GASTROENTEROLOGY
1992;103:1154-1161
Lactase Gene Expression During Early Development of Rat Small Intestine EDMOND ANTOON ROBERT
H. H. M. RINGS, PIET A. J. DE BOER, F. M. MOORMAN, ERIK H. VAN BEERS, JAN DEKKER, K. MONTGOMERY, RICHARD J. GRAND, and HANS A. BILLER
Center for Liver and Intestinal Research, Division of Pediatric Gastroenterology and Nutrition, and Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts
Expression of lactase messenger (m) RNA and protein in rat small intestine during fetal and postnatal development was analyzed using in situ hybridization and immunohistochemistry. Lactase mRNA was first identified at 18 days of development, and lactase protein was first detected at day 20. Lactase mRNA and protein were present along the entire villus. Lactase mRNA increased, reaching a maximum at day 20. Just before birth a decrease in lactase mRNA was observed. In newborn intestine, lactase mRNA was present only from the base of the villus up to the mid-villus region and was undetectable up to the villus tips. Lactase protein continued to be expressed along the entire villus. These data show that expression of lactase mRNA and protein do not parallel, indicating a posttranscriptional control in fetal development. Lactase gene transcription is initiated late in gestation concomitant with villus formation and is exclusively seen in villus epithelial cells. The restriction after birth of lactase mRNA expression to cells at the villus base suggests the occurrence of a previously unknown step in postnatal differentiation of the enterocyte.
L
actase-phlorizin hydrolase (EC 3.2.1.23, EC 3.2.1.62, subsequently referred to as lactase) plays a critical role in the nutrition of young mammals,‘*’ and the developmental pattern of its specific activity in many animal species and human populations is well documented. The specific activity of lactase is very high at birth and during nursing when milk is the sole nutrient; then around weaning a steep decline to low levels is seen in adults.3 As a consequence, adult animals and most human adults are considered to be lactase deficient. This mode of expression has been proven to be inherited through a single dominant autosomal locus. The molecular basis of the regulation of this biological phenomenon of a rapid increase before birth and a decrease of lactase-specific activity later in life is
still not clear. The influence of hormones and starvation on lactase messenger RNA (mRNA) and protein levels has recently been investigated.4*5 The transfection of full-length lactase complementary DNA (cDNA) into COS-1 cells to elucidate synthesis and transport has been the subject of a recent study.’ In addition, on the basis of sequence analysis, it has been suggested that lactase belongs to a superfamily of cellulases and P-glycosidases.7 Total intestinal lactase enzyme levels during maturation follow expression of lactase mRNA, suggesting that lactase total activity is predominantly regulated at a transcriptional level.*,’ Furthermore, it is known that total intestinal lactase activity in adult rats is maintained at the same level as in newborns, in contrast to the developmental pattern of decrease of lactase-specific activity.l’ Posttranslational alterations in glycosylation of the protein occur in a different pattern in time from the postweaning decline in lactase-specific activity, militating against a role for age-dependent changes in glycosylation in the decrease of lactasespecific activity observed during development.” In addition, manipulating posttranslational modifications of the protein, e.g., N-glycosylation processing, does not prevent the appearance ofactive lactase enzyme in the microvillus membrane.” The biosynthesis, posttranslational transport, and immunohistochemical localization of lactase have been characterized in several mammals.‘3-‘6 Human, rabbit, and rat lactase cDNAs have been cloned and sequenced.8*‘6n17 However, the appearance of lactase mRNA and protein in conjunction with morphological maturation and localization of lactase mRNA and protein in rat small intestine during development have not been studied. Recent in situ hybridizations, detecting mRNAs encoding different proteins expressed in enterocytes, including cytochrome PO 1992
by the American Gastroenterological 0016-5065/92/$3.00
Association
LACTASE
October 1992
4SOIIb1,‘s aminopeptidase N,” fatty acid binding have shown that protein, 2o and sucrase-isomaltase,21 the respective mRNAs are localized along the cryptto-villus axis in the intestine. The only developmental study of the intestine was performed by Tyner et of a-fetoprotein. al.” on the expression We describe the pattern of expression and topography of lactase mRNA and protein in rat small intestine during fetal and postnatal development using in situ hybridization and immunohistochemistry, respectively. The objective of the present study was to investigate the coordination of lactase mRNA and protein expression at the histological level and to understand the molecular basis of this expression during development.
Materials and Methods Animals Wistar rats were purchased from TN0 Breeding Laboratories (Zeist, The Netherlands) and housed at the Academic Medical Center animal facilities at constant temperature and humidity on a l&hour light/dark cycle. The dams had free access to standard chow and water. Fetal age was calculated from dated matings and neonatal age from the time ofbirth (22 days after conception). Fetal specimens were taken from 16-, la-, 20-, and 22day-old rat fetuses, and subsequent specimens were from O-day-old newborn rats before and 4 hours after nursing. Postnatal rat intestinal tissue samples were obtained from rats of 1, 4,9,18, and 25 days. Sections of three animals of the same age were studied. Tissue
Processing
Fetal samples were sections through whole fetuses. Total newborn intestines were rapidly dissected and fixed by immersion as described below. Tissue of older animals was from proximal jejunum, approximately 5 cm distal to the ligament of Treitz. Chemicals [a-35S]Deoxycytidine 5’-triphosphate (dCTP) (1350 Ci/mmol) was purchased from Du Pont-New England Nuclear (‘s-Hertogenbosch, The Netherlands) and nuclear research emulsion G5 from Ilford (London, England). Pronase (type XIV), and all other chemicals were purchased from Sigma (St. Louis, MO). Specifications
of Molecular
Probes
Lactase mRNA was detected using a 1.8-kilobase (kb) EcoRI-PstI insert of the cDNA lactasel-Bluescript clone, derived from a 2.3-kb rat lactase partial cDNA.‘This 1.8-kb insert corresponds to position 3839 through position 5694 of the full-length cDNA of rat lactase, as described by Duluc et alI7 As a positive control for the detection of mRNAs in the intestinal epithelium, PstI and EcoRI fragments of the 5.6-kb rat carbamoylphosphate synthetase (CPS) cDNA were used as previously described.23 The
GENE EXPRESSION
DURING DEVELOPMENT
1155
urea-cycle enzyme, CPS, is found in the mitochondria of both hepatocytes and enterocytes23-25 and is nucleus encoded. The agarose-purified (NuSieve LMP Agarose; FMC BioProducts, Rockland, ME) DNA probes were labeled overnight at 15’C with [a-35S]dCTP according to the multiprime labeling method,26 yielding fragments of approximately 100 bp.27 Localization Hybridization
of Lactase
mRNA
by In Situ
To localize lactase mRNA in rat small intestine, we performed in situ hybridization using previously published protoco1sz3 Tissues were fixed in 4% paraformaldehyde and further processed for preparation of paraffin sections. The 7-pm-thick paraffin sections were mounted onto 3-aminopropyltriethoxysilane-coated microscope slides. The in situ hybridization procedures for lactase and CPS were performed on closely adjacent sections to allow comparison of the hybridization patterns. Series of sections at each age were processed under identical conditions, with aliquots of probe from the same labeling reaction, to allow comparison of mRNA expression at different ages. Sections were deparaffinized in xylol, dehydrated in 100% ethanol, and air-dried. They were incubated in 0.2 mol/L HCl for 20 minutes, rinsed in sterile water for 5 minutes, and soaked in 2X SSC (1X SSC = 0.15 mol/L sodium chloride, 0.015 mol/L sodium citrate, pH 7.2) at 70°C for 10 minutes. Subsequently, they were incubated with pronase (250 pg/mL) in 50 mmol/L Tris and 5 mmol/L ethylenediaminetetraacetic acid (EDTA) (pH 7.5) at 37°C for 20 minutes. Pronase digestion was stopped by addition -of 0.2% glycine in phosphate-buffered saline (PBS) followed by two 30-second washes with PBS. Sections were then incubated in 4% paraformaldehyde in PBS for 20 minutes, rinsed in PBS, dehydrated in a graded series of ethanol, and air-dried. Hybridization was performed overnight at 44’C in 50% (vol/vol) deionized formamide, 0.1% (vol/vol) Triton X-100, 10% (wt/vol) dextran sulfate, 2X SSC, 0.02% (wt/ vol) bovine serum albumin, 0.02% (wt/vol) polyvinylpyrolidone and 0.02% (wt/vol) Ficoll, 10 mmol/L dithiothreitol, 200 ng/pL herring sperm DNA, and labeled probe. The probe concentration was approximately 0.5 ng/pL and contained 5 X IO4 cpm/pL specific activity. Sections were washed twice at 44’C for 15 minutes in 50% (vol/vol) formamide and 1 X SSC and twice for 10 minutes in 1 X SSC and finally in 0.1X SSC at the same temperature. Sections were dehydrated in a graded series of ethanol, containing 0.3 mol/L ammonium acetate, air-dried, and dipped in darkness into liquid emulsion (Ilford G5 emulsion, diluted 1:1.5 with glycerol/water: London, England). Dipped slides were stored at 4’C for 5 days. The emulsion was developed by dipping the slides for 4 minutes into developer at 18°C. Developer was freshly made, consisting of 0.45% (wt/vol) Amidol (4 - hydroxy - 1,3 - phenylenediammoniumdichloride; Merck, Darmstadt, Germany), 1.8% (wt/vol) Na,SO,, and 0.08% (wt/vol) potassium bromide, and filtered before use. Slides were then sequentially dipped for 1 minute into a stop bath containing water and
1156
RINGS ET AL.
