Terminal filament cell organization in the larval ovary of Drosophila melanogaster: ultrastructural observations and pattern of divisions

Roux's Arch Dev Biol (1996) 205:356-363 © Springer-Verlag 1996 Isabelle Sahut-Barnola • Bernard Dastugue Jean-Louis Couderc Terminal filament cell ...
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Roux's Arch Dev Biol (1996) 205:356-363

© Springer-Verlag 1996

Isabelle Sahut-Barnola • Bernard Dastugue Jean-Louis Couderc

Terminal filament cell organization in the larval ovary of Drosophilamelanogaster:ultrastructural observations and pattern of divisions Received: 23 August 1995 / Accepted in revised form: 7 December 1995

Abstract The adult ovary of Drosophila is composed of approximately twenty parallel repetitive structures called ovarioles. The ovarioles appear at the prepupal stage and their formation requires the presence of stacks of discshaped cells called the terminal filaments. Terminal filaments form in a progressive manner during the third larval instar. We have looked at the beginning of formation of both the terminal filaments and ovarioles at an ultrastructural level. Moreover, we studied the pattern of division of the terminal filament cell precursors using the base analog, BrdU. Two main waves of division are observed. The first wave consists of divisions of almost all the terminal filament cell precursors during a short period of time at the transition between the second and third larval instar. The second wave, in which the precursors carry out their final divisions before differentiating, occurs gradually, going from the medial to the lateral side of the ovary during the first half of the third larval instar. Key words Drosophila - Ovary morphogenesis - Mitotic wave • Pattern formation

Introduction The basis for understanding the development of complex structures such as the formation of an organ requires a thorough description of the pattern of divisions of the precursor cells, of the cell shape changes and of the sorting and spatial reorganization of cells during the formation of the organ. This can be done in a model system such as the morphogenesis of adult ovaries from larval primordia in Drosophila melanogaster. Each adult ovary of D. melanogaster consists of approximately twenty identical tube-like structures called ovarioles where oogenesis takes place (for a review, see Spradling 1993). This organization of the ovary into ovaI. Sahut-Barnola ( ~ ) • B. Dastugue - J.-L. Couderc INSERM U384, Laboratoire de Biochimie, UFR M6decine, F-63001 Clermont-Ferrand, France

rioles begins during the prepupal stage (King et al. 1968). Prior to this stage, the ovary is oval and contains layers of different cells which are arranged along the anterior-posterior axis. The germ cell population is sandwiched between anteriorly and posteriorly located somatic cell populations. The first morphological sign that indicates the beginning of the formation of ovarioles is the presence in the anterior region of the ovary of stacks of eight to nine disc-shaped cells called terminal filaments. Using different markers, such as enhancer trap lines expressing 13-galactosidase in terminal filament cell nuclei or antibodies against the BRIC ~ BRAC protein which is present in the nuclei of the terminal filament cells, it has been recently shown that the terminal filaments form during the third larval instar in a progressive manner medially to laterally across the ovary (Godt and Laski 1995; Sahut-Barnola et al. 1995). In a bric-&-brac mutant larval ovary, terminal filaments do not form and this leads to an absence of ovarioles in the adult ovary. This indicates that the terminal filaments are required for at least the initiation of ovariole formation. When terminal filaments are formed, the apical cells, which are located anterior to the terminal filaments, migrate from the anterior toward the posterior region of the ovary between these stacks of cells (AboYm 1945; King et al. 1968). This migration is accompanied by the deposition of a basement membrane, called the tunica propria, which encloses terminal filament cells and germ cells into individual tubes, the ovarioies. At this point, the germ cells no longer form a uniform population but are distributed into 18-20 ovarioles. In this paper, we look at an ultrastructural level at the start of two important events that lead to ovary morphogenesis, the early steps of terminal filament formation and tunica propria deposition at the beginning of ovariole development. After stack formation, terminal filament cells are mitotically inactive (King et al. 1968). We have studied the pattern of divisions of the terminal filament cell precursors and demonstrate that there are two different waves of division. The first occurs during a short period of time and affects almost all the terminal

357 f i l a m e n t cell precursors. The s e c o n d w a v e consists o f s p a t i a l l y o r d e r e d divisions starting f r o m the m e d i a l side and m o v i n g t o w a r d the lateral side o f the ovary over app r o x i m a t i v e l y 2 days.

