/. Embryol. exp. Morph. Vol. 63, pp. 233-242, 1981
233
Printed in Great Britain © Company of Biologists Limited 1981
Differentiation within the gonads of
Drosophila revealed by immunofluorescence
By DANNY L. BROWER, 1 R. J. SMITH 1
AND MICHAEL WILCOX 1
From the MRC Laboratory of Molecular Biology, Cambridge
SUMMARY
The antibody produced by the hybrid cell line DA.1B6 binds to the diploid epithelial
cells of Drosophila. In this paper, we describe the immunofluorescence-binding pattern of
the antibody to the gonads. A bright sheath of fluorescence extends from the seminal
vesicle onto the most proximal part of the adult testis. The only other significant binding
to the organ is to the apical cells of the germinal proliferation centre, which fluoresce
brightly in testes from adults and from third instar larvae. In the adult ovary, there is strong
binding to the cells of the follicular epithelium, although this binding is reduced in the
latter stages of follicle development. Soon after the formation of a follicle, a pair of epithelial
cells at each pole of the follicle can be seen to fluoresce much more brightly than the other
cells. This early differentiation is reflected in the morphogenetic behaviour of these polar
cells as the follicle develops. The anterior pair are among the 'border cells' which migrate
between the nurse cells to the anterior pole of the developing oocyte; and, when the follicular
epithelium around the oocyte becomes columnar, the posterior pair of cells do not elongate
as much as the surrounding cells.
INTRODUCTION
In an earlier paper we described a hybrid cell line, D A . 1B6, which produces
a monoclonal antibody that binds to the imaginal discs of Drosophila (Brower,
Smith & Wilcox, 1980). An immunofluorescence survey of the pattern of
binding of the antibody to tissues from different developmental stages revealed
that the antibody is generally specific for the diploid epithelia of Drosophila.
Epithelial cells from all three germ layers are recognized, but the binding
activity is lost when an epithelium becomes polyploid. This loss can be gradual,
for example, some polyploid larval cells do not lose the antigen completely
until well into larval life. Thus the disappearance of the antigen appears to be
a consequence of, rather than a prerequisite for, polyploidy.
In the earlier report, we gave a brief description of the pattern of antibody
binding to the gonads of Drosophila. This preliminary work yielded some
unexpected results. For example, previously undetected differentiations were
observed within the epithelia of the ovarian follicles. And, a very small patch
1
Authors' address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge
CB2 2QH, U.K.
234
D. L. BROWER, R. J. SMITH AND M. WILCOX
of cells at the distal tip of the testis, as opposed to a complete epithelium, was
observed to bind the antibody. This paper presents the results of a more
detailed analysis of these observations.
MATERIALS AND METHODS
The production of the antibody-secreting hybrid mouse cell line, DA.1B6,
has been described previously (Brower et aJ. 1980). Tissues dissected from
larvae and adults of Drosophila melanogaster (Barton wild-type strain) were
used for all the antibody-binding assays, with one exception: adult testes from
flies carrying the white mutation {w65a2S) were also examined as these organs
lack the autofluorescent pigment found in the outer sheath of the wild-type
testis. All antibody incubations and washes were carried out in RPMI (Gibco)
cell-culture medium. The tissues were incubated in DA.1B6 antibody for
45 min at 37 °C, washed three times at 4 °C, and then incubated in fluoresceinconjugated rabbit anti-mouse ]gG (Miles-Yeda) for 30 min at 37 °C. After
thorough washing, the tissues were either examined directly or, in some cases,
first fixed in 4 % formaldehyde/Z>0S0/?/j//tf Ringer for 30 min at 4 °C. This
latter treatment did not alter the fluorescence patterns observed but permitted
flattening of tissues without disruption of their integrity. Fluorescence microscopy was carried out with a Zeiss Universal microscope using epi- and transillumination.
For controls, all tissues were treated as above except that DA. 1B6 antibody
was omitted from the first incubation medium. All of the described fluorescence
of the testes and ovaries was shown to be dependent on the presence of DA. 1B6
antibody, except for the autofluorescence of the pigment layer of the adult
testis and the yolk in developing oocytes.
