Specialized Cell Types in the Human Fetal Small Intestine PAMELA C O L O N Y MOXEY AND JERRY S. TRIER Departments of Medicine, Harvard Medical School and Peter Bent Brigham Hospital, Boston, Massachusetts 021 15

ABSTRACT

In the present study we describe the time of appearance and morphological differentiation of specialized epithelial cells in human fetal small intestine (SB). Proximal and distal SB from 36 nonviable fetuses was studied by light and electron microscopy. During the 9- to 10-week period, villi lined by simple columnar epithelium replaced the stratified epithelial lining which was two to six cell layers thick. During this transition, distinctive junctional complexes and a single secondary lumen were identified in the deeper layers of stratified epithelium, and there was evidence of cellular degeneration of some superficial cells. Oligomucous and mature goblet cells were present in both the stratified and simple columnar epithelium. Crypt formation began proximally a t 10 t o 11 weeks and, within a week, crypts lined by undifferentiated crypt cells (UCC) could also be identified in the distal SB. These cells resembled adult UCC's except for the presence of large aggregates of glycogen, and the absence of large adult-type secretory granules (SG) until 16 weeks. At all ages SG's were smaller and less numerous than in adults. Paneth cells appeared with crypt development at 11 to 12 weeks. Unlike adult Paneth cells their SG's were structurally heterogeneous and frequently had cores with halos of differing density. Caveolated or tuft cells with dense bundles of microfilaments extending from microvilli into apical cytoplasm, apical granules, occasional caveolae, and a microvillus membrane denser than that of adjacent cells were identified by 16 weeks. Putative microfold ("M") cells were seen in the distal SB of a 17-week fetus. These cells had an unusual apical border with irregular projections, many small membrane bound vesicles in the cytoplasm, and were in direct contact with underlying lymphoid cells. The glandular cells of Brunner's glands a t 14 to 15 weeks resembled those of normal adult.

The architectural changes occurring during early morphogenesis of the mucosa of the human fetal small intestine have been described in classical histologic studies. Early in morphogenesis (between the fifth and eighth weeks of gestation) vacuoles develop within the stratified epithelium of the duodenum and luminal occlusion occurs (Cho, '31; Forssner, '07; Johnson, '10; Patzelt, '31; Tandler, 1900). Following vacuolization and occlusion, a discrete intestinal lumen reappears and mucosal folds, then villi, and finally crypts form between the eighth and twelfth weeks of gestation (Berry, 1900; Cho, '31; Forssner, '07; Garbarsch, '69; Johnson, '10; Patzelt, '36). Villus and crypt formation proceeds in a cranio-caudal direction, occurring in the distal small intestine about 1 week after i t begins in the proximal small intestine. Brunner's glands ANAT. REC.

(1978)191: 269-286.

were reported to appear in the duodenum a t 12 weeks (Johnson, '101, a t the end of the third month (Patzelt, '31) and as late as the fifth month (Baginsky, 1882). There are few ultrastructural studies which define the time of appearance and differentiation of specific cell types during organogenesis of the human fetal small intestine. The ultrastructure of the villous absorptive cell has been described at several gestational ages (Andersen et al., '64; Bierring et al., '64; Kelley, '73; Kovalchuk, '74; Varkonyi et al., '74). Some information relating to the ultras t r u c t u r e of fetal intestinal goblet cells (Kelley, '73; Lev and Weisberg, '69; Varkonyi e t al., '741, and undifferentiated crypt cells Received Sept. 15, '77. Accepted Jan. 13, '78. ' Supported by Research Grant AMDD-17537 from the National Institutes of Health, Bethesda, Maryland.

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(Kelley, '73) a t different gestational ages is also available. In addition, there has been detailed characterization of a variety of entero-endocrine cells using electron microscopy (Bartman, '72; Moxey and Trier, '77; Osaka, '75; Todd et al., '70; Varkonyi, '74) and immunofluorescence (Dubois e t al., '76a,b; Falck e t al., '67; Larsson e t al., '75; Polak e t al., '71). In contrast, no ultrastructural studies of undifferentiated crypt cells after ten weeks, of Paneth cells, of caveolated or tuft cells (Nabeyama and Leblond, '74), of microfold or "M" cells (Owen and Jones, '74) or of Brunner's gland epithelial cells have been reported in the fetal small intestine. In the present study we define the time of appearance and ultrastructural features of all epithelial cell types found in the human fetal small intestine between the ninth and twentysecond weeks of gestation. The entero-endocrine cells and villous absorptive cells are excluded since they are discussed in detail in separate papers (Moxey and Trier, '77; Moxey and Trier, in preparation). MATERIALS AND METHODS

