Cell Tiss. Res. 186, 413-422 (1978) Cell and Tissue Research �9 by Springer-Verlag 1978

Intercellular Bridges in Ovarian Follicle Cells of Drosophila melanogaster

Franco Giorgi

Institute of Histology and Embryology, University of Pisa, Pisa, Italy

Summary. Intercellular bridges have been detected in ovarian follicle cells of Drosophila melanogaster. These bridges occur widely between follicle cells of previtellogenic chambers, while, in vitellogenic chambers, they become restricted to the columnar follicle cells. Usually, only one bridge is detectable between adjacent follicle cells, but a single cell may form two cytoplasmic continuities.

The fine structure of the intercellular bridges is similar to that previously described in the development of Drosophila. The bridge wall consists of two layers of which the more external is more electron dense and thinner than the inner one.

The role played by the intercellular bridges in the determination of a synchronous differentiation of the linked follicle cells is discussed in relation to the known behaviour of these cells in the secretion of the egg covering precursors.

Key words: Oogenesis - Drosophila - Intercellular bridges - Synchronous development.

Introduction

Follicle cells in ovarian chambers of Drosophila melanogaster are cells of mesodermal origin which come to cover a 16-cell cluster of cystocytes during their migration through the germarium of the ovariole. As soon as the first egg chamber leaves the germarium, a layer of approximately eighty cells, all being more or less of the same size, surrounds a 16-cell cluster (King and Vanoucek, 1960).

At stage 6 - the staging characteristics are those worked out by Cummings and King (1969) - a maximum number of about one thousand and two hundred cells is reached. In following stages cell divisions cease in follicle cells and further development of the egg chamber is accomplished by stretching of the follicle layer.

Send offprint requests to: Dr. Franco Giorgi, Istituto di Istologia ed Embriologia, Via A. Volta 4,1-56100 Pisa, Italy

0302-766X/78/0186/0413/$2.00

414 F. Giorgi

W h e n vitel logenesis commences - stage 8 - the follicle cells become s t ructura l ly var ied: the follicle cells over lying the oocyte become columnar , while those over lying the nurse cells become ext remely thin (King, 1970).

U l t r a s t ruc tu ra l as well as a u t o r a d i o g r a p h i c evidence f rom several invest igat ions have so far ind ica ted tha t only co lumnar follicle cells are involved in the synthesis o f vitell ine m e m b r a n e and endochor ion ic precursors ( Q u a t t r o p a n i and Ander son , 1969; King, 1970; Cummings , Brown and King, 1971; Mahowa ld , 1972). I t has also been shown tha t bo th vitelline m e m b r a n e and endochor ion ic precursors are synthesized by the co lumnar follicle cells in a ra ther synchronized manner , all a r o u n d the deve loping oocyte (Giorgi , 1977).

The present paper repor ts on the occurrence o f intercel lular br idges within the follicle cell layer and a t t empts to corre la te these f indings with the behav iour exhib i ted by these cells dur ing ovar ian development .

Materials and Methods

Drosophila melanogaster were reared on standard Drosophila food and kept at a constant temperature of 25 ~ C with continuous illumination.

Ovaries were dissected from 2-3 day old flies while still immersed in the fixative. The ovaries were hence transferred to fresh fixative and kept there for 2-3 h at 4 ~ C. The fixative consisted of a mixture of 5 % glutaraldehyde and 5 % formaldehyde in 0.1 M cacodylate buffer at pH 7.2. Following a prolonged wash in the buffer, the ovaries were post-fixed in 1% osmium tetroxide in cacodylate buffer at pH 7.2 for 2h at 4~

A few ovaries were fixed for the same length of time in 1% osmium tetroxide without prefixation in aldehydes. After post-fixation in osmium, the ovaries were dehydrated in a graded series of alcohols, transferred to propylene oxide and finally embedded in Epon-Araldite mixture.

Polymerized blocks of Epon-Araldite mixture containing the separated ovarian chambers were sectioned using an LKB-Huxley ultramicrotome and thin sections were mounted on formvar coated copper grids. The sections were then stained with uranyl acetate and lead citrate and observed in a Elmiskop 101 electron microscope operating at 80 Kv.

Results

Each ovar i an chambe r in Drosophila melanogaster is composed o f three different types o f cells all o f which are clearly shown in F igure 1. These cells are c o m m o n l y referred to as nurse cells, follicle cells and oocyte. The nurse cells, which occupy the largest a rea o f the chambe r in previ tel logenic stages, can be identif ied by the varying m o r p h o l o g y assumed by the nucleolar mater ia l in their nuclei. The follicle cells form a layer o f un i fo rm thickness all a r o u n d the chamber . They usual ly exhibit an ell iptical nucleus in which the nuc leo lar mater ia l is f requently asymmetr ica l ly pos i t ioned. In the pos te r ior pa r t o f the chamber , the oocyte nucleus with a character is t ic roundish e n d o b o d y therein, can be clearly seen (Fig. 1).

