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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Dev Biol. 2014 May 9;392(1):52–61. doi: 10.1016/j.ydbio.2014.04.024

Regulation of broad by the Notch pathway affects timing of follicle cell development

Dongyu Jia 1, Yoichiro Tamori 1, George Pyrowolakis 2,3, Wu-Min Deng 1,*
PMCID: PMC4296560  NIHMSID: NIHMS593928  PMID: 24815210

Abstract

During Drosophila oogenesis, activation of Notch signaling in the follicular epithelium (FE) around stage 6 of oogenesis is essential for entry into the endocycle and a series of other changes such as cell differentiation and migration of subsets of the follicle cells. Notch induces the expression of zinc finger protein Hindsight and suppresses homeodomain protein Cut to regulate the mitotic/endocycle (ME) switch. Here we report that broad (br), encoding a small group of zinc-finger transcription factors resulting from alternative splicing, is a transcriptional target of Notch nuclear effector Suppressor of Hairless (Su(H)). The early pattern of Br in the FE, uniformly expressed except in the polar cells, is established by Notch signaling around stage 6, through the binding of Su(H) to the br early enhancer (brE) region. Mutation of the Su(H) binding site leads to a significant reduction of brE reporter expression in follicle cells undergoing the endocycle. Chromatin immunoprecipitation results further confirm Su(H) binding to the br early enhancer. Consistent with its expression in follicle cells during midoogenesis, loss of br function results in a delayed entry into the endocycle. Our findings suggest an important role of br in the timing of follicle cell development, and its transcriptional regulation by the Notch pathway.

Keywords: endocycle, mitotic cycle, Notch, broad

Introduction

The Notch signaling pathway has a wide range of functions in many different tissues in metazoan development (Cummings and Cronmiller, 1994; Klusza and Deng, 2011). The Drosophila egg chamber provides an excellent model for studying Notch signaling in development. Each egg chamber, the developmental unit of oogenesis, consists of sixteen germ-line cells-one oocyte and fifteen nurse cells-covered by a single layer of somatic follicle cells (Spradling, 1993). The Notch pathway mediates crucial interactions between the germ line and follicle cells to coordinate their development (Ruohola et al., 1991; Xu et al., 1992). An important function of Notch in oogenesis is to control the switches of different cell cycle programs in the follicle cells. First, its activation by germ-line-expressed Delta (Dl) induces a transition from the mitotic cycle (stages1-6) to three rounds of the endocycle (stages 7-10a). Next, its downregulation is necessary for transitioning into gene amplification (stages10b-13), through which specific genomic regions are selectively amplified, mostly for chorion (egg shell) production (Calvi et al., 1998; Deng et al., 2001; López-Schier and Johnston, 2001; Sun et al, 2008).

During the ME switch, the germ-line-expressed ligand Dl binds to the Notch (N) receptor in the FE, resulting in proteolytic cleavage of Notch by the gamma-secretase complex, which releases the intracellular domain of Notch (NICD) to be translocated into the nucleus (Deng et al., 2001; López-Schier and Johnston, 2001). Inside the nucleus, NICD interacts with the CBF1/Suppressor of Hairless/LAG-1 (CSL) transcription factor (Suppressor of Hairless [Su(H)] in Drosophila), turning it from a repressor into an activator, to induce the transcription of downstream genes (Deng et al., 2001; López-Schier and Johnston, 2001, 2002; Andersson et al, 2011). Acting downstream of Notch signaling during the ME switch are two transcription factors, Cut and Hindsight (Hnt) (Sun and Deng, 2005; 2007). Zinc finger protein Hnt is upregulated uniformly in main-body follicle cells by Notch signaling. It suppresses String, a G2/M transition regulator, thus preventing follicle cells from going into M phase. Cut, a homeodomain protein, is expressed during stages 1-6 to maintain the follicle cells in the mitotic cycle. Its downregulation by Notch signaling through Hnt is necessary for the follicle cells to enter the endocycle (Sun and Deng, 2005; 2007).

Here, we report the identification of broad (br) as a transcriptional target of Notch signaling in follicle cells during midoogenesis. br, alternatively spliced to produce four different isoforms of zinc-finger transcription factors (Br-Z1-Z4), has been known as an early ecdysone response gene that is essential for metamorphosis (Ashburner, 1974; DiBello et al., 1991). During late oogenesis, Br function is critical for dorsal chorionic appendage formation (Deng and Bownes, 1997; Tzolovsky et al., 1999; Ward et al., 2006). Two non-overlapping enhancers, brE (br-Early) and brL (br-Late), were identified that regulate br expression in response to epidermal growth factor receptor (EGFR) signaling during late oogenesis (Fuchs et al., 2012). Our studies reported here indicate that the upregulation of Br in the FE during midoogenesis is induced by Notch signaling via Su(H) binding to the brE enhancer region. Notch-induced Br regulates the timing of the ME switch.

Materials and Methods

Fly Strains and Genetics

The following fly Strains were used: N55e11 (amorphic allele), Dlrev10 (amorphic allele), Su(H)SF8 (hypomorphic allele loss of function allele), PsnC1 (loss of function allele), hntEH704a (loss of function allele) (Sun and Deng, 2005; 2007), brnpr-3 (amorphic allele; Belyaeva et al., 1980; Restifo and White, 1991; Gotwals and Fristrom, 1991), nctR46 (hypomorphic allele; Chung and Struhl, 2001), grkHK and grk2B6 (Neuman-Silberberg and Schüpbach, 1993), brE-GFP, brE-lacZ, brL-GFP, brL-lacZ, br631-lacZ, br631trun-lacZ (Fuchs et al., 2012), UAS-br-Z1 (Zhou and Riddiford, 2002), Gbe-lacZ (Furriols and Bray, 2001), UAS-br RNAi (Bloomington Drosophila Stock Center, BDSC#27272), UAS-EGFR RNAi (BDSC#25781), UAS-EcR RNAi (BDSC#9326, #9327), EcRE-lacZ (BDSC#4516), UAS-Notch RNAi (BDSC#28981), and w1118 was used as a wild-type control.

