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Distribution of polyanionic sites in the developing gonads and the dorsal mesentery of the chick embryo.

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THE ANATOMICAL RECORD 205321-329 (1983)
Distribution of Polyanionic Sites in the Developing Gonads and
the Dorsal Mesentery of the Chick Embryo
ELAINE DIDIER, PIERRE DIDIER, AND NOEL FARGEIX
Laboratoire de Biologie Animale (E.D.,
N.W, and Laboratoire de Zoologie
(ED.),UniuersitC de Clermont II, B P 45, 63170 Aubihre, France
ABSTRACT
The distribution of glycoconjugates was investigated in the
embryonic trunk mesoderm used as a substrate by migrating primordial germ
cells (PGCs) by means of ultrastructural cytochemistry. In both mesentery and
developing gonads polyanionic sites were abundant in epithelial and mesenchymal cell coats, basal laminae, and extracellular matrices (ECM). In the
latter, polyanions distributed on microfibrils and granules were associated
with collagen fibers, forming a n entangled network. No preferential association of this fibrillo-granular material with PGCs was observed, suggesting that
polyanions present in ECM likely act by promoting inflation of the extracellular spaces rather than by providing mechanical guides for the moving cells.
Beside its structural role in maintaining
the shape of the embryo, embryonic mesenchyme has morphogenetic functions either
by interacting with epithelia or by acting as
a substrate for migratory cells. Over the past
decade, a number of ultrastructural, cytochemical, and biochemical data have revealed that mesenchyme from a large variety
of embryonic organs is frequently characterized by a well-developed extracellular matrix
(ECM) mainly composed of collagen, glycosaminoglycans (GAG), and glycoconjugates
(proteoglycans and glycoproteins). In the
avian embryo such a matricial organization
has been described in several developmental
systems involving migrations of isolated or
clustered cells such as in cornea (Meier and
Hay, 1974; Trelstad et al., 1974; Toole, 1976,
1981); heart (Manasek, 1975; Orkin, 1978;
Markwald et al., 1979);sclerotome and axons
(Ebendal, 1977; Solursh et al., 1979a); myogenic cells (Jacob et al., 1979); neural crest
cells (Weston et al., 1978; Noden, 1978; Bolender et al., 1980; Le Douarin, 1980); and
primitive streak mesoderm (Solursh, 1976;
Sanders, 1979; Solursh et al., 1979b; Vanroelen et al., 1980). During morphogenesis and
differentiation one ECM has a strategic role
by interacting with the surface of migrating
cells (review in Hay, 1981).
During gonadal development, 'homing of
primordial germ cells (PGCs) to the early
genital ridges offers a model system for the
analysis of a great range of cel1:cell interac-
0 1983 ALAN R. LISS, INC.
tions (Nieuwkoop and Satusarya, 1979). Recently it was shown from ultrastructural
studies that the splanchnopleure of the gonadal area had a loose arrangement due to the
presence of wide interspaces between both
mesenchymal and epithelial cells. Such a cellular pattern was thought to be well suited
for splanchnopleure invasion by migrating
PGCs (Fargeix et al., 1981). Thus it was interesting to collect further information on
the ultrastructural organization and the cytochemical characteristics of the materials
present on the splanchnopleural cell surfaces
as well as in the extracellular compartment.
Both are thought to play a decisive role in
the preferential adhesivity involved in cell
migration (Rambourg, 1971;Mestres and Hinrichsen, 1974; Luft, 1976; Ebendal, 1977).
Standard procedures for the identification of
the glycoconjugate and collagen components
usually present in pericellular matrix have
been used and reveal the high polyanionic
content of the pathway followed by PGCs
moving towards the genital epithelia.
MATERIALS AND METHODS
Chick embryos were removed from fertile
eggs (Hubbard strain), incubated for 2-5
days, washed in Tyrode saline solution, and
staged (15-27) according to Hamburger and
Hamilton (1951). Whole trunks (stages 15Received October 7, 1981; accepted October 28, 1982
322
E. DIDIER, P. DIDIER. AND N. FARGEIX
23) or dissected gonadal areas (stages 24-27)
were fixed at 4°C unless otherwise specified.
All fixatives were prepared in 0.12 M cacodylate buffer a t pH 7.4. Specimens were immersed in glutaraldehyde (30 m i d , washed
three times in buffer, and then postfixed in
osmium tetroxide (1hr).
Routine fixation
Fourteen embryos were fixed in 3% glutaraldehyde, rinsed in buffer, and then postfixed
in 2% osmium tetroxide (1hr).
