Structural and biochemical differentiation of the guinea-pig colon during foetal development

Cell Tissue Res (1985) 242:197-209 Cell and Tissue Research �9 Springer-Verlag 1985

Structural and biochemical differentiation of the guinea-pig colon during foetal development Teresa Smith*, Kevin Christianson**, Raymond Moss*, and David Bailey** Departments of Electron Microscopy* and Surgery**, St. George's Hospital Medical School, London, Great Britain

Summary. We have studied some aspects of the morphological and biochemical differentiation of the foetal guinea-pig colonic epithelium. At day 40 the epithelium was organised in ridges and appeared pseudo-stratified. Folding of the epithelium, followed by villus formation, occurred between days 45 and 55, and by day 50 mucus-secreting goblet cells appeared at the bases of the colonic villi. By day 55 most epithelial cells, including goblet cells, possessed numerous microvilli which, by day 65, had become organised into well developed brush-borders. Between day 55 and term (day 65-68) mucosal depth increased markedly and the colon attained its final glandular morphology.

Biochemical studies showed the specific activities of the microvillar hydrolases to be much lower in the washed colon than in either foetal meconium or small intestine at all times during development. Furthermore, a membrane fraction highly enriched in microvillus hydrolase activities was prepared from foetal colonic meconium using techniques originally devised to isolate the foetal small intestinal microvillus membrane. This meconial subfraction was almost identical in polypeptide composition to the highly- purified foetal small intestinal microvillus membrane. Iden- tification of the colonic microvillus membrane was ham- pered by the absence of reliable membrane markers. Never- theless, a fraction 14-fold enriched in aminopeptidase activity was prepared from day 40 foetal colon and its polypeptide composition compared by SDS-PAGE to that of the small intestinal microvillus membrane at the same age.

Key words: Foetal colon Guinea-pig - Meconium - Mi- crovillus membrane

Surprisingly little is known about the biochemical development of the mammalian colon, most biochemical studies having focussed on the small intestine and its differentiation-specific microvillar hydrolases (reviewed by Grand et al. 1976). These 'markers ' have assisted in the isolation and characterisation of the small intestinal microvillus membrane as a pure fraction for biochemical analysis during both foetal and postnatal development. Although the

Send offprint requests to." Dr. D.S. Bailey, Department of Surgery, St. George's Hospital Medical School, Cranmer Terrace, London SWt7 ORE, Great Britain

luminal surface of the adult mammalian colon possesses a clearly defined brush-border membrane, the latter ex- presses fewer differentiation markers than that of the small intestine. Its isolation has therefore remained somewhat en- igmatic (Biber et al. 1983).

In the human, there is strong evidence that some characteristics of the foetal colon are shared by colonic neoplasms. Glycogen storage, a characteristic feature of the foetal intestine (Lev and Weisberg 1969; Rousset et al. 1979), is also associated with the growth of human intestinal carcinoma cell lines both in vitro and in vivo (Rousset et al. 1980). Sucrase-isomaltase, a small intestinal microvillar hydrolase (Semenza et al. 1983), is also expressed during early development of the human foetal colon and in some colonic tumours (Zweibaum et al. 1983). The demonstration of these and other oncofoetal characteristics and their obvious clinical importance (Goldenberg 1976) necessitates further detailed studies of mammalian colonic development.

Colonic development has been examined in several ex- perimental systems using the rat (Helander 1973, 1975; Brackett and Townsend 1980; Colony and Neutra 1983) and chick (Lira and Low 1977). However, some aspects of gastrointestinal development differ between species with long and short gestation periods. In animals with short gestation periods, such as the mouse and rat, much intestinal maturation takes place after birth when the young are suckling. In contrast, both the human and the guinea-pig have relatively long gestation periods (266 and 68 days respectively), during which time the foetal intestine achieves considerable maturity (Grand et al. 1976; Moog and Ortiz 1960; Bailey et al. 1984). The structural and enzymatic differentiation of the human foetal colon have been reported recently (Bell and Williams 1982; Lacroix et al. 1984).

In the present study we have used a variety of techniques to investigate certain aspects of the structural organisation of the guinea-pig colon during foetal and early postnatal development. Some biochemical studies of the enzymatic differentiation of the foetal guinea-pig colon are presented, together with an analysis of foetal intestinal meconium. We have also isolated a microvillus membrane-enriched fraction from the 40-day foetal colon. The polypeptides of this membrane fraction have been analysed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and have been compared with previous results from our laboratory concerning the foetal guinea-pig small intestinal microvillus membrane (Bailey et al. 1984).

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Materials and methods

Animals. Foetuses from timed-pregnant guinea-pigs (Dun- kin-Hartley strain) were exposed through a longitudinal incision in the maternal abdominal wall and a small incision in the uterine horn, under methoxyflurane anaesthesia (Penthrane, Abbott Laboratories, Queenborough, Kent). The entire foetal colon was excised, briefly washed with buffer (300 mM mannitol, 5 mM HEPES/KOH, pH 7.4) and immediately processed for light microscopy, electron microscopy or biochemical analysis.

