Morphogenesis of intestinal villi - Development

/ . Embryol exp. Morph. Vol. 34, 3, pp. 723-740, 1975 Printed in Great Britain

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Morphogenesis of intestinal villi II. Mechanism of formation of previllous ridges By DAVID R. BURGESS 1 From the Department of Zoology, University of California, Davis

SUMMARY Villi lining the avian intestine originate from longitudinal folds (previllous ridges) running the length of the embryonic intestine. The morphogenetic events that occur in the epithelium during initial ridge formation in the chick embryo duodenum were examined by light and electron microscopy. The epithelium, in cross-section, undergoes three stages prior to the formation of ridges; termed the circle (4^-6 days), the ellipse (6-8£ days), and the triangle (8^—9 days). At about 9 days of development three ridges form with three more forming one day later. The mechanisms responsible for folding of the epithelium were examined. Microdissection followed by organ culture demonstrated that constriction by the surrounding circular smooth muscle cannot account for folding of the epithelium. Mitotic pressure within the epithelium also cannot account for folding since there is no difference in the number of epithelial cells per cross-section between the ellipse and the triangle stages and the epithelial tube is not restricted from expanding. Active constrictions in groups of epithelial cells, mediated by bands of microfilaments, are thought to cause folding. Bundles of microfilaments are localized in the apical region of all epithelial cells at all stages studied and are localized in the basal region of those cells occupying the crests of the forming ridges. Cytochalasin B-treatment prevented ridge formation and disrupted the bundles of microfilaments. INTRODUCTION The development of shape or form, usually termed morphogenesis, is of prime interest in the study of development. An example of an epithelial sheet that undergoes dramatic change in form during development is the intestinal epithelium, which becomes shaped into finger-like villi that protrude into the lumen of the gut. The intestinal epithelium in the chicken does not form villi directly, but first forms longitudinal folds, termed previllous ridges, running the length of the intestine (Hilton, 1902). The processes by which sheets of epithelial cells form curved or lobulated structures have been under intensive investigation, and several possible mechanisms have been proposed to account for such morphogenetic events. One proposed mechanism involves mitotic pressure within an epithelium. According to this concept the addition, by mitosis, of cells to the epithelium would force it to fold or buckle z/the epithelium were confined by some external 1 Author's address: Friday Harbor Laboratories, Friday Harbor, Washington 98250, U.S.A.

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force such as surrounding tissues or mesenchyme (Zwann & Hendrix, 1973). Another proposed mechanism to account for folding of epithelial cell sheets is based on 'active' contractions by individual cells in the tissue. Such active cell contractions by a group of cells within a sheet can account for folding if: (1) contraction is restricted either to the apical or basal portion of the cells; and (2) the cells retain their adhesions within the epithelium. Localized contractions within individual cells are thought to be mediated by contractile intracellular microfilaments, 4-6 nm in diameter (see Wessells et al. 1971; Schroeder, 1973; Spooner, 1973). Contractile microfilaments in epithelial cells have been proposed to supply the force required for cell-shape changes responsible for amphibian neurulation (Baker & Schroeder, 1967; Schroeder, 1970; Burnside, 1971; Karfunkel, 1971), pancreas morphogenesis (Wessells & Evans, 1968), lens invagination (Wrenn & Wessells, 1969), morphogenesis of salivary epithelium (Spooner & Wessells, 1970, 1972) and morphogenesis of oviducal epithelia (Wrenn & Wessells, 1970; Wrenn, 1971). Several lines of evidence support the contention that intracellular microfilaments are involved in active cell constrictions which are thought to cause folding of epithelial cell sheets. There is a spatial and temporal correlation between the appearance of microfilament bundles and cell shape changes in folding epithelial sheets (Schroeder, 1970; Burnside, 1971). Also, microfilaments demonstrate a structural and biochemical similarity to actin, a contractile protein of muscle (Ishikawa, Bischoff & Holtzer, 1969; Pollard & Weihing, 1974). Cytochalasin B (CB) has been widely used in the investigation of cell motility because it inhibits many kinds of cell movements, as first described by Carter (1967). The list of folding in epithelia inhibited by CB includes morphogenesis of oviduct epithelium (Wrenn & Wessells, 1970; Wrenn, 1971), morphogenesis of salivary gland epithelium (Spooner & Wessells, 1970, 1972), and neurulation (Karfunkel, 1971). CB not only prevents folding in these systems, but also disrupts the structure of the bands of microfilaments within those epithelial cells that change shape during the folding process (Wrenn & Wessells, 1970; Wrenn, 1971; Cloney, 1972; Spooner & Wessells, 1972). The three-dimensional changes that occur in the duodenum of the chick embryo during previllous ridge formation have been described by a variety of techniques (Hilton, 1902; Coulombre & Coulombre, 1958; Grey, 1972). The first paper in this series described the very precise and predictable morphogenetic folding of the embryonic chick duodenum that leads to the establishment of definitive villi (Grey, 1972). The mechanisms responsible for the conversion of the tubular epithelium into an intricate pattern of previllous ridges have not, however, been explored. This paper describes the central events involved in the establishment of the first previllous ridges and reports on experiments designed to illuminate the mechanisms of ridge formation.