Materials and methods Incorporation and detection of bromo-deoxyuridine (BrdU) The base analogue BrdU, which is incorporated into replicating DNA, was added to standard Drosophila medium at a 1 mg/ml concentration. Larvae were fed with this medium for 3-24 h. To visualize incorporated BrdU, the larvae were dissected in phosphate-buffered saline (PBS) and fixed for 6 min in modified Carnoy's fixative (100% ethanol: glacial acetic acid, 6:2), washed three times in PBS and incubated for 30 rain in 3 M HC1 to denature the DNA. After this step, they were washed 2x15 min in PBS containing 0.3% Triton X-100. The larvae were then incubated overnight in PBS containing 0.3% Triton X-100 and a monoclonal antibody against BrdU (Becton-Dickinson) at a dilution of 1/50. After three washes of 30 rain in PBT (1 x PBS, 0.1% bovine serum albumin, 10% Triton) with 2% goat serum (Vector laboratories; ABC Kit), the larvae were incubated for 3 h in an anti-mouse secondary antibody (Vector laboratories; ABC Kit; preadsorbed to fixed larvae) diluted 1/400 in PBT. After washing in PBT for 10 min, a new fixation in 0.5% glutaraldehyde in PBS was performed for 10 rain, followed by two washes in PTW (lx PBS, 0.1% Tween 20). The ovaries were treated for 30 min with biotinylated horseradish peroxidase (HRP)-avidin complex (Vector laboratories, ABC Kit) diluted 1:50 in PTW and washed again in PTW 3x5 rain. The HRP reaction was performed in a staining solution containing 2 mg/ml diaminobenzidine tetrachloride, with or without 0.03% NiC1 in PTW, respectively leading to a black or maroon final staining. After 10 rain, 0.01% H202/ml was added. The tissues were washed in lx PBS and mounted in ethanol/glycerol (1/1). All the preparations were viewed on a Zeiss Axiophot microscope and photographed using Ektachrome 50 slide film at ASA 160. In a first series of experiments, we performed BrdU incorporations over several broad time periods during the larval stages to define the period of time in which the terminal filament cells were dividing. Then, using these preliminary results, we performed BrdU incorporations during more specific periods of time which are presented here. For each period of BrdU incorporation, 20-30 ovaries were examined.

Results

Beginning of the formation of terminal filaments and tunica propria To gain m o r e insight into the early steps o f t e r m i n a l filam e n t formation, w e have e x a m i n e d ultrathin sections o f the anterior region o f m i d third instar larval ovaries b y electron m i c r o s c o p y . In such ovaries, two kinds o f pattern are observed, d e p e n d i n g on the orientation o f the sections. On one side o f the ovary, the apical r e g i o n contains both e l o n g a t e d and r o u n d somatic cells l o c a t e d j u s t above the g e r m cells (Fig. 1A) and with no distinguishable organization. On the other side o f the ovary, a furrow is present on the surface, i n d i c a t i n g the r e g i o n w h e r e terminal filaments are b e g i n n i n g to f o r m (Fig. 1B, open arrow). This region contains two clusters o f t w o - t h r e e e l o n g a t e d and flattened cells. T h e extremities o f these s p i n d l e - s h a p e d cells are c o n n e c t e d together, defining att a c h m e n t sites (Fig. 1B, arrows). T h e s e sites have b e e n shown to be very rich in actin filaments and to contain large a m o u n t s o f the a d h e s i o n m o l e c u l e A r m a d i l l o ( G o d t and L a s k i 1995). T h e r e are several other cells with one end that is a l r e a d y attached to these sites. T h e s e cells s e e m to be e l o n g a t i n g to reach the other a t t a c h m e n t site o f this cluster as they will be i n c o r p o r a t e d into the clusters. This specific o r g a n i z a t i o n i m p l i e s that cells a d o p t a crescent shape giving an e y e - l i k e a p p e a r a n c e to such a group o f cells. T h e c h a n g e in cell shape, e l o n g a t i o n and m i g r a t i o n to form such e y e - s h a p e d clusters are the earliest steps in the f o r m a t i o n o f the t e r m i n a l filaments. L a ter, m o r e cells will j o i n these clusters to f o r m stacks o f about eight or nine blunt e n d e d cells with p e r f e c t l y a l i g n e d nuclei ( G o d t and L a s k i 1995; S a h u t - B a r n o l a et al. 1995). A p i c a l cells will then m i g r a t e b e t w e e n the term i n a l filaments, separating t h e m from each other and s u b s e q u e n t l y d i v i d i n g the g e r m cell population. A basem e n t m e m b r a n e , called the tunica propria, is laid d o w n