Microscopy. Specimens for LM, TEM and SEM were excised and immediately fixed in 50 mM cacodylate-buffered 3% glutaraldehyde (pH 7.2) for 12 h at room temperature. Post-fixation for 1 h in 1% osmium tetroxide in cacodylate buffer was followed by a buffer rinse and dehydration in graded ethanol/aqueous uranyl acetate. Propylene oxide acted as the intermediate fluid and specimens were embedded in Spurr's low viscosity resin. Sections were cut with a glass knife using a Reichert ultramicrotome. For TEM, sections were collected on 200 mesh copper grids, counter- stained with aqueous uranyl acetate and lead citrate (Stem- pak and Ward 1964; Sato 1968), and examined in a Philips 301 electron microscope. For LM, 2-~t resin-embedded sections were stained with hot toluidine blue (Richardson et al. 1960) for 60 sec and examined in a Nikon light microscope.

Specimens for SEM were post-fixed for 1 h using 50 mM cacodylate-buffered 1% osmium tetroxide, rinsed with buffer and dehydrated through graded concentrations of ethanol and ethanol/freon. Specimens were critical-point dried, mounted on aluminium stubs, sputter-coated with gold and examined in a Coates and Welter field emission scanning electron microscope.

To identify intracellular glycogen, 2-1a resin-embedded sections of 50-day-old foetal colon were stained with peri- odic acid-Schiff reagent (BDH, Southampton) both with and without pretreatment with a-amylase according to the method of Lev and Weisberg (1969).

Enzymatic determinations. Aminopeptidase (E.C. 3.4.11.2) and dipeptidyl peptidase IV (E.C. 3.4.14.X) assays were performed as described by Wachsmuth et al. (1966) and Nagatsu et al. (1979), respectively. Alkaline phosphatase (E.C. 3.13.3.1) was assayed according to the method of Engstr6m (1961) as modified by Graham and Ford (1983). Sucrase (E.C. 3.2.1.48), enterokinase (E.C. 3.4.21.9) and protein were assayed by the methods of Dahlqvist (1968), Baratti et al. (1973), and Lowry et al. (1951) respectively.

Microvillus membrane preparation from foetal colon. Vesicu- larised microvillus membranes were prepared from 40-day foetal guinea-pig colon by cation precipitation, performed as described by Kessler et al. (1978) with the modifications of Gains and Hauser (1981), followed by isopycnic sucrose density gradient centrifugation as described in detail by Bai- ley et al. (1984) for foetal guinea-pig small intestine. This technique is similar to that originally used to prepare both the human small intestinal microvillus membrane by Schmitz et al. (1973) and the human foetal colon microvillus membrane by Lacroix et al. (1984), with the exception that the divalent cation used is Mg 2+ not C a 2 +

Briefly, colons from 20 40-day-old foetal guinea-pigs were excised and opened along their length with fine surgical scissors. All further operations were carried out

at 4 ~ C. Gentle washing in homogenisation buffer (300 mM mannitol, 5 mM HEPES/KOH pH 7.4) to remove any loosely-associated meconium was followed by homogenisation in a Waring blender (full speed, 3 min, in 150 ml buffer) and filtration through 40-11 nylon mesh. Two fractions were produced by differential centrifugation, a total membrane fraction and a microvillus-enriched membrane fraction.

The total membrane fraction was prepared by direct centrifugation of one half of the filtered homogenate at high speed (Sorvall RC5B centrifuge, SS34 rotor, 20000 rpm, 30 rain). The microvillus-enriched membrane fraction was prepared by adjusting the other half of the homogenate to 10mMMgC12, allowing non-microvillus membrane fragments to aggregate for 30 min, and then re- moving the aggregated membranes by low speed centrifugation (Sorvall RC5B centrifuge, SS34 rotor, 5000rpm, 10 min). Microvillus-enriched membranes were then har- vested by high speed centrifugation of the resulting superna- rant (Sorvall RC5B centrifuge, SS34 rotor, 20000 rpm, 30 min). The resulting pellet was dispersed by passage through a gauge 19 syringe needle and applied to a 15-55% (w/w) sucrose density gradient. Isopycnic centrifugation (Beckman L8 centrifuge, SW40Ti rotor, 35000 rpm, 16 h) was followed by fractionation of the gradient into 1 ml fractions, and each fraction was assayed for aminopeptidase and alkaline phosphatase activities, as well as density (using an Abbe refractometer) and protein content. Equal volumes of certain fractions were then centrifuged (Beckman L8 centrifuge, 50Ti rotor, 45000 rpm, 90 min) and the pelleted material analysed by SDS-PAGE.

