The Function of the Broad-Complex During Drosophila melanogaster Oogenesis

Corresponding author: Mary Bownes, Institute of Cell and Molecular Biology, University of Edinburgh, Darwin Bldg., King's Bldgs., Mayfield Rd., Edinburgh EH9 3JR, United Kingdom., mary.bownes{at}ed.ac.uk (E-mail)

Communicating editor: T. SCHÜPBACH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Broad-Complex (BR-C) is an early ecdysone response gene that functions during metamorphosis and encodes a family of zinc-finger transcription factors. It is expressed in a dynamic pattern during oogenesis. Its late expression in the lateral-dorsal-anterior follicle cells is related to the morphogenesis of the chorionic appendages. All four zinc-finger isoforms are expressed in oogenesis, which is consistent with the abnormal appendage phenotypes resulting from their ectopic expression. We investigated the mechanism by which the BR-C affects chorion deposition by using BrdU to follow the effects of BR-C misexpression on DNA replication and in situ hybridization to ovarian mRNA to evaluate chorion gene expression. Ectopic BR-C expression leads to prolonged endoreplication and to additional amplification of genes, besides the chorion genes, at other sites in the genome. The pattern of chorion gene expression is not affected along the anterior-posterior axis, but the follicle cells at the anterior of the oocyte fail to migrate correctly in an anterior direction when BR-C is misexpressed. We conclude that the target genes of the BR-C in oogenesis include a protein essential for endoreplication and chorion gene amplification. This may provide a link between steroid hormones and the control of DNA replication during oogenesis.

THE regulation of the expression of structural genes is critical in morphogenesis. This requires differential expression of transcription factors, which in turn regulate the tissue-specific expression of structural genes. Drosophila oogenesis is ideal for the study of developmental gene regulation as it takes a fairly short time to develop from a stage-1 egg chamber to a mature egg and all the stages are morphologically well defined. Further, egg chambers, the developmental units of oogenesis, contain only the somatically derived follicle cells and the germline cells. The former undergo dramatic morphogenetic movements and eventually synthesize the yolk and then the eggshell, as well as interact with the germline cells to generate the two major axes of the egg and embryo.

The Broad-Complex (BR-C), a gene encoding a family of zinc-finger transcription factors (DIBELLO et al. 1991 ; BAYER et al. 1996), has been shown to be expressed in the follicle cells in a dynamic pattern, the late pattern being defined by two groups of dorsal-anterior follicle cells at stage 10B of oogenesis (DENG and BOWNES 1997 ; for the staging of oogenesis, refer to SPRADLING 1993 ). This dorsal-anterior expression pattern is specified by the Grk-DER and decapentaplegic (DPP) signaling pathways along the two major axes, and is associated with the function of the BR-C in dorsal appendage formation. The involvement of BR-C in dorsal appendage morphogenesis was shown by mutational analysis of BR-C partial "loss-of-function" mutants, and was supported by ectopic expression of BR-C "transgenes" during oogenesis. It is proposed that the BR-C may provide a link between pattern formation and cell behavior in morphogenesis (DENG and BOWNES 1997 ).

The BR-C has been previously identified as a key gene required for Drosophila metamorphosis. It is among the early ecdysone responsive genes, which are directly activated by the ecdysone receptor and coordinate the subsequent transcription of the tissue-specific "late genes" (ASHBURNER 1974 ; and for reviews see KISS et al. 1988 ; KARIM et al. 1993 ; ZHIMULEV et al. 1995 ; BAYER et al. 1996A ). The BR-C is located at chromosomal region 2B5. Genetically, the BR-C locus has three fully complementing functions: br (broad), rbp (reduced bristle number on palpus), and 2Bc, as well as a noncomplementing npr (nonpupariating) class (Figure 1C; BELYAEVA et al. 1980 ). Additionally, a number of BR-C alleles have been categorized to the 2Bab group. These alleles do not complement br or rbp mutations, but do complement 2Bc mutations (BELYAEVA et al. 1980 ). The nonpupariating mutations are probably null mutations, because alleles in this class fail to complement mutations in each of the three complementing groups. These mutants are also phenotypically indistinguishable from deletions of the locus. It has been shown by genetic analysis that BR-C is essential for the morphogenesis of imaginal discs. br⁺ function is primarily required in the elongation and eversion of appendages from imaginal discs as well as tanning and hardening of the larval cuticle. rbp⁺ function, on the other hand, is essential for muscle and bristle development. Additionally, both rbp⁺ and 2Bc⁺ functions are vital for histolysis of the larval tissues and gut morphogenesis. 2Bc⁺ function was shown to be essential in the fusion of discs to form a continuous adult epidermis (KISS et al. 1988 ). All three functions are also required for the reorganization of the central nervous system (CNS) (KISS et al. 1988 ; EMERY et al. 1994 ).

View larger version (29K): In this window In a new window Download PPT slide   Figure 1. Organization of the BR-C gene. (A) Molecular organization of the BR-C gene, which maps to the 2B5 region (adapted from CHAO and GUILD 1986 ). For simplicity and clarity the upstream half of BR-C, including the first exon, is not shown in the figure. Shaded boxes represent open reading frames; solid boxes represent untranslated regions of the BR-C transcripts. Two putative promoters have been previously described: P distal at nucleotide 120; and P proximal at nucleotides 163, 165, and 167 (DIBELLO et al. 1991 ; BAYER et al. 1996B ). The Z1–Z2 transcripts can be synthesized from either P120 or P165, while Z2 and Z4 initiate only at P165 and P163, respectively (BAYER et al. 1996B ). The linker regions for Z1 and Z3 domains are contiguous in the respective exon. (B) Organization of zinc-finger isoforms. Differentially spliced BR-C transcripts share a common core domain linked to one of the four (Z1–Z4) different pairs of C2H2 zinc-finger domains. The TNT, Q1, and Q2 linker sequences found in Z1 transcripts are contiguous in the Z1 exon, and generate by alternatively splicing three Z1 isoforms (BAYER et al. 1997 ). Arrows indicate the primer pairs used in RT-PCR analysis. (C) Complementation map of the BR-C based on BAYER et al. 1997 .

The BR-C encodes a family of C₂H₂ zinc-finger proteins (Z1, Z2, Z3, and Z4), which share a common aminoterminal (the BR-C "core") domain but differ in zinc-finger domains (DIBELLO et al. 1991 ; BAYER et al. 1996B ). The core contains a highly conserved amino-terminal motif, called the BTB or POZ domain, which appears to be involved in protein-protein interactions and is widely distributed among metazoans (DIBELLO et al. 1991 ; BARDWELL and TREISMAN 1994 ; ZOLLMAN et al. 1994 ). The core is alternatively spliced to one of the four zinc-finger domains (Figure 1), generating four classes of proteins, the Z1, Z2, Z3, and Z4 isoforms. Additionally, three variants of the Z1 isoform have been identified. They differ in the linker region between the core motif and the zinc-finger domain.