Fig. 1. Adult testis. The antibody binds to the seminal vesicle (sv) and to the
beginning of the first gyre of the testis (arrow). There is some autofluorescence
from the pigment of the outer layer of the testis. The bright patch at the apical end
of the organ is barely visible at this magnification (arrowhead), x 70.
Fig. 2. Proximal area of an adult testis. Thefluorescenceof the seminal vesicle (sv)
continues into the testis, but ends in the first gyre of the organ (arrow), x 275.
Figs. 3-5. Distal ends of adult testes. In both the side (Fig. 3) and end (Figs. 4, 5)
view of the tip of the testis, antibody binding is evident only to the apical cells
of the germinal proliferation centre, x 950.
Figs. 6 & 7. Larval testes. Side (Fig. 6) and end (Fig. 7) views of the antibody binding
to the apical cells of testes from third instar larvae, x 950.
Fig. 8. Larval testis. Opposite the apical cells (arrow) there is a faint fluorescent
patch (arrowhead) on the third instar testis. The organ is surrounded by fat body,
which accumulates some of the fluorescent antibody nonspecifically. x 200.
Differentiation in Drosophila gonads
235
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D. L. BROWER, R. J. SMITH AND M. WILCOX
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Fig. 9. Diagram of adult ovary, (a) 10-20 ovarioles are tightly packed into each
ovary. Two ovarioles are pulled away from the ovary here, and stretched to reveal
the individual egg chambers, or follicles, (b) A single ovariole, with the numbers
denoting the various stages of oogenesis. Thefluorescenceof the follicle epithelium
starts to fade at about stage 7, after these cells become polyploid. The polar cells
(p) are discernible by their exceptionally brightfluorescenceby stage 2. The anterior
pair of polar cells are among the border cells which migrate to the anterior end of
the oocyte at stage 9. The polar cells remain fluorescent until stage 11, when the
oocyte fills more than half of the egg chamber (not shown). ES, epithelial sheath;
G, germarium; TF, terminal filament; Ooc, oocyte (after King, 1970).
RESULTS
Testis: (For a detailed description of the structure and development of the
testis, see Miller, 1965; Bodenstein, 1965 and Lindsley & Tokuyasu, 1980).
Each adult testis consists of a coiled sperm tube about 2 mm long and 0-1 mm
Differentiation in Drosophila gonads
237
wide. At its distal end lies the germinal proliferation centre, comprising a
'hub' of 8-16 apical cells, to which adhere the spermatogonial stem cells and
cyst progenitor cells (Hardy, Tokuyasu, Lindsley & Garavito, 1979). Moving
proximally, one then finds a sequence of developmental stages of the germ
cells, with mature spermatozoa present where the testis merges into the seminal
vesicle.
In general, there are only very faint specks of fluorescence associated with
the wall of the adult testis. Proximally, however, there is considerable antibody
binding to a sheath of cells surrounding the testis, but this fluorescence is
restricted to only a part of the first gyre of the coiled organ (Figs. 1, 2). The
fluorescent sheath also appears to extend onto the seminal vesicle, which is
derived from the genital imaginal disc (Bodenstein, 1965). This distribution
suggests that the antibody is binding to the relatively thick epithelium adjacent
to the lumen of the sperm tube (Miller, 1965). This hypothesis is supported
by our observations of antibody fluorescence in sections of the gonad (not
shown).
At the distal tip of the testis there is a small patch of very bright fluorescence
(Fig. 3). When viewed end-on, we are typically able to count at least eight
fluorescent cells and sometimes as many as 15 within the patch (Figs. 4, 5).
This observation, together with the general morphology of the cells, leads us
to conclude that the antibody is binding to the apical cells of the germinal
proliferation centre (cf. Hardy et al. 1979).
In the testes of late third instar larvae, we see a similar bright hub of
fluorescence, again indicating binding to the apical cells (Figs. 6, 7). The only
other antibody binding we have detected is seen as a very faint web'like
fluorescence on the surface of the spherical organ opposite the apical cells
(Fig. 8).
Ovary: (For a detailed discussion of the Drosophila ovary, see King, 1970
and Mahowald & Kambysellis, 1980). Each ovary of an adult fly contains
10-20 parallel egg tubes, or ovarioles (Fig. 9). Distally, the terminal filament
of each ovariole attaches to the germarium which contains the dividing germ
cells and the precursor cells of the follicle epithelium (Johnson & King, 1972).