GASTROENTEROLOGY
then dipped in a fixation bath for 10 minutes by soaking them in 30% (wt/vol) Na,S,O, * 5H,O. Tissue was counterstained in 0.01% (wt/vol) toluidine blue and dehydrated. Sections were mounted in Malinol (Chroma, Stuttgart, Germany) and photographed with Agfa Pan APX 25 (15 DIN) film (Agfa-Gevaert, Leverkusen, Germany). Antibodies The monoclonal antibody against rat lactase” used for immunohistochemistry has been described previ0us1y.‘~ The antibody was used in the form of ascites from pristane-primed BALB/c mice. As a positive control for the detection of proteins in the enterocytes, a polyclonal anti-CPS antiserum was used.24 Immunodetection Intestine
of Lactase
in Rat Small
Lactase protein was detected by immunohistochemistry using the indirect unconjugated peroxidase anti-peroxidase (PAP) technique.” Tissues were fixed in methanol, acetone, and distilled water (40/40/20, vol/ vol/vol) and further processed for preparation of paraffin sections. The 7-pm paraffin sections were mounted on polylysine-coated microscope slides. Lactase and CPS were visualized on adjacent sections to allow direct comparison of the patterns. After deparaffination, sections were treated with hydrogen peroxide (3%, vol/vol in PBS) for 20 minutes to reduce endogenous peroxidase activity, followed by preincubation in TENG-T [lo mmol/L Tris, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.25% (wt/vol) gelatin, and 0.05% (vol/vol) Tween-20; pH 81 for 30 minutes to reduce nonspecific binding. Sections were incubated with ascites fluid containing the anti-lactase monoclonal antibody (diluted 1:6400 in PBS) or with serum-containingantibodies directed against CPS (diluted I:2000 in PBS). Monoclonal antibody binding was detected using rabbit anti-mouse immunoglobulin, goat anti-rabbit immunoglobulin, and rabbit-PAP complex, respectively. Polyclonal antibodies were detected using goat anti-rabbit immurespectively. noglobulin and rabbit-PAP complex, Negative controls included incubations without primary antibodies. All incubations were followed by washes in PBS for 5 minutes. The immunocomplex formed was visualized by incubation of the sections with 0.5 mg/mL 3,3’diaminobenzidine, 0.02% (vol/vol) hydrogen peroxide in 30 mmol/L imidazole, and 1 mmol/L EDTA (pH 7). Sections were mounted in Entellan (Merck). Control
Vol. 103. No. 4
more stringent conditions (higher hybridization and washing temperature at 50°C). Data are presented from the lower stringency hybridizations.
Results Morphological
Development
At 16 days of development, the intestine is lined by stratified epithelium; the lumen and villi are not yet present. At 18 days of development, a monolayer of cuboidal epithelial cells cover the developing villi in the more proximal part of the intestine. In contrast, at the same age in the more distal part of the intestine, no villi can be observed, although secondary lumina have appeared. After its formation around 18 days of development, the lumen is lined by an undifferentiated stratified epithehum. These observations are consistent with the previously described craniocaudal gradient, with the slower maturation of the distal intestine than the proximal intestine.30 At 20 days of development, the cuboidal epithelial cells have become more columnar and villus height is increasing. Crypts appear only after birth. Lactase Intestine
mRNA
and Protein
in the Fetal
At 16 days of development, when the mucosa is characterized by an undifferentiated, stratified epithelium, CPS mRNA is abundant in the intestinal epithelium (Figure 1A). In contrast, neither lactase
Experiments
The three different control experiments for in situ hybridization were performed as follows. In fetal and postnatal samples, control hybridizations were performed using pBR322 vector DNA labeled as described above. To exclude nonspecific hybridization with the 1.8-kb lactase probe, the fetal data of rats of 18 and 20 days of development were verified by individual hybridization to three subfragments of the 1.8-kb probe obtained by HinfI digestion (1000, 450, and 250 bp, respectively]. To confirm the specificity, in situ hybridization was also performed under
Figure 1. Detection of mRNA of CPS and lactase in cross sections of fetal rat at 16 days of gestation. (A) CPS mRNA expression in the intestinal epithelium. The multilayer of undifferentiated, stratified epithelium is heavily labeled by the CPS probe. (II) Lactase mRNA expression in the intestinal epithelium. In an adjacent tissue section, lactase mRNA cannot be detected in the intestinal epithelium. The arrows indicate corresponding sections of undifferentiated multilayered stratified epithelium in the two specimens (bar = 6.5 mm).
October 1992
LACTASE GENE EXPRESSIONDURING DEVELOPMENT
1157
lus enterocytes. The intervillus regions are devoid of lactase mRNA hybridization. In contrast, at 22 days of development, this pattern of mRNA expression is significantly altered, because there is an apparent decrease of lactase mRNA abundance just before birth (Figure 4B). Lactase mRNA and Protein Expression Postnatal Development
During
In contrast to the uniform expression of lactase mRNA in fetal intestine (shown above), expression in postnatal intestine undergoes topographic redistribution. As shown in Figure 5A and C, lactase mRNA becomes restricted to the lower portions of
Figure 2. Detection of lactase mRNA in fetal rat intestine at 18 days of gestation. (A) Standard hematoxylin and azophloxine staining of intestinal sections. In the proximal section (P), rudimentary villi lined by a single layer of cuboidal epithelium (open triangle) have formed. A more distal part of the intestine (D) still shows a multilayer of undifferentiated, stratified epithelium (closed triangle). (B) Expression of lactase mRNA. Lactase mRNA is expressed in the monolayer of epithelium in the proximal small intestine. Lactase mRNA is not detectable in the undifferentiated multilayered epithelium of the distal part of the intestine (bar = 0.1 mm).
mRNA (Figure 1B) nor lactase protein (not shown) can be detected in the intestine at this age. At 18 days of development (Figure 2), the proximal intestine is characterized by the presence of primitive villi, lined by a single layer of cuboidal epithelial cells (Figure 2A). The distal intestine continues to show a stratified multilayered epithelium. Lactase mRNA is first detectable at this age and is found in all of the epithelial cells lining the primitive villi (Figure 2B). No lactase can be detected at this age in the stratified multilayered epithelium of the distal intestine. Lactase mRNA is present at 18 days of development (Figure 3A), whereas lactase protein is not yet detectable (Figure 3B). Both mRNA and protein for CPS are detectable in these specimens (data not shown), as was also previously reported.23p25 At 20 days of development, when rapid villus growth has occurred, lactase mRNA is uniformly distributed in enterocytes lining the entire villus (Figure 3C). At this age, lactase protein is readily detected in the microvillous membranes of the enterocytes, with equal distribution from villus base to villus tip (Figure 3D). Lactase protein is also found in membranous material from sloughed cells in the lumen. Figure 4 contrasts lactase mRNA density at 20 and 22 days of development under the same hybridization conditions. Similar to Figure 3C, Figure 4A shows intense expression of lactase mRNA in all vil-
Figure 3. Detection of lactase mRNA and protein in fetal rat intestine at 18 and 20 days of gestation. (A) Lactase mRNA expression in 18-day intestine. Lactase mRNA is expressed in the newly formed epithelial monolayer (arrow). (B) Lactase protein expression at 18 days of gestation. No lactase protein is detectable in these same epithelial cells (arrow) at this age. (C) Lactase mRNA expression in 20.day intestine. The epithelial layer displays a uniform expression of lactase mRNA over the whole villus. (D) Lactase protein expression at 20 days of gestation. Brush borders of the enterocytes show staining for lactase protein from villus base to the villus tip (bar = 0.1 mm).