Glycoconjugate cytochemistry
Three procedures for identifying the glycoconjugates in electron microscopy (Rambourg, 1971; Schrevel, 1972; Trelstad et al.,
1974; Luft, 1976)were selected:
Cetylpyridinium chloride
Cetylpyridinium chloride (CPCL), which
selectively precipitates polyanionic molecules, may be used in order to improve the
ultrastructural demonstration of glycosubstances. Eight specimens were treated according to Markwald et al. (1979) with minor
modifications: tissues were first fixed in 3%
glutaraldehyde supplemented with 0.5%
CPCL at room temperature (30 min), carefully rinsed in buffer, and then osmicated as
described above.
coproteins. Treated sections were observed
without any subsequent counterstain. All observations were made with a Siemens Elmiskop 1A electron microscope.
Observations
At the level of the genital area, the
splanchnopleure mesoderm lining the coelomic cavity has differentiated into gonadal
anlages and the axial dorsal mesentery, respectively (Figs. 1,2);whereas the mesentery
is made up of a loosely arranged mesenchyme, limited on the left and right sides by
a single layer of cuboidal epithelial cells, the
gonads display three components: (1)a germinal epithelium (GE) composed of several
layers of elongated cells; (2) a subepithelial
dense mesenchyme, termed “stroma,” from
which the gonadal epithelioid cords arise
(Fargeix et al., 1981); and (3) a loosely arranged mesenchyme in the dorsal part of the
gonad which is in continuity with the mesentery (Fig. 1).In these tissues, many migratory (in mesenchyme) and postmigratory (in
epithelia) PGCs can be observed.
Routine fmation
The organization and the ultrastructural
characteristics of epithelial and mesenchyma1 cells in the genital area have been previously described from conventionally fixed
embryos (Fargeix et al., 1981). In the present
Ruthenium red
According to Luft’s procedure (Luft, 19711,
based upon the affinity of the polycationic
Figs. 1 and 2. Organization of the splanchnopleural
dye ruthenium red (RR)for the glycoconju- sheet
in the genital area at stage 23. GE, germinal
gates of cell surfaces and, ECMs, 18 embryos epithelium; LS, loose gonadal stroma; DS, dense gonadal
were immersed in 2.6% glutaraldehyde + stroma; M, dorsal mesentery; migrating (in mesen0.1% RR (30 min), rinsed in buffer and then chyme) and postmigrative (in epithelia) PGCs (arrows)
postfixed in 2% osmium tetroxide
0.075% are recognizable. Fig. 1, X250; Fig. 2, X540.
RR (1hr). After embedding in Epon 812 (PoFig. 3. Right gonad (stage 26). Interface between gerlysciences) thin sections from routinely fixed minal epithelium (GE) and stromal mesenchyme (S). Exand CPCL- or RR-treated specimens were tracellular materials are present as the epithelial basal
lamina (double arrows) and mesenchymal matrix (arcounterstained with uranyl acetate-lead cit- row).
~20,000.
rate.
+
Periodic acid-thiocarbohydrazide-silver
proteinate reaction
Fig. 4. Right gonad (stage 26). Interface betweeen GE
and stroma. Fibrillogranular material associated with
collagen fibrils (arrow) in the basal lamina (double arrows) and in a coated vesicle. ~40,000.
Some sections from seven routinely fixed
Fig. 5. Right gonad (stage 26). Stroma. High magniembryos were mounted on gold grids and
of the extracellular material; microfibrils and
then treated for the periodic acid-thiocarbo- fication
dense granules associated with bundles of collagen fihydrazide-silver proteinate reaction (PA-T- bers. ~56,000.
Ag) according to Thiery (1967). Incubations
Fig. 6. Left gonad (stage 26). Abundant extracellular
in thiocarbohydrazide (Eastman Kodak) were
prolonged for 48 or 72 hr, in order to attempt material around the stroma epithelioid cords. ~20,000.
to demonstrate some intra- and extracellular
Fig. 7. Dorsal mesentery (stage 23). Dense CPCL-prepolyholosides, glycosaminoglycans, and gly- cipitated material and striated collagen. x 120,000.
POLYANIONS IN DEVELOPING GONADS
323
324
E. DIDIER, P. DIDIER, AND N. FARGEIX
study, attention has been focused on the extracellular materials present in these tissues. They are specifically developed as basal
laminae underlying the epithelia and as an
organized ECM, interspersed among the
mesenchymal cells (Figs. 3-6).
Basal laminae in early chick embryos display the typical structure described by Sanders (1979); they consist of a fibrillogranular
lamina densa (20 nm thick) associated with
the plasma membrane of the epithelial cells
(through a 20- to 40-nm lamina internu) and
with fibrillar materials present at the epithelia-mesenchymal interface (Fig. 4). In both
loose and dense mesenchymal areas, large
amounts of extracellular material can be observed, around single mesenchymal cells
(Fig. 3) and around those which aggregate to
form the stromal cords (Fig. 6). This material
is composed of fine fibrils and electron-dense
granules associated with bundles of aligned
collagen fibers (Figs. 4, 5). The presence of a
similar material in and near coated vesicles
suggests that ECM is synthesized and then
exported by the epithelial and mesenchymal
cells (Fig. 4).
