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Development 120, 579-590 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Epithelium formation in the Drosophila midgut depends on the interaction of
endoderm and mesoderm
Ulrich Tepass* and Volker Hartenstein
Department of Biology, University of California Los Angeles, Los Angeles, CA 90024-1606, USA
*Author for correspondence
SUMMARY
The reorganization of mesenchymal cells into an epithelial
sheet is a widely used morphogenetic process in metazoans.
An example of such a process is the formation of the
Drosophila larval midgut epithelium that develops through
a mesenchymal-epithelial transition from endodermal
midgut precursors. We have studied this process in wild
type and a number of mutants that show defects in midgut
epithelium formation. Our results indicate that the visceral
mesoderm serves as a basal substratum to which endoder-
mal cells have to establish direct contact in order to form
an epithelium. Furthermore, we have analyzed the midgut
phenotype of embryos mutant for the gene shotgun, and the
results suggest that shotgun directs adhesion between
midgut epithelial cells, which is independent from the
adhesion between endoderm and visceral mesoderm.
INTRODUCTION
studies also show that the formation of primary and secondary
epithelia may be governed by different mechanisms. The genes
crumbs and stardust, for example, are required for the development of primary epithelia in the Drosophila embryo, but not
for the formation of the (secondary) midgut epithelium (Tepass
and Knust, 1990, 1993).
The development of the midgut epithelium in Drosophila
represents a model system for a systematic genetic approach
to study the mechanisms controlling epithelium formation. The
analysis of midgut development in wild type and various
mutants shows that the mesenchymal-epithelial transition that
leads to the formation of the midgut epithelium requires interactions between the endoderm and the adjacent visceral
mesoderm, as well as interactions among the endodermal cells
themselves. In flies with mutations that result in a complete
absence of visceral mesoderm (twist (twi); twi snail (sna)
double mutants) the midgut epithelium does not form. In
mutants with reduced visceral mesoderm or where the
endoderm is spatially separated from the visceral mesoderm
(tinman (tin); dorsal (dl) twi double heterozygotes; torso4021
(tor4021); folded gastrulation (fog)) only those endodermal
cells that directly contact the visceral mesoderm form an
epithelium. Finally, in shotgun (shg) mutant embryos the
contact between endoderm and visceral mesoderm is properly
established but endodermal cells do not form a columnar
monolayer suggesting that shg might control adhesion between
midgut epithelial cells.
The formation of epithelia is a fundamental step in metazoan
development. After a phase of rapid division, cells of the early
embryo form the blastoderm epithelium. Blastoderm cells, as
well as all later formed epithelia, are characterized by their
structural and functional apicobasal polarity and their twodimensional arrangement. Some of the blastoderm cells are
internalized during gastrulation and form the endoderm and
mesoderm. In many metazoans, including Drosophila, cells of
the endoderm and mesoderm lose their epithelial organization
and become mesenchymal during or shortly after gastrulation,
while the ectoderm remains epithelial. The various epithelia of
the later embryo are formed by two different mechanisms.
Primary epithelia originate directly (without any non-epithelial
intermediates) from the blastoderm. In Drosophila, mainly the
ectodermal epithelia (e.g. epidermis, fore- and hindgut, tracheal
system) belong to this class. By contrast, secondary epithelia
develop from mesenchymal intermediates by a process called
mesenchymal-epithelial transition (Tepass and Hartenstein,
1993). In this case, cells have to undergo profound changes in
their shape and spatial arrangement. A typical example of a
secondary epithelium is the Drosophila midgut epithelium, the
formation of which we have investigated in this study.
Recent studies carried out mainly in vertebrate model
systems suggest that both cell-cell and cell-substratum adhesion
triggers and stabilizes the reorganization from largely apolar
mesenchymal cells into a polarized epithelium (for review see
Rodriguez-Boulan and Nelson, 1989; Fleming, 1992). Evidence
from Drosophila indicates that interactions at the apical surface
of epithelial cells are also important for their development
(Tepass et al., 1990; Tepass and Knust, 1990, 1993). These
Key words: Drosophila, epithelium, mesenchymal-epithelial
transition, midgut
MATERIAL AND METHODS
Fly stocks and egg collections
The following mutations were used in this study. The strong twi
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U. Tepass and V. Hartenstein
alleles twi1D96 and twiHH07 (Nüsslein-Volhard et al., 1984), the strong
sna allele sna4.26 (Lindsley and Zimm, 1992), the strong dl allele dl1
(Nüsslein-Volhard et al., 1980), the strong tin alleles Df(3R)GC14 and
tinEC40 (Mohler and Pardue, 1984), the strong fog allele fogS4
(Wieschaus et al., 1984), the dominant tor allele tor4021 (Klingler et
al., 1988), the strong shg allele shgg317 (U. T., E. Gruszynski de Feo,
and V. H., unpublished data), and the intermediate shg allele shgIH
(Nüsslein-Volhard et al., 1984). As wild-type stock we used Oregon
R.
Flies were grown under standard conditions and crosses were
performed at room temperature or at 25°C. Egg collections were done
on yeasted apple juice agar plates. Embryonic stages are given
according to Campos-Ortega and Hartenstein (1985). The cross and
the egg collection to generate the dl twi double heterozygous embryos
were done at 30oC. Embryos were then stained with the anti-fasciclin
III antibody (see below) and embryos that had small gaps in the
visceral mesoderm were picked out for further examination.