Morphogenesis of intestinal villi. II

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MATERIALS AND METHODS

Embryos Eggs from a commercial line of White Leghorn chickens were obtained from a local hatchery and incubated in a forced-draft incubator. All embryos were sacrificed by decapitation and staged according to Hamburger & Hamilton (1951). The proximal end of the duodenum from the pylorus to the apex of the loop was used for all parts of the study. Organ culture Embryos were dissected in warm Hank's balanced salt solution (HBSS) and fragments 1-2 mm in length were cut from the proximal end of the duodenum. Tissues were cultured in Eagle's Minimal Essential Medium containing 10% fetal calf serum and 100 i.u./ml penicillin and 100 /*g/ml streptomycin (MEM) (Grand Island Biological Co. or Pacific Biological Co.). Fragments were cultured floating in MEM in 35 mm culture dishes (Falcon Plastics, Inc.) in an atmosphere of 5% CO2 in air. For some experiments varying amounts of mesenchyme were removed prior to culture. This was done either by dissection with tungsten needles or with sharpened fine forceps. Alternatively, treatment of stage-34 intestines with 1% trypsin in calcium- and magnesium-free Hank's balanced salt solution for 30 min at 4 °C allowed the successful separation of epithelium from mesenchyme. Fragments of intact duodena from stage-34 embryos were also cultured for 24 to 36 h in glucose-free MEM (GFMEM). GFMEM was prepared from Eagle's Minimal Essential Medium without glucose (Pacific Biological Co.) to which fetal calf serum was added to achieve a final concentration of 10%. The fetal calf serum had been dialyzed for 2 days against 0-85 % NaCl and for 1 day against glucose-free HBSS (GFHBSS). Effect of Cytochalasin B on epithelial morphogenesis In some experiments 1- to 2-mm segments of intact duodena from stage-34 and stage-36 embryos were cultured for 24-36 h in MEM. To some of these cultures Cytochalasin B (CB) (Imperial Chemical Industries, Ltd) was added to achieve a final concentration in the medium of 1-0/tg/ml. For control cultures a volume of dimethylsulfoxide (DMSO), the solvent for CB, equal to the volume of CB solution added to the experimental cultures was added to the control dish. In order to test for reversibility of the drug's effect, CB-treated fragments from stage-34 embryos that had been cultured for 24-36 h were washed three to four times with fresh MEM for 30 mins to 1 h and then reincubated for 24-36 h. Microscopy Tissues were fixed at room temperature in 2 % glutaraldehyde buffered in 0-1 M cacodylate buffer, pH 7-2, and postfixed in 1% osmium tetroxide.

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Tissues were embedded in Epon and sectioned with glass knives on a PorterBlum MT-1 ultramicrotome or with a diamond knife on a Porter-Blum MT-2 ultramicrotome. One-micrometer sections, stained with a solution of 1 % toluidine blue and 1% boric acid, were routinely taken for light microscopy. Thin sections were stained in 2 % aqueous uranyl acetate and lead citrate and examined with a Hitachi HU-11E electron microscope. Some tissues and cultured fragments were prepared for scanning electron microscopy. The tissues were fixed, postfixed, and dehydrated as usual, then dehydrated by passage through a graded series of amyl acetate. Final preparation was by the critical point method as utilized by Grey (1972). The fragments were coated with gold and silver, using a vacuum evaporator equipped with a rotating specimen holder, and examined in a Cambridge Stereoscan electron microscope. RESULTS