Cetylpyridinium chloride fixation
The fibrillogranular material described
above can be precipitated by CPCL; when
precipitated this material appears highly
contrasted, revealing its polyanionic nature.
In the mesenchymal ECM of the dorsal mesentery and gonads, microfibrils and densely
clustered granules are seen closely associated with a well-developed collagen network
(Figs. 7, 8) or as dense patches between adjacent mesenchymal cells (Fig. 10).At the level
of the epithelial basal laminae and in the
coated vesicles, CPCL-precipitated materials
are composed of minute granules with variable electron densities (Fig. 9). In the mesenchyme, cell outlines are decorated by flocculent dark deposits spaced along the plasma
membrane (Fig. 10)or arranged as a continuous cell coat (Fig. 11).A similar cell coat (30
nm thick) is demonstrated by CPCL on the
outer leaflet of the plasma membrane of gonadal and mesenterial cells (Figs. 12, 13) and
migratory and postmigratory PGCs (Fig. 13).
Ruthenium red fixation
The electron-dense deposits resulting from
RR binding to polyanion-osmium complexes
are very abundant in the genital area and
highlight ECM and cell outlines (Fig. 14).
The apical surfaces of mesenterial and gona-
dal epithelia appear to be decorated by finely
grained 30- to 50-nm thick cell coats (Figs.
14, 15). On the lateral faces of the epithelial
cells, RR particles are irregularly spaced
along the plasma membrane and accumulate
in the intercellular spaces (Fig. 17). RR-positive materials are very abundant in the mesentery as a heavily stained network including the epithelial basal lamina, the cell
coat of the mesenchymal and migrating germ
cells, and the wide ECM interspersed among
cells (Fig. 14). In the gonads, the distribution
and electron-density of RR particles are
rather different since spaced 20- to 40-nmdiameter dense granules can be observed associated with the fibrillar material present
at the epithelia-mesenchymal interface (Fig.
16) and the ECM surrounding the stroma
cords.
Periodic acid-thiocarbohydrazide-silver
proteinate reaction
As previously reported (Fargeix et al.,
19811, a-glycogen particles can be revealed in
the PGCs when grids are incubated in thiocarbohydrazide for 24 hr. In the present series, incubations were prolonged for an
additional 24 or 48 hr in order to attempt to
demonstrate GAG or proteoglycans and glycoproteins; no intra- or extracellular material other than glycogen (Fig. 19) could be
detected with this technique; cell coats as
well as epithelial basement membranes and
ECM (which had been shown to react with
CPCL or RR positively) failed to be stained
after the PA-T-Agreaction (Figs. 18,19).
Fig. 8. Left gonad (stage 23). Loose mesenchyme.
CPCL-precipitated extracellular material. x 60,000.
Fig. 9. Dorsal mesentery (stage 24). Aspect of the
epithelial basal lamina (double arrow) and of the me*
enchymal matrix (arrow) after CPCL treatment.
~60,000.
Fig. 10. Left gonad (stage 23). Stroma. CPCL has precipitated polyanions as a discontinuous cellcoat and
dense matricial material. ~20,000.
Fig. 11. Left gonad (stage 23). Stroma. CPCL-visualized cell-coat on the surface of a mesenchymal cell.
~40,000.
Fig. 12. Left gonad (stage 19). The apical membrane
of the GE is coated by CPCL-stained polyanionic materials. x 30,000.
Fig. 13. Left gonad (stage 19). Postmigrative germ cell
having penetrated the GE. x 12,000. Inset: CPCL-precipitated cell-coat on both germ cell (PGC)and epithelial
cell (GE). ~ 3 0 , 0 0 0 .
POLYANIONS I N DEVELOPING GONADS
325
326
E. DIDIER, P. DIDIER, AND N. FARGEIX
Fig. 14. Dorsal mesentery (stage 23). Abundant RRstained polyanionic materials associated with the plasma
membranes of the epithelial (E) and mesenchymal cells
(MI, as well as with the surface of a migrating germ cell
(PGC).
Heavy deposits in the well-developed extracellular matrix (ECM). ~ 4 , 5 0 0 .
POLYANIONS IN DEVELOPING GONADS
Fig. 15. Left gonad (stage 20). RR-stained cell coat of
the apical membrane of the villous GE. X40,OOO.
Fig. 17. Left gonad (stage 24). RR particles in the
epithelial interspaces. X 20,000.