For FLP/FRT clone induction (Golic and Lindquist, 1989; Xu and Rubin, 1993), previously described procedures were followed (Sun and Deng, 2005). To characterize the phenotypes, results were reported as A/B, meaning A number of clones out of B number total clones showed the phenotypes, clones from the same egg chamber were only counted once. To generate mosaic egg chambers expressing UAS constructs, the flip-out Gal4 (Pignoni and Zipursky, 1997) stock hsFLP;actin<CD2<Gal4,UAS-RFP/TM3,Sb was applied. Flip-out clones were induced by 30 minutes heat shock at 37°C for 2 days before dissection. Mosaic analysis with a repressible cell marker (MARCM) technique was applied to misexpress UAS constructs in mutant clones (Lee and Luo, 2001).

Immunohistochemistry and Image Analysis

Immunohistochemistry and image acquisition were carried out as previously described (Sun and Deng, 2005). The following antibodies were used: mouse anti-Br-Core (25E9) 1:30, mouse anti-Br-Z1 (3C11) 1:30, mouse anti-Hnt (1G9) 1:15, anti-CycB (F2F4) 1:50, anti-Cut (2B10) 1:15 (Development Studies Hybridoma Bank, USA), rabbit anti-β-Galactosidase 1:5000 (Sigma, USA), rabbit anti-PH3 1:200 (Upstate Biotechnology, NY, USA). Images were acquired with a Zeiss LSM 510 confocal microscope and processed in Photoshop and Image J. Signal intensity was measured by the Interactive 3D Surface Plot Plugin of Image J.

Program “Patser” to predict Su(H) binding sites

The common motif of the Su(H) binding site is YGTGDgAA (R=[AG], Y=[CT], M=[AC], D=[AGT], adjusted based on YGTGRGAAM (Crocker et al, 2010); YGTGDGAA (Rebeiz et al, 2002), and the third “g” in the sequence is critical for Su(H) binding (Bailey and Posakony, 1995; Barolo et al, 2000). The bioinformatic program “Patser” (Hertz and Stormo, 1999) and a position-specific scoring matrix (Krejci et al., 2009; Bernard et al., 2010) were applied to predict putative Su(H) binding sites. The cut-off value Patser score 6 was assigned. Within the predicted Su(H) binding site GTGGgAATGGgAA, the high-affinity motif GTGGgAA has a Patser score of 7.15, and the one with lower affinity, TGGgAA, has a Patser score of 4.94. The Patser scores of the two mutated Su(H) sites within br631c (with both “g”s substituted with “c”s at the two predicted Su(H) sites) were reduced to 3.38 and 1.17, respectively. The reduction of Patser scores is consistent with the previous research that this substitution could severely decrease in vitro binding affinity of Su(H) (Bailey and Posakony, 1995; Barolo et al., 2000).

Construction of the mutant reporter line, br631c-GFP

A wild-type linear DNA template of br631 was generated by digesting the br631 plasmid with restriction enzymes Xbal and Xhol. The site-directed PCR mutagenesis (Ho et al., 1989) was carried out with two complementary primers, 5′-GAGTATCAGTGGcAATGGcAATCCGATGGG -3′ and 5′-CCCATCGGATTGCCATTGCCACTGATACTC -3′, which contain mutations at the predicted Su(H) binding site (“g”s from both GTGGgAA and TGGgAA were replaced with “c”s to reduce Su(H) binding affinity).

The PCR mutagenesis result was confirmed by DNA sequencing. Mutated DNA fragments were digested by restriction enzymes Xbal and Xhol, and inserted into the destination vector which contains hs70 promotor, nuclear EGFP, polylinker and attB for site directed transgenesis (Fuchs et al., 2012). These resulting plasmids were then used to create transgenic flies (GenetiVision, Houston, TX, USA). The PhiC31/attB-mediated site specific integration technique was applied to insert the mutant reporter into chromosome position 68A4.

Chromatin immunoprecipitation (ChIP)-PCR

The ChIP protocol was modified from Li et al., 2011 (Li et al., 2011). 0.2 mL dissected ovaries were collected, then underwent Fix and DNA shearing (Bioruptor® UCD-200, Diagenode). Sonication settings were low power, 10 pulses, 30 seconds on then 30 seconds off each time, which could give rise to 50-350 bp fragments. Later on, the respective extracts were pre-cleared by pre-washed Protein A/G PLUS - Agarose beads (Catalog # SC-2003, Santa Cruz Biotechnology, USA). Each experimental sample (60μl DNA extract diluted with 560μl RIPA buffer) was incubated overnight with goat anti-Su(H) 1:200 (Catalog # SC-15813, Santa Cruz Biotechnology, USA). Control samples were treated with goat serum IgGs 1:200 (Catalog # 50-588-35, Millipore, USA). Antibody-bound extract was pulled down by pre-washed agarose beads, then washed and reverse-crosslinked. ChIP DNA Clean & Concentrator kit (Catalog # D5201, Zymo Research, USA) enriched DNA samples for the polymerase chain reaction (PCR). 30 cycles were carried out. PCR primer sequences were as described, br631-p forward: 5′ CCAGAAGCGGGTCTAATC 3′, br631-p reverse: 5′ TTCGCCAACGCTGATACG 3′, br631-pp forward: 5′ GTCTAGACGGGCCCCAAA 3′, br631-pp reverse: 5′ ATTACCCACTGTCCATTA 3′. Both forward and reverse primers of br631-p bind to genomic br631 sequences, amplifying both endogenous and transgenic br631 sequences (245-bp PCR products). The reverse primer of br631-pp still binds to genomic br631 sequence, its forward primer targets the transgenic plasmid sequence, selectively amplifying transgenic br631 sequence (a 285-bp product).

Results

The Br early pattern in follicle cells is induced by Notch signaling

Using a mouse monoclonal anti-Br-core antibody 25E9 (Emery et al., 1994), we confirmed that Br expression appears initially at a low level in the FE at stage 6, and is upregulated significantly at stage 7 (Fig. 1A, S1A; Deng and Bownes, 1997; Tzolovsky et al., 1999). High levels of Br expression persist in the main-body follicle cells until stage 10a, when the late Br pattern in two patches of anterior-dorsal follicle cells becomes prominent (Fig. S1A; Deng and Bownes, 1997; Tzolovsky et al., 1999; Fuchs et al., 2012). Br expression was not detected in the stretched cells and the polar cells (Fig. S1A, S1B). During late oogenesis, unlike its mRNA pattern which is restricted to the two-patch region that gives rise to the chorionic dorsal appendages, Br protein still has a low level of expression in other main-body cells except those in the T-shape region (Fig. S1A; Tzolovsky et al., 1999; Ward and Berg, 2005; Fuchs et al., 2012).

Figure 1.

