/ . Embryol. exp. Morph. Vol. 34, 3, pp. 695-705, 1975
Printed in Great Britain
695
Epithelio-mesenchymal interface during mouse
kidney tubule induction in vivo
By E. LEHTONEN 1
From the Third Department of Pathology and Electron Microscope Laboratory,
University of Helsinki, Finland
SUMMARY
Transmission and scanning electron microscopy were used to study the epithelio-mesenchymal interface between the interacting mouse ureter-bud and the metanephric mesenchyme.
The gap between the epithelial and mesenchymal cells varied in width. At the stalk of the
ureter-bud the interspace was often about 1 /im, but in the inductively active areas at the tips
of the branching ureter-bud epithelio-mesenchymal contacts were seen through discontinuities in the basal lamina. At these points the gap between the interacting cells was often less
than 20 nm, in places less than 10 nm. The amount of electron-dense, ruthenium-red-positive
material was greatest at the stalk of the ureter-bud, but only a small amount of extracellular
material was found between the interacting cells at the tips.
Whether epithelio-mesenchymal cell contacts play a role in kidney tubule induction is not
yet known, but their existence in the inductively active areas and their absence in inactive
zones suggests that they are morphogenetically significant. The finding also obviates the need
to postulate long-range transmission of inductive signals to explain this example of embryonic
induction.
INTRODUCTION
Differentiation of the metanephric kidney rudiment requires interaction between an epithelial component, the ureter-bud, and a mesenchymal component,
the metanephric mesenchyme (Grobstein, 1955; Saxen, 1971). This interaction
results in the formation of mesenchymal cell aggregates which continue to
differentiate into secretory tubules. The mechanism by which the tubuleinducing stimulus is transmitted has been studied in vitro by interposing filters
between the interacting tissues, on the assumption that this would prevent cell
contacts (Grobstein, 1956, 1957; Saxen et al. 1968). However, the rate of transmission of the tubule-inducing signal proved too slow to be explained by longrange diffusion of signal substances (Nordling, Miettinen, Wartiovaara &
Saxen, 1971). Filters do not necessarily prevent cell contacts (Wartiovaara,
Lehtonen, Nordling & Saxen, 1972), and a positive correlation was observed
between successful transfilter tubule induction and the establishment of membrane contact between processes from the interacting cells (Wartiovaara,
Nordling, Lehtonen & Saxen, 1974; Lehtonen, Wartiovaara, Nordling & Saxen,
1
Author's address: Third Department of Pathology, Haartmaninkatu 3, SF-00290
Helsinki 29, Finland.
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E. LEHTONEN
1975). Between the cell processes, there was no accumulation of extracellular
material.
These observations led us to suggest that, in this experimental transfilter
system, induction of tubules requires close association of the interacting cells.
As yet, there have been no morphological studies of the normal situation in vivo,
where the ureter-bud interacts with the metanephric mesenchyme. In the present
study I have examined whether the contact between ureter-bud and mesenchyme
is close enough to be compatible with the hypothesis presented.
MATERIALS AND METHODS
Metanephric kidney rudiments were removed from 12-day-old CBA/T6T6 x
BALB/C hybrid mouse embryos. At this stage the inducing ureter-bud has
branched only once within the metanephric mesenchyme. Therefore the kidney
rudiments of this stage were found technically most suitable for the study of
tissue interaction in question.
For transmission electron microscopy, kidney rudiments were fixed in 2-5%
glutaraldehyde in 0-1 M cacodylate buffer, pH 7-8, for 60 min at 4 °C, then
rinsed and postfixed in 1% osmium tetroxide in 0-1 M cacodylate buffer,
pH7-8, for 60 min at 4 °C.
Rudiments to be stained with ruthenium red were fixed for 12 h at room
temperature in 2-5% glutaraldehyde in 0-1 M cacodylate buffer, pH 7-8, which
contained 0-05% ruthenium red, then rinsed and postfixed in the dark for 3 h
at room temperature with 2% osmium tetroxide in 0-1 M cacodylate buffer,
pH 7-8, which also contained 0-05% ruthenium red (Luft, 1971). Because of the
poor penetration of the dye, most of the mesenchymal tissue was teased away
from the kidney rudiments, which left a thin layer of mesenchyme 2-10 cells
thick on the epithelium (see Fig. 5).
The fixed specimens were dehydrated in ethanol and orientated in a drop of
Epon 812 before polymerization on a siliconized object glass (SilicladR, Becton,
Dickinson and Co., Parsippany, N.J. 07054). Thin sections were stained for
90 min with 3% uranyl acetate in 50% ethanol and for 1 min with Reynolds's
lead citrate diluted to 20% with 0-01 N-NaOH and examined in a Jeol JEM100B transmission electron microscope. Thick Epon sections for light microscopy were stained with 1% toluidine blue in 1% borax.