Major developmental changes in the configuration of the intestinal epithelium Between 8 and 12 days of incubation the embryonic intestine constructs the previllous ridges upon which the definitive villi later form. These early events of ridge formation can be easily followed in cross-sections of the proximal duodenal loop. At about A\ days of incubation (stage 24) the intestinal epithelium appears in cross-section as a thick-walled circular tube with a small lumen (Fig. 1 A). From stage 26 to approximately stage 30 (5-7 days of incubation) the cross-sectional profile of the epithelium becomes elliptical and the lumen begins to expand (Fig. 1C-F). The circumference of the ellipse increases between stage 30 and stage 34 (7-8 days of incubation). Most of the increase occurs in the long axis; the width remains fairly constant (Table 1). During the 6-8 h required for the embryo to advance from stage 34 to stage 35 the elliptical tube of epithelium is transformed into a triangular-shaped tube (Fig. 1G). Immediately following its formation, the sides of the triangle appear to fold toward the lumen so that the first three previllous ridges are formed (Fig. 1H-J). During the 24-36 h of development after formation of the first three ridges, one previllous ridge usually forms in the location occupied by the valley

FIGURE 1

Cross-sections (1 ftm Epon sections) through duodenal fragments from embryos ranging from 4£ to 12 days of development. The cross-sectional shape of the epithelium at different stages of development is represented in this series of micrographs. All x 145. (A) Circle, stage-24 embryo; (B) small ellipse, stage-26 embryo; (C) small ellipse, stage-29 embryo; (D) elongated ellipse, stage-30 embryo; (E) elongated ellipse, stage-33 embryo; (F) elongated ellipse, stage-34 embryo; (G) forming triangle, stage-34 + embryo; (H) triangle, stage-35 embryo; (I) three-ridge, stage-36 embryo; (J) three-ridge, stage-36 embryo; (K) six-ridge, stage-37 embryo; (L) formed previllous ridges, stage-38-39 embryo.


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between two established ridges so that a total of six previllous ridges are present (Fig. IK). After this period ridge formation becomes more irregular. There are generally about eight previllous ridges formed by eleven days and sixteen by thirteen days (Fig. 1L and Grey, 1972). Table 1. Cross-sectional dimensions of epithelial tubes at different stages of development Embryonic stage*

Shape of lumen

4* (24) 5-7 (26-30)

Oval Small ellipse

7_8 (30-34)

Elongated ellipse

8-8* (34-35)

Triangle

Measurements Diameter Length Width Length Width Base Height

Average Number of (/*m) Range (/tm) samples 127 151 106 181 114 177 145

120-130 142-162 93-115 160-196 98-135 172-184 140-152

5 10 9 5

* The first numbers refer to days of incubation. The figures in parentheses are the Hamburger-Hamilton stage.

Formation of circular smooth muscle Coulombre & Coulombre (1958) suggested that contraction by the developing circular smooth muscle of the epithelial tube could cause the formation of the previllous ridges by forcing the epithelium to buckle. It was necessary, therefore, to determine if the forming circular smooth muscle has, at the time immediately prior to folding, the contractile machinery necessary for contraction. Up to the six-ridge stage there are about 15-20 layers of concentrically arranged, loosely packed mesenchymal cells around the epithelium. Beyond these loosely packed cell layers are 10-20 cell layers of tightly packed mesenchymal cells which will form circular smooth muscle (Fig. 1). By stage 34 (ellipse stage) organized contractile apparatuses are evident in many of these cells. The hypothesis that the band of smooth muscle produces folding of the epithelium was tested by removing the mesenchyme from one side of the elliptically-shaped epithelium in intestinal segments of stage-34 embryos (Fig. 2A). The mesenchyme was removed by dissection using sharpened fine forceps. This operation removed the forming circular smooth muscle but did not remove the two to six loosely packed mesenchymal cell layers adjacent to the epithelium. The circular band of smooth muscle was therefore prevented from being continuous: consequently it could exert little or no force on the intact epithelial tube. When such experimentally altered fragments were cultured for 24-36 h, all epithelial tubes folded to form three ridges (Fig. 2B). Loosely packed mesenchymal cells repopulated the region previously occupied by the