Figure 1

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The Br early pattern in follicle cells is induced by Notch signaling. In all panels, Br staining is shown in either green (A-G) or white (A”-G”). DAPI staining (blue in A and B) marks cell nuclei. (A) Br expression was absent in a stage 5 egg chamber, but upregulated dramatically at stage 7. PH3 staining in follicle cells was used to indicate early-stage (stages 1-6) egg chambers. (B) Br (green in B, white in B”) upregulation was concomitant with the expression of Notch activity reporter Gbe-lacZ (red in B, white in B′) in follicle cells during midoogenesis. (C-C”) Follicle cells covering the Dlrev10 germline clone (marked by absence of RFP) in a stage 9 egg chamber did not show detectable Br expression. (D-G”) N55e11 (stage 7, D-D”), nctR46 (stage 7, E-E”), PsnC1 (stage 7, F-F”), and Su(H)SF8 (stage 7, G-G”) follicle cell clones (all marked by absence of GFP or RFP and outlined with dotted lines) showed no expression of Br when compared with neighboring wild-type cells. The polar cells (arrows) did not express Br. Anterior is to the left. Bars, 10 μm.

Hnt is upregulated uniformly in main-body follicle cells around stage 7 by Notch signaling (Sun and Deng, 2007). Its expression pattern is very similar to the pattern of Br during midoogenesis (stages 6-9; Fig. 1A, S1A) that also coincides with Notch signaling activation, based on the expression of the Notch Gbe-lacZ reporter (Fig. 1B). Although based on remaining expression of Br in Notch loss-of-function clones at stage 10a, we originally thought that Br was not regulated by Notch signaling (Fig. S2A; Deng et al., 2001), reexamination of Br expression in a series of mutations with disrupted Notch signaling revealed a strong correlation between Notch activation and Br expression during midoogenesis (Fig. 1B-G″). Using the FLP/FRT system, we found that follicle cells covering the Dlrev10 germline clone did not show Br upregulation during the stages when wild-type cells normally had high levels of Br (12/12; Fig. 1C). Similarly, Br expression failed to be upregulated in N55e11 follicle cell clones during stages 6-9 (27/27; Fig. 1D). Likewise, follicle cells mutant for gamma-secretase components nicastrin (nctR46) (15/17, stages 6-9) or Presenilin (PsnC1) (22/31, stages 6-7; 16/38, stages 8-9) did not show Br upregulation during midoogenesis (Fig. 1E and 1F). Mutant clones of Notch signaling nuclear effector Su(H) (hypomorphic allele Su(H)SF8) also led to reduced expression of Br (14/24, stages 6-8; 3/23, stage 9; Fig. 1G). Together, these results suggest that canonical Notch signaling is required for upregulation of Br expression in follicle cells during midoogenesis. Interestingly, we found that stage 10 follicle cells with defective Notch signaling showed clear Br expression (Fig. S2A), similar to what was reported originally (Deng et al., 2001), suggesting that late Br expression in main-body follicle cells is probably independent of Notch signaling.

br has been shown to be one of the early induced genes by ecdysone receptor (EcR) signaling during metamorphosis (Ashburner, 1974; DiBello et al., 1991). High levels of EGFR signaling in dorsal anterior follicle cells suppress Br expression, whereas moderate levels of it in lateral follicle cells enhance Br expression in late oogenesis (Deng and Bownes, 1997; Fuchs et al., 2012). To determine whether these two pathways affect Br expression during the ME switch, we generated flip-out follicle cell clones with EcR RNAi or EGFR RNAi expression, and found that Br expression was normal in these knockdown cells (Fig. S2B, S2C). The efficacy of EcR RNAi was confirmed by downregulation of EcR activity reporter EcRE-lacZ (Schwedes et al., 2011) in EcR RNAi-expressing follicle cells (Fig. S2D). Mutant clones of an amorphic allele, EGFRf24 (Poulton and Deng, 2006), had proper Br upregulation, consistent with the EGFR knockdown result (Fig. S2E). In addition, egg chambers mutant for gurken (grk), which encodes the ligand and induces two rounds of EGFR activation in follicle cells during mid- and late-oogenesis respectively (Neuman-Silberberg and Schüpbach, 1993; González-Reyes et al., 1995; Roth et al., 1995; Ghiglione et al., 2002), still showed normal Br upregulation at stages 6-9 (Fig. S2F). Taken together, our results suggest that Br is upregulated by Notch signaling but not by EcR or EGFR signaling in follicle cells undergoing the endocycle.

Br is regulated by Notch signaling through the br early enhancer, brE, in follicle cells

The induction of Br expression by Notch signaling during the ME switch raised an interesting question whether this regulation is at the transcriptional level. Two non-overlapping enhancers, brE (br-Early) and brL (br-Late), have been identified that regulate Br expression in follicle cells (Fuchs et al., 2012). At stage 10, EGFR signaling induces brL but suppresses brE in the dorsal anterior follicle cells. The combined pattern of these two enhancer reporters resembles that of endogenous Br during this stage (Fuchs et al., 2012). To determine whether these enhancers mediate the regulation of Br expression by Notch signaling during the ME switch, we first compared the spatial and temporal patterns of these two enhancer reporters with endogenous Br expression during midoogenesis. As expected, brL reporter expression was not detected before stage 10a (Fig. S3A), whereas the brE reporter pattern was similar to that of endogenous Br, uniformly expressed in main-body follicle cells starting at stage 6 (Fig. 2A).

Figure 2.

Figure 2

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Notch signaling regulates brE reporter expression in follicle cells. In all panels, expression of the brE reporter is shown in either green (A-F) or white (A”-F”). (A-A”) Upregulation of Br (red in A, white in A′) was simultaneous with that of brE reporter activity (green in A, white in A”). DAPI staining (blue) marks cell nuclei. (B-B”) Follicle cells covering the Dlrev10 germline clone in a stage 8 egg chamber failed to upregulate brE expression. (C-F”) N55e11 (stage 7, C-C”), nctR46 (stage 7, D-D”), PsnC1 (stage 7, E-E”), and Su(H)SF8 (stage 7, F-F”) follicle cell clones (all marked by absence of RFP or GFP; outlined with dotted lines) showed no or significantly reduced expression of brE when compared with neighboring wild-type cells. Two green dots, in the outlined area in E, are not clone cells. The top one is a stalk cell, based on its location on the follicle cell's basal membrane. The bottom one is the signal from lower confocal layer, based on its weak GFP and lack of overlapping nucleus (data not shown). Anterior is to the left. Bars, 10 μm.