For scanning electron microscopy the kidney rudiments were fixed in 2-5%
glutaraldehyde in 0-1 M cacodylate buffer, pH 7-8, for 60 min at room temperature and embedded in paraffin. Sections were cut at 5-10/tm, deparaffinized
with xylene, and dried by the critical point method (Anderson, 1951). The
specimens were covered with a thin layer of carbon and gold in Balzers MicroBA3 evaporator and examined in a Jeol JSM-U3 scanning electron microscope.
Cell contacts in developing kidney
Fig. 1. Micrograph of a 12-day mouse kidney rudiment. Condensation of metanephric mesenchymal cells (M) takes place at the tips of the epithelial ureter-bud (E).
xlOO.
Fig. 2. Mesenchymal cells (M) at the onset of condensation round the tip of the
ureter-bud (E) in a 12-day kidney. Indications of cytoplasmic processes (arrows) are
seen in the epithelio-mesenchymal interspace. Thick Epon section stained with
toluidine blue, x 350.
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E. LEHTONEN
Cell contacts in developing kidney
699
RESULTS
Light microscopy
The first morphological event in the induction of kidney tubules is a condensation of mesenchymal cells round the tips of the branching ureter-bud
(Fig. 1). Examination under a high-power oil-immersion lens revealed a gap
with indications of cytoplasmic protrusions between the tip of the ureter-bud
and the aggregated mesenchymal cells (Fig. 2). The electron microscope studies
were primarily focused on this area.
Transmission electron microscopy of intact kidney rudiments
The intercellular space separating the epithelium from the mesenchyme varied
greatly in width. At the tips of the ureter-bud the space between the epithelial
and mesenchymal cell membranes was often only 15-20 nm, and in places less
than 10 nm (Fig. 3). No specialized cell junctions could be identified at the
points where actual contacts were visible between the mesenchymal and
epithelial cells.
Proximally to the tips of the ureter-bud the distance between the epithelial and
mesenchymal cells tended to increase. At the stalk of the ureter-bud it was
often about 1 /tm (Fig. 4) and epithelio-mesenchymal cell contacts were not
seen.
Electron-dense material was seen in the epithelio-mesenchymal interspace. The
amount of this material was usually smallest at the tip of the ureter-bud and
greatest at the stalk, where induction is not seen to take place. Against the stalk
of the ureter-bud the interspace contained both fibrillar structures and fuzzy
material (Fig. 4), whereas at the tips there were usually only small amounts of
fuzzy extracellular material. There the basal lamina coating the epithelial cells
was often discontinuous, and cell contacts were visible in gaps in the lamina.
Transmission electron microscopy of kidney rudiments stained with ruthenium red
Ruthenium red is an electron-dense polyvalent cation which interacts with
negatively charged groups at the cell surface. It has been used as an indicator
F I G U R E S 3 AND 4
Fig. 3. Electron micrograph showing cytoplasmic protrusions from both epithelial
(E) and mesenchymal (M) cells at the tip of the ureter-bud. The basal lamina (arrow)
coating the epithelial cells seems discontinuous, x 28000. Inset: electron micrograph
showing the area enclosed by the rectangle in Fig. 3. At the arrow (where the basal
lamina is discontinuous) the distance between the epithelial and mesenchymal cell
membranes is about 10 nm. x 100000.
Fig. 4. Electron micrograph of the stalk of the ureter-bud. Fibrillar and fuzzy
material (arrows) in the wide gap between the epithelial (E) and mesenchymal (M)
cells, x 23000.
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E. LEHTONEN
of acid mucopolysaccharides (Martinez-Palomo, 1970). Most of the mesenchymal tissue was teased away from the epithelium before staining to facilitate
penetration of the dye (Fig. 5). This treatment did not damage the general
ultrastructure of the epithelio-mesenchymal interface (compare Figs. 3 and 6).
Fig. 5. Micrograph of the tip of the ureter-bud. Most of the mesenchymal tissue is
teased away (see upper left corner) to facilitate penetration of ruthenium red stain.
Indications of the dye are seen in the epithelio-mesenchymal interface (arrows).
Toluidine blue staining, x 350.
Ruthenium red stained the intercellular spaces in both mesenchyme and
epithelium. At the epithelio-mesenchymal interface the thickness of the
ruthenium-red-positive material varied, tending to increase proximally to the
tips of the branching ureter-bud. No thick ruthenium-red-positive layers were
seen between the epithelium and mesenchyme at the tips of the ureter-bud
(Fig. 6). In places, areas of apparent membrane fusion were seen between the
apposed epithelial and mesenchymal cell membranes (Figs. 7, 8).