The egg chambers, or follicles, bud off from the basal end of the germarium
and pass down the ovariole as they develop. Each follicle consists of an oocyte
and 15 nurse cells surrounded by an epithelium of follicle cells. Early in
oogenesis, the follicular epithelial cells divide as the follicle enlarges, but they
eventually cease division (King & Vanoucek, 1960) and become polyploid
(Mahowald, Caulton, Edwards & Floyd, 1979). Before the secretion of the
vitelline membrane around the developing oocyte, there is a general migration
of follicle cells to produce a dense columnar epithelium over the oocyte itself
and a thin squamous epithelium over the nurse cells (King & Vanoucek, I960).
Also during this time, a group of 6-10 follicle cells (the 'border' cells) leaves
the anterior end of the epithelium and migrates between the nurse cells, to the
238
D. L. BROWER, R. J. SMITH AND M. WILCOX
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Differentiation in Drosophila gonads
239
anterior pole of the oocyte. Later, these 'border' cells will make the micropylar
apparatus at the anterior end of the egg. Finally, the follicle and nurse cells
degenerate, leaving the mature egg.
The overall pattern of binding of DA. 1B6 antibody to the adult ovary has
been described (Brower et al. 1980). Briefly, the antibody binds to the cells
of the follicular epithelium, including those of the stalks which connect the
follicles (Figs. 10, 11). Typically, the fluorescence becomes greatly reduced at
about the time the follicle cells become polyploid (stage 7). In preparations
where the outer epithelial sheath has become separated from the follicles, it
shows faint immunofluorescence (Fig. 12). There is no detectable antibody
binding to the terminal filament, the germ cells of the germarium or the developing
oocyte and nurse cells.
From very early in oogenesis, a pair of follicle cells at each pole of the egg
chamber shows especially high levels of binding (Figs. 13, 14). These pairs of
cells remain brightly fluorescent when the fluorescence of the other epithelial
cells diminishes during vitello genesis (Fig. 15). The anterior pair of these
* polar' cells are among the group of border cells that leave the follicular
epithelium at stage 9 and migrate between the nurse cells to the oocyte (Figs.
16, 17). Late in oogenesis, the posterior pair of polar cells can be distinguished
Fig. 10. Adult ovary. The cells of the follicular epithelium are strongly fluorescent.
One pair of polar cells can be distinguished by their especially bright fluorescence
(arrow). x260.
Figs. 11 & 12. Adult ovaries. These micrographs show dissected ovarioles that
have been stretched, separating the individual follicles. The arrowheads point to
fluorescence on the stalk cells between the follicles (Fig. 11) and the weak
fluorescence on the epithelial sheath (Fig. 12). There is no fluorescence on
the germ cells in the germarium (g), and thefluorescenceof the follicles is greatly
reduced after stage 7 (arrows). Fig. 11, x 145; Fig. 12, x 180.
Figs. 13 & 14. Adult ovary. These two micrographs are of the same ovariole taken
at different focal planes, and show that the anterior (Fig. 13) and posterior (Fig. 14)
polar cells can be distinguished in a follicle which has only recently separated from
the germarium (g). x 260.
Fig. 15. Adult ovary. This (stage 7) follicle illustrates the especially bright fluorescence
of both the anterior and posterior polar cells, x 260.
Fig. 16 & 17. Adult ovaries. The micrographs show stages in the migration of the
anterior polar cells, with the other border cells, out of the follicular epithelium.
In Fig. 16, the cells have just begun to migrate between the nurse cells. Fig. 17
shows a (stage 10) follicle in which the two brightly fluorescent polar cells can
be seen lying next to the oocyte. Some of the other, weakly fluorescent border
cells can be seen adjacent to the polar cells. Fig. 16, x 165; Fig. 17, x 400.
Fig. 18. Adult ovary. Following columnarization of the follicular epithelium,
the posterior pair of polar cells are only about two-thirds as tall as the rest of
the epithelial cells. x415.
Fig. 19. Larval ovary. In ovaries of late third instar larvae, there is a bright fluorescent cap opposite a fainter fluorescent band. These areas are sometimes seen
to be connected by a thin strand of fluorescent cells (arrow), x 265.