Some genetic studies suggest a one-to-one link between the specific complementing genetic functions and protein isoforms. However, other data suggest that the relationships between the complementing groups and protein isoforms are more complicated. For example, in the br²⁸ mutant, Z3 transcripts and protein levels are reduced and all Z1 isoforms are truncated. Clearer data on these relationships were provided by BAYER et al. 1997 , who showed that lethality associated with each of the complementing groups was rescued using heat-inducible transgenes. It was found that br⁺ function is only provided by the Z2 isoform. Despite this, there may be functional redundancy or regulatory dependency associated with rbp⁺ and 2Bc⁺ functions. It was found that Z1 transgenes provide full rbp⁺ function, while Z4 provides partial function. The 2Bc lethality is fully rescued by Z3 protein expression, and partially rescued by Z2 protein expression.

The two clusters of chorion genes on the X-chromosome and third chromosome, which are responsible for the production of large amounts of chorion protein in the follicle cells at very precisely defined points in late oogenesis, are selectively amplified above the level of the remainder of the follicle cell genome, which also endoreplicates to produce polyploid cells (SPRADLING and MAHOWALD 1980 ; ORR-WEAVER 1991 ). The third chromosome cluster is amplified 60- to 80-fold and the X-chromosome group 15- to 20-fold above the rest of the genome. This specific amplification depends on cis-acting sequences among the chorion genes (ORR-WEAVER and SPRADLING 1986 ; DELIDAKIS and KAFATOS 1989 ).

We know that the BR-C expression in the lateral-dorsal-anterior follicle cells during oogenesis is related to its function in dorsal appendage formation. However, we do not know what the early function of the BR-C is, when it is expressed in all follicle cells at stage 6 of oogenesis. Since the chorion genes encode major eggshell components, and rbp⁺ function has been reported to be necessary for chorion gene amplification during oogenesis (ORR et al. 1989 ; HUANG and ORR 1992 ), we investigate in this article the relationship between the BR-C and chorion gene amplification and expression.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila strains:
The following Drosophila melanogaster strains were used: Oregon R, br¹, br⁵, br⁶, rbp¹, rbp², 2Bc¹, 2Bc², npr⁶ (KISS et al. 1988 ); br^A47 (DENG and BOWNES 1997 ); w¹¹¹⁸, hs.Z1 (527-5; 708-1), hs.Z2 (CD5-1), hs.Z3 (797-3), hs.Z4 (Z4-11) (BAYER et al. 1997 ). The rbp² and Br¹ alleles are viable and were maintained as homozygotes. All other BR-C mutations were maintained over Binsn, an X-chromosome balancer carrying the markers Bar and singed. All stocks were maintained on standard cornmeal food at 26°.

Antibody staining of ovaries:
Ovaries were dissected from yeasted flies in Ringer's solution. The anterior parts of the ovaries were torn apart to facilitate antibody penetration. The ovaries were transferred to a microfuge tube containing 2% p-formaldehyde (in 1 x PBS) and fixed for 30 min at room temperature. The fixative was carefully removed and ovaries were washed in 1 ml of PTW [1.5% (v/v) Tween-20 in PBS] for 5 min. Then the ovaries were incubated in 1% (w/v) bovine serum albumin (Sigma, St. Louis) in PTW for 1 hr. Blocking was accomplished by incubation of the ovaries in PTW-NGS [5% (v/v) normal goat serum in PTW] for 2 hr at room temperature. The first antibody was added at 1:200 dilution in PTW and the incubation was carried out overnight at 4°. Residual antibody was washed away with three changes of PTW with 30 min of incubation per change. The HRP-conjugated secondary antibody (Promega, Madison, WI) was then added to the ovaries at 1:500 dilution and incubated for 2 hr at room temperature or overnight at 4°. Excess secondary antibody was removed with three PTW washes at 30-min intervals. Diaminobenzidine (DAB) staining solution (Sigma) was added and the staining was allowed to proceed for 10–30 min before washing with several changes of PBS to stop the reaction. Stained ovaries were mounted in PBS/glycerol (1:4) to allow microscopy.

Hoechst staining:
Ovaries were dissected in PBS and fixed in 4% p-formaldehyde (w/v in 1 x PBS) for 20 min. This was followed by washing in 1 x PBT [1% (v/v) Triton-X100 in PBS] for 30 min. The ovaries were then washed for 30 min in PBS and stained for 5 min in 1 µg/ml Hoechst 33258 (Sigma; dissolved in PBS). After washing in PBS for 2 hr to overnight, the ovaries were mounted in PBS/glycerol (1:4) and examined under a fluorescent microscope.

Preparation of the eggshell for dark-field microscopy:
Freshly laid eggs were collected from the apple juice plate and placed in a drop of Hoyer's mountant (Hoyer's mounting medium:lactic acid = 1:1) on a glass slide and covered by a coverslip. After an overnight incubation at 65° the slides were ready for dark-field microscopy.

RNA extraction and RT-PCR:
The BR-C transcript levels in ovaries were detected by reverse transcriptase (RT)-PCR as described previously (HODGETTS et al. 1995 ). Total RNA from ovaries and larvae (control RNA) was isolated using RNAeasy-Total RNA Kit (QIAGEN, Chatsworth, CA, no. 74104). The RNA (5 µg) was primed with oligo-p(dT)₁₅ and reverse transcribed using Superscript II (Gibco BRL, Gaithersburg, MD) following the supplier's protocol. For the subsequent DNA amplification, 5% of the first-strand reaction mix was used. To amplify each of the zinc-finger domains, appropriate primer pairs were added to the PCR mixture: a common primer for the core domain was combined with one of the four primers for the respective zinc-finger motif. The sequence data for the primers were obtained from DIBELLO et al. 1991 and HODGETTS et al. 1995 : core, 5'-ACAAGATGTTCCATGCAGCC-3'; Z1, 5'-TGCTGGTGCTGCTGGTGATA-3'; Z2, 5'-TCATCTCCATTTCGCCGGGA-3'; Z3, 5'-GATGGCGGTCGTCTTAAGCA-3'; Z4, 5'-GTGGTTGTTCAGCGAGTTCA-3'. In the PCR reaction QIAGEN Taq Polymerase and the protocol designed for use with Q-Solution was used. The PCR reaction was carried out as follows: one cycle at 94° for 4 min; 35 cycles, step one at 94° for 30 sec, step two at 60° for 30 sec, step three at 72° for 1.5 min; one cycle at 72° for 7 min.

