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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Dev Cell. Author manuscript; available in PMC Mar 1, 2008.
Published in final edited form as:
Dev Cell. Mar 2007; 12(3): 431–442.
doi:  10.1016/j.devcel.2007.02.003
PMCID: PMC1851662
NIHMSID: NIHMS19663

Hindsight Mediates the Role of Notch in Suppressing Hedgehog Signaling and Cell Proliferation

Summary

Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle. Here we show that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of Cubitus interruptus. Our studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation.

Keywords: endoreplication, cell growth, germ-line–soma interaction, polyploidy, signaling crosstalk, Pebbled, RREB, Hnt, Hh, Ci

Introduction

The Drosophila follicular epithelial cells, which form a monolayer to cover the germ-line cells during oogenesis, provide an excellent model system for the study of temporal regulation of cell proliferation and cell differentiation, because different-stage egg chambers are clearly distinguishable morphologically and positionally in the ovariole. Follicle cells undergo normal mitotic divisions that produce about 650 follicle cells covering 16 germ-line cells in each egg chamber up to stage 6. Afterward, they undergo a marked switch of cell-cycle programs from the mitotic cycle to three rounds of the endoreplication cycle (also called the endocycle) during stages 7–10A, in which cells duplicate their genomic DNA without cell division (Deng et al., 2001; Lopez-Schier and St Johnston, 2001). Coupled with the cell-cycle switch, follicle-cell differentiation occurs, as indicated by downregulation of immature-cell-fate markers, such as Fasciclin III (FasIII) and Eyes absent (Eya) (Lopez-Schier and St Johnston, 2001; Sun and Deng, 2005). At stage 10B, main-body follicle cells leave the endocycle and undergo amplification of specific genomic regions, which is marked by several 5-bromo-2-deoxyuridine (BrdU) incorporation foci in each nucleus (Calvi et al., 1998).

The mitotic cycle/endocycle (M/E) switch is triggered by the Notch pathway, an evolutionarily conserved pathway implicated in many biological and pathological processes (Deng et al., 2001; Lopez-Schier and St Johnston, 2001; Bray, 2006). Upon binding to its ligand, the transmembrane receptor Notch is cleaved through a conserved proteolysis that requires the function of Presenilin (Psn) (Struhl and Greenwald, 1999). The released Notch intracellular domain (NICD) enters the nucleus and converts Suppressor of Hairless (Su(H)) from a transcriptional repressor to an activator (Struhl and Adachi, 1998). At stage 6/7 of oogenesis, upregulation of the ligand for Notch signaling, Delta (Dl), in the germ-line cells activates the canonical Notch pathway in the follicle cells. Mutant clones of these signaling components result in continued follicle-cell proliferation and aberrant cell differentiation (Deng et al., 2001; Lopez-Schier and St Johnston, 2001).

We recently showed that the homeodomain gene cut, normally expressed in the mitotic follicle cells, is downregulated by Notch during the M/E switch (Sun and Deng, 2005). Repression of Cut in follicle cells is required for the expression of Fizzy-related (Fzr), an adaptor of the Anaphase Promoting Complex/Cyclosome (APC/C), which mediates the degradation of M-phase cyclins by the Ubiquitin Proteasome System (UPS; Sigrist and Lehner, 1997). Interestingly, Cut does not seem to regulate String (Stg), the Drosophila homolog of Cdc25 phosphatase that is a critical regulator of G2/M transition, suggesting that only part of Notch’s effect is mediated through Cut downregulation during the M/E switch. The mechanism through which Cut is suppressed by Notch is unclear, as NICD/Su(H) normally acts as a transcriptional activator, and a known Notch target, the transcriptional repressor Enhancer of Split (E(Spl)), is not required in the M/E switch (Deng et al., 2001; Lopez-Schier and St Johnston, 2001). Other transcription factors may therefore mediate the Notch function in suppressing Cut expression.