Tissue from 36 nonviable human fetuses 9 to 22 weeks of age was obtained following therapeutic abortion performed by hysterotomy. Studies were approved by appropriate institutional human subjects review committees. Proximal and distal small bowel were dissected and segments were placed immediately in complete Liebovitz L-15 medium (Grand Island Biological Co., Grand Island, New York) in a n effort to minimize degeneration prior to fixation. Developmental age was determined by crown-rump andlor crown-heel measurements ( P a t t e n , '53). Tissues remained in medium for 30 to 90 minutes after surgery and were then fixed. When sufficient tissue was available, pieces were prepared for both paraffin and epoxy embedment. When only a limited amount of tissue was available, samples were prepared only for epoxy embedment. Light microscopy ofparaffinembedded tissue Tissue from four fetuses of 10 to 18 weeks gestation was fixed in Bouin's fixative and embedded in paraffin. Sections were cut at 8 pm, mounted on glass slides, and stained with hematoxylin and eosin or the periodic acid Schiff (PAS) technique. Selected sections were treated with a-amylase (Saunders, '64) prior to PAS staining.

Light and electron microscopy of epoxy embedded material Small intestine from all 36 fetuses was embedded in epoxy resin for light and electron microscopy. Tissue from 28 of the 36 fetuses was fixed for one to two hours in 1%osmium tetroxide ( 0 ~ 0 ,in ) dichromate buffer, pH 7.4 (Dalton, '55), containing 0.9 mM CaCl, at 4°C and post fixed for one-half hour in 0.1 M phosphate buffered formalin, pH 7.2. Tissue from five fetuses was fixed for one to two hours in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, containing 0.45 mM CaCl,, washed in buffer for 2 to 24 hours and post-fixed in 1% cacodylate buffered OsO,, pH 7.4. Tissue from the remaining three fetuses was fixed by both methods. All tissue was dehydrated in graded ethanols and embedded in epoxy resin by the method of Luft ('61). Sections 1 p m thick were cut on a Porter Blum MT2-B microtome, mounted on glass slides and stained with methylene blue-azure I1 (Richardson et al., '60)or a modified PAS procedure. Thin sections of well oriented areas were cut with a diamond knife using a n LKB microtome and mounted on copper or gold grids. Tissue on copper grids was stained with uranyl acetate (Watson, '58) and lead citrate (Reynolds, '63). Tissue on gold grids was stained with silver methenamine with and without prior a-amylase digestion (Rambourg et al., '69) to detect glycogen at the ultrastructural level. Thin sections were examined with a Philips 300 electron microscope. Fig. 1 The intestinal lumen in this 1-pm epoxy section from the proximal small intestine of a 9- to 10-week fetus is lined by a highly stratified epithelium two to six cell layers thick. Primordial villi or folds are indicated by protrusions of the mucosa into the lumen (arrows). Richardson's stain. X 190. Fig. 2 This 1-pm epoxy section from the proximal small intestine of a 9- to 10-week fetus illustrates early villus formation. One short villus (arrow) is lined at its apex by a simple columnar epithelium and has a connective tissue core with blood vessels and smooth muscle cells. The epithelium lining the intervillous areas and mucosal protrusions remains stratified. Richardson's stain. x 170. Fig. 3 This l-Fm epoxy section of an 11- to 12-week fetus illustrates the penetration of cords of epithelial cells (arrows) into the mesenchyme which will ultimately develop into early crypts. PAS stain. X 230. Fig. 4 This 1-pm epoxy section from the well developed crypt region of a 19- to 20-week fetus illustrates the putative muscularis mucosa. This thin muscularis consists of aggregates of mesenchymal cells (arrows) oriented perpendicular to the crypts. Richardson's stain. X 490.