Markers and immunohistochemistry
The following markers were used in this study. The enhancer-trap line
B11-2-2 (Hartenstein and Jan, 1992) to label endodermal cells in wild
type and in twi mutants. The enhancer-trap line A490 (Bellen et al.,
1989) to label endodermal cells in wild-type and in fog and shg mutant
embryos (data not shown). These enhancer-trap lines express β-galactosidase that was detected with a polyclonal anti-β-galactosidase
antibody (Cappel; dilution 1:2000). The monoclonal anti-Fasciclin III
antibodies mAb6D6 (kindly provided by Seymour Benzer) and
mAb2D5 (Patel et al., 1987; kindly provided by Corey Goodman)
were used to label the median portion of the visceral mesoderm in
wild type and all examined mutants. Both antibodies were diluted 5or 10-fold. A polyclonal anti-muscle myosin antibody (kindly
provided by Dan Kiehart; dilution 1:200) was used to detect visceral
muscle in wild type and in tor4021 mutant embryos. Antibody stainings
and sections of stained embryos were done as described previously
(Tepass and Knust, 1993).
Other histological techniques
Embryos for examination in the transmission electron microscope
were prepared as described previously (Tepass and Hartenstein,
1993). Embryos for semi-thin sectioning were prepared in the same
way. 2 µm sections were cut on an LKB Ultrotom V and stained with
a toluidine blue/methylene blue/borate solution.
RESULTS
Structure and embryonic origin of the midgut
The Drosophila larval midgut is composed of two tissue layers,
an inner epithelial layer, which develops from the endoderm,
and a mesodermally derived outer layer of visceral muscle. The
primordia of these two tissues have established contact with
each other at stage 11 (extended germ band stage). At this
stage, the endoderm forms two mesenchymal cell masses
called the anterior and posterior midgut rudiment, respectively
(Fig. 1A).
Using cell-specific markers (U. T. and V. H. unpublished
data) three different cell types can be distinguished in the
midgut rudiments. The majority of cells of both midgut
rudiments form an epithelium during germ band retraction. We
suggest the name principle midgut epithelial cells (PMECs) for
this cell type. A smaller fraction of cells do not become part
of the midgut epithelium initially. This group of cells is
composed of two cell types, the adult midgut precursors
(AMPs) and the so called ‘large basophilic cells’ (LBCs). The
Fig. 1. Midgut development in wild-type embryos. (A-F) Midgut
epithelium visualized with the enhancer trap line B11-2-2.
(G-J) Visceral mesoderm labeled with an anti-fasciclin III antibody.
(A) Lateral view of a stage 11 embryo. Both the anterior (am) and
the posterior (pm) midgut rudiments form clusters of mesenchymal
cells that are attached to the stomodeum (st) and proctodeum (pr),
respectively. (B) Same stage, ventral view. The anterior midgut
rudiment has started to form two bilateral lobes (arrows).
(C) Dorsolateral view of stage 12 embryo in which both the anterior
(long arrows) and posterior (short arrows) midgut rudiments form a
Y-shaped structure (Poulson, 1950). (D) Lateral view of mid stage
12 embryo (at 55% germband retraction). The posterior midgut
rudiment bends around the posterior pole. Both rudiments have
closely approched each other and are about to fuse (arrow).
(E) Lateral view of stage 13 embryo. At the end of germ band
retraction both rudiments have fully fused and form a rectangular
plate (arrows) on each side of the embryo. (F) Lateral view of stage
17 embryo. The midgut (mg) forms an elongated convoluted tube
and has developed four gastric caeca (gc) anteriorly. The midgut is
confluent anteriorly with the foregut that consists of the pharynx
(ph), the esophagus (out of focal plane), and the proventriculus (pv);
posteriorly it abuts the hindgut (hg). (G) Dorsolateral view of mid
stage 12 embryo (compare with D). The fasciclin III-positive portion
of the visceral mesoderm forms a narrow band at the interior-dorsal
aspect of the germ band. (H) Dorsolateral view of a stage 13 embryo
(compare with E). The midgut epithelium (me) covers the visceral
mesoderm. (hg) hindgut epithelium. (I) Same stage as in H. This
ventral view shows the two bilateral bands formed by the visceral
mesoderm. Arrow points to the junction of foregut and midgut. Note
that the fasciclin III-positive visceral mesoderm extends onto the
caudal portion of the foregut epithelium. (cl) clypeolabrum. (J) Cross
section of the anterior midgut at stage 13. The midgut epithelium is
attached to the internal surface of the visceral mesoderm. Anterior is
to the left except in J where dorsal is up. Scale bars: (A,B,C,E,F,I)
100 µm; (D,G,H) 50 µm; (J) 30 µm.
LBCs, which derive only from the posterior midgut rudiment,
and the AMPs occupy a position in the center of the midgut
rudiments. When the PMECs organize into the midgut epithelium, AMPs and LBCs remain attached as mesenchymal cells
to the apical surface of the epithelium (Fig. 2G,H,I). The LBCs
integrate into the midgut epithelium at a later stage (stage
14/15; Reuter et al., 1990). The AMPs become transiently part
of the midgut epithelium during late embryogenesis; in the
larva, they assume a position at the basal surface of the epithelium (Hartenstein and Jan, 1992).