Next, we examined brE reporter expression in mosaic egg chambers with disrupted Notch signaling. In follicle cells covering Dlrev10 germline clones, brE expression was not detected during midoogenesis (9/9, stages 6-9; Fig. 2B). Consistently, brE expression failed to be upregulated in follicle-cell clones of N55e11 (15/15, stages 6-9) (Fig. 2C). Likewise, significantly reduced brE expression was also detected in follicle-cell clones of nctR46 (13/14, stages 6-9), PsnC1 (17/21, stages 6-9), and Su(H)SF8 (21/28, stages 6-9), respectively (Fig. 2D, 2E and 2F). These results indicate that Notch signaling is critical for brE reporter expression during midoogenesis. In contrast, disruption of Notch signaling had no effect on brL reporters, which remained silent throughout midoogenesis (Fig. S3B).

brE contains 2,855 bp of DNA sequence that is located in the fourth intron and part of exon 5 of the br gene (Fig.3A; Fuchs et al., 2012). To determine which DNA fragment within the brE enhancer region is important for the establishment of the early Br pattern in response to Notch signaling, we analyzed several shorter versions of this enhancer reporter (Fig. 3A). br631, which contains a 979-bp intronic subfragment in the brE region (Fig. 3A; Fuchs et al., 2012), retained a similar pattern to that of Br and the full-length brE reporter during stages 6-9 (Fig. 3B). Notch mutant clones failed to express the br631 reporter during these stages (Fig. 3C). Another truncated brE reporter, the 721-bp br631trun (Fuchs et al., 2012), which had the Mirror binding site removed from br631, also mimicked the Br and the full-length brE reporter patterns during stages 6-9 (Fig. 3D), indicating that the regulatory element responsive to Notch signaling is within this 721-bp region.

Figure 3.

Figure 3

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The Su(H) binding site is crucial for Notch regulated brE expression. (A) Schematics of the br genomic locus and genomic fragments (brE, br631, br631c and br631trun) used for br early enhancer reporter constructs. Exons are indicated by black boxes, introns are in white. Compared to br631, br631c has two point mutations aiming to reduce Su(H) binding affinity. br631trun is a truncated version of br631 in which the 3′ DNA sequence containing the Mirror-binding site has been deleted (Fuchs et al., 2012). (B-B′) The br631 reporter (green in B, white in B”) showed a similar pattern to that of Br (red in B, white in B′) in follicle cells during midoogenesis. (C–C”) A stage 7 N55e11 follicle-cell clone (marked by absence of RFP; outlined) showed greatly decreased br631 reporter expression (green in C and white in C”) when compared with neighboring wild-type cells. (D-D”) The br631trun reporter pattern (green in D, white in D”) also resembled that of Br (red in D, white in D′) during stages 6-7. (E-H”) Expression of br631c-GFP (green in E′) was significantly lower than endogenous Br expression (yellow in E”) and br631-lacZ expression (red in E) during midoogenesis (a stage 7 egg chamber in E-E”), when Notch signaling is active. However, br631c-GFP showed a high level of expression during late oogenesis (G′). The images in E-E” and G-G” were taken and processed with the same settings, signal intensity was measured by the Interactive 3D Surface Plot Plugin of Image J (F-F”, H-H”). (I-I′) ChIP samples, with goat IgGs, serve as the control groups. Goat anti-Su(H) antibody was used in the experimental groups to pull down Su(H)-associated DNA fragments. In the control samples, both the br631-p and br631-pp primer pair do not amplify br631 sequences from br631 (Lane 1-2 in I) and br631c (Lane 1-2 in I′) transgenic flies. In experimental samples, the br631-p primer pair amplified both endogenous and transgenic br631 sequences (245-bp PCR products; Lane 3 in I) and the br631-pp primer pair amplified the transgenic br631 sequence (a 285-bp product, Lane 4 in I) from br631 transgenic fly samples. The br631-pp primer pair failed to amplify the target sequence (Lane 4 in I′), whereas br631-p retained the ability to amplify the endogenous br631 sequence (Lane 3 in I′) from br631c transgenic fly samples (Fig.3I′, Lane 3). DAPI (blue) marks cell nuclei. Anterior is to the left. Bars, 10 μm.

The Su(H) binding sites in brE are critical for Notch regulation

Transcriptional activation of target genes by the Notch pathway is achieved after the cleaved Notch intracellular domain (NICD) turns Su(H) from a repressor into an activator (Barolo et al., 2000). Therefore, the Su(H) binding sites in the enhancer region of the target genes are critical for Notch induced expression (Bailey and Posakony, 1995). Applying the bioinformatic program “Patser” and position-specific scoring matrix (Hertz and Stormo, 1999; Krejci et al., 2009), we identified a sequence GTGGgAATGGgAA, which contains a high-affinity Su(H) binding motif GTGGgAA and a lower affinity site TGGgAA, within the 721-bp early enhancer region (X chromosome: 1516942-1517682, Fig. 3A). To determine whether this sequence is critical for Notch-mediated transcriptional activation, we generated a mutant construct, br631c, with both “g”s replaced with “c”s at the two potential Su(H) sites (GTGGcAATGGcAA) to reduce Su(H) binding affinity (Bailey and Posakony, 1995; Barolo et al., 2000). The mutated enhancer was then fused with the GFP open reading frame to generate transgenic reporter lines. As anticipated, compared to relatively high expression of br631 reporter (Fig. 3E, 3F) and Br protein (Fig. 3E″, 3F″) in midoogenesis, br631c reporter expression was severely reduced in follicle cells from stage 6 to 9 when Notch signaling is normally active (Fig. 3E', 3F′), suggesting that the Su(H) binding affinity to the enhancer is critical for Notch-induced brE reporter expression in follicle cells. The result also suggests that Su(H) binding in the brE region is involved in mediating transcriptional activation of Br by Notch signaling in endocycling follicle cells.

Interestingly, when egg chambers from stage 10 onward were examined, we found that the br631c reporter was expressed (Fig. 3G′, 3H′), similar to the late patterns of brE and br631 reporters (Fig. S1A′, 3G, 3H). Together with re-appearance of Br in Notch mutant cells after stage 9 (Fig. S2A), we conclude that the regulation of Br expression by Notch signaling in follicle cells is stage specific, and that late stage Br expression in main-body follicle cells is independent of Notch regulation.