As is a l ready known, nurse cells are all jo ined by r ing canals; two o f such canals are shown in F igure 1.

A n example o f the intercel lular br idges which have been detected within the follicle cell layer is given in the low magni f ica t ion mic rograph shown in F igure 1. I t is evident tha t the intercel lular br idges are much smaller than the r ing canals, the former being only 0.25 ta in average diameter .

Intercellular Bridges in Drosophila melanogaster 415

Fig. 1. Low magnification picture of part of a stage 5 ovarian chamber. Two ring canals (R) are visible between nurse cells (NC) and the oocyte (OO). Follicle cells (FC) envelop the cluster of nurse cells and oocyte; two intercellular bridges (arrows) are discernible within the follicle layer; no nucleolus. • 4000

416 F. Giorgi

Fig. 2. Two intercellular bridges ( ib) are clearly visible between three follicle cells. N follicle cell nucleus; chr chromatin material; no nucleolus, x 14,250

Fig. 3. An intercellular bridge joining two adjacent follicle cells. The bridge wall appears to consist of two layers of which the external one (large arrows) is more electron dense and thinner than the internal layer (small arrows). N follicle cell nucleus, x 28,500

Intercellular Bridges in Drosophila melanogaster 417

Intercellular bridges may occur anywhere along the plasma membrane of the follicle cells. Usually, it has been found that only one bridge is present between two adjacent follicle cells, but the same cell may form at least two cytoplasmic connections with adjacent cells (Fig. 2). Since the size of the intercellular bridges are such as to allow the passage of ribosomes or even organelles, this observation may suggest that follicle cells form a sort of cytoplasmic syncytium.

Intercellular bridges may either extend across the intercellular space interposed between adjacent cells as in the case of Figure 2 or may protrude into one of the two facing cells, if they are so closely apposed as to leave no intercellular space between (Fig. 3).

As far as the present observations go, the intercellular bridges seem to occur widely between follicle cells of previtellogenic stages, whereas in vitellogenic stages (viz.: from stage 8 up to the completion of oogenesis which occurs at stage 14) they are practically restricted to the columnar follicle cells. No intercellular bridge has so far been detected in the thin follicle cells which overlay the nurse cells from stage 9 onwards.

At high magnification, intercellular bridges appear to consist of a cylindrical structure with a wall of 600& in thickness and a core of cytoplasm which is continuous with both cells. The wall of the cylindrical bridge is further resolved in an outer dense layer, about 200A thick, and in an inner, much less dense, layer about 400 A thick (Fig. 3).

The fine structure of the intercellular bridges is better resolved in preparations of ovarian chambers fixed in osmium tetroxide and examined at high magnifi- cation. In longitudinal sections (Fig. 4), it can be seen that the less dense layer which binds to the bridge wall from the inner side presents a varying extension along the entire length of the bridge itself. This layer may also be filled at varying degrees of density with material of fibrous nature. Occasionally, empty looking areas are visible within the cytoplasmic core of the bridge (Figs. 2, 4). It is not known whether this is due to transfer of lipid droplets through the bridge or simply to artifacts arising from fixation.

Examination of intercellular bridges in cross sections has revealed the presence of a typical substructure within the outer thick layer of the wall bridge. This consists of a number of septa-like structures (100A wide) which are spaced at regular intervals along the bridge wall (Fig. 5). The same alternation of septa is not so evident in intercellular bridges examined in longitudinal section.

In an attempt to establish the earliest appearance of intercellular bridges within the ovariole, a careful search for them was made in a number of ovarian chambers of different stages. At every stage so far examined, intercellular bridges appear to exhibit a substructure similar to that already described. The only exception to this seems to occur within the germarium where intercellular bridges appear somehow variable in morphology. The part of the germarium where intercellular bridges have most frequently been encountered is the intermediate region. Here, intercellular bridges form between follicle cells which have just migrated from more lateral positions and have not yet come to cover a 16-cell cluster of cystocytes. In this location, intercellular bridges possess a tortuous wall and they usually exhibit a number of microtubules which run parallel to the longitudinal axis of the bridge. Figure 6 illustrates part of this region where the plasma membranes of the adjoining follicle cells form intricated interdigitations.