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smooth muscle; these invading mesenchyme cells did not, however, form smooth muscle. In all cultured fragments the epithelium overgrew the ends of the tube and covered the surface of the whole fragment, but this overgrowth appeared to have no effect on folding of the epithelium. The conclusion that smooth muscle is not required for folding of the epithelium is strengthened by evidence from a more radical experiment. Twelve ellipse-stage intestines from eight embryos were cut open lengthwise to form flat fragments with the epithelium on the surface. This surgery was done by slitting duodenal fragments open lengthwise with a sharpened tungsten needle cutting through both mesenchyme and one side of the epithelial tube. Immediately after such surgery the epithelial sheet was at first slightly curved (Fig. 3), but flattened out within five or six hours of culture. Ridge formation in these fragments was delayed by about 24 h as compared to cultured intact duodenal fragments. After culturing in MEM for 48 h the epithelia formed ridges in nine of the twelve fragments. Four of the fragments had formed six or more ridges (Fig. 4); the others formed two or three ridges. In most cases the ridges were shorter than normal and highly irregular in contour (Fig. 4). Whether the irregular ridges that form in these experimental cases do so by the same mechanism as the very regular ridges that normally form is not clear. It does seem possible, however, to rule out the notion that the growing epithelium simply buckled because it was prevented from spreading. Extensive spreading of the epithelium occurred not only in those fragments that formed ridges, but also in those that did not. Although smooth muscle was not required for epithelial folding, it was found that mesenchyme was required for folding morphogenesis. Intact epithelial tubes from early ellipse-stage intestines, as isolated by brief trypsin treatment, cultured for up to 48 h in MEM failed to undergo folding morphogenesis. Cellular dynamics in the epithelium associated with previllous ridge formation If the duodenal epithelium was restricted from expanding by the surrounding mesenchyme and smooth muscle concomitant with cell proliferation in the epithelium, the epithelial tube would be forced to buckle. The possibility that cell proliferation might play a role in the folding of the intestinal epithelium was therefore examined. In order to correlate the growth pattern (i.e. addition of cells) with the morphogenetic folding of the intestine, the number of cells in cross-sections of the epithelium was counted at all stages from the circle (4^ days) to the threeridge (10 days) stage. These data are summarized in Fig. 7. No significant increase in cell number occurs between 4y and 6\ days, i.e. during the period when the epithelium changes from the circle to the ellipse stage. Thus there appears to be no correlation between growth and shape of the epithelium during this period. Between 6\ and 8 days the cell number increases 38 %. This increase is accompanied by a lengthening of the ellipse. A major shape change 46-3

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occurs between 8 and 8^ days when the epithelium changes from elliptical to triangular in shape. There is, however, no addition of cells to the cross-sectional area during this period. A second prominent increase in the number of cells occurs between the ninth and tenth day of development; during this period the number of cells increases over 50%. This second saltation, unlike the first, occurs concomitantly with a shape change in the epithelium: the appearance of ridges in the epithelium. The possibility that mitotic pressure (during the second saltation) played a causal role in the formation of ridges was explored further. Mitotic pressure can effect the folding of an epithelium only if the epithelium is somehow confined to a restricted volume. An attempt was made, therefore, to estimate the volume available to the intestinal epithelium as it acquires the increased number of cells shown in Fig. 7. Such an estimate can be made by measuring the diameter of a circle drawn around the outer limits of the epithelium. Comparison of the triangle stage and the three-ridge stage shows, however, that a large increase in the diameter occurs between these two stages (an average of 162 jmn for six triangles and 216 /im for three three-ridges). Such an increase indicates that during the transformation of the triangle to the three-ridge stage, the epithelium is not completely restricted by the surrounding mesenchyme.

FIGURES

2-6

Fig. 2A. Drawing representing the fragment of tissue produced by removing half the mesenchyme from an ellipse-stage piece of intestine. Fig. 2B. Epithelial tube from ellipse-stage intestine cultured 24-36 h. The mesenchyme layers had been removed as shown in Fig. 2, and during the culture period loose-packing mesenchyme cells filled in the area that had been extirpated (black arrows). The circular smooth muscle did not invade the region that had been removed, but remained in its previous location (white arrows). A total of eight fragments from six embryos were altered and cultured for this experiment. All formed ridges, x 190. Fig. 3. Scanning electron micrograph of an ellipse-stage intestine that had been slit open lengthwise and fixed immediately. The epithelium (outlined by brackets) remains slightly curved as seen from the luminal surface in this view. The mesenchyme (mes) lies on either side of the epithelium, bordering it. x 260. Fig. 4. Scanning electron micrograph of a slit-open ellipse-stage intestine that had been cultured for 48 h. Numerous uneven ridges formed that were wavy and branching, x 250. Fig. 5. Thin section of the luminal area of an epithelial cell from an ellipse-stage intestine showing a fibrillar region (arrow) extending away from the intermediate junction region and terminating a short distance away in the cytoplasm. This region appears fuzzy in many cells indicating that the microfilament band had been cut in cross-section, x 42 500. Fig. 6. A glancing section of the luminal region of epithelial cells showing a tangentially cut intermediate junction region. A large microfilament band (arrows) with many organized filaments is seen running parallel with the junction in this region, x 25000.