Collection of larvae Eggs were collected on grape juice agar plates. The hatched larvae were collected and grown at 25 ° C in standard medium. The larvae were staged at the transition between the second and third larval instar. Staging of larvae was based on the descriptions of Ashburner (1989). Time is presented in hours after egg deposition (AED). Electron microscopy Larval and prepupal ovaries were dissected in a solution containing 3% glutaraldehyde in a 0.2 M cacodylate buffer (pH 7.4) and were kept at 4 ° C for 1 h. They were washed 3x10 min in cacodylate buffer and postfixed in 1% osmic acid in cacodylate buffer for 45 rain. Ovaries were washed 3x10 min in cacodylate buffer and dehydrated in an ethanol series (70, 95, 100%; 3x10 min each). Preparations were incubated in an 1/1 mixture of ethanol 100% and TAAB embedding resin (Taab Laboratories Equipment Limited) for 2x30 min and in resin overnight. Specimens were then kept at 37 ° C for 4-5 h and at 60 ° C for 3 days to allow the polymerization of the resin. Blocks were sectioned with a Reichert OMU2 microtome. Sections were mounted on grids and treated with uranyl acetate and lead citrate (Reynolds 1963). Sections were examined and photographed with a Hitachi HU 12A electron microscope.

Fig. 1A-D Beginning of terminal filament formation and of ovariole development. Electron micrographs of larval and prepupal ovaries, accompanied by an illustration showing shape and position of the apical cells located above germ cells. A and B Two different sections in the apical region of a wild type ovary at 96 h after egg deposition (AED). A In this section, both flattened cells and more round cells are located above the germ cell population (g), but no specific organization is visible. B Section showing two eye-shaped clusters. In the right and left clusters, three and two cells, respectively, are completely connected together by their tapered ends (arrows). Other somatic cells more or less elongated are surrounding these two clusters and might join them. The open arrow indicates the furrow present on the surface of the ovary in the region where terminal filaments are forming. C Section of a prepupal ovary (120 h AED) showing mature terminal filament formed of nine disc-shaped cells. These cells present blunt ends and are surrounded by a basement membrane, the tunica propria (tp). Forming tunica propria (arrowhead) is visible at the base of the stack. Two cells located at the posterior end of the stack are not elongated. D Higher magnification of a region of the ovary where the tunica propria is forming (g germ cell, a apical cells, bars 5 gm for A, B and C and 2 gm for D)

For legend of Fig. 1 see page 357

359

For legend of Fig. 2 see page 360

360 along the migration paths of the apical cells enclosing terminal filaments, germ cells and other somatic cells into tubes, the ovarioles (King et al. 1968). In order to visualize the deposition of the tunica propria, we have observed prepupal ovaries (120 h AED) at an ultrastructural level. Figure 1C shows a portion of such an ovary with a mature terminal filament containing eight or nine discshaped cells which are well ordered in a pile and two or three cells which are not elongated at its base, with germ cells located more posteriorly. Apical cells have begun their posterior migration and have separated the mature terminal filaments from each other. A thin basement membrane surrounds the stack except for the most anterior cell (Fig. 1C). At the bottom of the stack, the membrane is just being formed and looks very different. A large amount of high-electron-density material is first secreted in a very polarized fashion between the migrating apical cells and the cells they encounter in front of them,