418 F. Giorgi

Fig. 4. A high magnification picture of an osmium-fixed ovarian chamber of stage 6. Both layers of the bridge wall are clearly shown in the picture. N follicle cell nucleus; m mitochondrion, x 50,000

Fig. 5. A high magnification picture of an intercellular bridge seen in cross section. The outer layer of the bridge wall appears to consist of septa-like structures which are spaced at regular intervals, x 60,000

Intercellular Bridges in Drosophila melanogaster 419

Fig. 6. Part of the intermediate region of the germarium. Two intercellular bridges (ib) of unusual morphology are shown in the picture. The outer layer of one bridge (large arrows) is seemingly in continuity with a cell junction (J); mt microtubules, x 33,250

Fig. 7. Another plane of the intermediate region of the germarium. Only cell junctions (J) and microtubules (rot) are visible in this region; m mitochondrion, x 35,150

420 F. Giorgi

Apart from the presence of intercellular bridges, this region is also characterized by the presence of numerous desmosomes between interdigitating follicle cells (Fig. 7). In addition, the part of the cytoplasm close to the desmosomes is quite often found to contain a number of microtubules. Instances of actual continuity between the plasma membrane forming the desmosomes and the thick wall of the intercellular bridges have been encountered frequently. One such instance is also shown in Figure 6.

Discussion

Cytoplasmic continuity is widely recognized to play an important role in maintaining a synchronous development of the linked cells (Fawcett, 1961; Zamboni and Gondos, 1968). Instances where synchronization of development has been clearly shown to depend upon cell linkage have been frequently encountered among germ cells (Meyer, 1961; Anderson and Huebner, 1968; Steinert and Urbani, 1969; Koch and King, 1969; Ruby et al., 1969; Skalko, Kerrigan et al., 1972). Relatively fewer cases of intercellular bridges linking somatic cells have been reported so far in the literature (Fawcett et al., 1959; Schwalm and Bender, 1973; Arnold, 1974; Buck et al., 1976; Rickoll, 1976).

The evidence presented in this study indicates quite clearly that ovarian follicle cells in Drosophila melanogaster are continuous with each other by intercellular bridges. Cytoplasmic continuities similar to those now found in Drosophila ovary have been previously reported in the developing leg of Drosophila (Poodry and Schneiderman, 1971).

Koch and King (1963) have suggested that the accumulation of columnar follicle cells at one pole of the chamber in Drosophila is due to migration of those follicle cells which are originally located elsewhere within the follicle layer. It is of interest to recall here that the intercellular bridges are distributed at random within the follicle layer of previtellogenic ovarian chambers, while they become restricted to the region of the columnar follicle cells from stage 8 onwards. Unfortunately, at the present it is not known whether in previtellogenic chambers the intercellular bridges constitute a feature common to all follicle cells. One possibility, which cannot be ruled out, is that only part of the follicle cell population is in actual continuity, the other cells remaining isolated from each other throughout the period of oogenesis. This uncertainty makes it impossible to establish whether there could be any relationship between the migration of columnar follicle cells towards one pole of the chamber and the topographical distribution assumed by the intercellular bridges in succeeding stages of the ovarian development. It is, however, worthwhile noting that the intercellular bridges which are present in vitellogenic chambers are restricted to those cells which are capable of elaborating the various egg covering precursors. The existence of a cytoplasmic continuity between these cells could thus be interpreted as ensuring a synchronous secreting activity of the columnar follicle cells all around the developing oocyte.

So far the continuity between somatic cells has been found hard to interpret because of the known difficulty of attributing a functional role to cells which can- not be so easily staged as those of the germ line. Perhaps this is one of the reasons

Intercellular Bridges in Drosophila melanogaster 421

for considering intercellular bridges between somatic cells not so clearly involved in synchronization of development as those between germ cells (Arnold, 1974). Unlike the somatic systems so far studied, however, the intercellular bridges present in ovarian follicle cells of Drosophila may be interpreted similarly to those found in germ cells. This is because the association between germ and somatic cells within the same ovarian chamber makes it possible to stage the development followed by the follicle cells in relation to that of the chamber. In addition, the intercellular bridges of the follicle cell layer may be functionally interpreted with reference to a physiological condition, such as the secretion of the egg covering precursors, which can be ascertained morphologically and autoradiographically for all follicle cells involved (Giorgi, 1977).

