adaptive response by 10 days postoperatively, although adaptive increases in crypt cell proliferation were not attenuated and there were no differences in apoptotic indexes. The present study was designed to address the mechanisms by which vitamin A status effects adaptation by analyzing proliferation, apoptosis, and enterocyte migration in the early postoperative period (16 and 48 h) in vitamin A-sufficient, -deficient, and partially replenished sham-resected and resected rats. At 16 h postresection, apoptosis was significantly greater in the remnant ileum of resected vitamin A-deficient rats compared with the sufficient controls. Crypt cell proliferation was increased by resection in all dietary groups at both timepoints. However, at 48 h postresection, proliferation was significantly decreased in the vitamin A-deficient and partially replenished rats. By 48 h after resection, vitamin A deficiency also reduced enterocyte migration rates by 44%. This occurred in conjunction with decreased immunoreactive collagen IV at 48 h and 10 days postoperation. Laminin expression was also reduced by deficiency at 10 days postresection, whereas fibronectin and pancadherin were unchanged at 48 h and 10 days. These studies indicate that vitamin A deficiency inhibits intestinal adaptation following partial small bowel resection by reducing crypt cell proliferation, by enhancing early crypt cell apoptosis, and by markedly reducing enterocyte migration rates, which may be related to changes in the expression of collagen IV and other extracellular matrix components.     

Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.     

CELL POPULATION KINETICS IN THE RAT JEJUNAL CRYPT

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50 % peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1.43 ± 0.14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1.78 cell positions per hour. The crypt column length was 32.9 ± 0.2 cells and the column count was 22.3 ± 0.2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 ± 6 from direct observation of squashed, microdissected crypts. In each crypt 22.5 ± 0.5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0.62. A value of 0.61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.     

The effect of bile and of sodium taurocholate on the epithelial cell dynamics of the rat small intestine.

The absence of bile from the intestinal lumen of rats for a period of 48 hr led to: a decreased proliferative cell pool, reduced cell shedding, and a 50% decrease of the labeling index in the ileum. The constant duodenal perfusion of Na taurocholate for periods of 48, 60, 72, and 96 hr in animals with a biliary diversion was associated with: deepening of crypts and decreased crypt/villus ratios as well as with acceleration of the epithelial cell migration rate on the crypt-villus units. The data suggest that bile and bile acids constitute important regulatory factors influencing enterocyte proliferation, migration and loss.     

Cell population kinetics in the rat jejunal crypt.

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50% peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1-43 +/- 0-14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1-78 cell positions per hour. Tht crypt column length was 32-9 +/- 0-2 cells and the column count was 22-3 +/- 0-2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 +/- l from direct observation of squashed, microdissected crypts. In each crypt 22-5 +/- 0-5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0-62. A value of 0-61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.     

The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration

Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.