Analysis ofJoetal meconium. The separated colons and small intestines of several 60-day foetal guinea-pigs were carefully washed with homogenisation buffer (300 mM mannitol, 5 mM HEPES/KOH pH 7.4) to prepare mucosal and meconial fractions. All subsequent operations were carried out at 4 ~ C. Colonic meconium and meconium-free small intestinal tissue were then homogenised separately in homogenisation buffer using a Waring blender (full speed, 3 rain), and the homogenates adjusted to 10 mM MgC1 z. Aggrega- tion was allowed to proceed for 30 min, after which time the supernatant was clarified by low-speed centrifugation (Sorvall RC5B centrifuge, SS34 rotor, 5000 rpm, 10 rain). The resulting supernatants were then centrifuged at high speed (Sorvall RC5B centrifuge, SS34 rotor, 20000 rpm, 30 min), and the resulting pellets dispersed by passage through a gauge 19 syringe needle and applied to two identical 15-55% (w/w) sucrose density gradients. Isopycnic centrifugation (Beckman L8 centrifuge, SW28 rotor, 25000 rpm, 16 h) was followed by fractionation of the gradients into 3 ml fractions. Each fraction was assayed for aminopeptidase activity, as well as density and protein content. Specific fractions were then centrifuged (Beckman L8 ultracentrifuge, 50Ti rotor, 45000 rpm, 90 min) and the pelleted material analysed by SDS-PAGE.

SDS-polyacrylamide gel electrophoresis. One-dimensional SDS-PAGE was performed using slabs of 7.5% polyacrylamide as described by Laemmli (1970) under reducing con- ditions. Protein molecular weights were estimated by refer- ence to the migration of standard proteins (carbonic anhyd- rase, 29000 daltons; ovalbumin, 45 000 daltons; bovine serum albumin, 68 000 daltons; rabbit muscle phosphorylase

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Fig. I a--d. Foetal colon, day 40: a Colon organised in 3 clearly-defined layers, primordial muscle layer (M), submucosa (S) and epithelium (E), the latter staining quite strongly with toluidine blue. b Epithelium organised into 2 domains, one containing small closely-packed cells (A), the other containing groups of highly elongated cells (B), many of which possess apically-sited nuclei and protrude into the lumen, e Epithelium forms longitudinal ridges, d Transverse section through an epithelial ridge showing a lack of cytodifferentiation with extensive glycogen stores (G). Microvilli are more numerous at the cell margins, a) • 230; b) • 690; c) • 1000; d) • 3600

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Fig. 2a-e. Foetal colon, day 55: a By day 55, the epithelium forms distinct villi composed of toluidine blue-stained cells. The tips of adjacent villi may be fused (arrows), enclosing an intervillous space which appears distinct from the main intestinal lumen (L); b The surface of the epithelium has become highly contorted with 'knots ' of interconnecting luminally-positioned colonocytcs; c Basal regions of the crypt-villus unit (C) show signs of cytodifferentiation. Mucus- secreting goblet cells (cell 4) appear with glycogen-rich colonocytes (cells 7-9). Some cells show signs of recent division (cells 1 and 2). Luminal microvilli are scarce; d Colonic villi populated with apical cells distended with glycogen. At lower positions on the villus more compact cells are found some of which are secreting mucus (arrows); e Detail of epithelial surface from the basal region of the villus showing a goblet cell in the process of disgorging mucus. Mucus cells also possess microvilli, seen still adhering to the mucus droplets, a) x 65; b) x l 3 0 ; c ) x 670;d) x440 ;e ) x 2600

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Table l. Specific activities of the intestinal hydrolases aminopeptidase, alkaline phosphatase, sucrase, enterokinase and dipeptidyl peptidase IV in homogenates of buffer-washed small intestine and colon during foetal and post-natal development. Each value represents the mean (with range) for 3 guinea-pigs. N.D. denotes no detectable activity

Aminopeptidase Alkaline phosphatase Sucrase Dipeptidyl peptidase IV Enterokinase (p-moles pNA/h/mg) (mmoles pNP/h/mg) (p-moles glucose/h/mg) (p.moles pNA/h/mg) (nmoles trypsin/h/rag)

Foetal (day 40) Small intestine 1.03 (0.94-1.20) Colon 0.53 (0.42-0.61)

Foetal (day 60) Small intestine 1.53 (1.31-1.67) Colon 0.77 (0.73-0.83)

2.11 (0.74-2.80) 0.26 (0.19-0.37) 0.32 (0.28-0.38) 0.51 (0-1.52) 0.36 (0.11-0.79) 0.02 (0.02-0.02) 0.22 (0.19-0.28) n.d.

19.5 (18.3-21.4) 0.72 (0.67-0.75) 2.01 (1.93-2.14) 2.32 (2.12-2.47) 0.81 (0.51-1.02) n.d. 0.25 (0.15~0.37) n.d.