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D. R. BURGESS Coulombre & Coulombre (1958) reported that the diameter of the whole duodenum does not increase while the overall length of the proximal loop does increase from 6 to 12 days of development.

6

7

10

Days of incubation

Fig. 7. Graph plotting the number of cells per cross-sectional view of the intestinal epithelium versus age of the embryo. This graph demonstrates two phases of growth of the epithelium during the morphogenetic period studied. The line is drawn through the mean values with the ranges represented by the bars. The following number of epithelia were used in determining the values plotted: circle, 7; small ellipse, 10; elongated ellipse, 11; triangle, 12; three-ridge, 3.

Role of cell-shape changes in the formation of the first set of previllous ridges If cells in precise regions of the epithelium actively contracted to change their shape, folding of the epithelial sheet could occur. An ultrastructural analysis of the epithelial cells was therefore undertaken with particular attention focused upon the location of cytoplasmic microfilaments. The first question to be answered concerned the region within individual epithelial cells in which microfilaments were most abundant. Microfilaments are most conspicuous in the luminal neck where they appear to insert as large bundles into the intermediate junction region (Fig. 5), distal to the desmosomes with their associated tonofilaments. They are present in this region throughout the stages studied. In this apical region of flask-shaped cells, the bands of microfilaments primarily course around the apical perimeter of the cell at the level of the intermediate junction and usually do not connect the cell at directly opposite points on the surface. This arrangement is apparent from most micrographs, which demonstrate a fibrillar or fuzzy zone emanating from the region of the intermediate junction region and apparently ending in the cytoplasm a short distance away. However, when the tight junctionai


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region is cut tangentially over an extended area, a band of microfilaments can be observed running parallel with the membrane (Fig. 6). Bands of microfilaments are also localized along the bases of the epithelial cells, parallel with and immediately adjacent to the basal plasma membrane (Fig. 8). The second major question to be answered was whether the apical or basal groups of filaments were prominent only in cells in certain regions of the epithelium. No preferential localization could be observed in the apical band of microfilaments. When the tube is triangular in cross-section, apical microfilamentous bands are as frequent in the cells making up the sides of the triangle as in those cells in the corners. This general observation is true for the ellipse stage as well. Basal microfilaments are, however, most easily resolved as organized bands in the cells comprising the sides of the triangular-shaped tube as they buckle inward to form the first previllous ridges (Fig. 8). The basal surfaces of these cells are narrower in diameter than those of cells in corners of the triangle and are usually folded into extended pseudopods. Cytochalasin B was used to study the possible role of microfilaments in controlling active cell-shape changes, and thus the formation of previllous ridges. Control explants of whole duodenal fragments from stage-34 embryos cultured either in the presence or absence of DMSO formed three previllous ridges within 24-36 h. A common characteristic of all control cultures was the rapid outgrowth of the epithelium from the cut ends of the tubes. Aside from the tighter packing of peripheral mesenchyme in some fragments, the appearance of the epithelium and mesenchyme in control tissues cultured 24-36 h was indistinguishable from normal three-ridge-stage intestines at both the light and electron microscope level (Fig. 9B). Isolated ellipse-stage intestines grown in the presence of 1 ^g/ral CB did not fold (Table 2 and Fig. 9C). The general ultrastructural appearance of CB-treated intestinal fragments was generally comparable to that of normal intestines. The structure, location, and orientation of microtubules and desmosomal tonofilaments of CB-treated intestines did not differ from control cultures or normal tissue. The mesenchymal layers were generally less densely packed than normal. Some significant effects of CB were, however, noted. In contrast to control tissues, the epithelium in CB-treated fragments did not spread out from the ends of the tubes. Also, in CB-treated intestines the apical surfaces of the cells bulged into the lumen, reminiscent of normal cells in the circle stage of development. This bulging effect ranged from epithelium which could not be distinguished from control cultures to epithelium in which many of the cells appeared to have extruded portions of apical cytoplasm into the lumen. Particular attention was paid to the structure of the microfilament bands in the epithelium of CB-treated tissues. There was a subtle difference between the structure of these bands in the luminal region of CB-treated cells and those of

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control cultures. The response of microfilaments in the apical region of the cells was variable. The apical cytoplasm of many epithelial cells exposed to CB exhibited an increased granularity or denseness in place of organized bands of microfilaments (Fig. 10). In the apical region of some epithelial cells from CBTable 2. Effect of CB on the formation and maintenance of ridges in fragments of embryonic intestine in organ culture Experiment