BrdU labeling:
Ovaries were dissected at room temperature in 1 x Grace's medium (Flow Laboratories, no. 2700049) and incubated for 1 hr in 15 µM BrdU (Sigma) in Grace's medium (LILLY and SPRADLING 1996 ). After washing in EBR (Ephrussi Beadle Ringer) the ovaries were fixed for 20 min in 37% formaldehyde/buffer B/dH₂O (1:1:4; LIN and SPRADLING 1993 ), followed by 1 hr denaturing in 2 N HCl and neutralizing for 2 min in 100 mM Na tetraborate. The tissue was rinsed several times in PBT (PBS + 0.1% Triton X-100) and blocked for 1 hr in 5% NGS in PBT. After overnight incubation in 1:20 dilution of anti-BrdU antibody (Becton-Dickinson, San Jose, CA, no. 347580) detection was carried out with HRP-conjugated secondary antibody (1:25 dilution). Sigma Fast DAB peroxidase substrate (no. 4168) was used in the peroxidase color reaction. The latter was enhanced with 10 µl 1 M Ni SO₄ per 1 ml staining solution.

In situ hybridization to mRNA in ovaries:
The protocol is based on the procedure previously described (TAUTZ and PFEIFLE 1998 ) and modified as follows. The ovaries were dissected in Ringer's solution and fixed for 20 min in 4% p-formaldehyde in PBS. After rinsing the tissue in PBT it was treated for 10 min in methanol/0.5 M EGTA, pH 8 (9:1). The ovaries can then be stored in methanol at -20° for several months. The stored ovaries were rehydrated in PBT. The prehybridization was carried out for 1 hr at 45° in DNA Hybrix (50% deionized formamide, 5x SSC, 100 µg/ml sonicated salmon sperm DNA, 50 µg/ml Heparin, 0.1% Tween 20). The ovaries were hybridized overnight at 45° in DNA Hybrix containing digoxigenin-labeled probe (DIG DNA labeling and detection kit, Boehringer Mannheim, Indianapolis). For detection a 1:1000 dilution of anti-DIG-AP-conjugated Ab was used. The staining reaction was performed in 100 mM Tris pH 9.5, 50 mM MgCl, 10 mM NaCl, 0.2% Tween 20, 8 mM levamisole, 4.5 µl/ml NBT, and 3.5 µl/ml X-phosphate (Boehringer Mannheim). Anti-DIG-AP conjugate was preabsorbed with postfixed wild-type (Oregon R) ovaries at 4° overnight. The ovaries were mounted in a mixture of PBS/glycerol (1:4) for microscopy.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The BR-C protein distribution pattern during oogenesis:
In a previous article (DENG and BOWNES 1997 ), we reported that BR-C mRNA is expressed in follicle cells in a dynamic pattern. Its expression is first detected in all follicle cells at stage 6. During stage 10A, all the columnar cells, except the dorsal anterior follicle cells, contain the BR-C transcript. However, only two groups of dorsal-lateral-anterior follicle cells express the BR-C mRNA during stage 10B, marking the dorsal appendage secreting cells.

To investigate the function of the BR-C we need to establish whether or not the protein is distributed in a similar pattern to the mRNA during oogenesis. Antibodies that recognize the BR-core, Z1, or Z3 domains, respectively, were used to stain the whole-mount ovaries. Antibodies to the Z2 and Z4 isoforms have not been generated, so we were unable to check their expression pattern. Both the Z1 and BR-core antibodies exhibited similar staining patterns, while the Z3 antibody showed no staining during oogenesis. These observations are consistent with the results shown by RNA in situ hybridization; Z1 is the only zinc-finger isoform with expression at levels significantly high to be detected by in situ hybridization techniques during oogenesis.

The distribution pattern of the BR-C protein appears to be similar to that of its mRNA during stages 6–8 of oogenesis, when all follicle cells stain (Figure 2, A1). The protein is also detected in all columnar follicle cells except the dorsal anterior cells at stage 10 (Figure 2, A1–A3), similar to the pattern of mRNA distribution (DENG and BOWNES 1997 ). However, the follicle cells at the posterior pole appear to be stained at this stage (Figure 2, A1), which differs from the mRNA distribution pattern. The late distribution pattern of the BR-C protein and mRNA differs. A very strong signal is observed in two groups of the lateral-dorsal follicle cells at stages 11 and 12, but the posterior and ventral follicle cells are still stained (Figure 2B). The signal in the posterior and ventral region disappears around late stage 13, leaving only the dorsal-appendage-associated follicle cells stained (Figure 2C and Figure D). The differences between the distribution patterns of the protein and mRNA presumably reflect the fact that the half-life of the protein is much longer than that of the mRNA. By the time late BR-C transcription occurs at the lateral-dorsal-anterior follicle cells, the protein translated from the early BR-C transcripts remains at the posterior and ventral side, while the mRNA has been degraded. Thus, the early and late protein distribution patterns overlap to form a gradient-like pattern at stages 11 and 12. The same reasoning could also be used to explain why the protein, but not the mRNA, is detected in the follicle cells at the posterior pole during stage 10.

View larger version (140K): In this window In a new window Download PPT slide   Figure 2. The distribution pattern of the BR-C protein during oogenesis. (A1) Using BR-core antibody to stain the ovaries, signals are initially detected in all follicle cells at around stage 6 (arrowhead). Staining is also observed in all follicle cells at stage 7. During stage 10, columnar follicle cells over the oocyte are stained. However, staining is not seen at the dorsal anterior region. (A2) A closer look at the dorsal region. The dorsal gap is marked by a double-headed arrow, while the anterior gap is labeled by two curved lines. (A3) A ventral view of the same egg chamber. It appears that all ventral columnar cells are stained. (B) During stage 12, two groups of lateral-dorsal-anterior follicle cells are heavily stained, while the posterior and the ventral follicle cells are weakly stained. The dorsal gap between the two groups of the lateral-dorsal cells still exists (arrow). Expression in the posterior follicle cells becomes gradually weaker (C), until it disappears at approximately stage 13 (D). Expression is only detectable in the dorsal-appendage-associated follicle cells. The arrow shows a growing dorsal appendage.

Another feature of the BR-C protein distribution is that it only appears in the nuclei of the follicle cells (Figure 2), which is consistent with the fact that the BR-C encodes transcription factors.

rbp⁺ function is required for dorsal appendage formation:
The genetic organization of the BR-C is shown in Figure 1. BAYER et al. 1997 reported that Z1 provides the full rbp⁺ function. Since the Z1 zinc-finger isoform is expressed during oogenesis and is detectable by in situ hybridization, it is predicted that the rbp functional domain will be required. To test this, female homozygous viable rbp¹ and rbp² mutants were dissected to examine the eggshell phenotype. It was found that the dorsal appendages were abnormal, being shorter and rougher than the wild type, and the eggshells were much more fragile (Figure 3). Shortening of the dorsal appendages was observed in rbp² homozygous mutants.