Scanning electron microscopy
In scanning electron microscopy of thick deparaffinized sections the interspace between the epithelial and mesenchymal cells often appeared wider than
in transmission electron microscopy. This was probably due to the shrinkage
caused by the paraffin technique. At the tips of the branching ureter-bud the
epithelial cells were connected with the mesenchymal cells by thin intercellular
filaments (Fig. 9). Some of these filaments seemed to originate from epithelial,
others from mesenchymal cells. The gap between the apposed tissues was also
bridged by thicker cellular processes (Fig. 9).
Cell contacts in developing kidney
701
Fig. 6. Electron micrograph of the tip of the ureter-bud. Ruthenium red is seen
between both epithelial (E) and mesenchymal (M) cells and in the epithelio-mesenchymal interspace (arrow). Poststaining with uranyl and lead, x 10000.
Figs. 7, 8. Electron micrographs of the epithelio-mesenchymal interface at the tip of
the ureter-bud. Mesenchymal cells above, epithelial cells below. At points of apparent
focal fusion of the cell membranes (arrows) ruthenium red does not seem to penetrate
the epithelio-mesenchymal interface. The sections poststained with uranyl and lead,
x 160000.
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E. LEHTONEN
Fig. 9. Scanning electron micrograph of the tip of the ureter-bud. The area shown at
high magnification is indicated with a rectangle in the inset. The epithelial (E) and
mesenchymal (M) cells are connected by thin intercellular filaments and thicker
cellular processes (arrows), x 9000, inset x 1300.
DISCUSSION
At the tips of the ureter-bud, where induction is known to take place, the
interspace between the epithelium and the mesenchyme was often only 15-20 nm,
and in places less than 10 nm. At certain sites, areas of apparent membrane
fusion were seen between the epithelial and mesenchymal cell membranes.
Previously, we have shown that transfilter induction of kidney tubules is correlated with cell contacts between the interacting tissues within the filter (Wartiovaara et ah 191 A; Lehtonen et al. 1975). The occurrence of zones of close contact
Cell contacts in developing kidney
703
between probably interacting epithelial and mesenchymal cells has also been
reported in the developing cat (Pannese, 1962) and rat (Kallenbach, 1971) tooth,
mouse liver (Rifkind, Chui & Epler, 1969), rat duodenal mucosa (Mathan,
Hermos & Trier, 1972) and rat submandibular gland (Cutler & Chaudhry,
1973). Contacts have also been shown in the early chick embryo between
primary mesenchymal cells and epithelial cells of both the epiblast and the
hypoblast (Trelstad, Hay & Revel, 1967; Hay, 1968; Revel, 1974).
Certain types of intercellular junctions, i.e. gap junctions, have been reported
to allow passage of different molecules, including nucleotides, with molecular
weights of up to 1000 (Loewenstein, 1972; Pitts, 1972; see also McNutt &
Weinstein, 1973; Sheridan, 1974; Staehelin, 1974). Freeze-etching techniques are
necessary to demonstrate gap junctions (Friend & Gilula, 1972), but this was not
feasible in this study because of the small size of the developing kidney. However, in addition to close intercellular contacts local apparent fusions of cell
membranes between epithelial and mesenchymal cells were seen.
Whether epithelio-mesenchymal cell contacts play a role in kidney tubule
induction is not yet known, but their existence in the inductively active areas
and their absence in inactive zones suggests that they are morphogenetically
significant. The finding also obviates the need to postulate long-range transmission of inductive signals.
The epithelio-mesenchymal interaction in some systems seems to be dependent on the presence of extracellular matrix materials. Bernfield, Banerjee &
Cohn (1972) have shown that morphogenesis of the mouse salivary gland
requires the presence of interfacial glycosaminoglycans. Also in the developing
mouse kidney electron-dense material staining with ruthenium red was seen in
the epithelio-mesenchymal gap. In the areas where induction takes place, however, only a thin layer of extracellular material was seen, and cell contacts were
found without visible extracellular material between the interacting cells. It
therefore seems that the induction of mouse kidney tubules, unlike that of
salivary epithelium, is independent of extracellular matrix materials.
The results presented conform with previous results of transfilter studies on
the correlation between induction of kidney tubules and the presence of cell
contacts within the filter. In the normal situation in vivo, cell contacts occur
during epithelio-mesenchymal interaction, especially in the areas where cell
aggregation takes place. This lends additional, if only circumstantial, support
to the hypothesis that direct cell-to-cell interaction is essential for induction of
kidney tubules in the mouse.
The technical assistance of Miss Pirkko Leikas and Mrs Anja Tuomi is acknowledged.
Supported by the Finnish Culture Foundation, the Paulo Foundation, and the National
Research Council for Medical Sciences, Finland.
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E. LEHTONEN
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{Received 5 June 1975, revised 15 August 1975)