1158
RINGS ET AL.
the villi immediately after birth and before suckling. Lactase mRNA can only be detected from the villus base to the mid-villus region, whereas hybridization is undetectable from mid-villus to villus tip (Figure 5A). This pattern does not seem to be a result of suckling, because prevention of suckling does not influence the distribution of lactase mRNA expression (data not shown). There is a gradual restriction of lactase mRNA to the lower half of the villus during postnatal development. This is shown in intestine of g-day-old neonates (Figure 5C), and this restriction remains essentially unchanged at all subsequent ages studied, including adult animals (data not shown). Lactase mRNA hybridization in crypt cells was never observed. Despite the striking morphological difference of lactase mRNA shown in Figure 5A and C, lactase protein at birth and 9 days postnatally shows uniform expression from the villus/crypt junction along the whole villus to the villus tip, as shown in Figure 5B and D. These patterns remain constant throughout subsequent postnatal development of the intestine. These patterns were not influenced by suckling, as studied for animals of 0 days of age (data not shown). Lactase protein was never detected in crypt cells. No evidence for a mosaic pattern of expression of lactase mRNA was observed at any embryonic age, nor was there any mosaic pattern of lactase protein expression. To ascertain that technical factors do not explain
Figure 4. Detection of lactase mRNA in fetal rat intestine at 20 and 22 days of gestation. (A) Expression of lactase mRNA at 20 days of gestation. The fetal epithelial cells show a high expression of lactase mRNA. (B) Expression of lactase mRNA at 22 days of gestation. Under identical hybridization conditions, with the same probe as in (A), an apparent decrease of lactase mRNA transcripts is observed in the older intestine. Lactase mRNA is still uniformly distributed over the whole villus but not found in the intervillus regions (arrows) (bar = 0.1mm).
GASTROENTEROLOGY
Vol. 103.No. 4
Figure 5. Detection of lactase mRNA and protein in the small intestine of newborn (O-day) and suckling (g-day-old) rats. (A) Expression of lactase mRNA in newborn rats. Lactase mRNA expression is restricted to an area from the base to approximately half of the height of the villi (arrow). (B) Expression of lactase protein in newborn rats. Immunohistochemical staining shows lactase from the villus base to the tip. (C) Expression of lactase mRNA in suckling rats. At 9 days of age, lactase mRNA is detectable only in an area from the villus base to the midpoint of the villi (arrow). (D) Expression of lactase protein in suckling rats. At this age as well, lactase protein is identified from base to tip of the villi. Neither lactase mRNA nor protein is detectable in the crypt region (star) (bar = 0.1mm).
the absence of lactase mRNA and protein in crypt cells, hybridization with the CPS probe was used as a positive control. CPS has previously been shown to be localized to the crypts in postnatal rat intestine.24*25As shown in Figure 6A, when intestine was sectioned transversely, CPS was readily seen in crypts whereas lactase mRNA was undetectable. However, lactase mRNA was detectable at the villus base as shown in Figure 6B. Data obtained from the control experiments were as follows. In all ages studied, the pBR322 DNA gave no hybridization. Hybridization with the three HinfI subfragments of the 1.8-kb lactase probe yielded patterns identical to those with the original probe (not shown). The increased hybridization and washing temperature of 50°C did not change the hybridization pattern or diminish the intensity of signals.
October 1992
Figure 6. Detection of mRNA of CPS and lactase in adjacent crypt-rich tangential sections of a loop of small intestine of a &day-old rat. (A)Expression of CPS mRNA. Only the areas of the section which include the crypts (Cl show a signal for CPS. (B) Expression of lactase mRNA. In the same zone, lactase mRNA is not detectable. In the area that includes villus sections (V), lactase mRNA expression is found (arrow) (bar = 0.1mm).
Discussion In fetal rats formation of villi begins at 17 days of development, and tall, well-developed villi have formed in the proximal intestine by the 19th day of development.30 In contrast, discrete, well-defined crypts do not develop until shortly after birth. Hermos et a1.31 showed that some mitotic cells in the intestinal epithelium could be identified along the entire length of the villi in the rat fetal intestine as late as 1 day before birth. In the days before birth, the proliferating cells progressively become restricted to the bases of the villi and intervillus regions. The undifferentiated multilayer of stratified epithelial cells in the intestine at 16 days of development showed no lactase gene expression, whereas CPS mRNA was already present. Lactase mRNA was only identified at 18 days of development, either concomitant with or immediately after formation of a monolayer of cuboidal epithelial cells lining the villi of the fetal gut, suggesting that lactase expression is dependent on development of the enterocytes. The expression of lactase mRNA does not occur simultaneously throughout the intestine but in a proximalto-distal progression, as has been described for villus morphogenesis.32 There is a delay between the initial detection of lactase mRNA and detection of lactase protein. This may in part reflect technical differences or a discrepancy in sensitivity between the in situ and the immunohistochemical detection in paraffin sections. However, it also seems to represent a real physiological phenomenon, because with the same techniques lactase can be detected at 20 days of development
LACTASE GENE EXPRESSION DURING DEVELOPMENT
1159
using diluted antibody (1:6400), whereas it can not using the be detected at 18 days of development same or a loo-fold more concentrated antibody dilution. In addition, postnatal animals show both mRNA and protein in the enterocytes as soon as they exit the crypts. The time difference may represent the time required to accumulate significant amounts of protein in the fetal tissue or a delay in maturation of the translation machinery, as has been documented during oocyte maturation.33s34 Although in situ hybridization allows only semiquantative interpretation, the results of the present study seem to indicate that approximately 2 days before birth, the hybridization of lactase mRNA is most intense with expression along the entire villus. On the day of birth, the intensity of hybridization was decreased and hybridization became restricted to the lower half of the villus. In agreement with previous measurements of lactase activity, which peak in the perinatal period,3*‘0 this pattern suggests maximal transcription before birth, with decreased levels after birth. The restriction of mRNA expression from all fetal epithelial cells to only the enterocytes on the lower part of the villus after birth has not been reported previously and suggests the occurrence of an unknown step in postnatal differentiation of the enterocyte. The discrepancy between the expression of lactase mRNA and protein along the crypt/villus axis may be a result of changes in the half-life of the respective molecules after birth. This study, however, does not allow conclusions regarding the rate of translation of lactase mRNA into protein.35 Several possible explanations for this restriction of mRNA expression may be advanced. The most obvious stimulus, the changes associated with the onset of feeding, does not appear to be causative, because prevention of suckling did not alter the pattern. The shift from general cell proliferation to crypt-restricted proliferation and villus cell migration, coincident with the formation of the crypts, occurs around birth; however, no exact data are available. The observed change in lactase mRNA expression pattern may reflect the beginning of this shift to crypt-restricted proliferation and villus cell migration. Possible systemic hormonal stimuli from the process of birth have not yet been examined and remain a subject for further investigations. In adult intestine, there is a clear separation of proliferating crypt cells and differentiated villus cells. Numerous studies of enzyme levels have shown that none of the microvillus membrane enzymes such as sucrase-isomaltase, aminopeptidase N, or lactase are detectable below the crypt/villus junction. These enzymes are detected from the villus base and accumulate in characteristic patterns as the enterocytes migrate up the villus. In the crypts, a
1160 RINGS ET AL.
cells and a zone of nonproliferzone of proliferating ating cells, which do not yet show evidence of specialization, can be identified by DNA labeling.3s The nonproliferating crypt cells will move out of the crypts and differentiate into mature enterocytes. Although these moving cells begin to show differentiated proteins after the crypt/villus junction is passed, it is not clear when they begin to express the mRNA for these proteins. As shown in the present study, lactase mRNA is found in all enterocytes lining the villi of the fetal intestine, whereas no mRNA can be identified in the cells of the intervillus region. When crypt formation is completed after birth, lactase mRNA is only found from the base of the villi at the crypt/villus junction to the mid-villus region. This pattern is maintained during further development. Neither lactase mRNA nor protein was detected in the crypts at any age in the present study. A similar pattern in older animals was recently reported by Traber’l who showed that initiation of suerase-isomaltase mRNA expression in adult enterocytes occurred as the enterocytes reached the crypt/villus junction, with a steady decrease in expression as cells moved up the villus. In the postnatal intestine, the pattern for two apical membrane hydrolases, sucrase-isomaltase and lactase, appears very similars3’ However, other enterocyte proteins have very different patterns of expression. CPS mRNA is confined to the crypts in adult rats, although the protein is present in all of the villus enterocytes.23*24 In contrast, the expression of mRNA for transforming growth factor j3l increases steadily in the villus epithelial cells, with a maximum in the villus tip region3* As expected, there seems to be no global pattern of mRNA expression in intestinal epithelial cells. Thus, in the adult intestine, there seems to be four functional zones for lactase: a crypt zone of proliferating cells; a crypt zone of nonproliferating, undifferentiated cells; a villus zone of high mRNA expression and the beginning of protein accumulation; and a villus zone of decreased mRNA expression and maximal protein accumulation. The present study also indicates that in fetal intestine these zones are all contiguous. In addition, the adult phenotype becomes established during a brief perinatal period from 22 days of gestation to the day of birth. In immunohistochemical studies of the developmental pattern of two cytoplasmic proteins, fatty acid binding protein (FABP) and apolipoprotein AIV (apoAIV), Rubin et al. 39 observed that FABP but not apoAIV appeared initially in a patchy or mosaic pattern. They found that FABP was first detectable in scattered cells of the fetal intestinal epithelium, whereas adjacent cells did not express it. In contrast, apoAIV showed a uniform expression as soon as it
GASTROENTEROLOGY
Vol. 103, No. 4
appeared. With age, the mosaic pattern of FABP expression became a uniform distribution throughout the epithelium. We found no evidence for a mosaic expression of lactase, an apical membrane protein. These data suggest that the initiation of expression of lactase is mediated by a different mechanism from that of FABP, possibly the same or one similar to that for apoAIV. The mechanisms by which lactase expression is initiated and its pattern regulated are currently under investigation. References 1.