Next, we applied the chromatin immunoprecipitation (ChIP) technique to confirm that Su(H) binds to the br early enhancer (br631) region. To this end, two sets of primer pairs, br631-p and br631-pp, were designed (see Materials and Methods). br631-p recognizes genomic br631 sequences, thus amplifying both endogenous and transgenic br631 sequences. br631-pp, which has one primer targeting the plasmid DNA sequence, selectively amplifies only the transgenic br631 sequences. Consistent with the design purpose, after ChIP and PCR, the br631-p primer pair amplified both endogenous and transgenic br631 sequences (245-bp PCR products; Lane 3 in Fig. 3I), whereas the br631-pp primer pair selectively amplified transgenic br631 sequence (a 285-bp product: Lane 4 in Fig. 3I) from transgenic flies that carry the br631 transgene, indicating that Su(H) binds to both endogenous and transgenic br631 DNA sequences. In contrast, in transgenic flies that carry the mutant br631c transgene, which did not respond to Notch signaling, primer pair br631-pp failed to amplify the target sequence (Lane 4 in Fig.3I′), suggesting reduced binding affinity of Su(H) to br631c, whereas br631-p, as a positive control, retained the ability to amplify the endogenous sequence located in the br enhancer in these transgenic flies (Lane 3 in Fig.3I′). These results further support the idea that Notch signaling regulates br via Su(H) binding to the br631 enhancer region.

To determine whether regulation of brE by Notch signaling is conserved in other tissues or developmental stages, we first examined the expression patterns of Br and brE reporters in the wing imaginal disc. Although Br expression was detected ubiquitously in the epithelial cells of the wing disc (Fig. S4A), brE reporter expression was not detectable, even in the dorsal/ventral (D/V) boundary where Notch signaling is highly active (Fig. S4A, S4B; de Celis et al., 1996). The ubiquitous expression of Br in the disc was probably not regulated by Notch signaling, as N mutant cells did not affect Br expression (Fig. S4C, S4D). Next, we examined the polar/stalk cell precursor in the germarium where Notch is also active (Xu et al., 1992; Bender et al., 1993; Shyu et al., 2009), but no brE expression was detected (Fig. S4E). Together, these data suggest that the regulation of Br through brE by Notch signaling is tissue-specific.

The role of Br in the ME switch

To determine whether Br upregulation at the ME switch is associated with any role that Br may play during midoogenesis, we generated homozygous follicle-cell clones of a br amorphic allele brnpr-3 (Belyaeva et al., 1980; Restifo and White, 1991; Gotwals and Fristrom, 1991). These mutant clones showed no Br protein expression, confirming the null phenotype (Fig. S5A). In wild-type follicle cells, mitotic markers such as Cyclin B (CycB) and phospho-histone 3 (PH3) are expressed in oscillating patterns during the mitotic cycle, but disappear once the follicle cells enter the endocycle (Deng et al., 2001). In brnpr-3 mosaic egg chambers, we found that 55% of stage 7 follicle-cell clones had weakly elevated CycB (n = 75; Fig. 4A), and 4% of stage 7 mosaic egg chambers showed PH3-positive follicle cells (n = 76; Fig. 4B). In contrast, wild-type cells had no CycB or PH3 staining during the same stage (Sun and Deng, 2005; 2007). After stage 7, prolonged CycB and PH3 expression could no longer be detected in brnpr-3 mutant cells. These results suggest that Br function is required for prompt endocycle entry in follicle cells. We then examined whether misexpression of Br in follicle cells before stage 6 could drive them into the endocycle prematurely. The Br-Z1 isoform is the most abundant isoform of Br in follicle cells (Tzolovsky et al., 1999) and the antibody staining revealed that Br-Z1 shared the same expression pattern as the Br-Core domain in oogenesis (Fig. S5B). Using the flip-out Gal4/UAS technique (Pignoni and Zipursky, 1997), we found that Br-Z1 misexpression in follicle cells before stage 6, sufficiently blocked the expression of mitotic markers CycB (40/40, stages 4-6, Fig. 4C) and PH3 (40/40, stages 4-6, Fig. 4D). These Br-Z1-misexpressing follicle cells had much larger nuclei than neighboring wild-type cells (Fig. S5C). These results suggest that misexpression of Br-Z1 blocks mitotic division and drives premature endocycle entry in follicle cells.

Figure 4.

Figure 4

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The role of Br during the mitotic/endocycle switch. (A-B”) Stage 7 brnpr-3 follicle-cell clones (marked by the absence of RFP, red in A-B, white in A′-B′; outlined) showed elevated CycB (green in A, white in A”) and sporadic PH3 staining (green in B, white in B′). (C-D”) Mitotic markers, CycB (white in C”) and PH3 (white in D”), were suppressed in Br-Z1 misexpressing follicle cells (marked by RFP, white in C′and D′; outlined) during early oogenesis. DAPI marks cell nuclei. Anterior is to the left. Bars, 10 μm.

During the ME switch, Notch induces the expression of Hnt and suppresses Cut expression (Sun and Deng, 2005; 2007). To determine whether Br plays a role in the expression of these Notch targets, we examined the expression of Hnt and Cut in br mutant cells. In stage 7 brnpr-3 follicle-cell clones, Cut was weakly upregulated (42/63) and Hnt was downregulated (38/66) (Fig. 5A and 5B). No change of Cut or Hnt pattern was detected after stage 7 (Fig. S5D, S5E). Similarly, using a flip-out Gal4 to drive the expression of UAS-br RNAi randomly in follicle cells, we found that knockdown of Br resulted in weak upregulation of Cut and downregulation of Hnt at stage 7 (Fig. S5F, S5G).

Figure 5.