In contrast to Notch, Hedgehog (Hh) signaling appears to promote follicle-cell proliferation (Forbes et al., 1996a; Zhang and Kalderon, 2000; for a review of Hh signaling pathway, see Lum and Beachy, 2004). A Hh signaling reporter, patched-lacZ (ptc-lacZ), as well as the nuclear activator of Hh signaling, full-length Ci (Ci-155), is detected in follicle cells from the germarium to stage 6 and downregulated afterward (Forbes et al., 1996b; Zhang and Kalderon, 2000). In addition, mutation of a negative regulator of the Hh pathway, ptc, was reported to be able to keep the follicle cells in the mitotic cycle beyond stage 6 (Zhang and Kalderon, 2000). Mutation of shaggy (sgg), the Drosophila homolog of GSK3β, or protein kinase A (pka), both of which are required to phosphorylate Ci-155 and process it into Ci-75, causes a mild overproliferation phenotype in follicle cells (Zhang and Kalderon, 2000; Jordan et al., 2006).

In the work reported here, we identified a transcription factor, Hindsight (Hnt, encoded by pebbled), which mediates the role of Notch signaling in regulating follicle-cell proliferation and differentiation. Hnt, which has 14 zinc-finger domains and two coiled-coil domains, is a Drosophila homolog of Ras Responsive Element Binding protein (RREB-1; Yip et al., 1997; Zhang et al., 1999). Hnt is known to be involved in cell differentiation and maintenance of epithelial integrity in Drosophila retinal development (Pickup et al., 2002), and in the control of germ-band retraction and dorsal closure through downregulation of Jun kinase signaling in amnioserosa (Yip et al., 1997; Reed et al., 2001). Our results demonstrate that Hnt is upregulated by Notch signaling and mediates the Notch-dependent downregulation of Cut, Stg, and Hh signaling in follicle cells, thus promoting the M/E switch and cell differentiation.

Results

Hindsight Is Upregulated by Notch Signaling in Follicle Cells

To identify molecules that potentially mediate Notch function in promoting the M/E switch, we performed a small-scale screen of temporal patterns of protein expression in follicle cells with antibodies from the Developmental Studies Hybridoma Bank (DSHB). Hnt, which is recognized by monoclonal antibody 1G9 (Yip et al., 1997), was identified from this screen. Hnt was detected sporadically at around stage 6, then dramatically upregulated at stage 7 in the entire follicle-cell monolayer, except the polar cells and stalk cells (Figure 1A and data not shown). To determine the exact timing of Hnt upregulation, we double-labeled wild-type egg chambers with 1G9 and an antibody against phosphohistone H3 (PH3) to mark the M phase. No PH3 staining was detected in follicle cells that showed Hnt expression, and upregulation of Hnt was always associated with the disappearance of PH3 staining (Figure 1A), indicating that Hnt upregulation is concurrent with the M/E switch. High levels of Hnt expression persisted until about stage 10A. Afterward, Hnt expression was restricted to the anterior follicle cells (Figure S1B). Interestingly, Hnt expression was resumed in main-body follicle cells at around stage 13 (data not shown). No Hnt was detected in the germ-line cells, consistent with our finding that egg chambers with hnt germ-line clones developed normally (data not shown).

Figure 1
Hindsight is upregulated by Notch signaling in follicle cells. Hnt antibody staining is shown in red in A–E and white in A′–E′. In all figures, loss-of-function clones are marked by the absence of GFP and gain-of-function ...

The coincidence of Hnt upregulation and the Notch-induced M/E switch prompted us to examine the effect of Notch signaling on Hnt expression. In follicle cells covering Dlrev10 germ-line clones, Hnt expression was not detected during midoogenesis (stages 7-10A), when wild-type cells should have high levels of expression (Figure 1B). Consistently, Hnt expression was undetectable in the N55e11 or psnC1 follicle cells during stages 7–10A (Figure 1C and 1D). These cells also showed smaller nucleus size, indicating impaired M/E switch. In addition, follicle-cell clones of Su(H)SF8, a hypomorphic allele, revealed a lack of Hnt expression from stage 7 to 8 (Figure 1E). Compared to N55e11 clones, Su(H)SF8 clones had a less severe defect in Hnt expression as well as in nucleus size, because Hnt was detected in the clones at stages 9/10. Nonetheless, the loss of Hnt at the M/E transition suggests that the canonical Notch pathway is necessary for induction of Hnt expression in follicle cells during midoogenesis.