Henning SJ. Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol 1961;241:Gl99-
G214. 2. Flatz G. Genetics of lactose digestion
3.
4.
5.
6.
7.
a.
9.
10.
11.
12.
13.
14.
in humans. In: Harris H, Hirschhorn K, eds. Advances in human genetics. Volume 16. New York: Plenum, 1987:1-77. Doe11 RG, Kretchmer N. Studies of small intestine during development. I. Distribution and activity of beta-galactosidase. Biochim Biophys Acta 1962;62:353-362. Castillo RO, Glasscock GF, Noren KM, Reisenauer AM. Pituitary regulation of postnatal small intestinal ontogeny in the rat: differential regulation of digestive hydrolase maturation by thyroxine and growth hormone. Endocrinology 1991; 129:1417-1423. Freund JN, Duluc I, Raul F. Lactase expression is controlled differently in the jejunum and ileum during development in rats. Gastroenterology 1991;100:388-394. Naim HY, Lacey SW, Sambrook JF, Gething MH. Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein. J Biol Chem 1991;266: 12313-12320. Grabnitz F, Seiss M, Rticknagel KP, Staudenbauer WL. Structure of the j3-glucosidase gene bgIA of Clostridium thermocelfum. Sequence analysis reveals a superfamily of cellulases and B-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem 1991;200:301-309. Btiller HA, Kothe MJC, Goldman DA, Grubman SA, Sasak WV, Matsudaira PT, Montgomery RK, Grand RJ. Coordinate expression of lactase-phlorizin hydrolase mRNA and enzyme levels in rat small intestine during development. J Biol Chem 1990;265:6978-6983. Montgomery RK, Btiller HA, Rings EHHM, Grand RJ. Lactose intolerance and the genetic regulation of intestinal lactasephlorizin hydrolase. FASEB J 1991;5:2624-2832. Biiller HA, van Wassenaer AG, Raghavan S, Montgomery RK, Sybicki MA, Grand RJ. New insights into the lactase and glycosylceramidase activities of rat microvillus membrane (MVM) lactase-phlorizin hydrolase. Am J Physiol 1989; 257:G616-G623. Btiller HA, Rings EHHM, Pajkrt D, Montgomery RK, Grand RJ. Glycosylation of lactase-phlorizin hydrolase in rat small intestine during development. Gastroenterology 1990;96:667675. Biiller HA, Rings EHHM, Montgomery RK, Sasak WV, Grand RJ. Further studies of glycosylation and intracellular transport of lactase-phlorizin hydrolase in rat small intestine. Biothem J 1969;263:249-254. Naim HY, Sterchi EE, Lentze MJ. Biosynthesis and maturation of lactase phlorizin hydrolase in the human small intestinal epithelial cells. Biochem J 1987;241:427-434. Btiller HA, Montgomery RK, Sasak WV, Grand RJ. Biosynthe-
LACTASE
October 1992
sis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat. J Biol Chem 1987;262:1720617211.
29.
15. Yeh KY, Yeh M, Pan PC, Holt PR. Posttranslational
16.
17.
18.
19.
20.
cleavage of rat intestinal lactase occurs at the luminal side of the brush border membrane. Gastroenterology 1991;101:312-318. Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G. Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme. EMBO J 1988;7:2705-2713. Duluc I, Boukamel R, Mantei N, Semenza G, Raul F, Freund JN. Sequence of the precursor of intestinal lactase-phlorizin hydrolase from fetal rat. Gene 1991;103:275-276. Traber PG, Chianale J, Florence R, Kim K, Wojcik E, Gumucio JJ. Expression of cytochrome P45Ob and P450e genes in small intestinal mucosa of rats following treatment with phenobarbital, polyhalogenated biphenyls, and organochlorine pesticides. J Biol Chem 1988;263:9449-9455. Nor& 0, Dabelsteen E, Heyer PE, Olsen J, Sjijstriim H, Hansen GH. Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation. FEBS Lett 1989; 259:107-112. Iseki S, Kondo H. Light microscopic localization of hepatic fatty acid binding protein mRNA in jejunal epithelia of rats using in situ hybridization, immunohistochemical, and autoradiographic techniques. J Histochem Cytochem 1990;
30. 31.
32. 33. 34.
35.
36.
37.
38:111-115. 21. Traber PG. Regulation
38.
22.
39.
23.
24.
25.
26.
27.
of sucrase-isomaltase gene expression along the crypt-villus axis of rat small intestine. Biochem Biophys Res Commun 1990;173:765-773. Tyner AL, Godbout R, Compton RS, Tilghman SM. The ontogeny of a-fetoprotein gene expression in the mouse gastrointestinal tract. J Cell Biol 1990;110:915-927. Moorman AFM, Boer de PAJ, Geerts WJC, Zande van de LPGW, Charles R, Lamers WH. Complementary distribution of CPS (ammonia) and GS mRNA in adult liver detected by in situ hybridization. J Histochem Cytochem 1988;36:751-755. Gaasbeek Janzen JW, Westenend PJ, Charles R, Lamers WH, Moorman AFM. Gene expression in derivates of embryonic foregut during prenatal development of the rat. J Histochem Cytochem 1988;36:1223-1230. Moorman AFM, Boer de PAJ, Das AT, Labruyhre WT, Charles R, Lamers WH. Expression of mRNAs for ammonia-metabolizing enzymes in the developing rat: the ontogenesis of hepatocyte heterogeneity. Histochem J 1990;22:457-468. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984;137:266-267. Boer de PAJ, Labruytire WTh. Lab hint. Probe preparation for in situ hybridization. Boehringer Mannheim Biochemica 1990;1:5.
28. Quaroni A, Isselbacher
KJ. Study of intestinal
cell differentia-
GENE EXPRESSION
DURING DEVELOPMENT
1161
tion with monoclonal antibodies to intestinal cell surface components. Dev Biol 1985;111:267-279. Wessels A, Vermeulen JLM, Vir6gh Sz, KQlmen F, Morris GE, Nguen thi Man, Lamers WH, Moorman AFM. Spatial distribution of “tissue-specific” antigens in developing human heart and skeletal muscle. I. An immunohistochemical analysis of creatine kinase isoenzyme expression patterns. Anat Ret 1990;228:163-176. Mathan M, Moxey PC, and Trier JC. Morphogenesis of fetal rat duodenal villi. Am J Anat 1976;146:73-92. Hermos JA, Mathan M, Trier JS. DNA synthesis and proliferation by villous epithelial cells in fetal rats. J Cell Biol 1971;50:255-258. Koldovsky 0. Development of the functions of the small intestine in mammals and man. Basel, Switzerland: Karger, 1969. Wickens M. How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem Sci 1990;15:277-281. Wickens M. In the beginning is the end: regulation of poly(A) addition and removal during early development. Trends Biothem Sci 1990;15:320-324. Tsuboi KK, Kwong LK, D’Harlingue AE, Stevenson DK, Kerner JA, Sunshine P. The nature of maturational decline of intestinal lactase activity. Biochim Biophys Acta 1985;840:69-78. Gordon JI. Intestinal epithelium differentiation: new insights from chimeric and transgenic mice. J Cell Biol1989;108:11871194. Boyle JT, Celano P, Koldovsky 0. Demonstration of a difference in expression of maximal lactase and sucrase activity along the villus in the adult rat jejunum. Gastroenterology 1980;79:503-507. Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. Regulation of intestinal epithelial cell growth by transforming growth factor type p. Proc Nat1 Acad Sci USA 1989;86:1578-1582. Rubin DC, Ong DE, Gordon JI. Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic patterns of gene expression. Proc Nat1 Acad Sci USA 1989;86:1278-1282.
Received January 16,1992. Accepted April 14,1992. Address requests for reprints to: Edmond H. H. M. Rings, M.D., Division of Pediatric Gastroenterology and Nutrition, G8-260, Department of Pediatrics, Academic Medical Center, Meibergdreef 9,1105 AZ Amsterdam, The Netherlands. Supported in part by Inpharzam, Foundation Bevordering van Onderzoek in de Gezondheidszorg, Foundation de Drie Lichten, and Foundation Ludgardine Bouwman/Jan Dekker, The Netherlands (E.H.H.M.R.); Nutricia, The Netherlands (E.H.V.B., H.A.B.); National Institutes of Health Research Grant DK 32658; Center for Gastroenterology Research on Absorptive and Secretory Processes (NIH P30 DK 34928) (R.K.M., R.J.G.); and a NATO Collaborative Research Grant. E.H.H.M.R. is a clinical research fellow (KWO) at the Netherlands Organization for Scientific Research (NWO). The authors thank C. Gravemeijer for expert photographical assistance.