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Light microscopy Sections of the four 9- to 10-week fetuses (crown-rump length, 41-48 mm) confirmed that villus formation proceeds in a cranio-caudal direction. The small oval lumen of the distal small intestine was surrounded by stratified epithelium two to six cell layers thick in all four specimens. The stratified cells were characterized by large nuclei with prominent nucleoli and PAS positive deposits of glycogen which stained intensely with silver methenamine and were removed by prior digestion with a-amylase. This stratified epithelium was separated from the surrounding mesenchymal cells by a continuous basal lamina. At this age, however, the proximal intestine showed some architectural evidence of villus formation, although the degree of maturation varied among the four fetuses (figs. 1, 2). The earliest indication of mucosal remodeling was the appearance of subepithelial aggregates of mesenchymal cells which resulted in projections into the central lumen of the overlying stratified epithelium (fig. 1). In these regions the long axis of the mesenchyma1 cells a t the epithelial-mesenchymal interface tended to be oriented perpendicular to the basal lamina. Smooth muscle cells and blood vessels appeared within these mesenchymal invaginations a s their development progressed. Concomitantly, the degree of stratification of the epithelium lining these protrusions decreased. Initially, columnar epithelium lined only the apex of developing folds or villi but, as villi matured, columnar epithelium appeared along the sides (fig. 2). After ten weeks of gestation only the epithelium between villi remained stratified and the number and length of villi increased. Mitotic figures were abundant in all levels of the stratified epithelium. By 10 to 12 weeks, however, most were confined to the stratified intervillous regions and to the developing crypts, although occasional mitoses were evident on villi until 16 weeks of gestation. Crypt development proceeded in a craniocaudal direction during the eleventh to twelfth weeks. Initially, solid cords of epithelial cells extended from the base of adjacent villi into the underlying mesenchyme (fig. 3), but within a week a small lumen had developed. By the twelfth week the developing crypts were lined by simple columnar undif-

ferentiated cells. Putative Paneth cells with large supranuclear basophilic granules were occasionally seen a t the base of these early crypts. After villus and crypt development few major architectural changes occurred. The proximal small intestine of one 14- to 15-week fetus had well-developed Brunner’s glands with many large basophilic secretory granules in the apical cytoplasm. Also, between 17 and 20 weeks aggregates of specialized mesenchyma1 cells first appeared near the base of the crypts, suggesting the development of a thin, though discontinuous, muscularis mucosa (fig. 4). Electron microscopy The ultrastructure of the stratified epithelium of distal small intestine in 9- to 10-week fetuses differed from that of the proximal small intestine in two respects. First, extensive plications of some epithelial cell plasma membranes were much more prominent in the distal than in the proximal small intestine (fig. 5). Second, the continuous basal lamina applied to the deepest stratified epithelial cells differed in distal compared to proximal small intestine. In selected areas of the distal small intestine, two distinct laminae were seen along portions of the plasma membrane of some cells. In these regions, one basal lamina followed the general contour of the epithelial-mesenchymal interface whereas the other was closely applied t o invaginations along the basal plasma membrane (fig. 6). The individual cells of proximal and distal small intestine were relatively undifferentiated and mitoses were abundant (fig. 5). Profiles of centrally or basally located elongate nuclei usually revealed one to three prominent nucleoli. Aggregates of a-amylase sensitive glycogen which stained intensely with silver methenamine were present, most often in the paranuclear cytoplasm. The remainder of the cytoplasm contained abundant free ribosomes, small mitochondria, sparse amounts of rough endoplasmic reticulum (RER), and, in some sections, one to two small Golgi complexes. The apical surface of luminal cells had a few short irregular microvilli with a thin glycocalyx; a few dense granules and multivesicular bodies, presumed to be lysosomal, were occasionally seen in the apical cytoplasm. Zonula occludentes were seen along the apical aspect of adjacent luminal cells. Desmosomes were abundant along the lateral

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Fig. 5 Electron micrograph of the highly stratified epithelium of the distal small intestine from a 9- to 10-week fetus. Interdigitations of adjacent epithelial cells occur a t all levels of the epithelium but are most abundant among the basal cells where they are indicated by cross sectioned profiles of numerous cell processes (PI. Mitotic figures (M) are interspersed among the undifferentiated epithelial cells. L, central lumen. G , glycogen. X 3,700. Fig. 6 Higher magnification of the basal aspect of the stratified epithelium of the distal small intestine a t nine to ten weeks. There is duplication of the basal lamina in some areas. Where this occurs, one lamina follows invaginations of the basal plasma membrane while the other follows the general contour of the cell base. x 25,220.

