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. 2008 Mar 12;3(3):e1761.
doi: 10.1371/journal.pone.0001761.

The hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity

Jianzhong Yu  1 , John Poulton, Yi-Chun Huang, Wu-Min Deng
Affiliations

The hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity

Jianzhong Yu et al. PLoS One. .

Abstract

Specification of the anterior-posterior axis in Drosophila oocytes requires proper communication between the germ-line cells and the somatically derived follicular epithelial cells. Multiple signaling pathways, including Notch, contribute to oocyte polarity formation by controlling the temporal and spatial pattern of follicle cell differentiation and proliferation. Here we show that the newly identified Hippo tumor-suppressor pathway plays a crucial role in the posterior follicle cells in the regulation of oocyte polarity. Disruption of the Hippo pathway, including major components Hippo, Salvador, and Warts, results in aberrant follicle-cell differentiation and proliferation and dramatic disruption of the oocyte anterior-posterior axis. These phenotypes are related to defective Notch signaling in follicle cells, because misexpression of a constitutively active form of Notch alleviates the oocyte polarity defects. We also find that follicle cells defective in Hippo signaling accumulate the Notch receptor and display defects in endocytosis markers. Our findings suggest that the interaction between Hippo and classic developmental pathways such as Notch is critical to spatial and temporal regulation of differentiation and proliferation and is essential for development of the body axes in Drosophila.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The Hpo pathway is required for oocyte polarity formation.
(A) Stau:GFP (arrow) is localized to the posterior of wild-type stage-9 oocytes. (B) Large sav 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 hpo 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 sav 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 hpo 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 sav 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 sav and hpo mutants (red arrowheads). Loss-of-function clones are marked as the GFP-negative cells. Gain-of-function clones (UAS-Yki) 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.
Figure 2
Figure 2. Mutants of the Hpo pathway disrupt PFC differentiation.
The PFC markers pnt-lacZ (A) and 667/9-lacZ (C) are specifically expressed in the PFC after stage 6 in wild-type egg chambers. (B and D) hpo PFC clones fail to express pnt-lacZ (B) or 667/9-lacZ (D), in a cell-autonomous manner (arrows). (E) Activation of JAK-STAT signaling in the PFC (arrow) can be marked by the expression of dome-lacZ. (F) In sav PFC clones, expression of dome-lacZ is not affected (arrow). The AFC markers slbo-lacZ (G) and dpp-lacZ (I) are expressed in the AFCs in stage-9 wild-type egg chambers. In sav mutant PFC, no misexpression of slbo-lacZ (H) or dpp-lacZ (J) was detected.
Figure 3
Figure 3. Notch signaling is disrupted in PFC clones of Hpo pathway mutants.
(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 sav 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 sav (F) and wts (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 sav (J) or hpo (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 sav mutant PFC in this stage-7 egg chamber (arrow).
Figure 4
Figure 4. Rescue of Hpo phenotypes through overexpression of NICD.
GFP-positive sav clones were created by the MARCM technique. (A) Stau is mislocalized to the center of the oocyte when large sav clones are located at the posterior. (B) Stau is localized correctly to the posterior pole when NICD is expressed in sav PFC clones. (C) Grk was detected at the oocyte posterior when large sav PFC clones were generated . (D) Misexpression of NICD in sav PFC clones restored Grk at the dorsal-anterior corner of the oocyte.
Figure 5
Figure 5. Defective endocytosis in Hpo pathway–mutant follicle cells.
Both NECD and NICD accumulate in hpo (A,C) and sav (B,D) PFC clones of stage 9/10 egg chambers, including ectopic cytoplasmic puncta (arrowheads). (E) In hpo 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 hpo 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). hpo (G) and sav (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 hpo 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).
Figure 6
Figure 6. Expression of Hpo target genes in mosaic egg chambers.
(A,B) Upregulated Ex expression was found in hpo (A) and sav (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 ex (C) and cycE (D) was observed in sav follicle-cell clones (arrows). Note differences in expression of cycE-lacZ 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, diap-lacZ (arrow) was also upregulated in hpo PFC clones.
Figure 7
Figure 7. Apical-basal polarity in Hpo-defective follicle cells.
sav mutant follicle cells that remain in contact with the germline display correct localization of the apical marker, aPKC (A, blue arrows; compare to B which shows wildtype cell pattern (GFP-positive cells) as well as no obvious defects in neighboring clone cells). sav clone cells in outer layers of a multilayered clone, however, do not show apical accumulations (A, white arrows). Localization of the basal-lateral marker Dlg appears largely correct in both sav (C) and hpo (D) clones (white arrowheads), but see Results for further description.
Figure 8
Figure 8. mer mutation disrupts PFC fate and Notch signaling.
(A) mer 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 ex clones of this stage 7 egg chamber, although the penetrance was significantly lower (see Results). (C) Loss of pnt-lacZ expression was observed in mer PFC clones (arrow). (D) mer PFC clones accumulate high levels of NICD. Multilayering and small nuclei could also be seen in mer PFC clones (A’’’,D’’’, red arrowheads).
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