Figure 5

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Interactions between Br and other Notch signaling downstream targets. (A-B”) Stage 7 brnpr-3 follicle-cell clones (marked by the absence of RFP, red in A-B, white in A′-B′; outlined) showed elevated Cut (green in A, white in A”) and reduced Hnt (green in B, white in B”). (C-C”) hntEH704a follicle-cell clones (marked by the absence of GFP, red; outlined) in a stage 7 egg chamber showed decreased Br expression (green in C, white in C”). (D-E”) In Br-Z1 misexpressing follicle cells (marked by RFP, red in D and E, white in D′and E′; outlined), Hnt (green in D, white in D”) was induced and Cut (green in E, white in E”) was suppressed during early oogenesis. (F-F”) In Hnt misexpressing follicle cells (marked by RFP, red in F, white in F′; outlined), Br (green in F, white in F”) was induced during early oogenesis. (G-G”) In N mutant clones with Br-Z1 misexpression (marked by GFP, white in G′; outlined) by MARCM technique, Hnt (white in G”) was still induced during early oogenesis. (H-H”) Br-Z1 was misexpressed in hnt mutant clones (marked by the GFP, red in H, white in H′; outlined) by MARCM, Cut (green in H, white in J”) was still downregulated during early oogenesis. (I-J”) Br-Z1 misexpressed in N and hnt mutant clones (marked by the GFP, red in I and J, white in I′ and J′; outlined) by MARCM, failed to suppress prolonged Cut expression (green in I and J, white in I” and J”) during midoogenesis. (K) During the ME switch, germline-Dl-induced N activation upregulates Br and Hnt, and suppresses Cut. Downregulation of Cut is mainly through N-Hnt-mediated regulation; Br fine-tunes the process to help Hnt downregulate Cut, ensuring precise endocycle entry. DAPI marks cell nuclei. Anterior is to the left. Bars, 10 μm.

Intrigued by these results, we also examined Br expression in hnt mosaic egg chambers, and found that hnt mutant clones showed weakly reduced Br expression at stage 7 (31/44) (Fig. 5C). This lowered expression is probably not caused by transcriptional regulation through brE, as brE reporter expression was still present in stage 7 hnt clones (Fig. S5H). These data suggest that Br and Hnt have a small but indirect impact on each other's expression in follicle cells after entering the endocycle. We further examined whether misexpression of Br or Hnt could affect each other. Using the flip-out Gal4/UAS technique, we found that Br-Z1 misexpression in follicle cells before stage 6 induced early Hnt upregulation (42/42) (Fig. 5D) and Cut downregulation (35/35) (Fig. 5E), while Hnt misexpression before stage 6 could upregulate Br as well (29/38) (Fig. 5F). Taken together (Fig. 5A-5F), ectopic expression of either Br or Hnt in follicle cells promotes each other's expression in early oogenesis, even though they only have a small, indirect mutual impact in midoogenesis. Since Hnt is the result of Notch activation in follicle cells during midoogenesis, and downregulation of Cut by Notch signaling through Hnt is necessary for the follicle cells to enter the endocycle (Sun and Deng, 2005; 2007 and this study), we asked whether Br-misexpression-induced upregulation of Hnt and downregulation of Cut in early oogenesis depend on N-Hnt regulation. To address this question, we overexpressed Br-Z1 in Notch knockdown follicle cells, and found that Hnt was still upregulated before stage 7 (24/24) (Fig. 5G), suggesting Br-Z1 can upregulate Hnt independent of Notch signaling. In addition, Br-Z1 misexpression suppressed Cut in hnt mutant clones before stage 7 (13/13) (Fig. 5H), implying Br-Z1 can suppress Cut independent of Hnt in early follicle cells. These results (Fig. 5G, 5H) suggest that Br-misexpression is sufficient to independently induce premature follicle cell differentiation, marked by Hnt upregulation and Cut downregulation. Interestingly, when we overexpressed Br-Z1 in N or hnt mutant clones during stages 7-9, prolonged Cut expression in either the N (17/17) or hnt (20/20) mutant clones was not suppressed (Fig. 5I, 5J). Although the middle-stage follicle cells defective in Notch signaling are supposed to remain in a premature fate, our results suggest that these cells are still different from the wild-type early-stage follicle cells, at least in their ability to respond to Br misexpression. In summary, during the ME switch, germline-Dl-induced Notch activation upregulates Br and Hnt, and suppresses Cut. Downregulation of Cut is mainly through N-Hnt-mediated regulation; Br fine-tunes the process and assists Hnt to properly downregulate Cut, ensuring precise endocycle entry (Fig. 5K).

Discussion

Notch signaling plays a central role in follicle cell differentiation. The activation of the Notch pathway in the FE during midoogenesis triggers a switch from mitotic divisions to endoreplication cycles and induces cell differentiation that is essential for other developmental milestones in oogenesis. For example, proper Notch-induced cell differentiation is necessary for follicle cell-oocyte communication that establishes the oocyte polarity, and for the series of cell migrations at stage 9 (Xu et al., 1992; González-Reyes and St Johnston, 1998; Poulton and Deng, 2007; Wang et al., 2007; Xu and Gridley, 2012). Because of the crucial role of Notch signaling in oogenesis, its activation of downstream transcription factors is strictly regulated temporally and spatially. Its temporal pattern during the ME switch is modulated by the microRNA pathway, whereas the Hippo pathway promotes Notch signaling more prominently in the posterior follicle cells (Meignin et al., 2007; Polesello and Tapon, 2007; Yu et al., 2008; Poulton et al., 2011). At the transcriptional activation level, several co-factors, including COREST and SMRTER, also affect the ME transition. They may act downstream of NICD release and interact directly with co-repressors (Hairless, Groucho, CtBP) or activator Su(H) (Domanitskaya and Schüpbach, 2012; Heck et al., 2012). Downstream of Notch/Su(H) activation, several transcription factors have been identified that are involved in the regulation of the ME switch. These include the aforementioned Hnt and Cut, which are positive and negative targets of Notch signaling in the ME switch, respectively. Although they were implicated as direct Notch targets in the Notch-activated DmD8 cells and contain putative Su(H) binding sites identified by chromatin immunoprecipitation, whether these sites mediate direct transcriptional regulation by Notch signaling has not been verified in vivo (Krejci et al., 2009). Our in vivo study reported here revealed that Br is a transcriptional target of Notch/Su(H) in follicle cells. The regulation of Br resides in a predicted Su(H) binding site, mutation of two key nucleotides significantly abrogated the reporter activity of this enhancer in response to Notch signaling. This site is specifically regulated by Notch signaling to mediate br expression during oogenesis, and the regulation is tissue-specific, further confirming that Notch response is context-dependent during development.

Interestingly, brE expression persists after the follicle cells leave the endocycle and undergo gene amplification. This late expression is only suppressed in the dorsal anterior region by the EGFR pathway, which activates the late br pattern in the two-patch region through the brL enhancer. Because br RNA expression is restricted to the two-patch dorsal anterior region at stage 10b in follicle cells (Deng and Bownes, 1997; Fuchs et al., 2012), the late pattern of brE in mainbody follicle cells is not a reflection of the endogenous pattern of br transcription, and it is not subject to Notch regulation. Perhaps some other factors that have recognition sites in brE are responsible for the late brE pattern.