plasma membranes of luminal cells, but were also seen in the deeper epithelial cell layers. During the period of villus formation a t nine to ten weeks gestation, distinctive junctional complexes appeared between adjacent

cells in the deeper layers of the stratified epithelium of both proximal and distal small intestine (fig. 7). These membrane specializations, though uncommon, were characterized by an apparent increase in the electron

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Fig. 7 Basal third of the stratified epithelium of the distal small intestine a t nine to ten weeks. Note the presence of distinctive junctional complexes (long arrow) between adjacent cells deep in the stratified epithelium. The basal lamina is indicated by short arrows. X 9,600. Fig. 8 Higher magnification of the aggregate of junctional complexes seen in figure 7. The opposing plasma membranes which form the complexes are more electron dense than the surrounding membranes and the intercellular spaces are narrowed. Microfilaments are abundant in the adjacent cytoplasm. x 27,000.

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Fig. 9 Proximal SB of a 9- to 10-week fetus with a probable “secondary lumen” (arrow) located deep within the stratified epithelium. L, main lumen. X 3,050. Fig. 10 Higher magnification of the probable “secondary lumen” noted in figure 9. Well developed microvilli and a single cilium (short arrow) project into the lumen. Lysosome-like structures and a multivesicular body are present in the adjacent cytoplasm (long arrows). X 12,900.

opacity of the opposing plasma membranes, a decrease in width of the intercellular space, and the presence of many thin filaments in the adjacent cytoplasm (fig. 8 ) . In addition, a small lumen deep within the stratified epithelium was found in a single specimen of proximal small intestine (fig. 9). This “secondary lumen” (Mathan e t al., ’76) was surrounded by cells with long, regular microvilli, well developed junctional complexes just beneath the luminal surface of adjacent cells, and occasional lysosome-like structures in the adjacent cytoplasm (fig. 10).A single cilium-like structure projected into the secondary lumen from the apical cytoplasm of one of the cells forming the secondary lumen (fig. 10). Evidence of cellular degeneration was occasionally seen in superficial stratified cells during the period of villus formation. In these regions large putative cytolysosomes were pres-

ent in the superficial cells and cellular debris was sometimes observed in the lumen. Both immature and mature goblet cells were seen in proximal and distal small intestine between 9 and 22 weeks of gestation. At nine weeks many relatively undifferentiated goblet cells were present in all layers of the stratified epithelium. Their cytoplasm contained a few secretory granules whose size and density varied considerably, dilated strands of RER, a supranuclear Golgi complex, mitochondria, numerous free ribosomes and a few glycogen particles (fig. 11). More differentiated goblet cells were also present in the stratified epithelium. Their secretory granules generally were not sufficiently abundant to distend the cell into a typical “goblet” shape, and the electron density of individual granules varied considerably. Some of the fetal goblet cells both in the stratified and in

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Fig. 11 An immature goblet cell within the stratified epithelium of a 9- to 10-week fetus. The cytoplasm contains moderate amounts of dilated rough endoplasmic reticulum (arrows) and many small secretory granules of variable size and density. X 4,500. Fig. 12 A typical mature-appearing goblet cell found on a villus of proximal small intestine a t 16 weeks. The nucleus is located in the basal portion of the cell and the supranuclear theca is filled with many large secretory granules. X 3,200.

the simple columnar epithelium had secretory granules located primarily in the infranuclear cytoplasm. Once villi had formed most goblet cells were indistinguishable from those found on villi of normal adult small intestine (fig. 12). Goblet cells seemed to be more prevalent in the proximal than in the distal small intestine a t nine to ten weeks but this apparent difference decreased with increasing gestational age. The relative number of goblet cells in the small intestine appeared to increase with advancing gestational age. When crypts first appeared a t 11 to 12 weeks gestation they were lined primarily by undifferentiated crypt cells (UCC’s) and they remained the most abundant cell type in the crypts through the twenty-second week. Their structural features did not differ greatly either with their location in the proximal or distal small intestine or with advancing fetal

age. Fetal UCC’s, like those of the adult, were characterized by basal elongate nuclei with prominent nucleoli, a moderate number of small mitochondria, relatively little rough or smooth endoplasmic reticulum, a moderately well developed supranuclear Golgi complex, and abundant free ribosomes (fig. 13). Short microvilli lined the lumen and small putative secretory granules filled with homogeneous appearing electron lucent material were seen in the apical cytoplasm of many cells (fig. 14). Three differences were noted between UCC’s of fetal and adult human small intestine. First, fetal UCC’s contained large aggregates of glycogen in the paranuclear cytoplasm. Second, the relatively large dense secretory granules (up to 1.5 Fm in diameter) characteristic of adult UCC’s (Trier, ’63) were not seen until the sixteenth week of gestation (fig. 15). Initially, the dense secretory gran-