Fig. 2A-H Division of terminal filament cell precursors. Larvae were fed with bromo-deoxyuridine (BrdU) for different periods of time during the second and the third instars. Incorporated BrdU was detected with an anti-BrdU antibody in wild type prepupal (A, A', C, D, E, F, G) or early pupal (B) ovaries. A and A' Larvae fed with BrdU from 48 to 68 h AED; A' shows the left part of the ovary presented in A but at a different focus. Two neighbouring stained stacks are visible (arrowheads). The stack in A contains 4-5 stained terminal filament cells and in A' contains 3 more lightly stained terminal filament cells. Germ cells (black arrows) and a few somatic cells are also labelled in the ovary. B Larvae fed with BrdU from 68 to 71 h AED. The apical region of an early pupal ovary shows 80-90 stained terminal filament cells. These cells present different intensities of staining. C Larvae fed with BrdU from 71 to 79 h AED. Strongly stained terminal filament cells in 5 stacks (arrowheads) are located at the medial side of the ovary. A few lightly stained terminal filament cells are indicated with arrows with an open arrowhead. The insert shows a representative stack which contains lightly stained terminal filament cells (indicated with arrows with an open arrowhead). D Larvae fed with BrdU from 79 to 96 h AED. Strongly stained terminal filament cells in 7-8 stacks (arrowheads) are located in the lateral region of the ovary. A few lightly stained terminal filament cells are visible (arrows with an open arrowhead). The insert shows a stack in which labelled and unlabelled cells (indicated by dots) are mixed. E Larvae fed with BrdU from 73 to 76 h AED. Two-Three stacks containing a few stained terminal filament cells are located in the same region of the ovary. (Area between brackets corresponds to visible stacks with unlabelled terminal filament cells in this picture and also in pictures F, G, H). F and G Larvae fed with BrdU from 96 h AED to the prepupal stage. Stained terminal filament cells are present only in stacks located at the lateral side. More medially, some stained cells are also detected at the bottom of stacks (marked with white asterisk in the insert in F). The apical somatic cells that migrate between terminal filament are strongly labelled (marked with white lines in the insert in G). The incorporated BrdU, in the ovary presented in F, is only detected in the apical region. The basal region was not accessible to antibodies. H Larvae fed with BrdU from 105 h AED to the prepupal stage. No stained cell in terminal filaments are visible (area between brackets). Somatic cells located at the posterior region of the ovary show a strong staining. The lateral side of the ovary is located on the right in pictures C, F, G and in the front in picture D. The large stained nuclei visible in several pictures are from fat body cells. Black arrows indicate the germ cells that were replicating DNA at the time of BrdU treatment. For all figures, anterior is at the top, posterior at the bottom (Bars 20 gm)

forming a loose meshwork lining the contact side of these cells (Fig. 1C, arrowhead). This unorganized or fine fibrillar material, as seen at higher magnifications (Fig. 1D), can occupy up to 700 nm between cells. This material then assembles into a more and more organized sheetlike network that blends gradually to give the final thin sheet of about 60 nm that is the final tunica propria.

Precursors of the terminal filament cells divide during the transition between the second and third larval instar To analyse the pattern of terminal filament cell divisions, a series of pulse labelling experiments using the base analog bromo-deoxyuridine (BrdU) were performed. D r o s o p h i l a larvae were fed with BrdU for different periods of time ranging from the first larval instar to the prepupal stage. Before and after this period, the larvae were fed with standard medium without BrdU. Detection of incorporated BrdU was performed in dissected ovaries after the treatment at the prepupal (or even early pupal) stage when all the terminal filament cells are differentiated and integrated into stacks and are easily identifiable. It is when BrdU treatment occurs during the majority of the second larval instar (from 48 to 68 h AED) that we detect the first labelled terminal filament cells in prepupal ovaries, identified by anti-BrdU antibodies. Prior to this stage, we have not observed labelled terminal filament cells. Figure 2A and A ' show a typical prepupal ovary from an animal that has been fed with BrdU for 20 h during the second larval instar. Only a few terminal filament cells in the entire ovary are still labelled 52 h after BrdU treatment, indicating that these cells have differentiated before extensive cell divisions have diluted the incorporated BrdU below a detectable level during the long chase period without BrdU. When BrdU treatments are performed over short periods of time at the transition between the second and third larval instar, a dramatically different pattern is seen. An ovary from an early pupa that has been fed with BrdU for 3 h (between 68 and 71 h AED) shows many stained cells in all the terminal filaments (Fig. 2B). In such ovaries, 80-90 terminal filament cells are labelled indicating that almost all the terminal filament cell precursors were dividing during this short period of time and that these precursors have undergone only a few round of mitosis before differentiating. This is the window where we can observe the maximum number of labelled cells. We have never observed so many labelled cells by performing earlier (Fig. 2A and A') or later (see below) BrdU treatments, indicating that the rate of terminal filament cell precursor divisions is much lower during most of the second and third larval instars than during this short period at the transition between these two instars. Labelled cells are detectable in all the terminal filaments indicating that the precursors that form during this period of time will give rise to cells that will participate, after few divisions, in the formation of stacks throughout the terminal filament region of the ovary.