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D. L. BROWER, R. J. SMITH AND M. WILCOX
by their size; after the follicle cells surrounding the oocyte become columnar,
the polar cells are only about two-thirds as tall as their neighbours (Fig. 18).
Eventually, at about stage 11, the fluorescence of both pairs of polar cells
fades away.
In the ovaries of late third instar larvae, there is a bright cap of antibodybinding cells at one end and a band of less strongly fluorescent cells across the
other. These areas are sometimes seen to be connected by another strip of
fluorescent cells (Fig. 19).
DISCUSSION
DA.1B6 antibody is generally specific for the diploid epithelial cells of
Drosophila. This specificity is well illustrated in the pattern of binding to the
cells of the ovarioles. For example, the strong fluorescence of the follicular
epithelium is greatly reduced when these cells become polyploid during vitellogenesis. And, the non-epithelial cells, such as the germ cells, show no antibody
binding. Of particular interest, however, are the small groups of cells, in both
ovary and testis, that show especially bright fluorescence with the antibody.
One of these bright patches is seen at the distal tip of the adult testis (a
similar patch occurs at one end of the larval testis). Hardy et al. (1979) have
given a detailed description of the germinal proliferation centre of the adult
testis. This consists of a group of 8-16 apical cells surrounded by 5-9 stem
cells and 9-17 cyst progenitor cells. Based on the compact morphology of the
bright patch and the number of cells that stain, we conclude that the antibody
is binding only to the apical cells.
Hardy et al. suggest that the hub of apical cells 'is the centre of organization
of the entire contents of the testis, and its function is to support the stem cells
and the cyst progenitor cells for the orderly generation of spermatogenic
cysts'. Others have suggested that similar cells in other insects might control
the polarity of spermatogenesis by inhibiting the differentiation of the nearby
germ cells (reviewed by Roosen-Runge, 1977). Recent experiments have indicated such a function for the 'distal tip cells' in the gonads of the nematode,
Caenorhabditis elegans (Kimble & White, 1981). In the Drosophila testis, the
postulated inhibition could be mediated by direct communication between
neighbouring cells; following each division of the stem cells and cyst progenitor
cells, one set of daughters remains in direct contact with the apical hub while
the other proceeds with the processes of gametic differentiation. The apical
cells apparently do not undergo mitosis (Hardy et al. 1979).
In the adult ovary, there is a pair of brightly staining cells at both the anterior
and posterior poles of each follicle (Fig. 9). These polar cells are more fluorescent
than their neighbours at least as early as stage 2, shortly after the follicle has
separated from the germarium. This early differentiation is also indicated by
the observation that early in oogenesis, cells at each pole of the follicle show
little or no mitotic activity, in contrast to those of the rest of the follicular
Differentiation in Drosophila gonads
241
epithelium (Calvez, 1980). In addition, there is some ultrastructural evidence
for differentiation among the follicle cells in the proximal part of the germarium
(Koch & King, 1969).
We can only speculate on the function of the polar cells. It is possible that
the differential antibody binding simply reflects their involvement in the synthesis of specialized egg structures. For example, the anterior polar cells may
somehow control the migration of the border cells and the subsequent synthesis
of the micropyle by these cells (King, 1970). Similarly, the posterior pair may
be involved in the formation of the posterior protuberance of the chorion
(King & Koch, 1963). Such a mundane hypothesis fails, however, to explain
why the polar cells should differentiate so early in the developing egg chamber,
at a time when the other follicle cells, some of which will also make specialized
structures such as the dorsal appendages, are rapidly dividing.
A more intriguing hypothesis derives by analogy from the proposed role
of the brightly fluorescent apical cells of the testis. Thus, the pairs of polar
cells may be involved in the overall control of the polarity of the developing
follicle. This hypothesis is consistent with the early differentiation of the cells,
and with the events surrounding the migration of the anterior pair with the
border cells; the migration begins as the epithelium over the nurse cells is
becoming thin and relatively inactive, and, soon after the border cells reach
the oocyte, the columnar follicle cells migrate over its anterior surface to
surround them.
We would like to thank Judith Kimble for helpful discussions during the course of this
work, and Judith, Robert King, and Peter Lawrence for criticism of the manuscript. D.L.B.
was a fellow of the American Cancer Society.
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(Received 22 August 1980, revised 2 December 1980)