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Fig. 13 Undifferentiated crypt cells of a developing crypt in the proximal small intestine of a 10-to 11-week fetus. The cells a r e characterized by basally located elongate nuclei with prominent nucleoli, little formed endoplasmic reticulum, a small supranuclear Golgi complex (C), and numerous small mitochondria. Unlike the adult undifferentiated crypt cells, large aggregates of glycogen (GI a r e common and secretory granules and lysosomal elements are small. x 4,600. Fig. 14 A higher magnification of the area outlined in figure 13. Short irregular microvilli line the lumen and there are small putative secretory granules filled with homogeneous appearing electron lucent material (arrows) in the apical cytoplasm. X 10,500.

ules of fetal UCC’s were considerably smaller than those of adult cells and, although they increased in size and number with increasing gestational age, they were seldom as abundant or as large as in the adult. Third, the fetal UCC’s had fewer lysosome-like bodies in the apical cytoplasm. Differentiating Paneth cells were noted at the base of developing crypts of the proximal and distal small intestine as early as 11 to 12 weeks gestation. They were characterized by numerous supranuclear electron dense secretory granules and dilated strands of elongate

RER filled with electron lucent material (fig. 16). In favorable sections, they displayed an elaborate supranuclear Golgi complex. Between the twelfth and twenty-second weeks of gestation, the secretory granules of the differentiating Paneth cells increased in size and their contents often appeared heterogeneous. For example, some granules contained inclusions which resembled crystalline structures and myelin figures (fig. 181, whereas others contained central electron dense cores surrounded by paler halos (fig. 17). Brunner’s glands were noted in the prox-

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bundles in t h e apical cytoplasm, (3) abundant less dense fine filaments in the remaining cytoplasm and (4) a n apical plasma membrane t h a t was more electron dense than the apical plasma membrane of neighboring cells (figs. 20, 21). Apical vesicles and caveoli, abundant in caveolated cells of adults, were seen only rarely in the fetal cells. The only epithelial cell type not identified with certainty in t h e fetal small bowel was the microfold or “M” cell which overlies Peyer’s patches in the adult human ileum (Owens and Jones, ’74). In fetal ileum, accumulations of lymphocytes were unusual prior to the twentieth week of gestation. In t h e distal small intestine of a single 17-week fetus, however, a lymphoid aggregate within the lamina propria was associated with distinctive epithelial cells suggestive of adult microfold cells. These fetal cells were characterized by a n irregular apical border with some knobby surface projections (microfolds?) and many small, membranebound vesicles within the cytoplasm (fig. 22). Lymphoid cells were interposed between the basal lamina and t h e basal plasma membrane of these putative M cells (fig. 23). Thus, the epithelial cells and mesenchymal lymphoid cells were in direct contact with one another. DISCUSSION

Fig. 15 Apical cytoplasm of undifferentiated crypt cells from the proximal small intestine of a 15- to 16-week fetus. Well developed junctional complexes appear between adjacent cells and the small putative secretory granules containing electron lucent material (arrows) are still present. In addition, there are a few larger electron dense granules (SG) resembling secretory granules found in undifferentiated crypt cells of adults. X 13,965.

imal small intestine of a 14- to 15-week fetus. These glands were continuous with t h e base of the crypts and penetrated deep into the mesenchyme. The cytoplasm of t h e secretory cells of the glandular acini contained many prominent secretory granules, Golgi complexes and elongate strands of RER (fig. 19). The degree of differentiation of these cells suggests t h a t Brunner’s glands developed prior to 1 4 to 15 weeks. By 16 weeks of gestation t h e caveolated or tuft cell (Nabeyama and Leblond, ’741, was first seen on villi. These cells were identified by t h e presence of: (1)bundles of dense filaments which extended from the cores of t h e microvilli deep into t h e cytoplasm, (2) prominent granules located between these filament