1992;103:1154-1161
Lactase Gene Expression During Early Development of Rat Small Intestine EDMOND ANTOON ROBERT
H. H. M. RINGS, PIET A. J. DE BOER, F. M. MOORMAN, ERIK H. VAN BEERS, JAN DEKKER, K. MONTGOMERY, RICHARD J. GRAND, and HANS A. BILLER
Center for Liver and Intestinal Research, Division of Pediatric Gastroenterology and Nutrition, and Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts
Expression of lactase messenger (m) RNA and protein in rat small intestine during fetal and postnatal development was analyzed using in situ hybridization and immunohistochemistry. Lactase mRNA was first identified at 18 days of development, and lactase protein was first detected at day 20. Lactase mRNA and protein were present along the entire villus. Lactase mRNA increased, reaching a maximum at day 20. Just before birth a decrease in lactase mRNA was observed. In newborn intestine, lactase mRNA was present only from the base of the villus up to the mid-villus region and was undetectable up to the villus tips. Lactase protein continued to be expressed along the entire villus. These data show that expression of lactase mRNA and protein do not parallel, indicating a posttranscriptional control in fetal development. Lactase gene transcription is initiated late in gestation concomitant with villus formation and is exclusively seen in villus epithelial cells. The restriction after birth of lactase mRNA expression to cells at the villus base suggests the occurrence of a previously unknown step in postnatal differentiation of the enterocyte.
L
actase-phlorizin hydrolase (EC 3.2.1.23, EC 3.2.1.62, subsequently referred to as lactase) plays a critical role in the nutrition of young mammals,‘*’ and the developmental pattern of its specific activity in many animal species and human populations is well documented. The specific activity of lactase is very high at birth and during nursing when milk is the sole nutrient; then around weaning a steep decline to low levels is seen in adults.3 As a consequence, adult animals and most human adults are considered to be lactase deficient. This mode of expression has been proven to be inherited through a single dominant autosomal locus. The molecular basis of the regulation of this biological phenomenon of a rapid increase before birth and a decrease of lactase-specific activity later in life is
still not clear. The influence of hormones and starvation on lactase messenger RNA (mRNA) and protein levels has recently been investigated.4*5 The transfection of full-length lactase complementary DNA (cDNA) into COS-1 cells to elucidate synthesis and transport has been the subject of a recent study.’ In addition, on the basis of sequence analysis, it has been suggested that lactase belongs to a superfamily of cellulases and P-glycosidases.7 Total intestinal lactase enzyme levels during maturation follow expression of lactase mRNA, suggesting that lactase total activity is predominantly regulated at a transcriptional level.*,’ Furthermore, it is known that total intestinal lactase activity in adult rats is maintained at the same level as in newborns, in contrast to the developmental pattern of decrease of lactase-specific activity.l’ Posttranslational alterations in glycosylation of the protein occur in a different pattern in time from the postweaning decline in lactase-specific activity, militating against a role for age-dependent changes in glycosylation in the decrease of lactasespecific activity observed during development.” In addition, manipulating posttranslational modifications of the protein, e.g., N-glycosylation processing, does not prevent the appearance ofactive lactase enzyme in the microvillus membrane.” The biosynthesis, posttranslational transport, and immunohistochemical localization of lactase have been characterized in several mammals.‘3-‘6 Human, rabbit, and rat lactase cDNAs have been cloned and sequenced.8*‘6n17 However, the appearance of lactase mRNA and protein in conjunction with morphological maturation and localization of lactase mRNA and protein in rat small intestine during development have not been studied. Recent in situ hybridizations, detecting mRNAs encoding different proteins expressed in enterocytes, including cytochrome PO 1992
by the American Gastroenterological 0016-5065/92/$3.00
Association
LACTASE
October 1992
4SOIIb1,‘s aminopeptidase N,” fatty acid binding have shown that protein, 2o and sucrase-isomaltase,21 the respective mRNAs are localized along the cryptto-villus axis in the intestine. The only developmental study of the intestine was performed by Tyner et of a-fetoprotein. al.” on the expression We describe the pattern of expression and topography of lactase mRNA and protein in rat small intestine during fetal and postnatal development using in situ hybridization and immunohistochemistry, respectively. The objective of the present study was to investigate the coordination of lactase mRNA and protein expression at the histological level and to understand the molecular basis of this expression during development.
Materials and Methods Animals Wistar rats were purchased from TN0 Breeding Laboratories (Zeist, The Netherlands) and housed at the Academic Medical Center animal facilities at constant temperature and humidity on a l&hour light/dark cycle. The dams had free access to standard chow and water. Fetal age was calculated from dated matings and neonatal age from the time ofbirth (22 days after conception). Fetal specimens were taken from 16-, la-, 20-, and 22day-old rat fetuses, and subsequent specimens were from O-day-old newborn rats before and 4 hours after nursing. Postnatal rat intestinal tissue samples were obtained from rats of 1, 4,9,18, and 25 days. Sections of three animals of the same age were studied. Tissue
Processing
Fetal samples were sections through whole fetuses. Total newborn intestines were rapidly dissected and fixed by immersion as described below. Tissue of older animals was from proximal jejunum, approximately 5 cm distal to the ligament of Treitz. Chemicals [a-35S]Deoxycytidine 5’-triphosphate (dCTP) (1350 Ci/mmol) was purchased from Du Pont-New England Nuclear (‘s-Hertogenbosch, The Netherlands) and nuclear research emulsion G5 from Ilford (London, England). Pronase (type XIV), and all other chemicals were purchased from Sigma (St. Louis, MO). Specifications
of Molecular
Probes
Lactase mRNA was detected using a 1.8-kilobase (kb) EcoRI-PstI insert of the cDNA lactasel-Bluescript clone, derived from a 2.3-kb rat lactase partial cDNA.‘This 1.8-kb insert corresponds to position 3839 through position 5694 of the full-length cDNA of rat lactase, as described by Duluc et alI7 As a positive control for the detection of mRNAs in the intestinal epithelium, PstI and EcoRI fragments of the 5.6-kb rat carbamoylphosphate synthetase (CPS) cDNA were used as previously described.23 The
GENE EXPRESSION
DURING DEVELOPMENT
1155
urea-cycle enzyme, CPS, is found in the mitochondria of both hepatocytes and enterocytes23-25 and is nucleus encoded. The agarose-purified (NuSieve LMP Agarose; FMC BioProducts, Rockland, ME) DNA probes were labeled overnight at 15’C with [a-35S]dCTP according to the multiprime labeling method,26 yielding fragments of approximately 100 bp.27 Localization Hybridization
of Lactase
mRNA
by In Situ
To localize lactase mRNA in rat small intestine, we performed in situ hybridization using previously published protoco1sz3 Tissues were fixed in 4% paraformaldehyde and further processed for preparation of paraffin sections. The 7-pm-thick paraffin sections were mounted onto 3-aminopropyltriethoxysilane-coated microscope slides. The in situ hybridization procedures for lactase and CPS were performed on closely adjacent sections to allow comparison of the hybridization patterns. Series of sections at each age were processed under identical conditions, with aliquots of probe from the same labeling reaction, to allow comparison of mRNA expression at different ages. Sections were deparaffinized in xylol, dehydrated in 100% ethanol, and air-dried. They were incubated in 0.2 mol/L HCl for 20 minutes, rinsed in sterile water for 5 minutes, and soaked in 2X SSC (1X SSC = 0.15 mol/L sodium chloride, 0.015 mol/L sodium citrate, pH 7.2) at 70°C for 10 minutes. Subsequently, they were incubated with pronase (250 pg/mL) in 50 mmol/L Tris and 5 mmol/L ethylenediaminetetraacetic acid (EDTA) (pH 7.5) at 37°C for 20 minutes. Pronase digestion was stopped by addition -of 0.2% glycine in phosphate-buffered saline (PBS) followed by two 30-second washes with PBS. Sections were then incubated in 4% paraformaldehyde in PBS for 20 minutes, rinsed in PBS, dehydrated in a graded series of ethanol, and air-dried. Hybridization was performed overnight at 44’C in 50% (vol/vol) deionized formamide, 0.1% (vol/vol) Triton X-100, 10% (wt/vol) dextran sulfate, 2X SSC, 0.02% (wt/ vol) bovine serum albumin, 0.02% (wt/vol) polyvinylpyrolidone and 0.02% (wt/vol) Ficoll, 10 mmol/L dithiothreitol, 200 ng/pL herring sperm DNA, and labeled probe. The probe concentration was approximately 0.5 ng/pL and contained 5 X IO4 cpm/pL specific activity. Sections were washed twice at 44’C for 15 minutes in 50% (vol/vol) formamide and 1 X SSC and twice for 10 minutes in 1 X SSC and finally in 0.1X SSC at the same temperature. Sections were dehydrated in a graded series of ethanol, containing 0.3 mol/L ammonium acetate, air-dried, and dipped in darkness into liquid emulsion (Ilford G5 emulsion, diluted 1:1.5 with glycerol/water: London, England). Dipped slides were stored at 4’C for 5 days. The emulsion was developed by dipping the slides for 4 minutes into developer at 18°C. Developer was freshly made, consisting of 0.45% (wt/vol) Amidol (4 - hydroxy - 1,3 - phenylenediammoniumdichloride; Merck, Darmstadt, Germany), 1.8% (wt/vol) Na,SO,, and 0.08% (wt/vol) potassium bromide, and filtered before use. Slides were then sequentially dipped for 1 minute into a stop bath containing water and
1156
RINGS ET AL.