The midgut epithelium is surrounded by two layers of visceral
muscles, an inner layer of circular (i.e. transversal) fibers, and
an outer layer of longitudinal fibres (Tepass and Hartenstein,
1993). The circular fibers and their mesodermal precursors can
be specifically labeled with an antibody that recognizes fasciclin
III (Patel et al., 1987; Fig. 1G-J). The visceral mesoderm that
forms the circular fibers first appears as metamerically repeated
clusters in the dorsal mesoderm of trunk parasegments 2-13
(maxilla - seventh abdominal segment; Tremml and Bienz,
1989; Azpiazu and Frasch, 1993). During late stage 11, the
clusters on each side of the embryo fuse into a continuous band
at the interior-dorsal aspect of the mesoderm. At this stage, the
midgut rudiments come into contact with the bands of visceral
myoblasts and use them as tracks for migration and epithelium
formation (see below). The fasciclin III-positive visceral
mesoderm also covers the posterior portion of the foregut (Fig.
1I). The precursors of the longitudinal visceral fibres originate
in the mesoderm at the tail end of the embryo (own unpublished
Midgut epithelium formation
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observation). Finally, the anteriormost and posteriormost
portions of the mesoderm give rise to visceral fibres associated
with the foregut and hindgut, respectively. These cells can be
distinguished from the visceral muscles of the midgut by the
absence of anti-fasciclin III staining.
PMECs are in close contact with visceral mesoderm
during epithelium formation
Shortly before and during germ band retraction (late stage 11
and stage 12) the PMECs reorganize to form the midgut epithelium. Early morphogenetic movements during gastrulation and
germ band extension have placed the midgut rudiments in a
position where they are in contact with the anterior or posterior
ends of the visceral mesoderm, respectively. The PMECs
migrate along the visceral mesoderm (Fig. 1B,C) so that at
about 50% germ band retraction, the arms of the anterior and
posterior midgut rudiments meet and fuse with each other (Fig.
1D). By the end of germ band retraction (stage 13) two rectangular plates have formed (Fig. 1E).
At the same time when PMECs migrate along the visceral
mesoderm, they become epithelial. The outermost PMECs,
which directly contact the visceral mesoderm, are the first cells
to undergo this mesenchymal-epithelial transition (Fig. 2A-C).
They are organized in a regular monolayer and become
columnar. Subsequently, the more interior PMECs, which originally had no contact with the visceral mesoderm, send
processes between the already existing epithelial PMECs.
After these processes contact the visceral mesoderm, the corresponding cells also become columnar and intercalate with the
first formed epithelial cells (Figs 2B, 3B). At 50% germ band
retraction, most PMECs have formed a monolayer of columnar
cells covering the visceral mesoderm (Fig. 2D-F).
From mid stage 12 onwards the LBCs and the AMPs
become morphologically distinct from the PMECs. The LBCs
are larger, the AMPs are smaller than the PMECs. Both cell
types are found at the apical side of the epithelium formed by
the PMECs (Fig. 2D-I). At stage 13 all PMECs have assumed
a highly columnar shape with the exception of those cells that
lie beneath the LBCs, which form two clusters in the middle
of the developing midgut, and attain a squamous cell shape
during stage 13.
When PMECs move along the visceral mesoderm and start
to form an epithelium, their surface is characterized by
multiple slender processes (Fig. 3C). At this stage, no extracellular material or any kind of junctional specialization
between endodermal and mesodermal cells could be detected,
suggesting that the PMEC migration is mediated by direct cellcell contacts. After germ band retraction, the number of
filopodia between PMECs and visceral mesoderm diminishes
and adherens junctions begin to form (Fig. 3G). These
adherens junctions might be the precursors of the so-called
connecting hemi adherens junctions (Tepass and Hartenstein,
1993). Between neighboring PMECs, scattered spot adherens
junctions and gap junctions are the only cellular junctions
present during midgut epithelium formation. A circumferential
junction as seen in other epithelia is not differentiated by the
PMECs until very late in embryogenesis (mid stage 17) and
comprises then a smooth septate junction (Tepass and Hartenstein, 1993). Epithelium formation during stages 12 is accompanied by the appearance of prominent apicobasal bundles of
microtubules in the columnar PMECs (Fig. 3E,F).
In the hours following germ band retraction, the narrow
band of visceral mesoderm expands dorsally and ventrally to
form the circular visceral muscle fibres. The PMECs follow
this movement and change in shape from columnar to cuboidal
or squamous during stages 14 and 15 (Fig. 2J,K). Throughout
this process, the basal surface of the epithelial cells contacts
the visceral muscle. Fig. 4 provides a schematic overview of
the formation of the midgut epithelium.
Midgut epithelium formation requires the presence
of visceral mesoderm
Our analysis of midgut epithelium formation in the wild type
shows that all PMECs are in contact with the visceral
mesoderm while converting into epithelial cells. This observation suggests that the visceral mesoderm serves as a basal substratum to which the PMECs must attach in order to form an
epithelium. To test this hypothesis we analyzed midgut epithelium formation in the background of mutations that alter
specific aspects of visceral mesoderm development.