Neonatal (day 7) Small intestine 2.39 (2.06-3.03) 21.7 (18.5-25.6) 1.08 (0.96-1.30) 1.06 (0.88-1.27) 10.3 (9.05-11.20) Colon 0.64 (0.54-0.72) 0.89 (0.44-1.58) 0.04 (0.02-0.07) 0.15 (0.13-0.17) n.d.

Adult (day 40) Small intestine 4.20 (3.62-4.58) 32.7 (30.0 36.5) 3.37 (3.00-3.66) 2.22 (1.822.47) 11.9 (11.4-12.9) Colon 0.86 (0.81-0.89) 0.61 (0.56-0.66) 0.05 (0.05-0.05) 0.28 (0.24-0.32) n.d.

Table 2. Microvillus hydrolase activity in buffer-washed and unwashed 60-day foetal guinea-pig small intestine and colon. Each value represents the mean (with range) for 3 guinea-pigs. N.D. denotes no detectable activity

Specific activity Specific activity Specific activity Specific activity in unwashed small intestine in washed small intestine in unwashed colon in washed colon

Sucrase 1.16 (0.44-1.69) 0.72 (0.67-0.75) 0.34 (0.22-0.43) n.d. (p.moles glucose/h/mg) (100%) (62%) (29%) (0%) Enterokinase 2.84 (2.62-3.15) 2.32 (2.12-2.47) 1.07 (0-1.87) n.d. (nmoles trypsin/h/mg) (100%) (82%) (38%) (0%) Alkaline phosphatase 22.8 (18.1-26.9) 19.5 (18.3-21.4) 3.9 (1.0-7.8) 0.8 (0.5-1.0) (mmoles pNP/h/mg) (100%) (85%) (17%) (3%) Dipeptidyl peptidase IV 2.64 (2.35-3.03) 2.01 (1.93-2.14) 1.22 (0.94-1.39) 0.25 (0.15-0.37) (p-moles pNA/h/mg) (100%) (76%) (46%) (9%) Aminopeptidase 1.75 (1.6%1.85) 1.53 (1.31-1.67) 1.75 (1.46-2.18) 0.77 (0.73-0.83) (p-moles pNA/h/mg) (100%) (87%) (100%) (44%)

b, 97000 daltons; E. coli fl-galactosidase, 116000 daltons and rabbit muscle myosin, 205000 daltons, obtained from Sigma Chemicals, Poole, Dorset).

Results

Maturation of the guinea-pig colonic epithelium. Toluidine blue-stained sections of the foetal colon were examined by light microscopy (Figs 1, 2). At day 40, the epithelium consisted of a circular, relatively uniform array of pseudo-stratified cells (Fig. 1 a). At least two cell groupings were appar- ent. One group of cells appeared relatively compact, nes- tling close to the underlying mesenchymal layers. The other epithelial cell group consisted of elongated cells which pro- truded into the lumen of the developing colon. Some of the latter cells stained strongly with toluidine blue and contained extensive deposits of glycogen, the presence of which

was confirmed by histochemical techniques (data not shown; Lev and Weisberg 1969).

The developing colonic epithelium was also examined by both scanning and transmission electron microscopy (SEM and TEM respectively). At day 40, the colonic epithelium possessed ridges (Fig. 1 c) which corresponded to the bud-like villous projections seen by light microscopy (Fig. 1 a, b). The epithelial surface of such villous ridges consisted of a uniform pavement of cells whose apical surface bore variable numbers of microvilli. The latter were smaller than those of the neonatal or adult guinea-pig colon and showed little organisation into brush-border structures. Differentiation between the component cells Of the epithelial pavement, on the basis of their surface specialisation was evident by SEM, some cells being almost devoid o f microvilli. TEM of such early stages confirmed the presence of scattered microvilli on the surface of the cells constituting

Fig. 3a-d. Foetal colon at day 60 and neonatal colon at day 3: a At day 60, superficial villus cells form fan-shaped villus crests between which are openings leading into files of mucus-secreting goblet cells; b 3 days after birth, the colon has a similar morphology to that at day 60 of gestation, with goblet cells occupying the prominent glandular acini; e The surface of 3-day postnatal colon appears flat but is punctuated by numerous pits; d By day 3, numerous goblet cells have joined the microvillus-bearing colonocytes on the mucosal surface between the pits. a) x 420; b) x 500; c) • 200; d) x 1400

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the villous ridge (Fig. 1 d) and both SEM and TEM showed a higher concentration o f microvilli at the sites of contact between cells of the epithelial pavement. In addition, TEM showed that all the cells o f the epithelium at this early stage were packed with glycogen and demonstrated very little intracellular cytodifferentiation.