Hindsight Function Is Required for the Mitotic Cycle/Endocycle Switch

To examine the functional significance of Hnt upregulation in follicle-cell development, we recombined each of two hnt mutant alleles, hntXE81 and hntEH704a (Yip et al., 1997), into the FRT18D chromosome, separately. Both produced homozygous mutant clones in which antibody staining revealed no Hnt protein (Figure S1C). In the mutant follicle cells, DAPI staining revealed nuclei smaller than those in the neighboring wild-type cells after stage 7 (Figure 2A), indicating that loss of hnt function may have caused defects in the M/E switch, a process that is normally marked by the abrupt disappearance of mitotic markers, such as Cyclin A (CycA), Cyclin B (CycB), and PH3 (Deng et al., 2001). In hnt follicle-cell clones, both CycA and CycB continued to be expressed in an oscillating pattern after stage 6 (Figure 2B and 2C). PH3 was also detected in some of these mutant cells (Figure 2C). We quantified mosaic egg chambers that contained 10 or more hnt mutant follicle cells after stage 6 and found that 40% of stage 7 (n = 40), 19% of stage 8 (n = 43), and 4% of stage 9 (n = 28) mosaic egg chambers had PH3-positive follicle cells in the hnt clones. After stage 9, PH3-positive cells were no longer found in hnt mosaics. The extended presence of mitotic-cycle markers suggests that hnt mutant cells are kept in the mitotic cycle and fail to undergo the M/E switch at stage 6/7, when wild-type cells would.

Figure 2
Hindsight function is required for proper M/E switch. (A, A′) Genomic BrdU incorporation was detected in hnt mutant follicle cells (outlined) in a stage-10B egg chamber, but the adjacent wild-type follicle cells had the focus-like BrdU incorporation ...

Mitotic cells are diploid during most of the cell-cycle phases, whereas follicle cells in the endocycle can have up to 16 times the haploid genomic content. To determine whether hnt mutant follicle cells remain diploid, like mitotic cells, we compared the DNA content and the nucleus size of the mutant cells to those of the neighboring wild-type cells at different stages using high-resolution three-dimensional photomicroscopy (see supplemental materials and methods). The average DNA content of the hnt mutant cells was about half that of the wild-type cells during stages 8–12 (Figure 2F and Table S1). Consistently, the nuclei in the mutant clones were half the size of the nuclei in the twin spots (wild-type sister clones; Figure 2H). These data suggest that hnt mutant cells have approximately one additional round of mitotic cycle and may have switched into the endocycle at around stage 9, in agreement with the finding that PH3-positive cells decreased when hnt mosaic egg chambers became older. Furthermore, we found about 1.7 times more cells in the mutant clones than in the corresponding twin spots (Figure 2G). The difference from the expected two-fold increase following an extra round of mitosis might stem from cell death in some hnt mutant cells, as indicated by fragmented DNA and antibody staining of an apoptosis marker, cleaved Caspase-3, in these cells as early as stage 9 (Figure 2I).

To determine whether hnt mutant follicle cells can switch into the chorion-gene amplification stage correctly, we performed a BrdU-incorporation assay in hnt mosaic egg chambers. At stage 10B, wild-type cells show the focus-like BrdU incorporation pattern that marks chorion-gene amplification, but hnt mutant cells had the oscillating genomic BrdU incorporation characteristic of cells in the endocycle (Figure 2A), indicating a defect in the switch from the endocycle to gene amplification.

Misexpression of Hindsight Is Sufficient to Drive Follicle Cells to Enter the Endocycle Prematurely

To determine whether Hnt function is sufficient to drive the M/E switch, we induced misexpression of Hnt in follicle cells before stage 6 using the flip-out Gal4/UAS technique. hntEP55, a transgenic line with the UAS sequence inserted 585 bp upstream of the hnt gene, was confirmed by 1G9 antibody staining to be able to misexpress Hnt in follicle cells before stage 6 (Figure 3A and 3A′). Large clones with Hnt misexpression were rarely generated, and follicle-cell nuclei in the clones were much larger than those in the adjacent wild-type follicle cells (Figure 3A and 3A″), indicating a premature M/E switch. Antibody staining detected neither CycA (n = 60, Figure 3B) nor CycB (Figure 3C) in follicle cells with early Hnt misexpression.