GASTROENTEROLOGY
then dipped in a fixation bath for 10 minutes by soaking them in 30% (wt/vol) Na,S,O, * 5H,O. Tissue was counterstained in 0.01% (wt/vol) toluidine blue and dehydrated. Sections were mounted in Malinol (Chroma, Stuttgart, Germany) and photographed with Agfa Pan APX 25 (15 DIN) film (Agfa-Gevaert, Leverkusen, Germany). Antibodies The monoclonal antibody against rat lactase” used for immunohistochemistry has been described previ0us1y.‘~ The antibody was used in the form of ascites from pristane-primed BALB/c mice. As a positive control for the detection of proteins in the enterocytes, a polyclonal anti-CPS antiserum was used.24 Immunodetection Intestine
of Lactase
in Rat Small
Lactase protein was detected by immunohistochemistry using the indirect unconjugated peroxidase anti-peroxidase (PAP) technique.” Tissues were fixed in methanol, acetone, and distilled water (40/40/20, vol/ vol/vol) and further processed for preparation of paraffin sections. The 7-pm paraffin sections were mounted on polylysine-coated microscope slides. Lactase and CPS were visualized on adjacent sections to allow direct comparison of the patterns. After deparaffination, sections were treated with hydrogen peroxide (3%, vol/vol in PBS) for 20 minutes to reduce endogenous peroxidase activity, followed by preincubation in TENG-T [lo mmol/L Tris, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.25% (wt/vol) gelatin, and 0.05% (vol/vol) Tween-20; pH 81 for 30 minutes to reduce nonspecific binding. Sections were incubated with ascites fluid containing the anti-lactase monoclonal antibody (diluted 1:6400 in PBS) or with serum-containingantibodies directed against CPS (diluted I:2000 in PBS). Monoclonal antibody binding was detected using rabbit anti-mouse immunoglobulin, goat anti-rabbit immunoglobulin, and rabbit-PAP complex, respectively. Polyclonal antibodies were detected using goat anti-rabbit immurespectively. noglobulin and rabbit-PAP complex, Negative controls included incubations without primary antibodies. All incubations were followed by washes in PBS for 5 minutes. The immunocomplex formed was visualized by incubation of the sections with 0.5 mg/mL 3,3’diaminobenzidine, 0.02% (vol/vol) hydrogen peroxide in 30 mmol/L imidazole, and 1 mmol/L EDTA (pH 7). Sections were mounted in Entellan (Merck). Control
Vol. 103. No. 4
more stringent conditions (higher hybridization and washing temperature at 50°C). Data are presented from the lower stringency hybridizations.
Results Morphological
Development
At 16 days of development, the intestine is lined by stratified epithelium; the lumen and villi are not yet present. At 18 days of development, a monolayer of cuboidal epithelial cells cover the developing villi in the more proximal part of the intestine. In contrast, at the same age in the more distal part of the intestine, no villi can be observed, although secondary lumina have appeared. After its formation around 18 days of development, the lumen is lined by an undifferentiated stratified epithehum. These observations are consistent with the previously described craniocaudal gradient, with the slower maturation of the distal intestine than the proximal intestine.30 At 20 days of development, the cuboidal epithelial cells have become more columnar and villus height is increasing. Crypts appear only after birth. Lactase Intestine
mRNA
and Protein
in the Fetal
At 16 days of development, when the mucosa is characterized by an undifferentiated, stratified epithelium, CPS mRNA is abundant in the intestinal epithelium (Figure 1A). In contrast, neither lactase
Experiments
The three different control experiments for in situ hybridization were performed as follows. In fetal and postnatal samples, control hybridizations were performed using pBR322 vector DNA labeled as described above. To exclude nonspecific hybridization with the 1.8-kb lactase probe, the fetal data of rats of 18 and 20 days of development were verified by individual hybridization to three subfragments of the 1.8-kb probe obtained by HinfI digestion (1000, 450, and 250 bp, respectively]. To confirm the specificity, in situ hybridization was also performed under
Figure 1. Detection of mRNA of CPS and lactase in cross sections of fetal rat at 16 days of gestation. (A) CPS mRNA expression in the intestinal epithelium. The multilayer of undifferentiated, stratified epithelium is heavily labeled by the CPS probe. (II) Lactase mRNA expression in the intestinal epithelium. In an adjacent tissue section, lactase mRNA cannot be detected in the intestinal epithelium. The arrows indicate corresponding sections of undifferentiated multilayered stratified epithelium in the two specimens (bar = 6.5 mm).
October 1992
LACTASE GENE EXPRESSIONDURING DEVELOPMENT
1157
lus enterocytes. The intervillus regions are devoid of lactase mRNA hybridization. In contrast, at 22 days of development, this pattern of mRNA expression is significantly altered, because there is an apparent decrease of lactase mRNA abundance just before birth (Figure 4B). Lactase mRNA and Protein Expression Postnatal Development
During
In contrast to the uniform expression of lactase mRNA in fetal intestine (shown above), expression in postnatal intestine undergoes topographic redistribution. As shown in Figure 5A and C, lactase mRNA becomes restricted to the lower portions of
Figure 2. Detection of lactase mRNA in fetal rat intestine at 18 days of gestation. (A) Standard hematoxylin and azophloxine staining of intestinal sections. In the proximal section (P), rudimentary villi lined by a single layer of cuboidal epithelium (open triangle) have formed. A more distal part of the intestine (D) still shows a multilayer of undifferentiated, stratified epithelium (closed triangle). (B) Expression of lactase mRNA. Lactase mRNA is expressed in the monolayer of epithelium in the proximal small intestine. Lactase mRNA is not detectable in the undifferentiated multilayered epithelium of the distal part of the intestine (bar = 0.1 mm).
mRNA (Figure 1B) nor lactase protein (not shown) can be detected in the intestine at this age. At 18 days of development (Figure 2), the proximal intestine is characterized by the presence of primitive villi, lined by a single layer of cuboidal epithelial cells (Figure 2A). The distal intestine continues to show a stratified multilayered epithelium. Lactase mRNA is first detectable at this age and is found in all of the epithelial cells lining the primitive villi (Figure 2B). No lactase can be detected at this age in the stratified multilayered epithelium of the distal intestine. Lactase mRNA is present at 18 days of development (Figure 3A), whereas lactase protein is not yet detectable (Figure 3B). Both mRNA and protein for CPS are detectable in these specimens (data not shown), as was also previously reported.23p25 At 20 days of development, when rapid villus growth has occurred, lactase mRNA is uniformly distributed in enterocytes lining the entire villus (Figure 3C). At this age, lactase protein is readily detected in the microvillous membranes of the enterocytes, with equal distribution from villus base to villus tip (Figure 3D). Lactase protein is also found in membranous material from sloughed cells in the lumen. Figure 4 contrasts lactase mRNA density at 20 and 22 days of development under the same hybridization conditions. Similar to Figure 3C, Figure 4A shows intense expression of lactase mRNA in all vil-
Figure 3. Detection of lactase mRNA and protein in fetal rat intestine at 18 and 20 days of gestation. (A) Lactase mRNA expression in 18-day intestine. Lactase mRNA is expressed in the newly formed epithelial monolayer (arrow). (B) Lactase protein expression at 18 days of gestation. No lactase protein is detectable in these same epithelial cells (arrow) at this age. (C) Lactase mRNA expression in 20.day intestine. The epithelial layer displays a uniform expression of lactase mRNA over the whole villus. (D) Lactase protein expression at 20 days of gestation. Brush borders of the enterocytes show staining for lactase protein from villus base to the villus tip (bar = 0.1 mm).
1158
RINGS ET AL.
the villi immediately after birth and before suckling. Lactase mRNA can only be detected from the villus base to the mid-villus region, whereas hybridization is undetectable from mid-villus to villus tip (Figure 5A). This pattern does not seem to be a result of suckling, because prevention of suckling does not influence the distribution of lactase mRNA expression (data not shown). There is a gradual restriction of lactase mRNA to the lower half of the villus during postnatal development. This is shown in intestine of g-day-old neonates (Figure 5C), and this restriction remains essentially unchanged at all subsequent ages studied, including adult animals (data not shown). Lactase mRNA hybridization in crypt cells was never observed. Despite the striking morphological difference of lactase mRNA shown in Figure 5A and C, lactase protein at birth and 9 days postnatally shows uniform expression from the villus/crypt junction along the whole villus to the villus tip, as shown in Figure 5B and D. These patterns remain constant throughout subsequent postnatal development of the intestine. These patterns were not influenced by suckling, as studied for animals of 0 days of age (data not shown). Lactase protein was never detected in crypt cells. No evidence for a mosaic pattern of expression of lactase mRNA was observed at any embryonic age, nor was there any mosaic pattern of lactase protein expression. To ascertain that technical factors do not explain
Figure 4. Detection of lactase mRNA in fetal rat intestine at 20 and 22 days of gestation. (A) Expression of lactase mRNA at 20 days of gestation. The fetal epithelial cells show a high expression of lactase mRNA. (B) Expression of lactase mRNA at 22 days of gestation. Under identical hybridization conditions, with the same probe as in (A), an apparent decrease of lactase mRNA transcripts is observed in the older intestine. Lactase mRNA is still uniformly distributed over the whole villus but not found in the intervillus regions (arrows) (bar = 0.1mm).
