Download PDF

/ . Embryol exp. Morph. Vol. 34, 3, pp. 723-740, 1975
Printed in Great Britain
723
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.
46
E M B 34
724
D. R. BURGESS
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
725
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.
726
D. R. BURGESS
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.
Morphogenesis of intestinal villi. II
727
46-2
728
D. R. BURGESS
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
Morphogenesis of intestinal villi. II
729
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
730
D. R. BURGESS
Morphogenesis of intestinal villi. II
731
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.
732
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
Morphogenesis of intestinal villi. II
733
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
734
11 A
D. R. BURGESS
Morphogenesis of intestinal villi. II
735
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
8-11
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.
736
D. R. BURGESS
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
Morphogenesis of intestinal villi. II
737
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
738
D. R. BURGESS
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
739
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,
740
D. R. BURGESS
N. K. & EVANS, J. (1968). Ultrastructural studies of early morphogenesis and
cytodifferentiation in the embryonic mammalian pancreas. Devi Biol. 17, 413-446.
WESSELLS,
WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., WRENN,
J. T. & YAMADA, K. M. (1971). Microfilaments in cellular and developmental processes.
Science, N.Y. 171, 135-143.
J. T. (1971). An analysis of tubular gland morphogenesis in chick oviduct. Devi
Biol. 26, 400-415.
WRENN, J. T. & WESSELLS, N. K. (1969). An ultrastructural study of lens invagination in the
mouse. /. exp. Zool. 171, 359-368.
WRENN, J. T. & WESSELLS, B. K. (1970). Cytochalasin B: effects upon microfilaments
involved in morphogenesis of estrogen-induced glands of oviduct. Proc. natn. Acad.
Sci., U.S.A. 66, 904-908.
ZWANN, J. & HENDRIX, R. W. (1973). Changes in cell and organ shape during early development of the-ocular lens. Am. Zool. 13, 1039-1050.
WRENN,
(Received 29 April 1975, revised 4 July 1975)