Figure 3
Misexpression of Hindsight is sufficient to drive follicle cells to enter the endocycle prematurely. (A, A′, A″) A high level of Hnt protein was detected in the follicle cells with Hnt misexpression (arrowhead) in a stage-5 egg chamber, ...

In contrast, more than 50% of the wild-type follicle cells are CycA positive in egg chambers younger than stage 6 (Schaeffer et al., 2004). When follicle cells were in the mitotic cycle during stage 4 or 5 of oogenesis, each egg chamber contained on average 16 PH3-positive cells (888 PH3-positive cells in 57 egg chambers), which represent 4–6% of all cells (each egg chamber contains 250–400 follicle cells during stages 4–5). We counted 542 Hnt overexpression cells during stages 4–5 and found that none of them showed PH3 staining (Figure 3C); in theory, 21–33 cells should be positive for both PH3 and GFP. Ectopic Hnt expression at an early stage in oogenesis is therefore sufficient to stop the mitotic cycle prematurely.

Endocycle cells retain oscillating patterns of genomic BrdU incorporation and Cyclin E (CycE) expression (Follette et al., 1998). In Hnt-misexpressing follicle cells, BrdU incorporation analysis revealed an oscillating pattern of genomic DNA replication, similar to that in the adjacent wild-type cells (Figure 3D), and CycE was also detected (data not shown). Absence of CycA, CycB, and PH3 staining and the oscillating patterns of CycE staining and BrdU incorporation together suggest that ectopic Hnt is sufficient to promote a premature M/E switch in follicle cells.

Hindsight Is Involved in Downregulation of Cut and String

Downregulation of Cut by Notch signaling is necessary for proper M/E switch in follicle cells (Sun and Deng, 2005). To assess the potential requirement for Hnt in Cut downregulation, we examined Cut expression in hntXE81 mosaic egg chambers. As shown in Figure 4A, Cut was continuously expressed in hnt mutant cells in a cell-autonomous manner after stage 6 but was downregulated in wild-type cells. This result supports our hypothesis that Hnt is required for downregulation of Cut expression. In addition, we found that misexpression of Hnt in follicle cells before stage 6 was sufficient to downregulate Cut expression (Figure 4B).

Figure 4
Hindsight is involved in downregulation of Cut, string, and immature-cell–fate markers. (A) Cut was continuously expressed in hnt mutant clones in a stage-7 egg chamber (outlined), but not at all in adjacent wild-type cells. (B) Cut was downregulated ...

During the M/E switch, Cut downregulation is required for upregulation of Fzr at around stage 6/7, which contributes to the degradation of mitotic cyclins (Sigrist and Lehner, 1997; Sun and Deng, 2005). We used an enhancer-trap line, fzr-lacZ (Schaeffer et al., 2004), to monitor the transcription of fzr in follicle cells with Hnt misexpression by a heat-shock-inducible Gal4. Sporadic expression of Fzr-lacZ was detected in stage-4 egg chambers with Hnt misexpression, and this premature expression was always associated with downregulation of Cut (Figure 4C). This result indicates that Hnt is sufficient to upregulate Fzr, perhaps explaining the defects of CycA and CycB expression in egg chambers with Hnt misexpression, as well as hnt mutant follicle cells.

To determine whether Hnt upregulates Fzr expression directly or through downregulation of Cut during the M/E switch, we coexpressed Hnt and Cut with hs-Gal4 in a fzr-lacZ heterozygous background. Fzr-lacZ was never found to be expressed in these egg chambers before stage 6 and was significantly downregulated during midoogenesis (Figure 4D), as when Cut is overexpressed alone, suggesting that Cut is downstream of Hnt in the regulation of fzr expression.

Stg is downregulated by Notch signaling but independent of Cut function during the M/E switch (Deng et al., 2001; Sun and Deng, 2005). To determine whether Hnt also functions upstream of Stg, we used a stg-lacZ (pstgβ-R6.4) reporter to monitor the transcription of stg (Lehman et al., 1999). As previously shown, Stg-lacZ is normally expressed in the mitotic follicle cells from stages 4 to 6 and downregulated after stage 6 (Deng et al., 2001). In hntXE81 follicle cells, high levels of Stg-lacZ, similar to those in Notch mutant cells, were detected even after stage 6 (Figure 4E). Conversely, Stg-lacZ expression was never found in Hnt-misexpressing follicle cells during early oogenesis (n = 35; data not shown). Upregulated expression of Cut and Stg in hnt mutant cells, which were kept in the mitotic cycle (Figure 2), is consistent with the previous finding that simultaneous misexpression of Cut and Stg is sufficient to keep follicle cells in the mitotic cycle (Sun and Deng, 2005).