View larger version (67K): In this window In a new window Download PPT slide   Figure 3. Chorion defects in rbp mutants. (A) Wild-type chorionic appendages. (B) Appendages that are shorter and irregular in shape, produced by rbp1 females.

To test if rbp is the major functional domain involved in dorsal appendage formation we did a genetic analysis using the npr allele in crosses with br, rbp, and 2Bc alleles (Table 1). The cross between rbp¹ and npr⁶ produced only two males and no viable female heteroallelic mutants. Two rbp²/npr⁶ females were produced in the cross with rbp² flies. They lived for 2 days without laying any eggs. Then they were dissected to examine the ovarian phenotype. It was found that the ovaries were not completely developed and the few late stage oocytes formed had no appendages. Thus we were unable to examine rbp function by this method. The combination 2Bc/npr⁶ was found to be completely lethal, and as a result we could not establish if there is a function encoded by the Z3 (2Bc) isoform.

The function of Z2 was investigated by further genetic crosses. The cross between br⁵ and npr⁶ produced no viable heteroallelic flies, while the cross between br⁶ and npr⁶ generated 22 heteroallelic males, but no females that could be examined. It was observed that eggs produced by br¹/br^A47 and br¹/npr⁶ mothers have reduced dorsal appendages (DENG and BOWNES 1997 ), suggesting that the br functional domain is likely to be required for dorsal appendage formation. To test this hypothesis, eggs produced by br⁵/br¹ and br⁶/br¹ females were examined, and found to have normal dorsal appendages (data not shown). This observation, along with the fact that the br^del2 is actually an rbp allele (HUANG and ORR 1992 ), suggests that the br functional domain, and hence Z2, is not involved in dorsal appendage formation.

How can the phenotype of br¹/br^A47 and br¹/npr⁶ eggshells be explained if br is not the functional domain required for dorsal appendage formation? This could be understood if the br¹ mutant not only affected br function, but also affected rbp function. To test this possibility, the eggshell phenotype of eggs laid from br¹/rbp¹ mutants was examined. It was shown that eggs produced by the br¹/rbp¹ mothers have reduced dorsal appendages, similar to those produced by the br¹/br^A47 females. This indicates, therefore, that the br¹ is in fact a weak 2Bc or 2Bab allele, which fails to complement either rbp or br function. This suggests that rbp (which encodes Z1 and Z4) is a functional domain involved in dorsal appendage formation during oogenesis; however, we cannot rule out the involvement of Z3 from these experiments due to the failure of these crosses to generate adult females due to early lethality.

Ectopic BR-C expression induces ectopic dorsal appendage material:
Is BR-C function sufficient to direct the formation of the dorsal appendages? To address this, heat-inducible Z1 transgenic flies (hsp70/Z1) were used to examine the effect of ectopic BR-C expression during oogenesis. Following standard heatshock (37°, 30 min) and incubation at 26° for 2–48 hr, ovaries of the hsp70/Z1 females were analyzed (Table 2). Flies were dissected to examine the effect of the heatshock at 2, 3, 5, 9, 24, and 48 hr after heat treatment. The first abnormalities in the egg chambers were observed 3 hr after the heatshock. The eggs laid during the first 3 hr following heatshock also have a very high hatch rate, presumably being sufficiently differentiated at the time of the heatshock for ectopic BR-C expression to have no effect. The results over this period do not differ significantly for the control heatshocks (Table 2). The strongest effect was observed between 4 and 6 hr. It was observed that extra dorsal appendage material was produced in the dorsal-anterior region of the eggshells (Figure 4, B–F). In most cases, dorsal appendage material appeared in the dorsal gap between the two appendages. It condensed at the base of the dorsal appendages and less material was deposited in the appendages themselves. The dorsal appendages did not elongate properly (Figure 4, B–D), presumably due to a failed migration of the follicle cells. Different phenotypes have been observed, depending on the stage of the egg chamber at the time of heatshock (Figure 4 and Figure 5). Heatshock at the time of dorsal appendage formation, stage 11, leads to the "appendageless" phenotype or to small fused appendages (Figure 4B and Figure C). Appendages with abnormal shapes and/or different lengths have been observed on eggs at stages 12 and 13 at the time of heatshock (Figure 4, D–F). These observations indicate that ectopic Z1 can induce formation of ectopic dorsal appendage material. Nevertheless, the ectopic dorsal appendage material is restricted to the dorsal anterior eggshell, suggesting that the fate of the follicle cells is predetermined along the two major axes prior to the requirement for BR-C function in this process. It was hypothesized that the lack of BR-C expression in the dorsal-most follicle cells is due to high levels of expression of pointed (pnt) in those cells (DENG and BOWNES 1997 ). The data shown here indirectly support this hypothesis. In the dorsalmost follicle cells, there could be competition between the expression of Pnt and BR-C. When high levels of Pnt are expressed, BR-C expression is inhibited in these cells. However, in heatshock lines, the BR-C expression would overcome the inhibition by Pnt. Thus, dorsal appendage material can be synthesized by these cells.

View larger version (79K): In this window In a new window Download PPT slide   Figure 4. Ectopic expression of different BR-C zinc-finger isoforms during oogenesis. A–F, dark-field microscopy. (A) A wild-type egg after heatshock treatment. (B and C) Two distinctive hsp70/Z1 (527-5 and 708-1) transgenic lines exhibit a similar phenotype. Dorsal appendage materials are present in the dorsalmost region, resulting in fused, thickened, and shortened dorsal appendages. (D–F) A similar, but less severe, eggshell phenotype in hsp70/Z1 flies; see explanation in the text. Fused, thickened, and irregularly shaped appendages are also found in eggshells produced by the Z2, Z3, and Z4 transgenic lines.

View larger version (95K): In this window In a new window Download PPT slide   Figure 5. Ectopic BR-C expression affects the onset of chorion synthesis. A–D, Hoechst staining; C1–D1, dark-field microscopy. (A) A wild-type egg chamber at late-stage 11. The oocyte is larger than the nurse cell complex due to the onset of nurse cell dumping into the oocyte. (B) Wild-type egg chamber, late stage 12. Dumping is complete. C–C1 (late stage 11) and D–D1 (late stage 12) show that transgenic Z1–Z4 also causes inappropriate chorion synthesis (arrows), and this blocks dumping of the nurse cell cytoplasm into the oocyte during stage 11–12 of oogenesis.