In embryos mutant for twi1096 or in double mutant embryos
of the genotype twiHH07 sna4.26 the mesoderm and mesodermal
derivatives do not develop (Simpson, 1983; Grau et al., 1984).
The endoderm becomes internalized during gastrulation in
both mutants but does not form an epithelium (Fig. 5A,B).
To analyze midgut development in mutants that specifically
lack visceral mesoderm we examined tin mutant embryos
(Df(3R)GC14, tinEC40; Mohler and Pardue, 1984; Bodmer,
1993; Azpiazu and Frasch, 1993). A complete absense of the
fasciclin III-positive visceral mesoderm has been reported for
both tin alleles and also associated defects in midgut morphogenesis (Bodmer, 1993; Azpiazu and Frasch, 1993). We found,
however, that small clusters of fasciclin III visceral mesoderm
cells are present in tin mutants (Fig. 5E). Sectioned material
demonstrates that only those PMECs in contact with these
small islands of visceral mesoderm assume an epithelial
phenotype (Fig. 5F,G).
Similar results were obtained with twi1096 heterozygous
mutant embryos derived from heterozygous dl1 mothers. In
these animals, variable amounts of mesoderm are lacking
depending on the temperature at which the experiment is
performed (Simpson, 1983). The reduction in the number of
mesodermal cells leads to a reduction in cell number in all
mesodermally derived organs. In embryos of the cross dl1/+ ×
twi1096/+ raised at 30°C many embryos show small gaps in the
visceral mesoderm (Fig. 5C,D). Wherever these gaps occur, the
overlying midgut epithelium is absent too.
In twi and twi sna mutant embryos, the primary epithelia of
fore- and hindgut differentiate normally in the absence of
mesoderm (data not shown). Interestingly, the ectodermally
derived outer epithelial layer of the proventriculus, which is
commonly considered as part of the midgut, also differentiates
independently of visceral mesoderm (Fig. 5G).
Visceral mesoderm of the hindgut supports midgut
epithelium formation
In eggs collected from tor4021 heterozygous mothers the
segmented germ band is missing and tissues that derive from
the anterior and posterior pole are expanded (Sprengler and
Nüsslein-Volhard, 1993). The central part of tor4021 embryos
consists of hindgut epithelium whose apical (normally luminal)
surface faces outward, since it does not invaginate during gas-
Midgut epithelium formation
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Fig. 2. Formation of the midgut epithelium visualized in cross sections with the enhancer-trap line B11-2-2. (A-C) Early stage 12 embryo
(compare with Fig. 1C). (A,B) Sections through the anterior rudiment at a rostal (A) and caudal (B) level, respectively. (C) Section of the
posterior rudiment. Endodermal cells that are in contact with the visceral mesoderm (vm) have formed a monolayer of cuboidal to columnar
cells (arrowheads) while cells that are located interiorly (arrow in A) or lateral to the visceral mesoderm (arrow in C) form clusters of round
cells. Arrow in B points to a cell that apparently inserts between adjacent epithelial cells. Open arrow in C marks the LBCs. (D-F) Mid stage 12
embryo (compare with Fig. 1D). (D) Section of the anterior midgut rudiment; (E,F) sections of the posterior rudiment. Because the posterior
midgut rudiment is bent around the posterior pole it appears both dorsally and ventrally in E. In F the border between ventral and dorsal
portions is indicated by a dotted line. Most endoderm cells form an epithelium attached to the visceral mesoderm (arrowheads). Differences in
cell size become apparent at this stage. The small cells (short arrow) in D and F are AMPs. Large cells located internally in the posterior midgut
rudiment are LBCs (open arrow in E and F). The PMECs, which are of intermediate size, form the epithelium at this stage. (G-I) Late stage 13
embryo (compare with Fig. 1E). Dashed line in G indicates visceral muscle. (I) The posterior midgut and (H) the region where both rudiments
have fused. The PMECs form a highly columnar epithelium, except in the region covering the LBCs (open arrows) where the PMECs are
cuboidal or squamous (arrowheads in H). The AMPs (short arrows in G and I) are spread out over the whole apical surface of the developing
midgut. (J) Cross section of a stage 14 embryo. While spreading dorsally and ventrally, the cells of the midgut epithelium have assumed a
cuboidal shape. Fusion will occur first ventrally (arrow) then dorsally. (K) Section of a stage 15 embryo in which the midgut forms a closed
tube. At stage 17 (L) this tube has become convoluted (compare with Fig.1F). Dorsal is up in all panels. Other abbreviations: hg, hindgut. Scale
bars: (A-I) 30 µm; (J-L) 50 µm.
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U. Tepass and V. Hartenstein
Fig. 3. Histology of developing midgut epithelium. (A) Cross section of the anterior midgut rudiment (white arrow) attached to the visceral
mesoderm (black arrow) at mid stage 12 (compare with Fig. 1C). (B) TEM picture of the forming midgut epithelium (me; same stage as in A).