Between days 40 and 50, definite villi were formed. By day 55, mesenchymal cells had formed a central core within the villous ridge (Fig. 2 a) and the pseudo-stratified epithelium was changing to a simple columnar form. At this time, 3 definite cell domains could be discerned within the villus, a superficial domain dominated by glycogen-rich toluidine blue-stained cells which resembled the distended cells observed at earlier times during development, a domain of irregularly-sized, lightly toluidine-blue-staining cells occupying the basal regions of the epithelium and an inner cellu- lar core of mesenchymal origin consisting of toluidine-blue- negative cells. Both SEM and TEM confirmed that the superficial cells of the epithelium retained the appearance of the elongated cells observed at earlier stages, with microvillus-rich surfaces (Fig. 2d, e) and extensive intracellular glycogen deposits. Several intervillous connections could be seen both by LM (Fig. 2a) and SEM (Fig. 2b). Such structures may be associated with a growth-related expansion of the mucosal surface, reflecting villi undergoing crypt fis- sion.

At day 55 organised arrays of cells could be seen by TEM at the bases o f the villi (Fig. 2c). Some centrally- placed cells showed evidence of recent division, with irregularly-shaped nuclei containing diffuse chromatin aggregates (cells 1 and 2, Fig. 2c). Although at this stage most of the villus cells were immature, retaining extensive glycogen deposits, some basal cells had already differentiated. One cell type possessed numerous densely-staining ovoid granules in its apical cytoplasm (cell 4, Fig. 2c). Such cells are most probably mucus-secreting goblet cells. SEM demonstrated the presence of functioning goblet cells at the base of the villi at this stage (Fig. 2e). Another cell type possessed much smaller granules scattered throughout its cytoplasm (cell 5, Fig. 2c). The luminal surface of the basal crypt cells possessed very few microvilli, in sharp contrast to cells within the epithelium along the villus crests (Fig. 2d, e).

By day 60, the epithelium was dominated by goblet cells which occupied the inner basal region of the villi and whose proliferation confined the distended colonocytes to a superficial position on the villi (Fig. 3 a). At this time, the colonocytes formed an almost flat mucosal surface punctuated by openings of the mucosal glands through which mucus secretions reached the lumen. The morphological appearance of the colon at such late times in foetal development very closely resembled that of the early neonate (Fig. 3 b).

By day 65, the superficial colonocytes had lost most of their glycogen deposits and showed more intracellular organisation. The microvilli at this stage had formed a clearly defined brush-border with an associated terminal web. Files of goblet cells, organised as acini within this now glandular epithelium, dominated the lower regions of the mucosa. During late foetal and early neonatal development, mucus-secreting goblet cells appeared on the mucosal surface (Fig. 3 d).

Expression of microvillar hydrolase activities in the colon during development. Several enzymic activities characterise the microvillus membrane of the small intestine (Kenny

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Fig. 4. Fractionation of 40-day foetal colon tissue by Mg 2 +-treatment and isopycnic sucrose density gradient centrifugation: Mem- brane preparations from homogenates of washed foetal colon tissue, after Mg z +-treatment, were fractionated by isopycnic sucrose density gradient centrifugation. Fractions were analysed for protein content (A, expressed as gg/fraction), sucrose density (o, expressed as % w/w sucrose), aminopeptidase activity (e, expressed as nano- moles p-nitroaniline/h/fraction) and alkaline phosphatase (A, expressed as mmoles p-nitrophenol/h/fraction). Note especially the co-purification of aminopeptidase and alkaline phosphatase activities in a fraction of density 39.8% (w/w) sucrose (fraction 8, panel b)

and Maroux 1982). Amongst these are the enzymes aminopeptidase, dipeptidyl peptidase IV, sucrase, alkaline phosphatase and enterokinase. A recent report has shown some of these enzymes to be expressed in the developing human foetal colon (Lacroix et al. 1984). Nevertheless, at all times during guinea-pig development, the specific activities of such microvillar hydrolases were considerably lower in the colon than in the buffer-washed small intestine at the same age (Table 1).

A comparison of the specific activities of the microvillar hydrolases in the small intestine and colon at day 60, both with and without washing of the mucosal surface with homogenisation buffer showed that gentle washing removed considerable amounts of the detectable hydrolase activities from the colonic mucosa, but removed far less from the small intestinal mucosa (Table 2). In particular, enterokinase and sucrase activities in the colon were reduced to unde- tectable levels. In the adult guinea-pig, enterokinase secre- tion is confined to the duodenum and upper jejunum (Bett 1979), and its appearance in the foetal colon is most easily explained as a luminal artefact. Alkaline phosphatase and dipeptidyl peptidase IV were present in washed mucosal

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Fig. 5a-f. Polypeptides of the 40-day foetal colon microvillus membrane: Fractions at various stages of foetal colon microvillus membrane purification (see Fig. 4) were analysed by SDS-PAGE on 7.5% acrylamide slabs. Track (a), total membrane fraction from foetal colon, enriched only 1.8-fold in aminopeptidase activity; track (b), Mg 2 +-treated membrane, enriched 7-fold in aminopeptidase specific activity; tracks (e) and (e), fraction 8, Fig. 4, enriched 14.4-fold in aminopeptidase activity. Also shown are 28-fold purified adult guinea-pig small intestinal microvillus membrane (track d) and 25-fold purified 40-day foetal guinea-pig small intestinal microvillus membrane (track f). Molecular weights of some of the more prominent components are indicated

homogenates at demonstrable levels, although their specific activities were much lower in the colon than in the small intestine. Alone among the microvillar hydrolases studied, aminopeptidase showed considerable resistance to removal by gentle lavage.