To find out whether Hnt controls Cut and Stg expression by regulating Notch activity, we used a transgenic line E(spl)mβ-CD2 to examine Notch activity in hnt mutant clones. In wild-type egg chambers, Notch activity, as indicated by CD2 staining, was primarily detected in the polar cells of the follicle-cell layer before stage 6 but was present in all follicle cells from stage 7 to 10A. In hnt mutant follicle cells, E(spl)mβ-CD2 staining was not affected during midoogenesis (Figure 4F). We therefore conclude that Hnt does not interfere with Notch signaling and is a downstream executor of Notch signaling to downregulate Cut and Stg.

Hindsight Is Required for Follicle-cell Differentiation

Downregulation of Cut is necessary for follicle-cell differentiation, which is marked by reduced expression of FasIII and Eya during midoogenesis. In hntXE81 mosaic egg chambers, we found high levels of both FasIII and Eya in the mutant follicle cells after stage 6 but not in the adjacent wild-type cells (Figure 4G and 4H). Although FasIII is also expressed in polar cells, these cells do not express Eya. Coexpression of FasIII and Eya after stage 6 suggests that the hnt mutant follicle cells are in the immature cell fate, as is the case in Cut-misexpressing follicle cells (Sun and Deng, 2005). Hnt is therefore required for follicle-cell differentiation during the M/E switch. In contrast, downregulation of FasIII and Eya was detected in follicle cells with Hnt misexpression before stage 6 (Figure 4I and 4J), suggesting that these cells adopt a differentiated fate prematurely.

Notch Signaling Is Required for Downregulation of Hh Signaling during the M/E Switch

Hh signaling has been shown to be involved in follicle-cell proliferation and is downregulated during midoogenesis (Zhang and Kalderon, 2000). This involvement was confirmed through clonal analysis of fused (fu), a positive regulator of the pathway. fu mutant clones contained significantly fewer follicle cells than did the twin spots, suggesting a slower proliferation when Hh signaling is disrupted (Figure S2). The temporal pattern of Hh signaling was revealed by a triple labeling with antibodies against Cut, β-Galactosidase, and Ci-155 (recognized by 2A1 antibody) in the ptc-lacZ line, which showed that the expression patterns of these three markers were very similar. Both Ci-155 and Ptc-lacZ were detected in follicle cells during early oogenesis and significantly decreased after stage 6, concurrent with the downregulation of Cut during the M/E switch (Figure 5A–5D). The overlapping patterns of Cut, Ci-155, and Ptc-lacZ suggest that Hh signaling is active in the mitotic follicle cells but downregulated in cells undergoing the endocycle.

Figure 5
Notch signaling is required for downregulation of Hh signaling during the M/E switch. (A–D) Expression pattern of Ptc-lacZ (green in A and white in B), Ci-155 (red in A and white in C), and Cut (blue in A and white in D) in wild-type egg chambers ...

Because the M/E switch is induced by Notch signaling, we hypothesized that Notch signaling is required for the downregulation of Hh signaling. Indeed, we found high levels of Ci-155 protein, the Hh nuclear effector, in N55e11 follicle cells after stage 6 (Figure 5E). Ci-155 was also detected in follicle cells covering the Dlrev10 germ-line clones (data not shown) and Su(H)SF8 follicle cells (Figure 5F) after stage 6. In agreement with the Ci-155 staining, Ptc-lacZ, the Hh activity reporter, was also detected, although at a low level, in Su(H)SF8 follicle cells but not in the adjacent wild-type cells after stage 6 (Figure 5G). These results suggest that the canonical Notch pathway is involved in downregulating Hh signaling in follicle cells during the M/E switch.