More internally located endodermal cells (to the right) send processes (arrows) between neighboring cells that are attached to the visceral
mesoderm (vm). (C) Magnified view of the interface between visceral mesoderm and midgut epithelium seen in B. Many processes formed by
the endodermal cells are apparent (arrows). (D) Cross section of early stage 13 embryo. The midgut epithelium (white arrow) forms a highly
columnar, pseudostratified epithelium attached to the visceral mesoderm (black arrow). Note that the midgut epithelium dorsally and ventrally
protrudes over the surface provided by the visceral mesoderm; cells at the edges of the midgut plates (curved white arrows) bend around in
order to establish contact with the visceral mesoderm. Arrowhead points at a trachea. (E,G) Transmission electron micrographs of parts of
midgut epithelial cells at stage 13. Bundles of microtubules (arrows) extend from the apical (upward) to the basal pole of the cell (E,F).
Adherens junctions (arrow in G) form between the visceral mesoderm and the midgut epithelium. Scale bars: (A,D) 30 µm; (B) 3 µm; (C,E) 1
µm; (F,G) 250 nm.
trulation. Similarly, the posterior endoderm remains at the
surface at the tail end of the embryo (Fig. 6A). Visceral
mesoderm of the trunk, which normally gives rise to the
visceral muscles of the midgut, is absent (Fig. 6A). By contrast,
the posteriormost portion of the mesoderm is spared and
produces visceral muscle fibres which attach to the hindgut
epithelium (Fig. 6B). This posterior mesoderm seems to be
intrinsically different from the midgut associated mesoderm,
as suggested by the fact that it does not express fasciclin III.
Interestingly, the visceral muscles of the hindgut are able to
support the formation of a rudimentary midgut epithelium. A
fraction of the endoderm cells, which contacts the visceral
mesoderm underlying the hindgut, takes on epithelial characteristics (Fig. 6D).
Midgut epithelium formation
A
stage 11
pm
am
B
stage 12
E
C
D
stage 13
foregut and hindgut
epithelium
LBCs
PMECs (non-epithelial)
visceral mesoderm
(median portion)
visceral mesoderm (anterior
and posterior portions)
PMECs (epithelial)
AMPs
Fig. 4. Formation of the midgut epithelium in wild-type embryos.
Different cell types are indicated by different colours (see bottom of
diagram). (A) During stage 11, the anterior (am) and posterior (pm)
midgut rudiments form clusters of mesenchymal cells. These clusters
are attached to the fore- and hindgut primordia, respectively. The
anterior midgut rudiment contains AMPs and PMECs; the posterior
rudiment contains in addition LBCs. The median portion of the
visceral mesoderm that associates with the midgut and the posterior
part of the foregut (arrowhead) is not confluent with the anterior and
posterior parts of the visceral mesoderm (arrows). (B) The
endodermal cells of the midgut rudiments migrate over the visceral
mesoderm during germ band retraction (stage 12). Those PMECs
that are already in contact with the visceral mesoderm have become
epithelial and show a slender apicobasally elongated cell shape
(shown in cross section in C); more interior PMECs send processes
inbetween these epithelial PMECs to establish contact with the
mesoderm. (D) and (E) After germ band retraction (stage 13) all
PMECs, except those adjacent to the LBCs, form a columnar
epithelium. The LBCs and the AMPs are located at the inner (apical)
surface of the midgut epithelium. LBCs are concentrated in the
region where the midgut rudiments have fused, while the AMPs are
spread over the whole epithelium.
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Local contact between visceral mesoderm and
endoderm is required for midgut epithelium
formation
The amount of visceral mesoderm might play a critical role
by emitting a diffusible factor in limited amounts that is
required for epithelium formation. In an embryo with reduced
visceral mesoderm the concentration of such a factor might be
insufficient to support the mesenchymal-epithelial transition
of all endodermal cells. To weaken this argument and to
further support our conclusion that direct contact of visceral
mesoderm and endoderm is required for midgut epithelium
formation we studied midgut development in fogS4. This
mutation blocks gastrulation movements so that neither the
ventral furrow, which internalizes the mesoderm and part of
the anterior endoderm, nor the amnioproctodeal invagination,
which internalizes the posterior endoderm, are formed
(Zusman and Wieschaus, 1985; Sweeton et al., 1991). Despite
these early defects, all tissues except for the posterior midgut
epithelium develop normally. Thus, although the ventral
furrow does not form, the cells of the mesoderm and the
anterior endoderm are eventually internalized and assume a
proper position in relation to the ectoderm. Two normally
sized bands of visceral mesoderm are formed (Fig. 6E). Also
the anterior endoderm usually ends up in its proper position
and becomes attached to the visceral mesoderm. Subsequently, it forms a normal epithelium (Fig. 6F). The posterior
endoderm, on the other hand, fails to invaginate and remains
at the surface at the posterior pole of the embryo. Not contacting the visceral mesoderm, posterior endodermal cells
remain mesenchymal (Fig. 6G).
Cell-cell adhesion among PMECs is required for
midgut epithelium development
The gene shg (Nüsslein-Volhard et al., 1984) is required for
several aspects of epithelial morphogenesis in the Drosophila
embryo (U. T., E. Gruszinzky de Feo and V. H., unpublished
data). In the context of this paper we focus on the defects in
midgut development of shg mutant embryos. In embryos
mutant for the strong allele shg g317 , the visceral mesoderm
develops normally (Fig. 7A). The PMECs attach to and
spread over the visceral mesoderm but they do not become
columnar; instead, they maintain a rounded to cuboidal shape
(Fig. 7B,C) and do not form a monolayer (Fig. 7B). Similar
observations, although less well defined, have been made in
embryos carrying the intermediate shg IH allele. These
findings suggest that there exist two independent adhesion
systems required for midgut epithelium formation, one
between PMECs, the other between PMECs and visceral
mesoderm. shg mutations seem to delete specifically
adhesion between PMECs, while the second system appears
to be unaffected.