327
Fig. 18. Left gonad (stage 15). PA-T-Ag reaction. No
cellcoat is visualized on the apical membrane of an
epithelial cell (GE). Compare with Figure 15. ~36,000.
lar matrix are PA-T-Agnegative. Glycogen particles have
been stained by the PA-T-Ag in the cytoplasm of a germ
cell (PGC). X32,OOO.
328
E. DIDIER, P. DIDIER, AND N. FARGEIX
DISCUSSION
From these observations, it is seen that the
differentiating splanchnopleural cells of the
genital area display abundant surface and
extracellular materials during the period
considered.
The polyanionic compounds coating the epithelial and mesenchymal cells cannot be detected after conventional fixation. Their
demonstration requires the use of polycationic dyes (RR) or a n organic reagent
(CPCL). This cell coat is structurally similar
to those described previously in a variety of
celI types (Rambourg, 1971; Schrevel, 1972;
Luft, 1976); it adheres closely to the outer
leaflet of the plasma membrane, either as a
dense homogenous layer (after CPCL) or a
finely grained coat (with RR). Some variability in coat thickness (which ranges from
30nm to 50 nm) and in the density of the RR
particles occurs among adjacent cells as well
as on the surface of the same cell. Thus in
GE cells the apical (luminal) membrane displays a more heavily RR-contrasted coat than
the lateral and basal membranes. It is unclear whether such differences are due to difficulties in dye penetration or to some
variability in the nature and distribution of
polyanions over the cell surface. Differences
in the appearance of RR-stainable materials
could reflect a differential distribution of glycoconjugates on the surface of polarized epithelial cells (Spicer et al., 1979).
As in other embryonic mesenchyme, a wide
extracellular compartment is present in the
axial loose mesenchyme. Structurally this
ECM is composed of randomly orientated collagen fibers and fibrillogranular polyanionic
material, forming a n entangled network. The
possibilities of hydration of ECM due to the
electrophysiologic properties of polyanions
are thought to create a suitable substrate for
migrating cells (Toole, 1976; 1981). As no
preferential association of PGCs with the
ECM fibrillogranular material has been
noted, we suggest that collagenous and noncollagenous compounds do not act as actual
guides for migrating cells; it seems more
likely that the chemical composition and
structural characteristics of the ECM allow
PGCs that have ameboid properties to move
easily through the wide interspaces between
the splanchnopleural cells.
From the observations presented, it is not
possible to know the exact nature of the macromolecules present in the ECM and on the
cell surface since CPCL and RR bind nonspe-
cifically to the carboxyl groups of both acidic
GAG and dicarboxylic amino acids as well as
to sulfate or phosphate groups, present in
sulfated GAG and glycolipids, respectively.
However, according to Luft (1976) a strong
reactivity with RR would be indicative of
sialoglycoproteins and high-charge-density
GAG. These carbohydrate-rich molecules
usually do not react with the periodic acidSchiff (Rambourg, 1971) which explains the
inability of ECM to be stained with the PAT-Ag method. Furthermore, cytochemical
data obtained in light-microscopy indicate
among other substances the presence of acidic
and sulfated GAG in the ECM (Didier and
Sean Kim Eang, unpublished).
In the chick embryo, glycans and various
classes of glycoproteins (such as fibronectin
and endogenous lectins) have been shown to
play both structural and morphogenetic roles
in several developmental systems (Trelstad
et al., 1974; Toole, 1976; Ceri et al., 1979;
Critchley et al., 1979; Mayer et al., 1979;
Newgreen and Thiery, 1980; Sanders, 1980).
For instance, it has been suggested that hyaluronic acid provides a n appropriate substrate for cell migration by promoting ECM
inflation (Toole, 1976; Solursh et al., 1979a,
b). Fibronectin seems to play a decisive role
in preferential adhesivity (Le Douarin, 1980;
Newgreen and Thiery, 1980; Sanders, 1980).
Similarly, during gonadal development, synthesis and exportation of glycoconjugates in
the extracellular environment must be of importance since a concomitant inhibition in
PGC homing and a drastic reduction in RRstainable materials can be observed in pesticide-treated quail embryos (Bruel and David,
1981). As yet the molecular mechanisms involved in migration are not thoroughly
understood. According to the model proposed
by Shur (1977), surface transferases might
mediate interactions between the carbohydrate substrates and the surface oligosaccharide side chains of migrating cells. In PW
homing toward the developing gonads, possible interactions between the glycanic compounds of the mesodermal tissues and the
developmentally regulated galactosyl, glucosyl, and mannosyl residues present in migrating PGCs (Fargeix et al., 1980)remain to
be experimentally elucidated.
ACKNOWLEDGMENTS
We wish to thank Prof. J. Schrevel for
friendly advice and critical reading of the
manuscript.
POLYANIONS IN DEVELOPING GONADS
329
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