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Third larval instar

Terminal filament piling up

Fig. 3 Two main waves of divisions are required for the production of the terminal filament (TF) cells. The grey arrow symbolizes time of development after egg deposition (AED). The first wave of divisions is indicated with a black lozenge. Almost all the TF cell precursors divide during a short period of time between the second and third larval instar. The second wave of divisions is indicated with a black arrow and occurs throughout the first half of the third larval instar. Black cells in the schematized ovaries indicate the cells that have been formed at three time points in the first half of the third larval instar. Cells located in the medial side of the ovary derive from precursors which divide earlier than the precursors which give rise to cells in terminal filaments located at the lateral side of the ovary. The striped arrow indicates the time when the formation of the terminal filaments occurs as detected by the piling-up of terminal filament cells and expression of the LB27 marker (Sahut-Barnola et al. 1995). Anterior of prepupal ovaries is up and lateral is to the right When BrdU incorporation occurs during this period of time, the terminal filament cells are labelled at very different intensities, varying from a very strong to a very faint, barely detectable, labelling (Fig. 2B). These different levels of staining probably reflect the different numbers of cell cycles that terminal filament cell precursors have undergone following the period of BrdU incorporation before differentiating. Cells labelled with the lowest intensity (Fig. 2B) are just above the sensitivity level of our detection. Had they undergone another round of mitosis, they probably would no longer have been detectable. This could suggest that all precursors that will form terminal filament cells divide during this period of time.

Most terminal filament cells undergo final mitoses during the first half of the third larval instar When BrdU treatment occurs for long pulses during the first half of the third larval instar, we observe stained ter-

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minal filament cells in several (5-8) terminal filaments which are mostly clustered on one side of the prepupal ovary (Fig. 2C and D). Labelled and unlabelled cells are randomly arranged in the terminal filaments (Fig. 2C and D, and especially insert Fig. 2D) indicating that the cells within one stack are not forming at the same time. Staining is strong for most of the stained cells. This suggests that cells that replicate during this period of BrdU treatment probably divide only one or two time(s) following this period, then differentiate and incorporate into newly forming terminal filaments. The presence of a few lightly stained cells (Fig. 2C and D, and especially insert Fig. 2C) indicates that there are also some terminal filament cell precursors that replicate DNA at this stage but divide several times before differentiating. By applying a short (3 h) BrdU pulse treatment during the first half of the third larval instar (Fig. 2E), we observe that only a few (5-7) terminal filament cells are labelled. This is a very different pattern of labelling than that observed when such a short pulse of BrdU is given at the transition between the second and third larval instars (Compare Fig. 2B, E). This observation confirms the idea that precursors of terminal filament cells divide at a lower rate during the third larval instar than at the transition between the second and third larval instar. To determine the time at which the last terminal filament cell divisions occur, BrdU was given for long pulses during the second part of the third larval instar. In ovaries treated from 96 h AED to the prepupal stage, some terminal filaments with several labelled cells are visible in the lateral region of the ovary (Fig. 2F, G). This indicates that the terminal filament cells that are incorporated in the most distal stacks are the last to form. We can also observe the presence of 1-3 stained cells at the base of stacks that are located more medially (insert

362 Fig. 2F). These cells will probably become the germarial tip cells (GTC) located between the most posterior terminal filament cell and the germline stem cells at the tip of an adult germarium. These cells, therefore, form later than the terminal filament cells located just anterior to them. Following BrdU treatment during the last 15 h of the third larval instar, no terminal filament cells are labelled, although many other cells are (Fig. 2H). Thus, all of the terminal filament cells arise before 105 h of development AED.