Previous data show that high levels of Hh signaling resulting from a ptc mutation in follicle cells cause proliferation beyond stage 6 (Zhang and Kalderon, 2000), raising the question of whether Hh signaling can antagonize the activation of Notch signaling at stage 6/7. We therefore examined the expression of the mitotic markers (CycA and PH3), immature-cell-fate markers (FasIII, Eya, and Cut), and the Notch target (Hnt) in ptcS2 follicle-cell clones. ptc mutant cells tended to form multiple layers near the ends of the egg chamber; a monolayer was maintained in the lateral regions. In ptcS2 follicle cells in contact with the germ-line cells, neither immature-cell-fate markers nor mitotic markers were expressed after stage 6, but these markers were continuously expressed in the tumor-like cells that were not in direct contact with the germ-line cells and whose nuclei were much smaller (Figure S3A–S3C, S3E, and data not shown). In contrast, the Notch target, Hnt, was expressed in the ptcS2 cells in contact with germ-line cells, but not in those protruding out of the follicle-cell layer after stage 6 (Figure S3D). Together these data indicate that ptcS2 follicle cells can have Notch activated as long as they are in contact with the germ-line cells. 2A1 antibody staining revealed high levels of Ci-155 expression in all ptcS2 clones before stage 7 and those protruding out of the follicle-cell layer in older egg chambers, but not in those in contact with the germ-line cells and with Hnt expression (Figure S3F). In addition, ectopic expression of full-length Ci-155 after stage 6 had no effect on Hnt expression, indicating normal Notch activity (data not shown). These data suggest that Notch signaling is superimposed on the follicle cells during the M/E switch and is not antagonized by Hh signaling.

Hindsight Mediates Notch-dependent Downregulation of Hh Signaling through Transcriptional Repression of Cubitus Interruptus

To determine the relationship between downregulation of Hh signaling and Hnt upregulation in follicle cells, we applied 2A1 antibody to hntXE81 mosaic egg chambers. High levels of Ci-155 were continuously detected in hnt mutant follicle cells after stage 6 but not in the adjacent wild-type cells (Figure 6A). Ptc-lacZ was also detected in mutant cells after stage 6 (data not shown), as in Su(H) clones. In contrast, no Ci-155 was detected in Hnt-misexpressing follicle cells in egg chambers at early stages, when the wild-type cells have high levels of Ci-155 (Figure 6B). Because Hnt expression is induced by Notch signaling, these observations suggest that Hnt mediates the role of Notch in the downregulation of Hh signaling during the M/E switch.

Figure 6
Hindsight mediates Notch-dependent downregulation of Hh signaling through transcriptional repression of ci. (A, A′) Ci-155 was still detected at high levels in hnt mutant follicle cells (outlined) in a stage-9 egg chamber but not in the adjacent ...

It has been reported that PKA is required for Ci phosphorylation, which is necessary for proteolysis of Ci-155 into Ci-75 (Price and Kalderon, 1999), and pka mutant follicle cells accumulate ectopic Ci-155 (Bai and Montell, 2002). We found that Ci-155 accumulation in pka follicle cells occurred during stages 1–7, but after stage 8, no Ci-155 accumulation was detected (Figure 6C and data not shown), so active proteolysis of Ci-155 occurs specifically in early wild-type oogenesis. In contrast, Ci-155 upregulation in hnt clones was observed during midoogenesis and continued until after stage 10 (Figure 6A and data not shown). No such upregulation was detected in stage 1–6 hnt clones (data not shown). This difference suggests that Hnt regulation of Hh signaling is probably not through proteolysis of Ci-155, which requires PKA function. To determine whether Hnt regulates ci transcriptionally, we stained for CiAbN, an antibody against the N-terminal region of Ci protein (recognizing both Ci-155 and Ci-75; Aza-Blanc et al., 1997), in hnt mosaics. hnt follicle cell clones had high levels of CiAbN after stage 7, whereas wild-type cells had nearly undetectable staining (Figure 6D). Furthermore, we used, as a reporter for ci transcription, an enhancer trap ci-lacZ, which shows even expression of Ci-lacZ in wild-type follicle cells until stage 7, then downregulation (Forbes et al., 1996b). High levels of Ci-lacZ expression were found in hnt clones and persisted after stage 10 (Figure 6E), when the wild-type follicle cells had no detectable Ci-lacZ expression. In contrast, Ci-lacZ expression was not changed in pka clones, even in early oogenesis, when high levels of Ci-155 protein accumulated (Figure 6F). These results suggest that Hnt is involved in suppression of ci transcription in follicle cells.