GASTROENTEROLOGY
Vol. 103.No. 4
Figure 5. Detection of lactase mRNA and protein in the small intestine of newborn (O-day) and suckling (g-day-old) rats. (A) Expression of lactase mRNA in newborn rats. Lactase mRNA expression is restricted to an area from the base to approximately half of the height of the villi (arrow). (B) Expression of lactase protein in newborn rats. Immunohistochemical staining shows lactase from the villus base to the tip. (C) Expression of lactase mRNA in suckling rats. At 9 days of age, lactase mRNA is detectable only in an area from the villus base to the midpoint of the villi (arrow). (D) Expression of lactase protein in suckling rats. At this age as well, lactase protein is identified from base to tip of the villi. Neither lactase mRNA nor protein is detectable in the crypt region (star) (bar = 0.1mm).
the absence of lactase mRNA and protein in crypt cells, hybridization with the CPS probe was used as a positive control. CPS has previously been shown to be localized to the crypts in postnatal rat intestine.24*25As shown in Figure 6A, when intestine was sectioned transversely, CPS was readily seen in crypts whereas lactase mRNA was undetectable. However, lactase mRNA was detectable at the villus base as shown in Figure 6B. Data obtained from the control experiments were as follows. In all ages studied, the pBR322 DNA gave no hybridization. Hybridization with the three HinfI subfragments of the 1.8-kb lactase probe yielded patterns identical to those with the original probe (not shown). The increased hybridization and washing temperature of 50°C did not change the hybridization pattern or diminish the intensity of signals.
October 1992
Figure 6. Detection of mRNA of CPS and lactase in adjacent crypt-rich tangential sections of a loop of small intestine of a &day-old rat. (A)Expression of CPS mRNA. Only the areas of the section which include the crypts (Cl show a signal for CPS. (B) Expression of lactase mRNA. In the same zone, lactase mRNA is not detectable. In the area that includes villus sections (V), lactase mRNA expression is found (arrow) (bar = 0.1mm).
Discussion In fetal rats formation of villi begins at 17 days of development, and tall, well-developed villi have formed in the proximal intestine by the 19th day of development.30 In contrast, discrete, well-defined crypts do not develop until shortly after birth. Hermos et a1.31 showed that some mitotic cells in the intestinal epithelium could be identified along the entire length of the villi in the rat fetal intestine as late as 1 day before birth. In the days before birth, the proliferating cells progressively become restricted to the bases of the villi and intervillus regions. The undifferentiated multilayer of stratified epithelial cells in the intestine at 16 days of development showed no lactase gene expression, whereas CPS mRNA was already present. Lactase mRNA was only identified at 18 days of development, either concomitant with or immediately after formation of a monolayer of cuboidal epithelial cells lining the villi of the fetal gut, suggesting that lactase expression is dependent on development of the enterocytes. The expression of lactase mRNA does not occur simultaneously throughout the intestine but in a proximalto-distal progression, as has been described for villus morphogenesis.32 There is a delay between the initial detection of lactase mRNA and detection of lactase protein. This may in part reflect technical differences or a discrepancy in sensitivity between the in situ and the immunohistochemical detection in paraffin sections. However, it also seems to represent a real physiological phenomenon, because with the same techniques lactase can be detected at 20 days of development
LACTASE GENE EXPRESSION DURING DEVELOPMENT
1159
using diluted antibody (1:6400), whereas it can not using the be detected at 18 days of development same or a loo-fold more concentrated antibody dilution. In addition, postnatal animals show both mRNA and protein in the enterocytes as soon as they exit the crypts. The time difference may represent the time required to accumulate significant amounts of protein in the fetal tissue or a delay in maturation of the translation machinery, as has been documented during oocyte maturation.33s34 Although in situ hybridization allows only semiquantative interpretation, the results of the present study seem to indicate that approximately 2 days before birth, the hybridization of lactase mRNA is most intense with expression along the entire villus. On the day of birth, the intensity of hybridization was decreased and hybridization became restricted to the lower half of the villus. In agreement with previous measurements of lactase activity, which peak in the perinatal period,3*‘0 this pattern suggests maximal transcription before birth, with decreased levels after birth. The restriction of mRNA expression from all fetal epithelial cells to only the enterocytes on the lower part of the villus after birth has not been reported previously and suggests the occurrence of an unknown step in postnatal differentiation of the enterocyte. The discrepancy between the expression of lactase mRNA and protein along the crypt/villus axis may be a result of changes in the half-life of the respective molecules after birth. This study, however, does not allow conclusions regarding the rate of translation of lactase mRNA into protein.35 Several possible explanations for this restriction of mRNA expression may be advanced. The most obvious stimulus, the changes associated with the onset of feeding, does not appear to be causative, because prevention of suckling did not alter the pattern. The shift from general cell proliferation to crypt-restricted proliferation and villus cell migration, coincident with the formation of the crypts, occurs around birth; however, no exact data are available. The observed change in lactase mRNA expression pattern may reflect the beginning of this shift to crypt-restricted proliferation and villus cell migration. Possible systemic hormonal stimuli from the process of birth have not yet been examined and remain a subject for further investigations. In adult intestine, there is a clear separation of proliferating crypt cells and differentiated villus cells. Numerous studies of enzyme levels have shown that none of the microvillus membrane enzymes such as sucrase-isomaltase, aminopeptidase N, or lactase are detectable below the crypt/villus junction. These enzymes are detected from the villus base and accumulate in characteristic patterns as the enterocytes migrate up the villus. In the crypts, a
1160 RINGS ET AL.
cells and a zone of nonproliferzone of proliferating ating cells, which do not yet show evidence of specialization, can be identified by DNA labeling.3s The nonproliferating crypt cells will move out of the crypts and differentiate into mature enterocytes. Although these moving cells begin to show differentiated proteins after the crypt/villus junction is passed, it is not clear when they begin to express the mRNA for these proteins. As shown in the present study, lactase mRNA is found in all enterocytes lining the villi of the fetal intestine, whereas no mRNA can be identified in the cells of the intervillus region. When crypt formation is completed after birth, lactase mRNA is only found from the base of the villi at the crypt/villus junction to the mid-villus region. This pattern is maintained during further development. Neither lactase mRNA nor protein was detected in the crypts at any age in the present study. A similar pattern in older animals was recently reported by Traber’l who showed that initiation of suerase-isomaltase mRNA expression in adult enterocytes occurred as the enterocytes reached the crypt/villus junction, with a steady decrease in expression as cells moved up the villus. In the postnatal intestine, the pattern for two apical membrane hydrolases, sucrase-isomaltase and lactase, appears very similars3’ However, other enterocyte proteins have very different patterns of expression. CPS mRNA is confined to the crypts in adult rats, although the protein is present in all of the villus enterocytes.23*24 In contrast, the expression of mRNA for transforming growth factor j3l increases steadily in the villus epithelial cells, with a maximum in the villus tip region3* As expected, there seems to be no global pattern of mRNA expression in intestinal epithelial cells. Thus, in the adult intestine, there seems to be four functional zones for lactase: a crypt zone of proliferating cells; a crypt zone of nonproliferating, undifferentiated cells; a villus zone of high mRNA expression and the beginning of protein accumulation; and a villus zone of decreased mRNA expression and maximal protein accumulation. The present study also indicates that in fetal intestine these zones are all contiguous. In addition, the adult phenotype becomes established during a brief perinatal period from 22 days of gestation to the day of birth. In immunohistochemical studies of the developmental pattern of two cytoplasmic proteins, fatty acid binding protein (FABP) and apolipoprotein AIV (apoAIV), Rubin et al. 39 observed that FABP but not apoAIV appeared initially in a patchy or mosaic pattern. They found that FABP was first detectable in scattered cells of the fetal intestinal epithelium, whereas adjacent cells did not express it. In contrast, apoAIV showed a uniform expression as soon as it
GASTROENTEROLOGY
Vol. 103, No. 4
appeared. With age, the mosaic pattern of FABP expression became a uniform distribution throughout the epithelium. We found no evidence for a mosaic expression of lactase, an apical membrane protein. These data suggest that the initiation of expression of lactase is mediated by a different mechanism from that of FABP, possibly the same or one similar to that for apoAIV. The mechanisms by which lactase expression is initiated and its pattern regulated are currently under investigation. References 1.