Although Br has a prominent expression pattern in follicle cells during the endocycle stages, the function of br in the ME switch is subtle. Mutational studies only revealed a defect in the timing of endocycle entry, the mitotic markers show extended expression only briefly after they are supposed to be switched off. We suspect that Br does not have a direct impact on the expression of cell cycle regulators, and the role of Br in endocycle entry might be through its mild effect on Hnt, the zinc-finger protein that has a prominent role in the ME switch (Sun and Deng, 2007). In br mutant cells, Hnt expression is weakly disrupted at stage 7 and recovers afterwards, which correlates with the timing of the delayed endocycle entry. In contrast to the weak loss-of-function phenotype, misexpression of Br-Z1, the prominent form expressed in follicle cells (Tzolovsky, 1999), induces a premature entry into the endocycle, suggesting that although the Br function is not essential for endocycle entry, it can trigger the endoycle entry when misexpressed in early follicle cells. Interestingly, Hnt and Cut were also prematurely upregulated and downregulated, respectively, in these Br-Z1 misexpressing cells, and their regulation is Notch-independent. However, proper Cut downregulation after stage 7 still requires N-Hnt regulation, suggesting Br and Hnt are not functionally redundant, and Hnt is the main downstream factor of Notch regulating endocycle entry. We speculate that endogenous Br functions only during the transitional stage of the ME switch to accelerate Cut downregulation and Hnt upregulation, which would help prevent cells becoming “confused” between two cell cycle regimes.

Supplementary Material

01

Figure S1. The expression patterns of Br and brE during oogenesis. (A-A‴) Expression of Br (white in A) was detected as early as stage 6, then highly induced from stage 7 in the mainbody follicle cells. In the stretched cells, Br staining was gradually decreased from stage 9 and could no longer be detected at stage 10a (arrowhead in A). At stage 10b, Br was upregulated in the two-patch region (arrows). Its expression remained in main-body follicle cells, except those in the T-shape region (circled area in a stage 10b egg chamber in A). Expression of the brE reporter (white in A′) was similar to that of Br from the germarium to stage 9. During later stages, brE showed expression in the stretched cells (stage 10a egg chamber in A′), but not in the two-patch and T-shape regions (stage 10b egg chamber in A′). PH3 (white in A″) expression indicates early-stage egg chambers (stages 1-6). DAPI (white in A‴) marks cell nuclei. (B-C′) Br was not expressed in polar (arrow) and stalk cells (arrowhead). A101-lacZ reporter (red in B, white in B′) specifically marks the polar cells where no Br expression was detected (green in B, white in B‴). Stalk cells (arrowheads) linking two egg chambers, which already had upregulated Br in mainbody follicle cells, did not show Br expression, either. DAPI (blue in B, white in B″, C′) marks cell nuclei. Anterior is to the left. Bars, 50 μm.

02

Figure S2. The expression of Br in different genetic backgrounds. (A-A′) N55e11 follicle cell clones (marked by absence of RFP; outlined) at stage 10b showed expression of Br (green in A, white in A′). (B-C′) Follicle cells with EcR RNAi (stage 8 egg chamber in B, marked by RFP; outlined) and EGFR RNAi (stage 9 egg chamber in C, marked by RFP; outlined) expression showed normal Br expression (green in B and C, white in B′ and C′). (D-D′) Follicle cells with EcR RNAi (marked by RFP; outlined) expression showed downregulated EcRE-lacZ (green in D, white in D′). (E-E″) Stage 8 EGFRf24 follicle-cell clones (marked by the absence of RFP, red in E; outlined) showed normal Br expression (green in E, white in E′). (F-F′) Trans-heterozygous grkHK/grk2B6 egg chambers showed normal Br expression (green in F, white in F′) at stage 6. DAPI (blue in F) marks cell nuclei. Anterior is to the left. Bars, 10 μm.

03

Figure S3. brL expression before stage 10b. (A-A′) brL reporter expression (green in A, white in A′) was not detected before stage 10b. DAPI (blue in A) marks cell nuclei. (B-B′) Stage 7 N55e11 follicle cell clones (marked by absence of RFP; outlined) had no expression of brL (green in B, white in B′), similar to neighboring wild-type cells. Anterior is to the left. Bars, 10 μm.

04

Figure S4. Tissue-specific regulation of brE expression by Notch signaling. (A-A”) Br expression (red in A, white in A′) was detected ubiquitously in the epithelial cells of the wing disc, whereas brE reporter expression (green in A, white in A”) was absent. (B-B”) brE reporter expression (green in B, white in B”) was not detected in cells with active Notch signaling (marked with Cut staining, red in B, white in B′) in the wing disc. (C-D”) Knockdown of Notch signaling in the posterior compartment of the wing disc (UAS-N RNAi driven by en-Gal4, marked with GFP, green in C and D, white in C′ and D′) reduced the downstream Cut expression (red in D, white in D”), but showed no significant change of Br expression (red in C, white in C”). (E-E”) In the germarium of egg chambers, no Br (red in E, white in E′) or brE (green in E, white in E”) expression was detected. DAPI (blue in A-E) marks cell nuclei. Anterior is to the left. Ventral is up. Bars, 10 μm.

05

Figure S5. The role of Br in the mitotic/endocycle switch. (A-A”) Stage 7 brnpr-3 follicle cell clones (marked by absence of RFP; outlined) showed no expression of Br (green in A, white in A”). (B-B”) The expression of Br-Z1 (blue in B, white in B”) was upregulated at stages 6/7. DAPI (blue in B, white in B′) marks cell nuclei. (C-C”) Follicle cells over-expressing Br-Z1 (marked by RFP) showed larger nuclei than neighboring wild-type cells. DAPI (blue in C, white in C”) marks cell nuclei. (D-E”) Stage 8 brnpr-3 follicle-cell clones (marked by the absence of RFP, red in D-E, white in D′-E′; outlined) showed elevated Cut (green in D, white in D”) and reduced Hnt (green in E, white in E”). (F-G”) Stage 7 follicle cells with br RNAi expression (marked by RFP; outlined) showed weakly upregulated Cut expression (green in F, white in F”) and mildly reduced Hnt expression (green in G, white in G”). The two cells indicated by an arrow in F” are polar cells with Cut expression during midoogenesis. (H-H”) A stage 7 hntEH704a follicle-cell clone (lack of RFP; outlined) showed normal brE expression (green in F, white in F”). Anterior is to the left. Bars, 10 μm.