The observations described here on the time of appearance and ultrastructural features of t h e epithelial cells of the human fetal small intestine between t h e ninth and twenty-second weeks of gestation confirm and extend earlier reports. Prior to villus formation at nine to ten weeks, t h e stratified epithelium contained three cell types: undifferentiated epithelial cells, goblet cells and entero-endocrine cells. All other epithelial cell types known to occur in adult human small intestine, including villous absorptive cells, undifferentiated crypt cells, Paneth cells, caveoFig, 16 An immature Paneth cell located a t the base of a developing crypt in the proximal small intestine of an 11- to 12-week fetus. The cytoplasm contains dilated strands of rough endoplasmic reticulum (arrows), a Golgi complex (GI, and abundant supranuclear electron dense secretory granules of variable size. X 9,400. Fig. 17 Secretory granules of a Paneth cell from the proximal small intestine of a 20- to 22-week fetus. Many of the granules contain central electron dense cores surrounded by paler halos. X 17,300. Fig. 18 Secretory granules of a Paneth cell from the proximal small intestine of a 22-week fetus. The granule content is heterogeneous and some contain inclusions resembling crystalline structures (short arrows) and myelin figures (long arrow). X 16,100.

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entero-endocrine cells were present along the basal layer of the stratified epithelium. The morphogenesis of duodenal villi in fetal rats has recently been reported in detail (Mathan et al., ’76). The structural features observed included: (1) the formation of specialized junctional complexes in the deeper layers of the primitive stratified epithelium within which small “secondary lumina” develop, (2) eventual fusion of these lumina with the main duodenal lumen by their continued growth coupled with exfoliation of degenerative superficial epithelial cells and (3) upward growth of mesenchyme towards the lumen forming epithelial-lined protrusions which project into the lumen. Many of these structural features were noted in the stratified epithelium of the limited amount of young human fetal tissue available to us (9-10 weeks). For example, although infrequent, there were distinctive junctional complexes between adjacent cells of the deeper layers of the stratified epithelium in both proximal and distal human fetal intestine which were structurally comparable to those observed in fetal rats (figs. 7, 8). Similarly, an apparent secondary lumen was seen in a single specimen of human fetal proximal intestine. Considering its location deep within the stratified epitheFig. 19 Brunner’s gland cells deep in the mesenchyme lium (fig. 9) it seems likely that this lumen of the proximal small intestine of a 14- to 15-week fetus. was physically separated from the main luThe cytoplasm contains Golgi complexes (arrows), elonmen and not merely a continuous extension of gate strands of rough endoplasmic reticulum, and promi. it. Study of additional tissue following innent secretory granules. x 5,100. jection of a macromolecular marker into the lated or tuft cells, putative microfold or “M” main lumen would provide definitive proof (Mathan et al., ’76). Finally, there was evicells and Brunner’s gland cells appeared by dence in the human material of degeneration the beginning of the second trimester. The undifferentiated epithelial cells, many of superficial stratified cells during the period of which were in mitosis, were the most abun- of villus formation. These ultrastructural obdant of the three cell types found in the servations confirm and extend the earlier stratified epithelium at nine to ten weeks. The findings (Berry, 1900; Cho, ’31; Forssner, ’07; goblet cells, consistently reported by others as Johnson, ’10) that architectural remodeling to the first specialized epithelial cell type to ap- increase the intestinal mucosal surface occurs pear in fetal small intestine (Garbarsch, ’69; during the first trimester. Thus, i t seems likely that the structural Jirasek et al., ’65; Kelley, ’73; Lev and Weisberg, ’69; Lev e t al., ’72; Schmidt, ’051, changes which accompany morphogenesis of were seen in the youngest fetuses available to intestinal villi during the last trimester of us (9-10weeks). Many of the goblet cells in the pregnancy in the fetal rat also occur as villi stratified epithelium were immature and re- form in the human fetus during the first trisembled the oligomucous cells found in small mester of pregnancy. That certain structural intestinal crypts of adult mammals (Cheng specializations seen in abundance during viland Leblond, ’74; Troughton and Trier, ’69); lus formation in developing rat intestine were others resembled the mature goblet cells less common in developing human intestine found on intestinal villi of adult human small may be explained in part by the fact that the intestine (Trier, ’68).Additionally, as reported morphogenesis of villi requires only one to two recently (Moxey and Trier, ’77), two types of days in the fetal r a t whereas i t takes place

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Fig. 20 Two adjacent tuft or caveolated cells on the upper third of a villus from the distal small intestine of a 20-to 22-week fetus. The apical plasma membrane appears more electron dense than that of adjacent villous absorptive cells. Bundles of dense filaments extend from the cores of the microvilli deep into the apical cytoplasm (short arrows). Also, dense apical granules (long arrows) are prominent and fine filaments fill the cytoplasmic matrix. x 9.100. Fig. 21 A higher magnification of the area outlined in figure 20 showing the electron dense apical plasma membrane with i t s adherent glycocalyx. Small dense granules are adjacent to filaments which extend from the cores of the microvilli into the apical cytoplasm. x 12,300.