Partial purification of the microvillus membrane from the early foetal colon. Biochemical studies of the developing colon were limited by two considerations, the amounts of colonic tissue obtained from each foetus (a major problem during the first month of gestation) and the increasing cellu- lar complexity of the epithelium (evident after day 55 of gestation). We chose to examine the colon at day 40 of gestation when the epithelium consisted of a relatively uniform population of undifferentiated cells, free of mucus- secreting goblet cells. In the following studies, aminopeptidase (E.C. 3.4.11.2), a widely occurring apical cell surface membrane marker enzyme in epithelial ceils (Louvard 1980) as well as microvillar hydrolase (Kenny and Maroux 1982), has been used as a marker of the intestinal microvillus membrane (see Discussion).

A total membrane preparation from 40-day old foetal colonic mucosa, when analysed by SDS-PAGE, showed prominent polypeptide components of 210000 and 200000 daltons as well as several lower molecular weight components (40000; 52000; 54000 and 62000 daltons; Fig. 5, track a), but was not highly enriched in aminopeptidase activity (specific activity 2.1 lamoles pNA/h/mg membrane protein).

Magnesium treatment followed by sucrose density gradient centrifugation resulted in the co-purification of both alkaline phosphatase and aminopeptidase in a fraction of isopycnic density 39.8% (w/w) sucrose (fraction 8, Fig. 4b). The specific activity of aminopeptidase in this fraction (7.6. txmoles pNA/h/mg membrane protein) showed an en-

richment above that of the total homogenate of 14.4 fold. This enrichment is, however, less than that which we have consistently obtained from small intestinal microvillus membrane preparations from foetuses of the same age using similar techniques (21-25 fold, Bailey et al. 1984).

An analysis of this membrane fraction by SDS-PAGE (Fig. 5c, e) showed several proteins of identical mobility to those present in highly purified microvillus membrane vesicles prepared from small intestine at the same age (Fig. 5 f). Some of these components, most noticeably those of molecular weight 47000 (actin) and 80000 daltons, are probably cytoskeletal proteins present as structural components of both microvillus membranes. However, the 205 000 dalton polypeptide specifically enriched in the foetal small intestinal microvillus membrane (Fig. 5 f) was not similarly enriched in the early foetal colon microvillar membrane. Moreover, 2 polypeptides, molecular weights 140000 and 150000 daltons, appeared unique to the colonic membrane fraction.

An analysis of intestinal meconium. Microvillus membrane vesicles are usually isolated by cation precipitation techniques (Schmitz et al. 1973; Kessler et al. 1978; Gains and Hauser 1981), not always preceeded by mucosal washing (Kessler et al. 1978; Sepulveda and Burton 1982; Lacroix et al. 1984). In the foetal gut such washing may be impor- tant, due to the presence within the foetal intestine of luminal meconium. This contains hydrolase activities such as the disaccharidases (Antonowicz et al. 1975) which are normal constituents of the microvillus membrane of the small intestinal epithelium.

Unfractionated meconium contained several major polypeptides including one of molecular weight 68 000 (Fig. 7, track a). This protein did not appear in either the precipitat- ing or hydrolase-enriched fractions during Mg 2+ precipita-

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Fig. 6. Fractionation of foetal meconium by Mg 2 + treatment and isopycnic sucrose density gradient centrifugation : Homogenates of both foetal meconium and buffer-washed small intestinal tissue from 60-day foetuses were subjected to Mg 2+ treatment and sucrose density gradient centrifugation as described in the text. Gra- dients were assayed for aminopeptidase activity, protein content and sucrose density (A). The distribution of aminopeptidase activity in fractionated foetal meconium (e, 100 units=31 gmoles p-nitroaniline/h/fraction) is compared to that in the purified microvillus membrane derived from small intestinal tissue (o, 100 units = 7 gmoles p-nitroaniline/h/fraction). Note the absence of soluble aminopeptidase activity in both cases, and the slightly broader density distribution of meconial aminopeptidase activity

tion and may correspond to soluble foetal albumin or ~- foetoprotein (AFP), both of which have previously been reported as components of amniotic fluid ingested by the foetus (Jones et al. 1981).