Results

Ellipse-stage intestines cultured 24-36 h in: (1) MEM+DMSO (control) (2)MEM + CB (3) MEM + CB, wash, MEM + DMSO (recovery from CB) Intestines with established ridges cultured 24 h in: (1) MEM + DMSO (control) (2)MEM + CB

Number of fragments folded* 25/30 5/50 7/151 Number maintaining ridges 9/9 12/13

* Folding is defined as the development either to the triangle stage or to the distinct three-ridge stage. t Those that recovered formed abnormal-looking triangle stages. treated fragments a web of microfilaments with many properly oriented microfilaments was present. Basal bands of microfilaments were not observed in CBtreated epithelia and the region normally occupied by such bands appeared as a fuzzy zone.

FIGURES

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Fig. 8. Basal region of epithelial cell occupying the crest of a forming ridge of a three-ridge-stage intestine. A band of organized microfilaments (arrows) is present running parallel with the basal plasma membrane, x 52000. Fig. 9. (A) An ellipse-stage intestine prior to culture. (B) A triangle-stage intestine formed while cultured in MEM + DMSO for 24 h. The epithelium folded normally while the mesenchyme became more tightly packed. (C) An ellipse-stage fragment cultured for 24 h in 1 /tg/ml CB. Folding of the epithelium did not proceed during this period. (A), (B), and (C) x 180. Fig. 10. Masses of finely granular material in the region normally occupied by microfilament bands in an epithelial cell from ellipse-stage intestine cultured for 24 h in the presence of 1 /tg/ml CB. x 32000. Fig. 11. (A) Thick section of a triangle-stage intestine dissected free of enveloping circular smooth muscle and most of the mesenchyme and cultured in MEM for 6 h. The triangle form was maintained in preparations like this one and in triangleand three-ridge-stage intestines isolated free of mesenchyme with the use of trypsin. x 240. (B) Six-ridge-stage intestine cultured for 24 h in the presence of 1 /tg/ml CB. Although large intercellular spaces formed in the mesenchyme the structure of formed ridges was maintained in 12 of 13 intestinal fragments cultured in CB. x 185.

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To determine if the CB effects were due to a general toxic effect not observable ultrastructurally, tissues were allowed to recover from CB treatment. Of 15 intestinal fragments (from 10 embryos) allowed to recover from CB treatment, only seven fragments recovered to form ridge-like structures. In those that did recover, the diameter of the epithelial tube and the height of the epithelial cells was decreased. The epithelium did, however, recover to the extent that it grew out the cut ends of the tubes to cover the mesenchyme. CB has also been shown to inhibit sugar transport across the plasma membrane (Kletzien & Perdue, 1973). In order to test whether CB prevented ridge formation by preventing uptake of glucose by the epithelial cells, fragments of intestines without ridges were cultured in glucose-free MEM (GFMEM). After 24-48 h, eight of nine fragments from six embryos had formed ridges in GFMEM. The epithelial cells appeared normal in all respects, but the mesenchyme appeared abnormal in that large numbers of intercellular spaces appeared. That folding of the epithelium occurred in the absence of glucose suggests that the effect of CB in preventing folding of the intestinal epithelium is independent of an effect on transport of glucose. Stability of previllous ridges Although the data presented in the preceding sections indicate that the mechanism of formation of ridges is an intrinsic property of the epithelium, there remained the possibility that the circular muscle layers act to maintain ridges, once they have formed. In order to test this possibility the mesenchyme layers were removed either manually, a procedure that leaves some mesenchyme adherent to the epithelium, or enzymically, a procedure that produces epithelial tubes that are completely free of mesenchyme. Three- and six-ridge-stage epithelial tubes that were freed from mesenchyme by either of these techniques maintained their original shape for up to 6 h in culture (Fig. 11 A). Longer times were not examined. Established ridges were also stable in the presence of CB at a concentration of 1 -0 /£g/ml. Thirteen fragments of whole three- to six-ridge-stage intestines were cultured for 24 h in the presence of CB. The structure of the ridges was maintained in 12 of 13 cultured fragments (Fig. 11B).