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Fig. 22 Putative microfold or “M” cell from the distal small intestine of a 17-week fetus. The apical surface of the cell is irregular with some cytoplasmic projections and the cytoplasm contains abundant membrane bound vesicles. Lymphoid cells (L) are in direct contact with the basal plasma membrane of the “ M ’ cell. x 7,700.

over a period of one to two weeks in human fetuses. Additionally, in fetal and neonatal rats, epithelial cell processes project through the basal lamina into the lamina propria and contact mesenchymal cells (Mathan e t al., ’72). Similar processes were not observed in human fetal intestine between 9 and 22 weeks.

Crypts appeared first in t h e proximal and then in t h e distal fetal small intestine about one week after villi had formed. Our observations a r e consistent with the hypothesis of Koelliker (1879) t h a t intestinal crypts originate as downgrowths of t h e epithelium into t h e mesenchyme from t h e base of adjacent villi. The ultrastructure of the undifferenti-

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Fig. 23 The interface between surface epithelial cells (El and the adjacent lymphoid cells (L) in the region where microfold-like cells were seen. Unlike most epithelial-mesenchymal interfaces, there is a direct contact between epithelial and mesenchymal cells with no separation by a basal lamina. Note t h a t a basal lamina (arrows) surrounds mesenchymal cells in this region. X 6,700.

ated cells which line these early crypts is similar to t h a t of undifferentiated crypt cells found in normal adult small intestine. The three major structural features which distinguish fetal from adult undifferentiated crypt cells were: (1) the abundance of cytoplasmic glycogen, (2) fewer and generally smaller secretory granules and (3) fewer cytoplasmic lysosome-like structures. The reported time of appearance of Paneth cells in human fetal intestine varies considerably, ranging from 13 weeks to 7 months (Garbarsch, '69; Lewin, '69; Patzelt, '31; Schmidt, '05; Wheeler and Wheeler, '64). These earlier reports were based entirely on light microscopic studies. In contrast, our findings indicate t h a t Paneth cells appeared immediately after crypt formation a t 11 to 12 weeks gestation. The earliest recognizable

human fetal Paneth cells resembled immature Paneth cells found in the crypts of adult small mammals (Troughton and Trier, '69). They contained less formed RER and smaller secretory granules in their cytoplasm than mature Paneth cells (Behnke and Moe, '64; Troughton and Trier, '69). Between 12 and 16 weeks gestation, the secretory granules of human fetal Paneth cells increased in size and their contents displayed striking heterogeneity. This contrasts markedly with the homogeneous internal structure of the large secretory granules of Paneth cells of normal human adults (Trier, '63). The significance of this structural heterogeneity of the secretory granules in fetal Paneth cells is unknown but it is not without precedent. Granules with dense cores and pale halos or pale cores and dense halos similar to those seen in fetal