According to the presently accepted view, the origin of intercellular bridges is explained as being due to an incomplete cytokinesis (Fawcett, 1961). Remnants of the meiotic spindle are also known to play a role in the formation of the wall bridges, as for instance in the ring canals of nurse cells in Drosophila melanogaster (Koch and King, 1969). Although it cannot be ruled out that this could also be the case for intercellular bridges in ovarian follicle cells of Drosophila, other possibilities should, however, be taken into account. In particular one would need to explain why forming intercellular bridges can only be detected in the germarial region of the ovary, although cell divisions are known to continue up to the completion of stage 6 (Koch and King, 1963). The presence of desmosomes in close proximity of the wall bridge would also be another point to deserve attention along with the presence of microtubules in both these structures.

A careful study of several serial sections and a building of a three-dimensional model could help to clarify this point. All that one can speculate at the present is that the intercellular bridges arise in the immediate vicinity of desmosomes but that the modalities of the process of bridge formation remain to be worked out.

References

Anderson, E., Huebner, E.: Development of the oocyte and its accessory cells of the Polychaete Diopatra cuprea (Bosc.). J. Morph. 126, 163-198 (1968)

Arnold, J.M.: Intercellular bridges in somatic cells: cytoplasmic continuity of blastoderm cells of Loligo pealei. Differentiation 2, 335-341 (1974)

Buck, R.C., Ohara, P.T., Daniels, W.H.: Intercellular bridges in chick blastoderm studied by SEM and TEM. Experientia (Basel) 32, 505-507 (1976)

Cummings, M.R., Brown, N.M., King, R.C.: The cytology of the vitellogenic stages of oogenesis in Drosophila melanogaster. I. General staging characteristics. J. Morph. 128, 427-442 (1969)

Fawcett, D.W.: Intercellular bridges. Exp. Cell Res. (Suppl.) 8, 174-187 (1961) Fawcett, D.W., Ito, S., Slautterback, D.: The occurrence of intercellular bridges in groups of cells

exhibiting synchronous differentiation. J. biophys, biochem. Cytol. 5, 453-460 (1959) Giorgi, F.: An EM autoradiographic study of ovarian follicle cells in Drosophila melanogaster with

special reference to the formation of egg coverings. Histochemistry, in press (1977) King, R.C.: Ovarian development in Drosophila melanogaster. New York-London: Academic Press.

1970 King, R.C., Koch, E.A.: Studies on ovarian follicle cells of Drosophila. Quart. J. micr. Sci. 104, 297-320

(1963) ...... King, R.C., Vanoucek, E.G.: Oogenesis in adult Drosophila melanogaster. X. Studies on the behavior of

the follicle cells. Growth 24, 333-338 (1960)

422 F. GiorN

Koch, E.A., King, R.C.: Further studies on the ring canal system of the ovarian cystocytes of Drosophila melanogaster. Z. Zellforsch. 102, 129-152 (1969)

Mahowald, A.P.: Ultrastructural observations on oogenesis Drosophila. J. Morph. 137, 29M8 (1972) Meyer, F.G.: Interzellul/ire Brticken (Fusome) im Hoden und im Ei-N/ihrzellenverband von Drosophila

melanogaster. Z. Zellforsch. 54, 238-251 (1961) Poodry, C.A., Schneiderman, H.A.: Ultrastructure of the developing leg of Drosophila melanogaster.

Wilhelm Roux' Archiv 166, 1M4 (1970) Quattropani, S.L., Anderson, E.: The origin and structure of the secondary coat of the egg of Drosophila

melanogaster. Z. Zellforsch. 95, 495 510 (1969) Rickoll, W.L.: Cytoplasmic continuity between embryonic cells and the primitive yolk sac during early

gastrulation in Drosophila melanogaster. Develop. Biol., 49, 304-310 (1976) Ruby, J.R., Dyer, R.F., Skalko, R.G.: The occurrence of intercellular bridges during oogenesis in the

mouse. J. Morph. 127, 307-340 (1969) Schwalm, F.E., Bender, H.A.: Early development of the kelp fly Coelopa frigida (Diptera). II.

Morphology of cleavage and blastoderm formation. J. Morph. 141, 235-255 (1973) Skalko, R.G., Kerrigan, J.M., Ruby, J.R., Dyer, R.F.: Intercellular bridges between oocytes in the

chicken ovary. Z. Zellforsch. 128, 31M1 (1972) Steinert, G., Urbani, E.: Communications intercellulaires dans les ovarioles de Dytiscus marginalis L. J.

Embryol. exp. Morph. 22, 45-54 (1969) Zamboni, L., Gondos, B.: Intercellular bridges and synchronization of germ cell differentiation during

oogenesis in the rabbit. J. Cell Biol. 36, 276 (1968)

Accepted May 23, 1977