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.

In an earlier paper we described a hybrid cell line, DA. 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 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.

The production of the antibody-secreting hybrid mouse cell line, DA.1B6, has been described previously (Brower et al. 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 (w65a25) 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 fluorescein-conjugated rabbit anti-mouse IgG (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/ Drosophila 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.

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 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).

Fig. 1

Adult testis. The antibody binds to the seminal vesicle (sv) and to the beginning of the first gyre of the testis Differentiation in Drosophila gonads (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), × 70.

Fig. 1

Adult testis. The antibody binds to the seminal vesicle (sv) and to the beginning of the first gyre of the testis Differentiation in Drosophila gonads (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), × 70.

Fig. 2

Proximal area of an adult testis. The fluorescence of the seminal vesicle (sv) continues into the testis, but ends in the first gyre of the organ (arrow), × 275.

Fig. 2

Proximal area of an adult testis. The fluorescence of the seminal vesicle (sv) continues into the testis, but ends in the first gyre of the organ (arrow), × 275.

Fig. 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, × 950.

Fig. 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, × 950.

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).

Fig. 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, × 950.

Fig. 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, × 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. × 200.

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. × 200.

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, 1960). 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 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.

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. The fluorescence of the follicle epithelium starts to fade at about stage 7, after these cells become polyploid. The polar cells (p) are discernible by their exceptionally bright fluorescence by 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).

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. The fluorescence of the follicle epithelium starts to fade at about stage 7, after these cells become polyploid. The polar cells (p) are discernible by their exceptionally bright fluorescence by 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).

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.

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), × 260.

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), × 260.

Fig. 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 the fluorescence of the follicles is greatly reduced after stage 7 (arrows). Fig. 11, × 145; Fig. 12, × 180.

Fig. 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 the fluorescence of the follicles is greatly reduced after stage 7 (arrows). Fig. 11, × 145; Fig. 12, × 180.

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 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.

Fig. 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). × 260.

Fig. 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). × 260.

Fig. 15

Adult ovary. This (stage 7) follicle illustrates the especially bright fluorescence of both the anterior and posterior polar cells, × 260.

Fig. 15

Adult ovary. This (stage 7) follicle illustrates the especially bright fluorescence of both the anterior and posterior polar cells, × 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, × 165; Fig. 17, × 400.

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, × 165; Fig. 17, × 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. × 415.

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. × 415.

In the ovaries of late third instar larvae, there is a bright cap of antibody-binding 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).

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). × 265.

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). × 265.

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 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.

Bodenstein
,
D.
(
1965
).
The postembryonic development of Drosophila
.
In Biology of Drosophila
(ed.
M.
Demerec
).
New York
:
Hafner
.
Brower
,
D. L.
,
Smith
,
R. J.
&
Wilcox
,
M.
(
1980
).
A monoclonal antibody specific for diploid epithelial cells in Drosophila
.
Nature, Lond
.
285
,
403
405
.
Calvez
,
C.
(
1980
).
Reduced mitotic activity in anterior and posterior follicle cells of the egg-chamber of Drosophila melanogaster
.
Dros. Inform. Serv
.
55
,
22
23
.
Hardy
,
R. W.
,
Tokuyasu
,
K. T.
,
Lindsley
,
D. L.
&
Garavito
,
M.
(
1979
).
The germinal proliferation centre in the testis of Drosophila melanogaster
.
J. Ultrastruct. Res
.
69
,
180
190
.
Johnson
,
J. H.
&
King
,
R. C.
(
1972
).
Studies on fes, a mutation affecting cystocyte cytokinesis, in Drosophila melanogaster
.
Biol. Bull. mar. biol. Lab., Woods Hole
143
,
525
547
.
Kimble
,
J. E.
&
White
,
J. G.
(
1981
).
On the control of germ cell development in Caeno-rhabditis elegans. Devl Biol
. (In the Press.)
King
,
R. C.
(
1970
).
Ovarian Development in Drosophila melanogaster
.
New York
:
Academic Press
.
King
,
R. C.
&
Koch
,
E. A.
(
1963
).
Studies on the ovarian follicle cells of Drosophila
.
Q. JI microsc. Sci
.
104
,
297
320
.
King
,
R. C.
&
Vanoucek
,
K. G.
(
1960
).
Oogenesis in adult Drosophila melanogaster. X. Studies on the behavior of the follicle cells
.
Growth
25
,
333
338
.
Koch
,
E. A.
&
King
,
R. C.
(
1969
).
Further studies on the ring canal system of the ovarian cystocytes of Drosophila melanogaster
.
Z. Zellforsch. mikrosk. Anat
.
102
,
129
152
.
Lindsley
,
D. L.
&
Tokuyasu
,
K. T.
(
1980
).
Spermatogenesis
.
In The Genetics and Biology of Drosophila, vol. 2d
(ed.
M.
Ashburner
&
T. R. F.
Wright
).
London
:
Academic Press
.
Mahowald
,
A. P.
,
Caulton
,
J. H.
,
Edwards
,
M. K.
&
Floyd
,
A. D.
(
1979
).
Loss of centrioles and polyploidization in follicle cells of Drosophila melanogaster
.
Expl Cell Res
.
118
,
404
410
.
Mahowald
,
A. P.
&
Kambysellis
,
M. P.
(
1980
).
Oogenesis
.
In The Genetics and Biology of Drosophila, vol. 2d
(ed.
M.
Ashburner
&
T. R. F.
Wright
).
London
:
Academic Press
.
Miller
,
A.
(
1965
).
The internal anatomy and histology of the imago of Drosophila melanogaster
.
In Biology of Drosophila
(ed.
M.
Demerec
).
New York
:
Hafner
.
Roosen-Runge
,
E. C.
(
1977
).
The Process of Spermatogenesis in Animals
.
London
:
University Press
.