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human material a r e found regularly in Paneth cells of adult mice (Hally, '58; Staley and Trier, '65) and granules with crystal-like inclusions (fig. 18) have been described in Paneth cells of young rats (Behnke and Moe, '64). The caveolated or tuft cells were seen only on villi and have not, to our knowledge, been described in human fetal small intestine. As in the adult the fetal intestinal caveolated cells are rare. In the adult they are characterized by prominent wider and longer microvilli than adjacent epithelial cells, extensions of microvillar filaments which project deep into the apical cytoplasm, prominent caveoli and cytoplasmic granules a t their apical surface, and a n abundance of fine cytoplasmic filaments (Isomaki, '73; Nabeyama and Leblond, '74). The putative fetal caveolated cells resembled those of the adult in all respects except that caveoli were uncommon and, though their microvilli stained more intensely than adjacent cells, they sometimes appeared neither wider nor longer. These structural differences may relate to gestational age or reflect a functional difference in utero, although the function of these cells even in the adult intestine is not known. The microfold or "M" cells of the adult human small intestine (Owen and Jones, '74) are always found in epithelium overlying lymphoid follicles of Peyer's patches. Like the putative fetal microfold cells noted in this study, adult microfold cells are cuboidal or squamoid in shape, have prominent Golgi complexes, abundant vesicles and vacuoles in their apical cytoplasm, lack a terminal web and have irregular apical surfaces probably lined by thin folds rather than true microvilli (fig. 22). Recent studies by Owen ('77) provide evidence that these cells transport potentially antigenic macromolecular substances to the underlying lymphoid tissue in adult rats. Whether microfold cells have such a function in fetal gut cannot be determined from our limited observations. As in the fetal small intestine a close association of specialized epithelial cells and underlying aggregates of lymphoid tissue has been described in the human fetal appendix (Bockman and Cooper, '75; Jones e t al., '72). In a single 17-week fetus with a n accumulation of lymphoid elements beneath the epithelium we noted translocation of the basal lamina from the basal surface of the epithelium into a n aggregate of lymphoid elements (fig.

23). This has not been described in previous studies concerning the development of aggregates or individual lymphoid elements in human fetal appendix or intestine (Bockman and Cooper, '75; Cho, '31; Cornes, '65; Haar, '77; Jones e t al., '72). The importance of this direct contact of lymphoid and epithelial cells in the fetal intestine is unknown though it may be important in normal immunological development. LITERATURE CITED Anderson, H., F. Bierring, M. Matthiessen, J. Egeberg and F. Bro-Rasmussen 1964 On the nature of the meconium corpuscles in human foetal intestinal epithelium: 11. A cytochemical study. Acta Path. Microbiol. Scand., 61: 377-393. Baginsky, A. 1882 Untersuchungen uber den Darmkanal des menschlichen Kindes. Virchows Arch., 89: 64-94. Bartmann, J. 1972 Ultrastructure of human foetal small-intestinal mucosa in early gestation. J. Pathol., 106: 8-9. Behnke, O., and H. Moe 1964 An electron microscope study of mature and differentiating Paneth cells in the rat, especially of their endoplasmic reticulum and lysosomes. J. Cell Biol., 22: 633-652. Berry, J. S. 1900 On t h e development of the human intestine. Anat. Anz., 17: 242-249. Bierring, F., H. Anderson, J. Egeberg, F. Bro-Rasmussen and M. Matthiessen 1964 On t h e nature of the meconium corpuscles in human foetal intestinal epithelium. I. Electron microscopic studies. Acta Path. Microbiol. Scand., 61: 365-376. Bockman, D. E., and M. D. Cooper 1974 Early lympho-epithelial relationships in human appendix. A combined light and electronmicroscopic study. Gastroenterology, 68: 1160-1168. Cheng, H., and C. P. Leblond 1974 Origin, differentiation, and renewal of the four main epithelial cell types in the mouse small intestine. 11. Mucous cells. Am. J. Anat., 141: 481-502. Cho, D. 1931 Histological investigation of the digestive tract of t h e human fetus. 11. Development of small intestines. Jap. J. Obstet., 14: 324-330. Cornes, J. S. 1965 Number, size and distribution of Peyer's patches in t h e human small intestine. I. The development of Peyer's patches. Gut, 6: 225-229. Dalton, A. J. 1955 A chrome-osmium fixative for electron microscopy. Anat. Rec., 121: 281. Dubois, P. M., C. Paulin and J. A. Chayvialle 1976b Identification of gastrin-secreting cells and cholecystokinin secreting cells in the gastrointestinal tract of the human fetus and adult man. Cell Tiss. Res., 175: 351-356. Dubois, P. R., C. Paulin and M. P. Dubois 1976a Gastrointestinal somatostatin cells in the human fetus. Cell Tiss. Res., 166: 179-184. Falck, B., R. HBkanson, C. Owman and N. Sjoberg 1967 Monoamine-storing cells of the enterochromaffin type in gastrointestinal tract of human fetus. Acta. Physiol. Scand., 71: 403-404. Forssner, H. 1907 Die angeborenen Darm-und Oesophagustresien. Eine entwicklungsgeschichtliche und pathologisch anatomische Studie. Anat. Hefte Wiesg., 34: 1-163. Garbarsch, C. 1969 Histochemical studies on the early development of the human small intestine. Acta Anat. (Basel), 72: 357-375.

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