Expression of Hpo target genes in mosaic egg chambers.

<p>(A,B) Upregulated Ex expression was found in <i>hpo</i> (A) and <i>sav</i> (B) mutant follicle cells (arrows). Note that a stronger upregulation of Ex was found in the PFC clone (B). (C,D) Upregulated expression of lacZ markers for the Hpo pathway target genes <i>ex</i> (C) and <i>cycE</i> (D) was observed in <i>sav</i> follicle-cell clones (arrows). Note differences in expression of <i>cycE-lacZ</i> at boundaries between wildtype and clone cells at posterior (white arrow), as well as more anterior locations (blue arrow) in this stage 7 egg chamber. (E) In addition, <i>diap-lacZ</i> (arrow) was also upregulated in <i>hpo</i> PFC clones.</p

Defective endocytosis in Hpo pathway–mutant follicle cells.

<p>Both NECD and NICD accumulate in <i>hpo</i> (A,C) and <i>sav</i> (B,D) PFC clones of stage 9/10 egg chambers, including ectopic cytoplasmic puncta (arrowheads). (E) In <i>hpo</i> follicle-cell clones (indicated by loss of GFP, false-colored blue in panel E, white in E’’), Hrs (red) accumulates at the apical region and overlaps significantly with NICD (false-colored in green to faciliate determination of colocalization by yellow signal, as shown in E'). (F) Some ectopic NICD is also found to colocalize with Rab7:GFP positive vesicles (white arrowheads) in <i>hpo</i> follicle cell clones (visualized by loss of lacZ in blue in panel F, white in F’’). Note in wildtype cells the Rab7:GFP-positive vesicles do not appear to contain Notch protein (red arrowheads). <i>hpo</i> (G) and <i>sav</i> (H) mutant PFC also contain discrete cytoplasmic as well as membrane-associated accumulations of Domeless protein. Staining of the endocytic marker FM4-64FX was significantly higher in <i>hpo</i> mutant follicle cells of stage 10 egg chambers, regardless of position in the FE (I cross-section, J top view-clone outlined in dashed yellow line).</p

Notch signaling is disrupted in PFC clones of Hpo pathway mutants.

<p>(A and C) PH3 and Cyclin B are expressed sporadically in immature follicle cells during early stages (S1–S6) in wild-type egg chambers. In <i>sav</i> mutants, staining of PH3 (B) and Cyclin B (D) was occasionally found in mutant PFC after stage 6 (arrows). (E) In wild-type egg chambers, Cut is expressed in follicle cells until about stage 6. (F and G) Prolonged Cut expression was found in <i>sav</i> (F) and <i>wts</i> (G) PFC clones at stages 8–10 of oogenesis. (I) Hnt is expressed in follicle cells after stage 6 in the wild type. No Hnt expression was found in <i>sav</i> (J) or <i>hpo</i> (K) mutant PFC in stage-8 egg chambers. (L) Lack of Hnt staining was also occasionally observed in anterior and lateral hpo clones in stage 7 egg chambers (arrows). Overexpression of Yki caused prolonged Cut expression (H) and decreased Hnt expression (M). (N) The E(spl):CD2 Notch activity reporter, visualized by CD2 staining, is upregulated in follicle cells during stages 7–10A of oogenesis in wild-type egg chambers. (O) Lack of CD2 staining was observed in <i>sav</i> mutant PFC in this stage-7 egg chamber (arrow).</p

The Hpo pathway is required for oocyte polarity formation.

<p>(A) Stau:GFP (arrow) is localized to the posterior of wild-type stage-9 oocytes. (B) Large <i>sav</i> follicle-cell clones cause a complete mislocalization of Stau-GFP (white arrow) toward the oocyte center, and the oocyte nucleus (blue arrow) remains at the posterior. (C) A stage 9 egg chamber with large <i>hpo</i> PFC clone also shows mislocalization of Stau:GFP toward the center of the oocyte (arrow). (E) Stau (arrow) is mislocalized away from the region adjacent to the <i>sav</i> clones when the PFC are partially mutated. (F) Oocyte nucleus and Grk (arrow) are localized to the dorsal anterior corner of wild-type stage-9 oocytes. (G) Large <i>hpo</i> follicle-cell clones cause mislocalization of the oocyte nucleus and Grk (arrow) at the oocyte posterior. Overexpression of Yki also caused Stau (D, arrow) and Grk (H, arrow) mislocalization. (I) Plus ends of microtubules, visualized with Kin:β-Gal (arrow) localization at the posterior of a wild-type stage-9 oocyte. (J) A stage-9 egg chamber with a large <i>sav</i> follicle-cell clone showing abnormal Kin:β-Gal (arrowhead) localization in the center of the oocyte, as well as mislocalization of the oocyte nucleus (blue arrow). Multilayering and small nuclear phenotypes can be observed in PFC clones of both <i>sav</i> and <i>hpo</i> mutants (red arrowheads). Loss-of-function clones are marked as the GFP-negative cells. Gain-of-function clones (<i>UAS-Yki</i>) are GFP-positive. All clones are additionally highlighted by yellow lines to indicate the affected follicle cells, except in a few cases of complete or almost complete follicle cell clones. In all Figures, posterior is to the right. Nuclei are marked in most figures by DAPI staining in blue.</p

Mutants of the Hpo pathway disrupt PFC differentiation.