Although Z1 seemed to be the sole BR-C zinc-finger isoform expressed at high levels during oogenesis when analyzed by in situ hybridization, we tested Z2, Z3, and Z4 to determine if they exhibit a similar phenotype when ectopically expressed during oogenesis. Thus, hsp70/Z2, hsp70/Z3, and hsp70/Z4 flies were heatshocked and the eggshell phenotype was examined (Table 2). It was found that ectopic dorsal appendage material is produced in the dorsal-anterior region of the eggshells by all three transgenic lines. This phenotype is similar to that exhibited by eggs of the hsp70/Z1 flies after heatshock, suggesting that all of the four zinc-finger isoforms could be functional in dorsal appendage formation during oogenesis.

It is apparent from Table 2 that heatshock has the strongest effect on chorion morphology and egg viability in hsp70/Z1 flies. Z2–Z4 recovered viability to ~95% in 2 days, while Z1 recovered only to 72% during that time period. It was also found that heatshocked Z1, Z2, and Z4 flies lay abnormal eggs (Table 2). The ectopic expression of BR-C in Z3 flies was found to disrupt the process of egg development soon after the heatshock. We observed that some 20% of all laid eggs have aberrant micropyles, due to excess chorion formation. This could prevent the sperm entering the egg and hence subsequent development would fail due to lack of fertilization. Another possible explanation is that ectopic expression of BR-C can disrupt some other Bric-a-brac/Tramtrack/Broad Complex (BTB)-containing protein that can dimerize with the BR-C and thus modulate its function.

Heatshock alone causes eggshell defects. The data of the control experiments with the heatshocked wild-type OrR flies and w¹¹¹⁸, the host line for the transgenic flies, is presented in Table 2. We observed in the few abnormal eggs wide-branched dorsal appendages of approximately normal length. It is quite clear that the results of misexpressing BR-C in oogenesis significantly affects the eggshell.

Other zinc-finger isoforms are expressed in oogenesis:
Although only Z1 expression was clearly observed by in situ hybridization we observed defects in chorion formation and morphology by overexpressing all four zinc-finger isoforms available. It became essential, therefore, to establish if this was due to some degree of functional redundancy between the isoforms with respect to eggshell development or if the other zinc fingers are, in fact, expressed at lower levels in oogenesis. To check this we used RT-PCR using primers for Z1, Z2, Z3, and Z4 and the core DNA binding domain. The organization of the zinc-finger isoforms in relation to the BR-C is shown in Figure 1. The primers used are illustrated in Figure 1B and should generate products of 974 bp, 780 bp, 728 bp (Z1); 320 bp (Z2); 784 bp (Z3); and 1082 bp (Z4), respectively, based on published data (HODGETTS et al. 1995 ; BAYER et al. 1996B ). The results clearly show that all four zinc fingers are expressed in oogenesis (Figure 6). The identity of the PCR-generated products was confirmed with Southern blot analysis.

View larger version (77K): In this window In a new window Download PPT slide   Figure 6. PCR analysis of the BR-C transcripts in Drosophila melanogaster (wild-type, Oregon R) ovaries. Total RNA from ovaries was reverse transcribed and the subsequent cDNA PCR amplified using the primer pairs shown with arrows below the exon map (Figure 1B). All four PCR reactions were carried out with a common primer located at the 3' end of the core domain of BR-C. The other primers are located just within, or immediately 3' to, the respective Z1–Z4 domain. M, marker–1-kb ladder (Gibco BRL); Ov, ovaries; L, larvae (whole organism) used as a control. The primer sets generate the following products from transcribed larvae RNA: 974 bp, 780 bp, 728bp (Z1); 320bp (Z2); 784bp (Z3); 1082 bp (Z4). The PCR does not generate the 728-bp Z1 product from transcribed ovarian RNA, indicating the lack of this transcript during oogenesis.

It seems likely therefore that, as in metamorphosis, all zinc-finger isoforms are expressed and function to regulate downstream gene expression. However, only Z1 is expressed at a high-enough level to detect the spatial distribution of the RNA and protein in oogenesis.

Ectopic BR-C expression during mid-oogenesis affects endoreplication and chorion gene amplification:
Ectopic BR-C expression appears to induce premature production of the chorion. Figure 5C and Figure D, shows that the chorion is already present in stage-11 egg chamber. This could isolate the oocyte from the nurse cells and physically prevent dumping of the nurse cell components into the oocyte. This could result from an altered pattern of transcription and translation of the chorion genes, or from abnormalities in amplification of the chorion genes, or both. We investigated, therefore, whether the alterations in BR-C expression affected the timing or pattern of chorion gene amplification. Since the chorion is synthesized by most follicle cells, this function could be related to the earlier expression of the BR-C. To monitor amplification we investigated the incorporation of BrdU in the follicle cell nuclei of wild-type ovaries and in ovaries misexpressing various isoforms of the BR-C.

In wild-type ovaries, after eight mitotic cell divisions, the endoreplication phase of the follicle cell development of oogenesis begins (stage 6), and is completed by stage 10B; during this process the entire nucleus is labeled by BrdU. The endoreplication is asynchronous in wild-type and w¹¹¹⁸ (the host strain used to produce the transgenic lines) ovaries and occurs in both nurse cells and follicle cells (Figure 7A and Figure C). We observed a continuous endoreplication in the nurse-cell-associated follicle cells at stage 10B (Figure 7C). This is followed by the chorion gene amplification phase when 4 spots of incorporation are seen per nucleus in the follicle cells overlying the oocyte (Figure 7C, Figure G, and Figure K). These 4 spots represent amplification of the two clusters of chorion genes (Figure 7G and Figure K). Two are always larger, presumably due to the higher level of amplification of the cluster on chromosome 3 compared to the X-chromosome cluster (ORR-WEAVER and SPRADLING 1986 ; DELIDAKIS and KAFATOS 1989 ). This amplification was first observed at the border between the oocyte and nurse cells and it soon spread to the rest of the follicle cells. When the BR-C isoforms are misexpressed, there is prolonged and synchronized endoreplication until late stage 10B, followed by specific amplification of genes in each nucleus (Figure 7D, Figure F, Figure H, and Figure L). These results are observed from 3.5 to 4.5 hr after heatshock. We also observe extra spots of incorporated BrdU in the nuclei (Figure 7H and Figure L). There were three possible explanations for this: either the heatshock could be responsible, the homologues of the chromosomes could have separated due to a defect in the cell cycle, or there could be amplification of DNA at additional sites in the genome. The host flies used to produce the transgenic lines, w¹¹¹⁸, were heatshocked and still showed 4 spots per nucleus, so heatshock itself was not responsible for the results. We counted the number of spots per nucleus and found 6 or 12 spots in ~80% of the nuclei. Occasionally we observed up to 28 spots. If the cell cycle was affected, and the homologues had separated, we would expect to see many more than 28 spots per nucleus due to the polyploidy of the follicle cells. If the amplification sites varied we could not predict the numbers, and indeed it may well be variable. This suggests that there are other sites in the genome induced to replicate by BR-C overexpression. We conclude that the endoreplication of DNA and the amplification of the chorion genes depends upon the BR-C encoded proteins or an unknown protein that is encoded by one of the downstream targets of the BR-C. This observation is consistent with the report that a mutation in the BR-C locus causes premature arrest of chorion gene amplification (HUANG and ORR 1992 ).