Downregulation of Ci during Midoogenesis also Requires Tramtrack Function

Mutation of another zinc-finger transcription factor, tramtrack (ttk), results in extended expression of PH3 and CycB and small follicle-cell nuclei after stage 6 (Jordan et al., 2006), a phenotype similar to that of hnt, but the Ttk expression pattern differed from that of Hnt (Figure S1D). One isoform of Ttk, Ttk69, was steadily expressed in follicle cells before stage 10 (Figure S1D), and no obvious change was found in Notch clones during midoogenesis (Figure 7A), whereas the other, Ttk88, showed no expression in follicle cells (data not shown). We therefore stained Ttk69 in hntXE81 and Hnt in ttk1e11 mosaic egg chambers. The expression level of Ttk69 in hnt mutant clones was not significantly different from that in wild-type cells (Figure 7B). Also, Hnt staining in ttk mutant follicle cells revealed no defects (Figure 7C), suggesting that ttk and hnt do not depend on each other for their expression in follicle cells.

Figure 7
Downregulation of Ci during midoogenesis also requires Tramtrack function. (A, B, A′, B′) Ttk69 antibody staining was still detected in Notch mutant clones (A, A′), as well as hnt mutant clones (B, B′); no significant difference ...

To address further whether loss of ttk resembles the loss of hnt phenotype, we examined ci expression in ttk mosaics. As in hnt clones, high levels of Ci-155, as well as CiAbN, were detected in ttk mutant cells after stage 6 (Figure 7D and 7E). In addition, we found upregulation of Ci-lacZ expression in ttk clones (data not shown), which, along with Ci protein expression defects, indicates that ttk is also required for ci transcriptional repression. Although Ttk69 was expressed in early oogenesis, no change of Ci protein was found in ttk1e11 clones before stage 6 (data not shown), indicating that Ttk-mediated ci downregulation requires Hnt.

Discussion

How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. Our data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. We show that Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Figure 7D–7E; Jordan et al., 2006).

Hindsight Function in Follicle-cell Cycle Regulation

Our previous studies showed that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed (Sun and Deng, 2005). Our current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle.

Our finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots (Deng et al., 2001), suggesting an additional cell cycle also takes place. Further test of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may be not the sole mediator of the Notch effect, for example Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later (data not shown), suggesting that Hnt function is also required for chorion-gene amplification.

The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells (Forbes et al., 1996a; Zhang and Kalderon, 2000; Figure S3). Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots (Figure S2). The nuclear sizes of fu mutant cells were similar to those of the wild type at the same developmental stage; and no fragmentation of the chromosomes was observed (data not shown). Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of the full-length Ci in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis (data not shown). Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells.

Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells but is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt (data not shown). Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Zhang et al., 2003; Date et al., 2004).

An interesting observation from our studies and that of Jordan et al. (2006) is that ttk clones have a phenotype similar to that of hnt clones. Jordan et al. (2006) reported that, as in Notch regulation of Hnt, ttk is possibly downstream of Notch, but our analysis of Notch mutants in stage 1–10 egg chambers showed no obvious change in Ttk expression. We also found that Hnt has no role in regulating ttk expression. Our findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis (Figure S1D). The phenotypic similarity between hnt and ttk mutants suggested to us that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye (Xiong and Montell, 1993). Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear.

Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process (Sigrist and Lehner, 1997). Second, nurse-cell endoreplication does not require Hnt, as no obvious defect was detected in hnt germ-line clones (data not shown). The specific role of Hnt in follicle-cell–cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation.

Interaction of Notch and Hedgehog Signaling

Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S-phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway (Baonza and Freeman, 2005; Firth and Baker, 2005). Hh and Notch therefore act sequentially and positively during the SMW; whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis but is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling, as mutation of the negative regulator of Hh pathway ptc in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germ-line cells. These ptc mutant cells show no accumulation of Ci-155 (Figure S3F), consistent with our finding that Notch signaling suppresses ci transcription through Hnt. The ptcS2 cells that were out of contact with germ-line cells remained in the mitotic cycle because they could not receive Delta signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state.

Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration (Figure S4A-S4C). When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% (n = 250) of egg chambers showed defects in border-cell migration (Figure S4D-S4I). Notch signaling, as well as ttk, has been reported to be required for border-cell migration (Wang et al., 2006; Wang et al., 2007). We found that Hnt was expressed in the border cells and depended on Notch signaling (Figure S4J and data not shown). The occasional hnt border-cell clones we observed also showed defects in border-cell migration (data not shown), so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells.

Experimental Procedures

Fly Strains and Generation of Mosaics

Strains N55e11, Dlrev10, Su(H)SF8, and psnC1 were described by Sun and Deng (2005). hntXE81 and hntEH704a were obtained from H. D. Lipshitz (Yip et al., 1997) and recombined to the FRT18D chromosome (Maines et al., 2004). Because these two alleles exhibited similar phenotypes, we described only the hntXE81 phenotype in the experiments. FRT82B ttk1e11/TM6 Hu was from D. Montell (Wang et al., 2006). FRT42D ptcS2, DCOH2 FRT40A, and fumH69 FRT 18D were obtained from J. Horabin (Horabin et al., 2003). The following transgenic lines were used in our study: slbo-Gal4, UAS-mCD8-GFP (Wang et al., 2006), UAS-Ci (a gift from J. Horabin), ci-lacZ (ciDplac allele from Bloomington Stock Center, BSC), fzr-lacZ and hntEP55 (BSC), ptc-lacZ (Zhang and Kalderon, 2000), string-lacZ (pstgβ-R6.4; Lehman et al., 1999) and the Notch activity reporter E(spl)mβ-CD2 (obtained from L. L. Dobens; de Celis et al., 1998). w1118 was used as a wild-type control. Clone induction followed previously described procedures (Sun and Deng, 2005; supplemental materials and methods).

Immunohistochemistry and BrdU Labeling

Immunohistochemistry, BrdU labeling, and image acquisition were carried out as previously described (Sun and Deng, 2005). The following antibodies were used: mouse anti-Hnt (1G9) 1:15, anti-FasIII (7G10) 1:15, anti-CycB (F2F4) 1:50, anti-Cut (2B10) 1:15, anti-EYA (10H6) 1:10, anti-Lamin (ADL84.12) 1:30, anti-Singed (sn7C) 1:30 (DSHB), mouse anti-BrdU 1:50 (BD Bioscience), mouse anti-CD2 1:50 (AbD Serotec), rabbit anti-β-Galactosidase 1:5000 (Sigma, USA), rabbit anti-PH3 1:200 (Upstate Biotechnology, NY), rabbit anti-cleaved Caspase-3 (Asp 175) 1:100 (Cell Signaling Technology), rabbit anti-CycA 1:500 (a gift from C. Lehner), and guinea pig anti-CycE 1:500 (a gift from T. Orr-Weaver). 2A1 antibody (rat monoclonal antibody recognizing only Ci-155) was obtained from R. Holmgren and T. B. Kornberg (Motzny and Holmgren, 1995; Aza-Blanc et al., 1997), and rabbit anti-CiAbN (recognizing the N terminal region of Ci protein) was a gift from J. Horabin (Aza-Blanc et al., 1997).

Supplementary Material

01

Acknowledgments

We are grateful to A. B. Thistle, J. Horabin, H. W. Bass, and J. Poulton for critical reading of and comments on the manuscript; special thanks are due to H. W. Bass for supporting the three-dimensional photomicroscopic analysis; to S. Qian, L. F. Shyu, and other members of the Deng lab for technical help and discussions on this project; and to L. L. Dobens, B. Edgar, R. Holmgren, J. Horabin, D. Kalderon, T. B. Kornberg, C. Lehner, H. D. Lipshitz, D. Montell, T. L. Orr-Weaver, L. A. Raftery, H. Ruohola-Baker, D. Stein, G. Struhl, the DSHB, and the Bloomington and Szeged Stock Centers for generously providing us antibodies and fly stocks. We thank K. Riddle and the Biological Science Imaging Facility at Florida State University for helping us acquire the images using the confocal microscope. WMD is supported by a Scientist Development Grant from the American Heart Association, Florida/Puerto Rico affiliate; National Institutes of Health Grant R01 GM072562-01A2; and the FSU College of Arts and Sciences setup fund.

Footnotes

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