<p>The PFC markers <i>pnt-lacZ</i> (A) and <i>667/9-lacZ</i> (C) are specifically expressed in the PFC after stage 6 in wild-type egg chambers. (B and D) <i>hpo</i> PFC clones fail to express <i>pnt-lacZ</i> (B) or <i>667/9-lacZ</i> (D), in a cell-autonomous manner (arrows). (E) Activation of JAK-STAT signaling in the PFC (arrow) can be marked by the expression of <i>dome-lacZ</i>. (F) In <i>sav</i> PFC clones, expression of <i>dome-lacZ</i> is not affected (arrow). The AFC markers <i>slbo-lacZ</i> (G) and <i>dpp-lacZ</i> (I) are expressed in the AFCs in stage-9 wild-type egg chambers. In <i>sav</i> mutant PFC, no misexpression of <i>slbo-lacZ</i> (H) or <i>dpp-lacZ</i> (J) was detected.</p

<i>mer</i> mutation disrupts PFC fate and Notch signaling.

<p>(A) <i>mer</i> clones lead oocyte nucleus mislocalization (blue arrow), and misexpression of Cut (white arrow) in the PFC clones of this stage 10 egg chamber. (B) Similar defects can also be observed in <i>ex</i> clones of this stage 7 egg chamber, although the penetrance was significantly lower (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001761#s2" target="_blank">Results</a>). (C) Loss of <i>pnt-lacZ</i> expression was observed in <i>mer</i> PFC clones (arrow). (D) <i>mer</i> PFC clones accumulate high levels of NICD. Multilayering and small nuclei could also be seen in <i>mer</i> PFC clones (A’’’,D’’’, red arrowheads).</p

Rescue of Hpo phenotypes through overexpression of NICD.

<p>GFP-positive <i>sav</i> clones were created by the MARCM technique. (A) Stau is mislocalized to the center of the oocyte when large <i>sav</i> clones are located at the posterior. (B) Stau is localized correctly to the posterior pole when NICD is expressed in <i>sav</i> PFC clones. (C) Grk was detected at the oocyte posterior when large <i>sav</i> PFC clones were generated . (D) Misexpression of NICD in <i>sav</i> PFC clones restored Grk at the dorsal-anterior corner of the oocyte.</p

EcR regulation of BR-Z1 before and after the prepupa-to-pupa transition.

<p>(A–B′) DN-EcR-expressing clones (green) displayed BR-Z1 (red) downregulation at third-instar larval stages (A, A′) and upregulation 20–24 h APF (B, B′). (C–F) BR-Z1 (red) expression detected in wing discs of <i>en-GAL4>UAS-DN-EcR; Tub-GAL80^ts/+</i> 21–22 h APF. White arrowheads indicate anterior-posterior boundaries. BR-Z1 expression in the posterior compartment (bottom) was unaffected when animals were incubated at 18°C from larva to pupa (C), or at 18°C except during larva-to-prepupa transition (−4 to 3 h APF) at non-permissive temperatures (37°C for one hour and 29°C for six hours) (D). BR-Z1 levels were upregulated in pupae incubated at non-permissive temperatures 8–12 h APF (E) or 13–21 h APF (F). (G) A diagram showing the temperature regimen in animals shown in Figure C–F.</p

Association of CSN subunits and EcR in BR-Z1 repression.

<p>(A) The BR-Z1 mRNA level in wing discs 20–24 h APF, assayed by semi-quantitative RT-PCR, was upregulated in wing discs of <i>CSN2</i> RNAi driven by <i>en-GAL4</i> compared to <i>en-GAL4/+</i> control. <i>rp49</i> levels served as internal controls. (B–C′) <i>EcRE-lacZ</i> (red) expression was upregulated in <i>CSN4^null</i> (B, B′) and <i>CSN5^null</i> (C, C′) clones 20–24 h APF, but unaffected in <i>CSN5^null</i> clones 6–8 h APF (D, D′). (E–E″) Both EcRA (red) and Myc-CSN2 (green) localized in nuclei in wing disc 20–24 h APF. (F) Western blots showing co-precipitations of Myc-EcRA (upper panel) or Myc-EcRB1 (lower panel) in Flag immunoprecipitates of Flag-USP, CSN2, CSN4 or CSN5 in S2 cell extract. Co-precipitation was not detected in Flag-GFP. * represents the non-specific bands. (G, G′) BR-Z1 levels (red) in double MARCM clones for <i>CSN5^null</i> and <i>DN-EcR</i> (green) were not further elevated compared to <i>CSN5^null</i> or <i>DN-EcR</i> single clones (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004760#pgen-1004760-g003" target="_blank">Figure 3B</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004760#pgen-1004760-g006" target="_blank">6B</a>).</p

BR-Z1 upregulation is required for Sens upregulation at the PWM of <i>CSN</i> mutants.

<p>(A–B′) Upregulation of BR-Z1 (red) in <i>CSN4^null</i> (A, A′) and <i>CSN5^null</i> clones (B, B′) 20–24 h APF. White arrowheads indicate the anterior-posterior boundary. (C–F) Transient overexpression of BR-Z1 in bristle lineage cells via <i>neur-GAL4</i> and <i>Tub-GAL80^ts</i>. (C–E) Ectopic expression of Sens (red in C), accumulation of Hnt (red in D) and ectopic formation of Elav-positive neurons (red in E) were observed at the PWM 22–24 h APF. Prepupae grown at 18°C were shifted to 37°C for one hour at 8–10 h APF, and then incubated at 29°C until dissection. (F) Bristles with dome-shape sockets (indicated by arrows) were observed at the PWM. Inset: enlarged figure showing single bristle. The experiment was carried out similarly to (C–E) except for incubation at 29°C for 12 hours, and returning back to 18°C until eclosion. (G–H′) Knockdown of <i>CSN2</i>, in MARCM clones (green), induced Sens (red) upregulation at the PWM 20–24 h APF (G, G′), which was abolished in <i>CSN2</i> RNAi <i>br^npr1</i> double mutant MARCM clones (green) (H, H′).</p