At later stages (stage 14-17) when the visceral mesoderm
extends dorsoventrally, all PMECs of shg mutant embryos
become attached to the visceral mesoderm and gradually adopt
a more wild-type-like appearance. In our ultrastructural examination of the midgut in fully differentiated shg mutant
embryos no difference from wild type could be found (data not
shown; for a description of the ultrastructure of the larval
midgut see Tepass and Hartenstein, 1993).
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U. Tepass and V. Hartenstein
Fig. 5. Midgut development in
the absence of visceral
mesoderm. (A) Stage 15 twi1D96
embryos labeled with the
enhancer trap line B11-2-2. In
the absence of visceral
mesoderm, the anterior (am) and
posterior (pm) midgut rudiments
remain solid clusters of
mesencyhmal cells. The anterior
midgut rudiment in twi is smaller
than in wild type. (B) Cross
section of a stage 16/17 twiHH07
sna4.26 double mutant embryo.
The posterior midgut rudiment
forms an irregular cluster of cells
(arrow). (C) and (D) Stage 13 dl1
twi1D96 double heterozygote
embryos. Small gaps (arrow in
C) appear in the visceral
mesoderm. No midgut
epithelium forms adjacent to
these gaps (arrow in D).
(E) Dorsolateral view of stage 13
tinEC40 mutant embryo stained
with the anti-fasciclin III
antibody which reveals small
clusters of remaining visceral
mesoderm (arrows). (F) Cross
sections of the anterior midgut
rudiment of a stage 13 tin mutant
embryo (Df(3R)GC14). Only
endodermal cells that contact
visceral mesoderm cells (arrow)
have assumed a columnar cell
shape (apical cell poles are
indicated by arrowheads). By
contrast, the endodermal cells on
the right side, where no visceral
mesoderm is present, form an
irregular cluster of mesenchymal
cells. (G) Cross section of a stage
17 tin mutant embryo
(Df(3R)GC14). Most endodermal
cells form irregular clusters of
mesenchymal cells (large arrow).
Only small patches of midgut
epithelium associated with visceral muscle have differentiated (inset; small arrow points at the thin layer of visceral muscle). G also shows a
longitudinal section of the proventriculus. Although the visceral muscle is missing the outer epithelial layer of the proventriculus has formed
normally (curved arrow). Arrowhead points to the visceral muscle of the esophagus. Other abbreviations: ep, epidermis; sg, salivary gland; sm,
somatic muscle; tr, trachea; vc, ventral cord.
DISCUSSION
The visceral mesoderm provides a basal substratum
required for midgut epithelium formation
In the present study the mesenchymal-epithelial transition that
leads to the formation of the midgut epithelium has been
analysed in wild type and in a number of mutant embryos in
which the visceral mesoderm is absent, reduced in size, or
where endodermal and mesodermal cells are spatially
separated (for a summary diagram see Fig. 8). Our findings
demonstrate that endodermal cells have to establish local
contact with the visceral mesoderm, which serves as a basal
substratum for both migration and epithelium formation. Even
small patches of visceral mesoderm are sufficient to induce
endodermal cells contacting them to become epithelial as seen
in tin mutant embryos. The fog mutant phenotype shows
further that midgut epithelium formation does not depend on
the fusion of the anterior and posterior midgut rudiments. This
is consistent with our observation on normal midgut development where the transition of mesenchyme into epithelium has
been almost completed before both midgut rudiments fuse.
In tor4021 mutant embryos the segmented germ band,
including the visceral mesoderm of the midgut, is absent (for
review see Sprenger and Nüsslein-Volhard, 1993). In these
embryos we found that ‘hindgut-specific’ mesoderm is able to
Midgut epithelium formation
587
Fig. 6. Midgut epithelium in
tor4021 (A-D) and fogS4 (E-G)
mutant embryos. (A) Stage 13
tor4021 mutant embryos labeled
with the anti-fasciclin III
antibody (compare with Fig.
1H,I). The enlarged posterior
midgut rudiment (pm) is located
at the posterior pole of the
embryo. The hindgut is also
strongly enlarged compared to
wild type (black bar). Note
staining in the clypeolabrum (cl)
and in a small patch of the
hindgut epithelium (arrowhead).
The Fasciclin III-positive
visceral mesoderm is absent.
(B) Late tor4021 mutant embryo
stained with an anti-muscle
myosin antibody. The hindgut
(hg) forms a large tube filled with
yolk (yo). Visceral muscle fibers
are attached to part of the basal
surface of the hindgut epithelium
(arrows). (C) Cross section of the
posterior pole of a late tor4021
embryo showing mesenchymal
organization of midgut cells.
(D) Cross section of the hindgut
region of the same embryo as in
C. The hindgut forms a columnar
epithelium that is covered basally
with a thin layer of visceral
muscle (long arrow; compare
with B). Some endodermal cells
have spread over the inner
surface of the visceral muscle
and form an epithelial sheet.