View larger version (106K): In this window In a new window Download PPT slide   Figure 7. Chorion gene amplification. BrdU incorporation associated with endoreplication and amplification. (A) BrdU incorporation in the nuclei of wild-type Drosophila (Oregon R) ovaries and w1118, the host strain used for the production of the BR-C transgenic lines at stage 10A. The endocycles are not synchronized. Thus, just some of the nuclei are positive. (CALVI et al. 1998 ). (B) Overexpression of a BR-C (Z1) transgene in ovaries at stage 10A. Most of the nuclei show synchronized endoreplication. (C) Normal synchronized amplification in wild type and w1118 at stage 10B. By that time the endoreplication associated with the main body follicle cells is completed. (D) BrdU incorporation in the nuclei of heatshocked BR-C (Z1) transgenic ovaries, stage 10B. Strong BrdU incorporation is present in the nurse cells. (G) Higher magnification of wild-type and w1118 nuclei at stage 10B to show the four spots of amplification normally occurring in wild-type ovaries. (H) BrdU incorporation in the nuclei of BR-C (Z1) expressing ovaries 3 hr after the heatshock, stage 10B. Some of the nuclei contain extra spots of replication. The amplification pattern overlaps with the labeling due to continued endoreplication. (E–K) The nuclei of heatshocked w1118 ovaries still show four spots of amplification. (F–L) The nuclei of BR-C (Z1) transgenic ovaries 4.5 hr after heatshock exhibit a multispotted pattern.

Ectopic BR-C expression in relation to chorion gene expression:
The chorion is produced by the columnar follicle cells to provide a shell around the egg. Later in oogenesis, two groups of cells migrate anteriorly to produce the chorionic appendages and very large amounts of chorion material. Ectopic expression of the Z1 isoforms leads to chorionic appendage deposition by extra cells lying at the anterior of the egg filling in the middorsal gap observed in wild-type eggs. Often the follicle cells fail to migrate anteriorly over the remaining nurse cells at stage 11 and they are present, therefore, at a more posterior position. The pattern of chorion gene expression was compared in wild-type ovaries and in those expressing the Z1 isoform ectopically. In the wild type, we observe a high concentration of chorion transcripts in all follicle cells at stage 9, prior to their translation; they then become inactive and transcripts are again seen in stages 11–14 (Figure 8A, Figure C, Figure E, and Figure G). In the ovaries with ectopic BR-C gene expression, examined 3.5–4.5 hr after the heatshock, the same high concentration of chorion transcripts is observed in anterior follicle cells, even though their location in relation to the nurse cells is more posterior. This suggests the expression pattern is not dependent on the BR-C along the anterior-posterior axis (Figure 8B, Figure D, Figure F, and Figure H). Moreover, posterior follicle cells do not produce substantially more chorion material even though they express Z1 protein after heatshock. The fact that more dorsal cells produce appendage material and express the chorion genes means that BR-C expression in the most dorsal anterior cells does induce additional chorion production. Thus we observed two different effects. Initially the ectopic expression of BR-C prevents the migration of the dorsal follicle cells in an anterior direction. Then the midline cells that normally express chorion protein at that stage start depositing chorion material in the wrong location. This results in the production of aberrant dorsal appendages. It is possible that the BR-C activates downstream genes which, in turn, activate the chorion genes. In anterior cells high levels of BR-C expression "win" over trans-acting repressors, but in posterior cells they do not. The in situ hybridization results and the observed characteristic phenotypes following ectopic BR-C expression are both consistent with this observation. Alternatively, BR-C could be essential for the cell migrations to position the follicle cells and endoreplication of the chorion genes but not in regulating chorion gene expression.

View larger version (100K): In this window In a new window Download PPT slide   Figure 8. In situ hybridization to chorion RNA in ovaries. (A, C, E, G) In situ hybridization to wild-type (Oregon R) Drosophila ovaries with a DIG-labeled probe hybridizing to chorion mRNA. The chorion probe, 7C8, was generated from cDNA encoding the c38 chorion protein, which maps to the X chromosome. Similar results were obtained using the alternative 7B7 probe (cDNA encoding c15 protein), which maps to a site on the third chromosome. For more information about the probes see SPRADLING et al. 1980 . (B) In situ hybridization to heatshocked Drosophila BR-C (Z1) transgenic ovaries with 7C8 probe, stage 11. At that stage an increase in the amount of chorion mRNA is observed at the border between the nurse cells and the oocyte. The misexpression of the BR-C leads to abnormal migration of the anterior follicle cells. (D) In situ pattern of chorion mRNA expression in heatshocked BR-C (Z1) transgenic ovaries, stage 12. A gap (arrows) is formed at the anterior pole as a result of failed migration of the follicle cells. (F) Strong expression of the chorion gene clusters in heatshocked BR-C (Z1) transgenic ovaries at stage 13. The variations in the egg chamber shape result from the heatshock-induced misexpression of BR-C. (H) Strong continuous expression of chorion mRNA in heatshocked BR-C (Z1) transgenic ovaries, stage 14. The anterior gap is not present because the nurse cells have completed dumping and the follicle cells have migrated correctly by the time of heatshock.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The BR-C complementing groups and zinc-finger isoforms:
It has been shown by genetic analysis that rbp⁺ function is required for dorsal appendage formation, and it was observed that Z1 is the sole zinc-finger isoform expressed at high levels in the appendage-producing cells. These observations are compatible with the report that Z1 provides rbp⁺ function (BAYER et al. 1997 ). It was also found that rbp⁺ function is partially provided by Z4, but is not provided by Z2 and Z3 (BAYER et al. 1997 ). However, heatshock-induced expression of all four zinc-finger isoforms (Z1, Z2, Z3, and Z4) leads to a similar phenotype of extra dorsal appendage material production in the dorsal gap, indicating that the different transcripts may substitute for each other functionally in dorsal appendage formation.

The homozygous viable mutant br¹was the first mutant identified in the BR-C locus. It exhibits a broad wing phenotype and fails to complement other mutations that are categorized in the br complementation group. However, the complementation analysis presented in this article suggests that the br¹ mutations also partially remove rbp function. Therefore, it is in fact a 2Bab allele. It is known the 2Bab mutations cause reduction of both Z1 and Z2 expression. Thus in the br¹/br^A47 and br¹/npr⁶ females, both Z1 and Z2 are reduced. The reduction of Z1 levels results in the reduction of dorsal appendages, while no effect is produced by the reduction of Z2 levels. This is why no defects were observed in eggs produced by br¹/br⁵ and br¹/br⁶ females.