Broad is directly regulated by Notch signaling during Drosophila midoogenesis.

The regulation of Broad by Notch is through the Su(H) binding sites.

Broad, together with Hindsight and Cut, plays a role in the mitotic/endocycle switch.

Acknowledgments

We would like to thank Stanislav Y. Shvartsman and Lily Cheung for sharing information about brE and fly stocks prior to publication, Sarah Bray for fly stocks and sharing the bioinformatic program “Patser”. We thank Beth Alexandra, Myra Hurt, Jamila Horabin, Nicholas Leake, Kimberly Riddle, Tomas J. Fellers, Cheryl Pye, “Patser” developers, the Molecular Cloning Facility and the Biological Science Imaging Facility at Florida State University for technical help. The DSHB, the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) and the Bloomington Stock Center for providing us antibodies and fly stocks. Jianjun Sun, Jen Kennedy, Yi-Chun Huang, Pang-Kuo Lo, William Palmer for critical reading of, and comments on, the manuscript. W.-M. D. is supported by NIH grant R01GM072562 and National Science Foundation IOS-1052333.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Figure S1. The expression patterns of Br and brE during oogenesis. (A-A‴) Expression of Br (white in A) was detected as early as stage 6, then highly induced from stage 7 in the mainbody follicle cells. In the stretched cells, Br staining was gradually decreased from stage 9 and could no longer be detected at stage 10a (arrowhead in A). At stage 10b, Br was upregulated in the two-patch region (arrows). Its expression remained in main-body follicle cells, except those in the T-shape region (circled area in a stage 10b egg chamber in A). Expression of the brE reporter (white in A′) was similar to that of Br from the germarium to stage 9. During later stages, brE showed expression in the stretched cells (stage 10a egg chamber in A′), but not in the two-patch and T-shape regions (stage 10b egg chamber in A′). PH3 (white in A″) expression indicates early-stage egg chambers (stages 1-6). DAPI (white in A‴) marks cell nuclei. (B-C′) Br was not expressed in polar (arrow) and stalk cells (arrowhead). A101-lacZ reporter (red in B, white in B′) specifically marks the polar cells where no Br expression was detected (green in B, white in B‴). Stalk cells (arrowheads) linking two egg chambers, which already had upregulated Br in mainbody follicle cells, did not show Br expression, either. DAPI (blue in B, white in B″, C′) marks cell nuclei. Anterior is to the left. Bars, 50 μm.

NIHMS593928-supplement-01.jpg (869.3KB, jpg)
02

Figure S2. The expression of Br in different genetic backgrounds. (A-A′) N55e11 follicle cell clones (marked by absence of RFP; outlined) at stage 10b showed expression of Br (green in A, white in A′). (B-C′) Follicle cells with EcR RNAi (stage 8 egg chamber in B, marked by RFP; outlined) and EGFR RNAi (stage 9 egg chamber in C, marked by RFP; outlined) expression showed normal Br expression (green in B and C, white in B′ and C′). (D-D′) Follicle cells with EcR RNAi (marked by RFP; outlined) expression showed downregulated EcRE-lacZ (green in D, white in D′). (E-E″) Stage 8 EGFRf24 follicle-cell clones (marked by the absence of RFP, red in E; outlined) showed normal Br expression (green in E, white in E′). (F-F′) Trans-heterozygous grkHK/grk2B6 egg chambers showed normal Br expression (green in F, white in F′) at stage 6. DAPI (blue in F) marks cell nuclei. Anterior is to the left. Bars, 10 μm.

NIHMS593928-supplement-02.jpg (2.2MB, jpg)
03

Figure S3. brL expression before stage 10b. (A-A′) brL reporter expression (green in A, white in A′) was not detected before stage 10b. DAPI (blue in A) marks cell nuclei. (B-B′) Stage 7 N55e11 follicle cell clones (marked by absence of RFP; outlined) had no expression of brL (green in B, white in B′), similar to neighboring wild-type cells. Anterior is to the left. Bars, 10 μm.

NIHMS593928-supplement-03.jpg (487.6KB, jpg)
04

Figure S4. Tissue-specific regulation of brE expression by Notch signaling. (A-A”) Br expression (red in A, white in A′) was detected ubiquitously in the epithelial cells of the wing disc, whereas brE reporter expression (green in A, white in A”) was absent. (B-B”) brE reporter expression (green in B, white in B”) was not detected in cells with active Notch signaling (marked with Cut staining, red in B, white in B′) in the wing disc. (C-D”) Knockdown of Notch signaling in the posterior compartment of the wing disc (UAS-N RNAi driven by en-Gal4, marked with GFP, green in C and D, white in C′ and D′) reduced the downstream Cut expression (red in D, white in D”), but showed no significant change of Br expression (red in C, white in C”). (E-E”) In the germarium of egg chambers, no Br (red in E, white in E′) or brE (green in E, white in E”) expression was detected. DAPI (blue in A-E) marks cell nuclei. Anterior is to the left. Ventral is up. Bars, 10 μm.

NIHMS593928-supplement-04.jpg (1.4MB, jpg)
05

Figure S5. The role of Br in the mitotic/endocycle switch. (A-A”) Stage 7 brnpr-3 follicle cell clones (marked by absence of RFP; outlined) showed no expression of Br (green in A, white in A”). (B-B”) The expression of Br-Z1 (blue in B, white in B”) was upregulated at stages 6/7. DAPI (blue in B, white in B′) marks cell nuclei. (C-C”) Follicle cells over-expressing Br-Z1 (marked by RFP) showed larger nuclei than neighboring wild-type cells. DAPI (blue in C, white in C”) marks cell nuclei. (D-E”) Stage 8 brnpr-3 follicle-cell clones (marked by the absence of RFP, red in D-E, white in D′-E′; outlined) showed elevated Cut (green in D, white in D”) and reduced Hnt (green in E, white in E”). (F-G”) Stage 7 follicle cells with br RNAi expression (marked by RFP; outlined) showed weakly upregulated Cut expression (green in F, white in F”) and mildly reduced Hnt expression (green in G, white in G”). The two cells indicated by an arrow in F” are polar cells with Cut expression during midoogenesis. (H-H”) A stage 7 hntEH704a follicle-cell clone (lack of RFP; outlined) showed normal brE expression (green in F, white in F”). Anterior is to the left. Bars, 10 μm.

NIHMS593928-supplement-05.jpg (2.8MB, jpg)

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