Meconium, fractionated by sucrose density gradient centrifugation after Mg 2+-treatment (Fig. 6) exhibited substantial aminopeptidase activity, ranging in specific activity from 14 to 23 gmoles pNA/h /mg protein (fractions 6 and 9 respectively, Fig. 6). This compared favourably with its specific activity in purified small intestinal microvillar membrane vesicles (17 gmoles pNA/h /mg membrane protein). Aminopeptidase, already present at a specific activity of 2.7 gmoles pNA/h /mg in the unfractionated meconium, was enriched 9-fold after Mg 2+-treatment and sucrose density gradient fractionation. Activity banded at an isopycnic density (33-41% (w/w) sucrose) broadly similar to that of vesi- cularised microvillus membranes from the washed small intestine (39% w/w sucrose). Such gradient-purified meconial fractions had a polypeptide composition (Fig. 7b, c) which was almost identical to that of purified microvillar membrane preparations from foetal small intestinal tissue of the same age (Fig. 7d).

Discussion

In this paper, we have described the major changes in morphology which occur during foetal development of the guinea-pig colon. Previous work from our laboratory de- scribing corresponding changes in the guinea-pig small intestine (Bailey et al. 1984) serves to highlight several pro- nounced differences in the developmental programmes of the 2 tissues.

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Fig. 7a-e. Polypeptides of the foetal meconium at day 60 of gestation: Various fractions derived from both foetal meconium and foetal small intestine by Mg 2§ treatment and sucrose density gradient centrifugation (Fig. 6) were analysed by SDS-PAGE on 7.5 % acrylamide slabs. Track a, untreated foetal meconium; track b, fraction 6 from the meconial gradient, Fig. 6; track c, fraction 9 from the meconial gradient, Fig. 6; track d, purified microvillus membrane vesicles from buffer-washed foetal small intestine, fraction 9 from the corresponding gradient, Fig. 6. Also shown for comparison are the polypeptides of the adult guinea-pig small intestinal microvillus membrane (track e). The molecular weights of the major polypeptides are also indicated. Note especially the dis- tinctive polypeptide profile of unfractionated meconium (track a), and the similarities in polypeptide compositions between tracks b, c and d

The colonic epithelium changes from an initial, simple pseudostratified structure into its final complex glandular form in several distinct stages, which broadly correspond to those observed in the human (Bell and Williams 1982). The villous ridge appears by day 45, giving place to well defined villi by day 55. During this time, the pseudostrati- fled epithelium changes to a simple cuboidal epithelium. Mucus-secreting goblet cells appear by day 55, and by term this cell type dominates the epithelium.

In contrast, the guinea-pig small intestine shows an earlier development. Already by day 40, tall villi are present in the duodenum. These possess mesenchymal cores, although those in the ileum do not. In the colon, similar mesenchymal infoldings do not occur until day 50-55 (Fig. 2).

Another marked difference between the 2 tissues is the much lower abundance of goblet cells in the small intestine than the colon. During foetal colonic development goblet cell differentiation is a major part of the developmental programme. The identification of the physiological stimulus promoting goblet cell differentiation has been the subject of intensive research in vitro, and certain tumour cell lines (for example, HT29) can be induced to differentiate into absorptive enterocytes and goblet cells by metabolic condi-

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tioning (Pinto et al. 1982). Such mechanisms may also oper- ate at a developmental level.

The morphology of the adult colon and small intestine differ in that the adult colon does not posses villi. However, we have observed villus formation in the foetal colon, as have several other workers (Johnson 1913; Bell and Wil- liams 1982; Colony and Neutra 1983). A point which re- quires further clarification is the generation of the final adult morphology, during the process of which separate villi are converted into a flat mucosal floor, punctuated by intramucosal pits or crypts (Fig. 3 c).

Our studies using SEM parallel those of other workers. The clinical use of SEM to investigate human colonic dis- ease, recently reviewed by Siew (1983), has demonstrated an inverse correlation between cell surface microvillus density and colonic malignancy (Cooke et al. 1984). Our studies have clearly shown a lower incidence of luminal microvilli at early times during development of the foetal guinea-pig colon. Indeed, variation in the concentration of surface microvilli may occur between adjacent cells within the epithelium, presumably reflecting varying degrees of differentiation between neighbouring cells. SEM represents a powerful tool with which to examine such changes in cell surface morphology.

Our attempts to isolate the foetal colonic microvillus membrane, using methods which we have successfully used to isolate that of the foetal small intestine, have been com- plicated by the presence of colonic meconium and the absence of demonstrable marker enzymes. In the latter re- spect, the foetal guinea-pig colon resembled the adult guinea-pig colon (Biber et al. 1983).

It was clear from our early experiments that considerable luminal activities of the microvillar hydrolases were present in the foetal colon, but that most were effectively removed by gentle washing with homogenisation buffer which did not disrupt the surface of the colonocytes. The luminal material is meconium, some of which passes from the small intestine into the colon by peristalsis and has a yellow colour due to bile pigments secreted from the developing liver. Inspection of the gut at various stages during development showed a progressive movement of the yellow meconium through the small intestine towards the colon. By day 60 of gestation, the colon contained substantial amounts of meconium which was difficult to remove entire- ly from the mucosal surface.