DISCUSSION

The morphogenetic changes in the epithelium and mesenchyme of the early chick embryo intestine are quite precise and predictable. The epithelium exhibits three distinct stages during the establishment of the first three previllous ridges; these stages have been termed the circle, ellipse, and triangle. The observation that villar morphogenesis begins with the formation of three previllous ridges is consistent with the finding of Hilton (1902). These findings conflict with those of Coulombre & Coulombre (1958) who reported that the


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embryonic chick duodenum initially forms only two previllous ridges. Since ridge formation begins with a starting number of three, the Coulombre's contention that ridges are added in a geometric progression (2, 4, 8, 16, 32) needs to be re-examined. The apparent discrepancy in the number of initial previllous ridges as reported in this study versus that reported by the Coulombres may be due to the region of the developing duodenum examined by the Coulombres. Nevertheless, there appears to be an average of eight previllous ridges in the 11-day duodenum (Grey, 1972). The transition to this stage from the threeridge stage has not been studied. This investigation sought to determine the location of the force(s) that generates the previllous ridges. The first question, therefore, was whether the folding mechanism or force was extrinsic to the epithelium (i.e. located in surrounding mesenchymal or muscular layers) or, alternatively, whether the folding mechanism resides within the intestinal epithelium itself. Several lines of evidence militate against the notion that the force is extrinsic to the epithelium. Folding occurs when the continuity of the circular layer of smooth muscle is disrupted (Fig. 2). Folding occurs even when the epithelial tube is slit open lengthwise to permit culture of the intestinal fragment as a flat sheet (Fig. 4). The diameter of the epithelial tube also increases as folding occurs (Fig. 1); although this observation does not, of itself, rule out the possibility that the folding force is extrinsic, it does establish that the space available to the epithelium does not remain fixed throughout the period of folding. These experiments and observations, taken together, provide clear evidence that a mechanism for folding based on mechanical constriction, as proposed by Coulombre & Coulombre (1958), is not applicable to the initial formation of previllous ridges. The question was then addressed as to what factors intrinsic to the epithelium could be responsible for folding. Mitotic pressure within the epithelium seems to be an unlikely mechanism, since certain periods of folding occur without a concomitant increase in cell number (Fig. 7). For cell proliferation to be responsible for folding, the epithelium would also have to be restricted from expanding. Since: (1) ridges form when the epithelial tube is slit open lengthwise, thereby permitting the epithelium to migrate laterally beyond its normal limits, and (2) the circular sheath of mesenchyme surrounding the epithelium expands in diameter during folding, there is no evidence to suggest that the epithelium is restricted to a fixed diameter during folding. Several lines of evidence are consistent with the hypothesis that cytoplasmic microfilaments are responsible for epithelial folding. Microfilaments are present in the basal region of only those cells on the apices of the folding ridges. The presence of microfilaments in only those cells with narrow basal ends is consistent with the notion that microfilaments are responsible for the narrowing and buckling of these cells, and that the cellular constrictions are responsible for folding of the epithelium. Similar basal buckling was observed in salivary

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gland epithelial cells during cleft formation (Spooner & Wessells 1970, 1972). By contrast, apical microfilaments are present in all cells of the epithelium during all stages studied. Intracellular microfilaments have been localized in a number of other folding epithelial sheets, including the neural plate (Schroeder, 1970; Burnside, 1971; Karfunkel, 1971), forming glands in the oviduct (Wrenn, 1971), developing lens (Wrenn & Wessells, 1969) and the developing salivary gland (Spooner & Wessells, 1970, 1972). In all these cases except for the salivary gland and the intestinal epithelium, microfilaments are preferentially localized in those regions of the cells which change shape and only in those cells whose shape change could bring about the final conformation of the epithelium. An interesting point is that in the neural plate, and also in the lens, cell-shape changes need occur only once in order to establish the correct final shape of the organ. In the cases of the epithelium from the salivary gland and the intestine, both of which undergo numerous foldings, apical microfilaments are present in all epithelial cells and are not preferentially localized in cells whose changes in shape could account for folding morphogenesis. One explanation of the presence of microfilaments in all cells of an epithelium during folding is that all cells in such continually folding epithelia should possess microfilaments, since all cells ultimately change shape. Folding in such epithelia cannot be controlled by the mere presence or absence of microfilaments. Instead, the control mechanism must involve the selective stimulation of contraction of microfilaments in only certain cells. Further evidence that microfilaments are responsible for active cell constrictions leading to folding of the intestinal epithelium comes from experiments using CB on cultured ellipse-stage intestines. CB inhibited folding of the epithelial cells. The locations previously occupied by microfilament bands were replaced by highly granular dense material. The apparent dissolution of bands of microfilaments and the concomitant prevention of folding in the intestine implicates microfilaments as being responsible for folding of previllous ridges. Similar correlations have been noted in other developing epithelia (Spooner & Wessells, 1972). The failure of CB to abolish microfilaments in all cells of the intestinal epithelium is inconsistent with the reports of the effects of this agent in other developing epithelia. The reason for this difference is unclear. CB apparently does not act by disrupting sugar transport since ridge formation proceeds normally in medium lacking glucose, indicating that the effects of CB on sugar transport and on folding are independent. The forces that act to maintain established ridges once they are formed are not well understood. It has been proposed that collagen fibers surrounding the developing salivary epithelium act to stabilize established clefts (Bernfield & Banerjee, 1972). Isolated three-ridge-stage intestinal epithelia, however, maintain their folded configuration even with little or no mesenchyme or other extracellular materials present. This result indicates a degree of intrinsic stability