Divisions of other cell types present in the prepupal'ovary We have also looked at the division of other cells of the larval ovary.When BrdU incorporation occurs during the second and third larval instars, germ cells and many somatic cells other than the terminal filament cells are detected with an anti-BrdU antibody at the prepupal stage (Fig. 2A, H). Therefore, both somatic cells and germ cells multiply during the larval period, as indicated by King et al. (1968). Unfortunately, when BrdU was incorporated during the second larval instar, the large number of subsequent cell divisions diluted the DNA marker too much to be detectable in many cells at the prepupal stage. Many cells of the larval ovary are difficult to identify without the help of markers, therefore only two cell populations could be clearly identified and the time of their division estimated. When BrdU incorporation occurs between 96 h AED and the prepupal stage, the apical cells, which have migrated between the terminal filaments, are particularly strongly labelled (Fig. 2G and insert), while the cells of the terminal filaments are not. This indicates that the migrating cells divide after the cells of the terminal filaments which they separate. One can also observe that the migration of the apical cells begins earlier on the medial side than on the lateral side of the ovary, starting as soon as the first terminal filaments have been formed (Fig. 2G). When BrdU incorporation occurs between 105 h AED and the prepupal stage, the most posterior cells of the ovary are strongly stained, indicating intense mitotic activity by these cells at this stage (Fig. 2H). These cells will probably give rise to cells that participate in the elaboration of the structure that allows the connection between the ovary and oviduct, which is derived from the genital imaginal disc.

Discussion Formation of the terminal filaments is a morphogenetic process that sweeps across the larval ovary from the medial towards the lateral side (Godt and Laski 1995; Sahut-Barnola et al. 1995). It begins with the development of side by side eye-shaped clusters of terminal filament cell precursors, which arise from a population of

undifferentiated cells. This specific organization suggests that one (or a few) elongated cell is able to attract neighbouring cells at one end and to maintain cells connected to this point. The cell then extends to reach the other end of the cluster so that it is connected by both ends. This hypothesis involves strong cell-cell attachment and important cell shape changes that fit well with the observation that actin and the ~-catenin related protein armadillo are abundantly localized at the ends of cells in eyeshaped clusters (Godt and Laski 1995). As soon as a terminal filament is formed, some apical cells migrate and components of the tunica propria are secreted between the migrating cells and, first, the terminal filament and then the germline cells. This suggests that the basement membrane is probably secreted by the migrating apical cells (King et al. 1968). The tunica propria is first secreted as an unorganized or fine fibrillar material that later blends to form a thinner and very dense sheet, giving the tunica propria its shape and rigidity. Identifying the components of this structure will help to clarify which cells are synthesizing them and what role this particular basement membrane plays. The tunica propria may provide strength to maintain the structure of the ovariole during egg development or act as a molecular filter. It could also have adhesion properties that could be important in pupal ovaries during formation of the basal stalks (Godt and Laski 1995) or in adult ovaries - like the extracellular matrix in the C. elegans gonad (Maine and Kimble 1990) - for preventing the germline stern cells from differentiating. The formation of terminal filaments in Drosophila larval ovaries is the first sign of subdivision of the ovary into ovarioles. In this paper, we have studied the pattern of division of the terminal filament cell precursors using the base analog BrdU. We observe very different patterns of division depending on the period of time when the labelling is performed. This excludes the possibility that the formation of terminal filament cells results from simple exponential divisions of a few precursors. The main steps of this process are summarized in Fig. 3. Two main waves of division take place to produce the terminal filament cells. The first wave occurs during a short period of time at the transition between the second and third larval instars, when a large number (if not all) of the terminal filament cell precursors divide, giving' rise to an increased number of terminal filament precursors. This is the only period that we have been able to identify during which almost all of the terminal filament precursors divide at approximately the same time. The progeny of these precursors will be distributed throughout the region that the terminal filaments will populate. The second wave of division extends over the first half of the third larval instar. During this period of time, the terminal filament precursor cells undergo one or two last division(s) before they differentiate. These divisions occur at different times during the second wave, depending on the cell's position within the ovary. We observed that cells that divide at the same time will belong to stacks located in the same region of the ovary. More precisely, cells