Short arrows: AMPs that have
moved to the basal surface of the
midgut epithelium in this late stage tor4021 embryo as they do in wild type. (E) Stage 13 fog mutant embryo labeled with the anti-fasciclin III
antibody. Two continuous bands of visceral mesoderm, indicated by arrows, have assumed a normal position in the embryo and follow the
twisted path of the germ band. (F) Section of the anterior midgut in a late fog mutant embryo. Cells of the anterior midgut rudiment have spread
over the visceral muscle and form a regular epithelium (me). (G) Section of the posterior midgut rudiment of the same embryo as in (F). The
posterior endoderm retains its mesenchymal organization.
induce epithelium formation in endoderm cells that come into
contact with it. This observation bears on the question of the
specificity of the interaction between endoderm and visceral
mesoderm. As suggested by the analysis of embryos that lack
visceral mesoderm, other tissues (e.g., epidermis, nervous
system, and mesodermal derivatives such as somatic muscle
and fat body) cannot serve as a basal substratum for the endodermal cells. This indicates a high degree of specificity in the
interaction between the endoderm and the visceral mesoderm.
On the other hand, within the visceral mesoderm, all portions
may be capable of sustaining epithelium formation of the
endoderm.
The interaction between the endoderm and the visceral
mesoderm that leads to the establishment of the midgut epithelium is the first of several interactive processes between these
two tissues. It has previously been reported that regional
expression of a number of homeotic genes in the visceral
muscle is critical for midgut morphogenesis, including the
formation of the midgut constrictions and the gastric caeca
(Bienz and Tremml, 1988; Tremml and Bienz 1989; Reuter
and Scott, 1990). Furthermore, the visceral muscle emits diffusible signals that are involved in the late differentiation of
midgut epithelial cells (Immerglück et al., 1990; Reuter et al.,
1990; Panganiban et al., 1990).
Midgut epithelium development passes through
three phases characterized by different degrees of
intercellular adhesion
The reported observations on midgut development suggest that
the midgut epithelium undergoes three phases, which differ in
the amount of intercellular adhesion (Fig. 9). The initial phase
of midgut development, during which the highly columnar
epithelium emerges from the mesenchymal midgut rudiments,
may be characterized by strong adhesion. Experimental
evidence from vertebrate systems (injection of the adhesion
molecule N-cadherin into Xenopus embryos; Takeichi, 1991)
588
U. Tepass and V. Hartenstein
Fig. 7. Midgut phenotype in shgg317
mutant embryos. (A) Mid stage 12 shg
mutant embryo stained with fasciclin III
antibody to visualize the normally
developed visceral mesoderm. (B) Cross
section of the anterior midgut in a stage
13 embryo. The cell shape of the
endodermal cells (white arrow) has
remained round or cuboidal, instead of
columnar as in wild type (compare with
Fig. 3D). Many PMECs form a second
layer of cells (short arrows indicate a
cluster of PMECs). (C) A TEM section
of the midgut epithelium of a stage 13
shg mutant. Note the symmetrical cell
shape in the midgut epithelium (me) at
this stage (compare with Fig. 3). The
cells are, however, tightly attached to
the visceral mesoderm (vm). Scale bars:
(A) 100 µm; (B) 9 µm; (C) 3 µm.
A
stage 13
wt
B
twi
C
tin
dl/+;twi/+
D
tor D
E
fog
F
shg
indicates that high levels of adhesion are correlated with a
columnar cell shape, while a low adhesion level correlates with
a squamous or cuboidal epithelium. According to these
findings, the highly columnar morphology of early midgut
epithelial cells in Drosophila suggest a high level of intercellular adhesion. We propose that the strong intercellular
adhesion between the PMECs depends on the function of the
shg gene. The PMECs in shg mutant embryos do not become
columnar but keep a round to cuboidal shape instead (Fig. 8F).
shg might be involved in the adhesion process itself, or it might
control the cellular response (i.e., polarization of the cytoskeleton) to the adhesion event that leads to the reorganization of
the cell structure into an asymmetric columnar shape. Surprisingly, the drastic early defects in epithelial cell morphology in
shg mutants have no apparent consequences for the terminal
differentiation of the midgut epithelium. The functional significance of the highly columnar cell shape of the PMECs
might be related to the limited substratum surface provided by
the visceral mesoderm that forms a narrow band at stage 13.
Thus, a small basal cell surface is a prerequisite for the PMECs
to fit onto the surface area provided by the visceral mesoderm.
Fig. 8. Synopsis of the mutant phenotypes as seen shortly after
germband retraction. (A) Gut in a stage 13 wild-type embryo (for
explanations and colour code see Fig. 4). (B) The endodermal cells
remain mesenchymal in twi mutants (or twi sna double mutants), in
which the visceral mesoderm is absent. (C) In tin mutants (upper
part) only small patches of visceral mesoderm develop (arrow).
PMECs attached to these patches of visceral mesoderm form a
columnar epithelium, while all other PMECs remain mesenchymal.