The mutant phenotypes clearly show the need for the BR-C in chorionic appendage formation. PCR experiments have shown that all zinc-finger isoforms are in fact expressed in oogenesis, but as yet we have no evidence that they perform different functions. Neither do we know the spatial and temporal distribution of Z2–Z4, which are not present at sufficiently high levels for detection by in situ hybridization. Overexpression studies using transgenic flies carrying heatshock-controlled Z1 and Z4 isoforms lead a failure of proper migration of the follicle cells that will secrete the appendages, premature chorion deposition, and abnormal appendage formation.

Relationship between the BR-C and chorion production:
We have shown here that the BR-C is important for controlled DNA replication in oogenesis. Overexpression does not affect the timing of the onset of endoreplication and amplification, but endoreplication is prolonged beyond that observed in wild-type ovaries and it leads to additional replication sites in the genome. These additional sites presumably share sequence similarities with the cis-acting sites regulating chorion gene amplification. This suggests that the BR-C is a key regulator of endoreplication and chorion gene amplification. The early BR-C expression pattern is in all the follicle cells and it is presumably at this stage that it is involved in this function. The expression of the BR-C is first observed in wild-type flies at stage 6 and it is also at stage 6 that the endoreplication cycles begin. Since we did not observe premature endoreplication with BR-C overexpression, presumably other components essential for endoreplication are absent until stage 6 of oogenesis. The active role of the BR-C in endoreplication is also apparent from the fact that we observed prolonged incorporation of BrdU in the nurse cell nuclei when the BR-C is overexpressed. This presumably results in expression of the BR-C in the nurse cells, where it is normally not expressed. This shows that the proteins encoded by the BR-C can function to prolong replication of DNA even in cells where it is not normally used to control this process. It also suggests that an alternative regulator for DNA replication to the BR-C is used in the nurse cells. In normal development, the later BR-C expression, which is maintained in the dorsal-anterior cells making the appendages, is probably needed for cell migration, chorion deposition, and other follicle cell differentiation events.

It is possible that the initial activation of the BR-C in all follicle cells is regulated by the ecdysone/USP heterodimer (YAO et al. 1992 ; HORNER et al. 1995 ) as is observed in metamorphosis. There is also some evidence that in intermoult puffs a second heterodimer with DHR38 (a nuclear receptor related to NGF1-B from mammals) can compete for the same binding sites (CRISPI et al. 1998 ), so it is also possible that other DNA binding proteins, besides the ecdysone receptor, will be involved in BR-C expression. This would suggest a role for ecdysone during these stages of oogenesis. Later, the BR-C could be repressed in specific follicle cells by competitive and cooperative interactions with other gene products initiated by the grk and dpp signaling pathways. There is a significant amount of evidence that ecdysone and juvenile hormone [which binds to ultra spiracle proteins (USP)] are important for the progress of oogenesis (WILSON 1982 ; BOWNES 1989 , BOWNES 1994 ) and we have recently shown that there is a control point in oogenesis that regulates whether egg chambers will proceed with development or undergo apoptosis, which is regulated by the balance of juvenile hormone and ecdysone (SOLLER et al. 1999 ). However, there is little evidence as to precisely what role these hormones have in regulating oocyte development and egg chamber differentiation. We have shown that the ecdysone receptor is present in the follicle cells at the time BR-C is activated (D. MAUCHLINE, W.-M. DENG and M. BOWNES, unpublished results) by antibody staining.

Once activated, as we have shown, the BR-C gene is involved in endoreplication, the selective amplification of the chorion genes, and in the subsequent morphogenesis of the chorionic appendages. CALVI et al. 1998 have recently shown that the selective amplification of the chorion genes is closely linked with the cell cycle and the cycles of endoreplication that occur in the follicle cells earlier. Somehow the chorion genes escape the rereplication controls that influence other parts of the genome. Our BrdU labeling experiments confirm their results on the timing of endoreplication and chorion amplification and the close association between endoreplication and selective amplification. Using overexpression of the BR-C we see not only the two extra sites they mentioned that may represent another chorion gene amplified for a function in later oogenesis, but also a number of additional sites. These may be sites with sequence similarity to the cis-acting sites regulating amplification. CALVI et al. 1998 propose that there are amplification complexes located at chorion genes. Whether the BR-C encoded proteins are associated with these complexes or regulate the synthesis of one or more of their components remains to be elucidated. We have confirmed the link between endoreplication and chorion amplification and shown that it involves the BR-C. This may therefore provide a crucial link between hormones and the control of the cell cycle, and hence of differentiation, of the egg chamber during oogenesis.

In summary, our working model would be that the BR-C is activated by ecdysone in all follicle cells at stage 6 of oogenesis where its key function is the control of endoreplication, and then selective amplification. Later, when it is turned off in all but the anterior-dorsal follicle cells that will secrete the appendages it has a second set of functions and is involved in the migration of cells and morphogenesis of the chorionic appendages. Recently this link between ecdysone, the BR-C, and morphogenesis has also been described for the progression of the furrow in the developing eye imaginal disc of Drosophila (BRENNAN et al. 1998 ).

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195.
³ Present address: Genetisches Institut der Justus-Liebig-Universitaet, Heinrich-Buff-Ring 58-62, D-35392, Giessen, Germany.

	ACKNOWLEDGMENTS

We thank C. Bayer and J. Fristrom for sending us heatshock BR-C flies and BR-C alleles, L. Restifo for rpb² fly stocks, Gregory Guild for sending anti-BR-C core and Z1 and Z3 antibodies, Alan Spradling for chorion gene probes, and Brian Calvi for help with developing the BrdU labeling techniques. We also thank D. Zhao and other colleagues in M. Bownes' lab for helpful discussions and Sheila Milne for typing the manuscript. W.-M.D. and G.T. were supported by Darwin Trust scholarships. This research was supported by the Wellcome Trust.

Manuscript received May 7, 1999; Accepted for publication July 30, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ASHBURNER, M., 1974  Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibitors of protein synthesis. Dev. Biol. 39:141-157[Medline].

BARDWELL, V. J. and R. TREISMAN, 1994  The POZ domain: a conserved protein-protein interaction motif. Genes Dev. 8:1664-1677[Abstract/Free Full Text].

BAYER, C., L. VON KALM and J. W. FRISTROM, 1996a Gene regulation in imaginal disc and salivary gland development during Drosophila metamorphosis, pp. 321–361 in Metamorphosis. Academic Press, San Diego.