363 replicating DNA during the beginning of the third larval instar are mainly incorporated into terminal filaments located on the medial side of the ovary. In contrast, cells replicating at the end of the first half of the third larval instar participate in the formation of terminal filaments located on the lateral side of the ovary (Fig. 3, upper part). This suggests that the second wave of division spreads from the medial to the lateral side, preceding the wave of terminal filament formation. Many terminal filaments contain cells that are labelled at different intensities or are even unlabelled. This indicates that not all the cells that will belong to a particular terminal filament form at exactly the same time. Moreover, the position of the labelled (or unlabelled) cells in a terminal filament is random with no specific pattern, indicating that cells dividing at the same time can be incorporated at different places and as part of different terminal filaments. This suggests, as previously indicated by mosaic analysis (Godt and Laski 1995), that cells which form one terminal filament are recruited from a dividing cell population rather than generated by the clonal divisions of a precursor cell. The mosaic experiments also showed two observations that fit with our model for the divisions of the terminal filament cell precursors (Fig. 3). First, when mitotic recombination is induced at the early second larval instar, it usually occurs in only one precursor of the terminal filament cells. This cell gives rise to a large number of progeny that will become part of numerous mosaic stacks. This indicates that the number of terminal filament cell precursors is low (3-5 cells per ovary) at this time but that they divide during the second larval instar. Second, when mitotic clones are induced in the early third larval instar, the ovaries contain mosaic stacks located preferentially in the medial region, and when mitotic clones are induced in the mid third larval instar, the ovaries contain mosaic stacks located in the lateral region of the ovary. This observation suggests, as does our data, that the cells that form the medial terminal filaments arose before the cells that form the ones on the lateral side of the ovary (Fig. 3, upper part). The second wave of divisions preceds the wave of terminal filament formation

and so provides a continuous supply of new cells in the developing third instar ovary. Patterning in the apical region of the larval ovary depends on successive waves that spread from the medial towards the lateral side. This applies to the division of the terminal filament cells, their clustering, the formation of stacks, the formation of the germarial tip cells in the newly formed terminal filaments and the migration of the apical cells that will allow secretion of the tunica propria. Acknowledgements We thank Dorothea Godt and Frank Laski for fruitful discussions during this work and very helpful comments on the manuscript, and Sarah Cramton for critically reading the manuscript. We thank Michel Bourges for allowing us access to the electron microscope and for helpful discussions. We are expecially grateful to Annie Fraisse, Monique Oron and Josiane Payen for their technical assistance for electron microscopy. We thank Laury Arthaud for her excellent day-to-day technical assistance.

References AboYm AN (1945) D6veloppement embryonnaire et post-embryonnaire des gonades normales et agam6tiques de Drosophila melanogaster. Rev Suisse Zool 52:54-154 Ashburner M (1989) Drosophila. A laboratory handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, New York Godt D, Laski FA (1995) Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric &brac. Development 121:173-187 King RC, Aggarwal SK, Aggarwal U (1968) The development of female Drosophila reproductive system. J Morphol 124:143166 Maine EM, Kimble J (1990) Genetic control of cell communication in C. eIegans development. BioEssays 12:265-271 P~eynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208212 Sahut-Barnola I, Godt D, Laski FA, Couderc JL (1995) Drosophila ovary morphogenesis: analysis of terminal filament formation and identification of a gene required for this process. Dev Biol 170:127-135 Spradling AC (1993) Developmental genetics of oogenesis. In: Bate M, Martinez-Arias A (eds) The development of Drosophila melanogaster, vol 1. Cold Spring Harbor Press, Cold Spring Harbor, New York, pp 1-70

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