Preliminary analysis of unfractionated foetal meconium by 1-dimensional SDS-PAGE showed the presence of large amounts of a 68000 dalton polypeptide (Fig. 6a), either AFP or foetal albumin. These proteins are major components of amniotic fluid (Jones et al. 1979). We have not yet pursued a full characterisation of meconium by 2-dimensional SDS-PAGE, one way to unequivocally distin- guish between AFP and foetal albumin. It would be inter- esting to know whether amniotic fluid is passing through the foetal intestine at early stages of physiological development since human amniotic fluid is known to contain several hormones which are trophic for intestinal maturation, including steroids (Homoki et al. 1983), insulin (Casper and Benjamin i980; Spellacy et al. 1973), and epidermal growth factor (Dailey et al. 1978). Furthermore, extracts from rat amniotic fluid have been reported to induce intestinal crypt formation (Calvert et al. 1983).

In addition to soluble components such as AFP/albumin, we have isolated a particulate fraction from meconium

which is both enriched in microvillus hydrolase activities and which has an almost identical polypeptide composition to that of the foetal small intestinal microvillus membrane at the same age (Fig. 7). This suggests the presence of luminal microvillar fragments which may travel through the developing intestinal tract at the onset of peristalsis. These undoubtedly would pose a problem in studies of microvillus membrane differentiation in the colon, since the latter membrane may be contaminated by adsorbed polypeptides derived from the small intestinal microvillus membrane, trans- ported into the colon via the meconium. Even sophisticated techniques such as indirect immunodetection to identify antigenic determinants expressed at the microvillus membrane (Zweibaum et al. 1983) would be susceptible to misin- terpretation if meconial components bound extensively to the colonic membrane. In fact, if this were true, the only reliable way to identify endogenous microvillar components in the colonic epithelium would be by biosynthetic studies in segments of foetal colon isolated from intestinal contin- uity.

We chose to examine day 40 of colonic development in order to minimise meconial adsorption problems, making the assumption that peristalsis was unlikely to occur in the absence of well defined muscle layers (Fig. 1). Microvillar membrane preparation, using methods which are reportedly successful for the human foetal colon (purification factor not reported, Lacroix et al. 1984) proved much less successful than we had expected, purifying the putative colonic membrane only 14 fold above the homogenate on the basis of aminopeptidase activity enrichment. Exactly similar methods using small intestinal tissue of the same age have proved highly successful in our hands, resulting in 21-25 fold purification of this membrane (Bailey et al. 1984). We know that microvilli are indeed quite abundant in the colon at day 40, suggesting that the cause for this relatively poor purification must reside in the methodology employed. Ca- tion precipitation methods rely on the presence of negative- ly-charged side-chains expressed at the membrane surface (Booth and Kenny 1974). I f the colonic microvillus membrane does not possess such a high concentration of sialic- acid rich glycoproteins at the membrane surface, such cation-precipitation methods for microvillus membrane preparation from the colon may not be as effective as they are for preparing the microvillus membrane of the small intestine.

In our preparation, we noticed the copurification of aminopeptidase and alkaline phosphatase in a membrane fraction which may correspond to the foetal colonic microvillus membrane (Fig. 4). In many species, alkaline phosphatase is a marker for the foetal colonic microvillus membrane (Lev and Orlic 1974; Colony and Neutra 1983). Aminopeptidase has also been reported as a marker for the apical plasmamembrane domain of several epithelial cells (discussed by Louvard 1980). Moreover, in our experiments, aminopeptidase was to a large extent retained by the colon after gentle washing (Table 2), suggesting that this enzyme may be a true component of the epithelium. However, its presence in the foetal colonic microvillus membrane has not yet been demonstrated using histochemical techniques.

Analyses of the colonic microvillus membrane fraction and the microvillus membrane of the small intestine at day 40 by SDS-PAGE (Fig. 5) illustrated basic similarities in the structure of the 2 intestinal microvillus membranes, but

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also pointed to specific differences in polypeptide composition. Fur ther characterisation of the component polypeptides of the colonic microvillar membrane awaits its purification to homogeneity.

In conclusion, our results have illustrated the development of the foetal guinea-pig colon at histological, enzymic and molecular levels. The observed developmental changes, when compared with those occurring in the small intestine at the same age, highlight the early structural and functional divergence of these 2 regions of the gastrointestinal tract.

Acknowledgements. We would like to thank Prof. J. Hermon-Tay- lor for his advice and encouragement during this work. Financial support was provided by the Wellcome Trust, the Nuffield Founda- tion and the Cancer Research Campaign of Great Britain.

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Accepted March 10, 1985

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Structural and biochemical differentiation of the guinea-pig colon during foetal development