BAYER, C. A., B. HOLLEY, and J. W. FRISTROM, 1996b  A switch in broad-complex zinc-finger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs. Dev. Biol. 177:1-14[Medline].

BAYER, C. A., L. VON KALM, and J. W. FRISTROM, 1997  Relationships between protein isoforms and genetic functions demonstrate functional redundancy at Broad-Complex during Drosophila metamorphosis. Dev. Biol. 187:267-282[Medline].

BELYAEVA, E. S., M. G. AIZENZON, V. F. SEMESHIN, I. KISS, and K. KOCZYA et al., 1980  Cytogenetic analysis of the 2B3/4-2B11 region of the X-chromosome of Drosophila melanogaster. I. Cytology of the region and mutant complementation groups. Chromosoma 81:281-306[Medline].

BOWNES, M., 1989  The roles of juvenile hormone ecdysone and the ovary in the control of Drosophila vitallogenesis. J. Insect Physiol. 35:409-414.

BOWNES, M., 1994  The regulation of the yolk protein genes, a family of sex differentiation genes in Drosophila melanogaster.. Bioessays 16:745-752[Medline].

BRENNAN, C. A., M. ASHBURNER, and K. MOSES, 1998  Ecdysone pathway is required for furrow progression in the developing Drosophila eye. Development 125:2653-2664[Abstract].

CALVI, B. R., M. A. LILLY, and A. C. SPRADLING, 1998  Cell cycle control of chorion gene amplification. Genes Dev. 12:734-744[Abstract/Free Full Text].

CHAO, A. T. and G. M. GUILD, 1986  Molecular analysis of the ecdysterone-inducible 2B5 `early' puff in Drosophila melanogaster.. EMBO J. 5:143-150[Medline].

CRISPI, S., E. GIORDANO, P. P. D'AVINO, and M. FURIA, 1998  Cross-talking among Drosophila nuclear receptors at the promiscuous response element of the ng-1 and ng-2 intermolt genes. J. Mol. Biol. 275:561-574[Medline].

DELIDAKIS, C. and F. C. KAFATOS, 1989  Amplification enhancers and replication origins in the autosomal chorion gene cluster of Drosophila. EMBO J. 8:891-901[Medline].

DENG, W.-M. and M. BOWNES, 1997  Two signalling pathways specify localised expression of the Broad-Complex in Drosophila eggshell patterning and morphogenesis. Development 124:4639-4647[Abstract].

DIBELLO, P. R., D. A. WITHERS, J. W. FRISTROM, and G. M. GUILD, 1991  The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics 129:385-397[Abstract].

EMERY, I. F., V. BEDIAN, and G. M. GUILD, 1994  Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis. Development 120:3275-3287[Abstract].

HODGETTS, R. B., W. C. CLARK, S. L. O'KEEFE, M. SCHOULS, and K. CROSSGROVE et al., 1995  Hormonal induction of Dopa decarboxylase in the epidermis of Drosophila is mediated by the Broad-Complex.. Development 121:3913-3922[Abstract].

HORNER, M. A., T. CHEN, and C. S. THUMMEL, 1995  Ecdysteroid regulation and DNA binding properties of Drosophila nuclear hormone receptor superfamily members. Dev. Biol. 168:490-502[Medline].

HUANG, R. Y. and W. C. ORR, 1992  Broad-Complex function during oogenesis in Drosophila melanogaster.. Dev. Genet. 13:277-288[Medline].

KARIM, F. D., G. M. GUILD, and C. S. THUMMEL, 1993  The Drosophila Broad-Complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis. Development 118:977-988[Abstract].

KISS, I., A. H. BEATON, J. TARDIFF, D. FRISTROM, and J. W. FRISTROM, 1988  Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster.. Genetics 118:247-259[Abstract/Free Full Text].

LILLY, M. A. and A. C. SPRADLING, 1996  The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev. 10:2514-2526[Abstract/Free Full Text].

LIN, H. and A. C. SPRADLING, 1993  Germline stem cell division and egg chamber development in transplanted Drosophila germaria. Dev. Biol. 159:140-152[Medline].

ORR, W. C., V. K. GALANOPOULOS, C. P. ROMANO, and F. C. KAFATOS, 1989  A female sterile screen of the Drosophila melanogaster X chromosome using hybrid disgenesis: identification and characterization of egg morphology mutants. Genetics 122:847-858[Abstract/Free Full Text].

ORR-WEAVER, T. L., 1991  Drosophila chorion genes: cracking the eggshell's secretes. Bioessays 13:97-105[Medline].

ORR-WEAVER, T. L. and A. C. SPRADLING, 1986  Drosophila chorion gene amplification requires an upstream region regulating s 18 transcription. Mol. Cell. Biol. 6:4624-4633[Abstract/Free Full Text].

SOLLER, M., M. BOWNES, and E. KUBLI, 1999  Control of oocyte maturation in Drosophila. Dev. Biol. 208:337-351[Medline].

SPRADLING, A. C., 1993 Developmental genetics of oogenesis, pp. 1–70 in The Development of Drosophila melanogaster, Vol. 1, edited by M. BATE and A. M. ARIAS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SPRADLING, A. C. and A. P. MAHOWALD, 1980  Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster.. Proc. Natl. Acad. Sci. USA 77:1096-1100[Abstract/Free Full Text].

SPRADLING, A. C., M. E. DIGAN, and A. P. MAHOWALD, 1980  Two clusters of genes for major chorion proteins of Drosophila melanogaster.. Cell 19:905-914[Medline].

TAUTZ, D. and C. PFEIFLE, 1998  A nonradioactive in-situ hybridisation method for the localisation of specific RNAs in Drosophila embryos reveals a translational control of the segmentation gene hunchback.. Chromosoma 98:81-85.

WILSON, T. G., 1982 A correlation between juvenile hormone deficiency and vitellogenic oocyte degeneration in Drosophila melanogaster. Wilhem Roux's Arch. Entw. Mec. Org. 1919: 257–263.

YAO, T-P., W. A. SEGRAVES, A. E. ORO, M. MCKEOWN, and R. M. EVANS, 1992  Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell 71:63-72[Medline].

ZHIMULEV, I. F., E. S. BELYAEVA, O. M. MAZINA, and M. L. BALASOV, 1995  Structure and expression of the br-c locus in Drosophila melanogaster (Diptera: Drosophilidae). Eur. J. Entomol. 92:263-270.

ZOLLMAN, S., D. GODT, G. G. PRIVE, J. L. COUDERC, and F. A. LASKI, 1994  The BTB domain, found primarily in zinc-finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl. Acad. Sci. USA 91:10717-10721[Abstract/Free Full Text].

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