In dl twi double heterozygotes (lower part of C) the visceral
mesoderm has small gaps that are not crossed by midgut epithelium
(arrow). (D) In tor4021 (torD) mutant embryos some cells of the
posterior endoderm use the ‘hindgut specific’ visceral mesoderm as
substratum. (E) In the fog mutant, the posterior midgut rudiment fails
to invaginate and does not contact the visceral mesoderm. It does not
form an epithelium. (F) Midgut phenotype of shgg317 mutant
embryos. Endodermal cells spread normally over the visceral
mesoderm but do not develop a columnar, monolayered
organization.
Midgut epithelium formation
589
strong adhesion among epithelial cells is evidently necessary
to resist the stretching forces which arise during the peristaltic
movements of the midgut.
Fig. 9. Hypothetical phases of midgut development which are
characterized by various degrees of intercellular adhesion. (A) At
stage 13, intercellular adhesion is high, leading to a columnar cell
shape of the PMECs. (PMECs, white; LBCs, stippled; AMPs, black;
visceral mesoderm, gray bar). (B) From stage 14 to mid stage 17
intercellular adhesion is low. Midgut cell shape is cuboidal to
squamous; occasionally cells have detached from each other. LBCs
and AMPs integrate into the epithelium and the epithelium
rearranges from a short sac into a long and narrow tube. (C) High
intercellular adhesion starts with the differentiation of a smooth
septate junction (black bars) at mid stage 17. See text for further
explanations.
This view is corroborated by the observation that in shg mutant
embryos where the PMECs remain rounded or cuboidal, many
PMECs do not initially establish contact with the visceral
mesoderm.
During stage 14 the cell shape of the PMECs changes from
columnar to cuboidal or squamous, marking the beginning of
a phase of reduced intercellular adhesion that extends until mid
stage 17. During this phase, cells of the midgut epithelium may
detach from each other (Reuter and Scott, 1990; own unpublished observations). The monolayered arrangement of the
cells is ensured by the adhesion of the endodermal cells to the
surrounding visceral muscle. The phase of reduced intercellular adhesion between midgut epithelial cells might be
important for (i) the intergration of the LBCs and the AMPs,
which initially remain mesenchymal, into the midgut epithelium (Reuter et al., 1990; Hartenstein and Jan, 1992) and (ii)
the rearrangement of the midgut epithelium from a short saclike structure into the slender, elongated tube, the larval
midgut. A strong adhesion to the visceral muscle and a reduced
adhesion among the midgut epithelial cells themselves might
play a permissive role during these morphogenetic changes.
This view is corroborated by the observation of Newman and
Wright (1981) that midgut morphogenesis is arrested when the
midgut constrictions form in embryos mutant for the gene
lethal(1)myospheroid [that encodes the β chain of the
Drosophila PS-integrins (MacKrell et al., 1988)], where the
attachment between visceral muscle and midgut epithelium is
disrupted.
The third phase of midgut development is again characterized by strong cell-cell adhesion. It begins during mid stage 17
with the differentiation of a smooth septate junction that
extends over the apicolateral 40-60% of the cell surface
(Tepass and Hartenstein, 1993). This junctional specialization
is typical for the arthropod midgut epithelium and it is
generally believed to provide strong intercellular adhesion. The
Cell-cell and cell-substratum adhesion systems are
required for epithelium formation
In vertebrates it has been shown that cell-cell and cell-substratum adhesion systems are involved in both formation and
maintenance of the epithelial structure and act independently
in organizing the epithelial phenotype (Rodriguez-Boulan and
Nelson, 1989; Wang et al., 1990; Fleming, 1992). Like
epithelia in vertebrates, the formation of the midgut epithelium
in Drosophila depends apparently on cell-substratum and cellcell adhesion events. Preliminary observations (U. T., unpublished) suggest that the Drosophila PS-integrins, which are
expressed at the interface of the midgut epithelium and the
visceral mesoderm (Leptin et al., 1989), provide cell substratum adhesion during midgut epithelium formation. A further
candidate molecule is a newly identified integrin β chain that
is specifically expressed in the midgut epithelium (βν; Yee and
Hynes, 1993). That cell-cell adhesion, which might depend on
shg function, is involved in the formation of the midgut epithelium has been discussed above. Cell-cell and cell-substratum
adhesion events presumably initiate the reorganization of the
cell structure in a directed assembly process that leads, for
example, to the polarization of the microtubules that has been
observed in vertebrate epithelia (e.g. Bacallao et al., 1989) as
well as in the Drosophila midgut epithelium (this work). A
polarized array of microtubules, in turn, appears to be
important for the selective transport of proteins and organelles
to the apical or basolateral membrane domains that is critical
for the maintenance of the epithelial phenotype. The formation
of the midgut epithelium in Drosophila provides us with a
model system to study the geneyic mechanisms that control
early events in the reorganization of mesenchymal cells into
polarized epithelial cells.
We are grateful to Silvia Yu for technical assistance, to Dan
Kiehart, Seymor Benzer and Corey Goodman for providing antibody
probes and to Judy Lengyel, Steve Crews, Lisa Fessler, Christiane
Nüsslein-Volhard, Mary-Lou Pardue, Rolf Bodmer, and the Bloomington Stock Center for providing fly stocks. We thank Dorothea Godt
and members of the Hartenstein laboratory for critical reading of the
manuscript. This work was supported by NIH Grant NS29367 to V.
H. and by a HFSPO fellowship to U. T.
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(Accepted 7 December 1993)