Morphogenesis

of intestinal villi. II

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to the folded intestinal epithelium. Such stability is unaffected by CB treatment. There are a number of problems that remain unresolved. At the tissue level the central remaining questions concern the factors that regulate the number of ridges and their time of appearance. At the cellular level it is not known what factors control the placement and activation of contraction of microfilaments in the epithelial cells. Further work on the developing intestine may provide answers to some of these questions. The author would like to thank Drs Ursula K. Abbott, John H. Crowe and Robert D. Grey for helpful suggestions during the course of this work and Dr Grey for critical reading of the manuscript. The author also thanks Mr John Mais and Ms Carolyn Ishikawa for excellent technical assistance. REFERENCES P. C. & SCHROEDER, T. E. (1967). Cytoplasmic filaments and morphogenetic movement in the amphibian neural tube. Devi Biol. 15, 432-450. BERNFIELD, M. R. & BANERJEE, S. D. (1972). Acid mucopoly-saccharide (glycosaminoglycan) at the epithelial-mesenchymal interface of mouse embryo salivary glands. /. Cell Biol. 52, 664-673. BURNSIDE, B. (1971). Microtubules and microfilaments in newt neurulation. Devi Biol. 26, 416-441. CARTER, S. B. (1967). Effects of cytochalasins on mammalian cells. Nature, Lond. 213, 261-264. CLONEY, R. A. (1972). Cytoplasmic filaments and morphogenesis: effects of cytochalasin B on contractile epidermal cells. Z. Zellforsch. mikrosk. Anat. 132, 167-192. COULOMBRE, A. J. & COULOMBRE, J. L. (1958). Intestinal development. I. Morphogenesis of the villi and musculature. J. Embryol. exp. Morph. 6, 403-411. GREY, R. D. (1972). Morphogenesis of intestinal villi. I. Scanning electron microscopy of the duodenal epithelium of the developing chick embryo. /. Morph. 137, 193-214. HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, 49-92. HILTON, W. A. (1902). The morphology and development of intestinal folds and villi in vertebrates. Am. J. Anat. 1, 459-504. ISHIKAWA, H., BISCHOFF, R. & HOLTZER, H. (1969). Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. /. Cell Biol. 43, 312-328. KARFUNKEL, F. (1971). The role of microtubules and microfilaments in neurulation in Xenopus. Devi Biol. 25, 30-56. KLETZIEN, R. F. & PERDUE, J. F. (1973). The inhibition of sugar transport in chick embryo fibroblasts by cytochalasin B. /. biol. Chem. 248, 711-719. POLLARD, T. D. & WEIHING, R. R. (1974). Actin and myosin and cell movement. Critical Rev. Biochem. 2, 1-65. SCHROEDER, T. E. (1970). Neurulation in Xenopus laevis. An analysis and model based upon light and electron microscopy. /. Embryol. exp. Morph. 23, 427-462. SCHROEDER, T. E. (1973). Cell constriction: contractile role of microfilaments in division and development. Am. Zool. 13, 949-960. SPOONER, B. S. (1973). Microfilaments, cell shape changes, and morphogenesis of salivary epithelium. Am. Zool. 13, 1007-1022. SPOONER, B. S. & WESSELLS, N. K. (1970). Effects of cytochalasin B upon microfilaments involved in morphogenesis of salivary epithelium. Proc. natn. Acad. Sci., U.S.A. 66, 360-364. SPOONER, B. S. & WESSELLS, N. K. (1972). An analysis of salivary gland morphogenesis: role of cytoplasmic microfilaments and microtubules. Devi Biol. 27, 38-54. BAKER,

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(Received 29 April 1975, revised 4 July 1975)