Henning SJ. Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol 1961;241:Gl99-
G214. 2. Flatz G. Genetics of lactose digestion
3.
4.
5.
6.
7.
a.
9.
10.
11.
12.
13.
14.
in humans. In: Harris H, Hirschhorn K, eds. Advances in human genetics. Volume 16. New York: Plenum, 1987:1-77. Doe11 RG, Kretchmer N. Studies of small intestine during development. I. Distribution and activity of beta-galactosidase. Biochim Biophys Acta 1962;62:353-362. Castillo RO, Glasscock GF, Noren KM, Reisenauer AM. Pituitary regulation of postnatal small intestinal ontogeny in the rat: differential regulation of digestive hydrolase maturation by thyroxine and growth hormone. Endocrinology 1991; 129:1417-1423. Freund JN, Duluc I, Raul F. Lactase expression is controlled differently in the jejunum and ileum during development in rats. Gastroenterology 1991;100:388-394. Naim HY, Lacey SW, Sambrook JF, Gething MH. Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein. J Biol Chem 1991;266: 12313-12320. Grabnitz F, Seiss M, Rticknagel KP, Staudenbauer WL. Structure of the j3-glucosidase gene bgIA of Clostridium thermocelfum. Sequence analysis reveals a superfamily of cellulases and B-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem 1991;200:301-309. Btiller HA, Kothe MJC, Goldman DA, Grubman SA, Sasak WV, Matsudaira PT, Montgomery RK, Grand RJ. Coordinate expression of lactase-phlorizin hydrolase mRNA and enzyme levels in rat small intestine during development. J Biol Chem 1990;265:6978-6983. Montgomery RK, Btiller HA, Rings EHHM, Grand RJ. Lactose intolerance and the genetic regulation of intestinal lactasephlorizin hydrolase. FASEB J 1991;5:2624-2832. Biiller HA, van Wassenaer AG, Raghavan S, Montgomery RK, Sybicki MA, Grand RJ. New insights into the lactase and glycosylceramidase activities of rat microvillus membrane (MVM) lactase-phlorizin hydrolase. Am J Physiol 1989; 257:G616-G623. Btiller HA, Rings EHHM, Pajkrt D, Montgomery RK, Grand RJ. Glycosylation of lactase-phlorizin hydrolase in rat small intestine during development. Gastroenterology 1990;96:667675. Biiller HA, Rings EHHM, Montgomery RK, Sasak WV, Grand RJ. Further studies of glycosylation and intracellular transport of lactase-phlorizin hydrolase in rat small intestine. Biothem J 1969;263:249-254. Naim HY, Sterchi EE, Lentze MJ. Biosynthesis and maturation of lactase phlorizin hydrolase in the human small intestinal epithelial cells. Biochem J 1987;241:427-434. Btiller HA, Montgomery RK, Sasak WV, Grand RJ. Biosynthe-
LACTASE
October 1992
sis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat. J Biol Chem 1987;262:1720617211.
29.
15. Yeh KY, Yeh M, Pan PC, Holt PR. Posttranslational
16.
17.
18.
19.
20.
cleavage of rat intestinal lactase occurs at the luminal side of the brush border membrane. Gastroenterology 1991;101:312-318. Mantei N, Villa M, Enzler T, Wacker H, Boll W, James P, Hunziker W, Semenza G. Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme. EMBO J 1988;7:2705-2713. Duluc I, Boukamel R, Mantei N, Semenza G, Raul F, Freund JN. Sequence of the precursor of intestinal lactase-phlorizin hydrolase from fetal rat. Gene 1991;103:275-276. Traber PG, Chianale J, Florence R, Kim K, Wojcik E, Gumucio JJ. Expression of cytochrome P45Ob and P450e genes in small intestinal mucosa of rats following treatment with phenobarbital, polyhalogenated biphenyls, and organochlorine pesticides. J Biol Chem 1988;263:9449-9455. Nor& 0, Dabelsteen E, Heyer PE, Olsen J, Sjijstriim H, Hansen GH. Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation. FEBS Lett 1989; 259:107-112. Iseki S, Kondo H. Light microscopic localization of hepatic fatty acid binding protein mRNA in jejunal epithelia of rats using in situ hybridization, immunohistochemical, and autoradiographic techniques. J Histochem Cytochem 1990;
30. 31.
32. 33. 34.
35.
36.
37.
38:111-115. 21. Traber PG. Regulation
38.
22.
39.
23.
24.
25.
26.
27.
of sucrase-isomaltase gene expression along the crypt-villus axis of rat small intestine. Biochem Biophys Res Commun 1990;173:765-773. Tyner AL, Godbout R, Compton RS, Tilghman SM. The ontogeny of a-fetoprotein gene expression in the mouse gastrointestinal tract. J Cell Biol 1990;110:915-927. Moorman AFM, Boer de PAJ, Geerts WJC, Zande van de LPGW, Charles R, Lamers WH. Complementary distribution of CPS (ammonia) and GS mRNA in adult liver detected by in situ hybridization. J Histochem Cytochem 1988;36:751-755. Gaasbeek Janzen JW, Westenend PJ, Charles R, Lamers WH, Moorman AFM. Gene expression in derivates of embryonic foregut during prenatal development of the rat. J Histochem Cytochem 1988;36:1223-1230. Moorman AFM, Boer de PAJ, Das AT, Labruyhre WT, Charles R, Lamers WH. Expression of mRNAs for ammonia-metabolizing enzymes in the developing rat: the ontogenesis of hepatocyte heterogeneity. Histochem J 1990;22:457-468. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1984;137:266-267. Boer de PAJ, Labruytire WTh. Lab hint. Probe preparation for in situ hybridization. Boehringer Mannheim Biochemica 1990;1:5.
28. Quaroni A, Isselbacher
KJ. Study of intestinal
cell differentia-
GENE EXPRESSION
DURING DEVELOPMENT
1161
tion with monoclonal antibodies to intestinal cell surface components. Dev Biol 1985;111:267-279. Wessels A, Vermeulen JLM, Vir6gh Sz, KQlmen F, Morris GE, Nguen thi Man, Lamers WH, Moorman AFM. Spatial distribution of “tissue-specific” antigens in developing human heart and skeletal muscle. I. An immunohistochemical analysis of creatine kinase isoenzyme expression patterns. Anat Ret 1990;228:163-176. Mathan M, Moxey PC, and Trier JC. Morphogenesis of fetal rat duodenal villi. Am J Anat 1976;146:73-92. Hermos JA, Mathan M, Trier JS. DNA synthesis and proliferation by villous epithelial cells in fetal rats. J Cell Biol 1971;50:255-258. Koldovsky 0. Development of the functions of the small intestine in mammals and man. Basel, Switzerland: Karger, 1969. Wickens M. How the messenger got its tail: addition of poly(A) in the nucleus. Trends Biochem Sci 1990;15:277-281. Wickens M. In the beginning is the end: regulation of poly(A) addition and removal during early development. Trends Biothem Sci 1990;15:320-324. Tsuboi KK, Kwong LK, D’Harlingue AE, Stevenson DK, Kerner JA, Sunshine P. The nature of maturational decline of intestinal lactase activity. Biochim Biophys Acta 1985;840:69-78. Gordon JI. Intestinal epithelium differentiation: new insights from chimeric and transgenic mice. J Cell Biol1989;108:11871194. Boyle JT, Celano P, Koldovsky 0. Demonstration of a difference in expression of maximal lactase and sucrase activity along the villus in the adult rat jejunum. Gastroenterology 1980;79:503-507. Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. Regulation of intestinal epithelial cell growth by transforming growth factor type p. Proc Nat1 Acad Sci USA 1989;86:1578-1582. Rubin DC, Ong DE, Gordon JI. Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic patterns of gene expression. Proc Nat1 Acad Sci USA 1989;86:1278-1282.
Received January 16,1992. Accepted April 14,1992. Address requests for reprints to: Edmond H. H. M. Rings, M.D., Division of Pediatric Gastroenterology and Nutrition, G8-260, Department of Pediatrics, Academic Medical Center, Meibergdreef 9,1105 AZ Amsterdam, The Netherlands. Supported in part by Inpharzam, Foundation Bevordering van Onderzoek in de Gezondheidszorg, Foundation de Drie Lichten, and Foundation Ludgardine Bouwman/Jan Dekker, The Netherlands (E.H.H.M.R.); Nutricia, The Netherlands (E.H.V.B., H.A.B.); National Institutes of Health Research Grant DK 32658; Center for Gastroenterology Research on Absorptive and Secretory Processes (NIH P30 DK 34928) (R.K.M., R.J.G.); and a NATO Collaborative Research Grant. E.H.H.M.R. is a clinical research fellow (KWO) at the Netherlands Organization for Scientific Research (NWO). The authors thank C. Gravemeijer for expert photographical assistance.
