1. Volume 22, Issue 5, Pages 389-396 (March 2012)
Drosophila Pez Acts in Hippo Signaling to Restrict Intestinal Stem Cell Proliferation 
Ingrid Poernbacher, Roland Baumgartner, Suresh K. Marada, Kevin Edwards, Hugo Stocker 
Current Biology 
Volume 22, Issue 5, Pages (March 2012)
DOI: /j.cub
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2. Figure 1
Pez Is a Binding Partner of Kibra in S2 Cells and Regulates Growth
(A and B) Full-length Kibra coimmunoprecipitates with full-length Pez (A) and vice versa (B). Pez-HA (A) (or Kibra-HA in B) and Kibra-FLAG (A) (or Pez-FLAG in B) were cotransfected in S2 cells and HA immunoprecipitates were blotted for Kibra-FLAG (A) or Pez-FLAG (B), respectively. HA-GFP was used as a negative control.
(C–E) The small body size of Pez2/Df flies (D) is rescued by the presence of a single Pez genomic rescue construct (E). The scale bar represents 500 μm.
(F) Statistical analysis of fly dry weight (n = 16). Pez2/Df females are significantly lighter (0.13 ± 0.024 mg) than control flies (0.35 ± 0.019 mg; p = 4.84E-22). This decrease in weight is suppressed in the presence of a single Pez genomic rescue construct (0.39 ± 0.05 mg; p = 8.46E-15). Analyses were done with a Student's t test (two-tailed, unpaired). Error bars indicate the standard deviations.
(G–J) Eyes (G and H) and wings (I and J) overexpressing the indicated UAS transgenes under the control of eyeless (ey)-Gal4 (G and H) or MS1096-Gal4 (I and J). The scale bars represent 100 μm.
(K–M) Optical cross-sections through the center of a control adult intestine (K) and a Pez mutant adult intestine (L). Overgrowth of the Pez mutant adult intestine is rescued by the presence of a single Pez genomic rescue construct (M). The scale bar represents 100 μm.
(N) The Pez locus (drawn to scale) spans 10 kb and contains seven exons (filled boxes). Pez1 is an EMS allele and Pez2 is a deletion. The region covered by the Pez genomic rescue construct is indicated.
See also Figure S1.
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3. Figure 2
Pez Influences ISC Proliferation via Control of the EGFR and JAK/STAT Pathways
(A and B) Control adult midgut (A–A″) and Pez mutant adult midgut (B–B″) expressing esg-lacZ (red) and stained for pH3 (green) and DNA (blue). The scale bar represents 50 μm.
(C) Quantification of pH3-positive cells per adult midgut of the indicated genotypes (n = 13). Pez mutant midguts contain significantly more mitotic cells (26.77 ± 10.81) than controls (6.92 ± 4.37; p = 1.5E-05). This increase is suppressed by the presence of a Pez genomic rescue construct (10.77 ± 5.75; p = 7.5E-05). Analyses were done with a Student's t test (two-tailed, unpaired). Error bars indicate the standard deviations.
(D and E) Control (D–D″) and Pez mutant (E–E″) adult midgut expressing Dl-lacZ (red) and Myo1A-GAL4 > UAS-GFP (green) and stained for Pros (blue). The scale bar represents 50 μm.
(F) Induction of the genes encoding the cytokine Upd3 and the EGFR ligand Vn in Pez mutant adult midguts measured by RT-qPCR. Numbers indicate fold of activation over heterozygous control guts. Error bars indicate the standard deviations. Myo1A is used as an internal control.
(G–J) Pez mutant adult midguts show an increase in EGFR activity as monitored by staining for dpERK (green) (G, G′, H, H′) and in STAT activity as monitored by expression of a 10xSTAT-dGFP reporter (green) (I, I′, J, J′). esg-lacZ is shown in red and DAPI in blue. The scale bars represent 50 μm.
See also Figure S2.
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4. Figure 3 Pez Acts through Yki
(A–E) The small body size of Pez mutant flies (B and C) as well as the overabundance of pH3-positive cells (D; statistical analysis as in Figure 2C; n ≥ 10; p = 3.6E-07) and the induction of vn (E) seen in Pez mutant adult midguts are rescued by removal of a single copy of yki. The scale bar represents 500 μm. Error bars indicate the standard deviations.
(F and G) Pez mutant adult midguts show an increase in Yki activity as monitored by expression of the Diap1 enhancer element diap1-GFP4.3 (green). Expression of Dl-lacZ is shown in red. The scale bar represents 100 μm.
(H) Pez mutant adult midgut stained for Pdm1 (blue) and expressing Dl-lacZ (red) and diap1-GFP4.3 (green). The scale bar represents 100 μm.
(I and J) MARCM control clones (I–I″) and MARCM clones of Pez mutant cells (J–J″) (labeled with LacZ in white) in the adult midgut epithelium 10 days after induction. Expression of diap1-GFP4.3 is shown in green. The scale bar represents 100 μm.
See also Figure S3.
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5. Figure 4
Pez Acts in ECs and Does Not Require the FERM and PTP Domains to Exert Its Function in These Cells
(A and B) The overabundance of pH3-positive cells (A; statistical analyses as in Figure 2C; n ≥ 10; p = ; p = ) and induction of vn (B), normally observed in Pez mutant adult midguts, are rescued by Myo1A-Gal4-driven knockdown of yki as well as by Myo1A-Gal4-driven expression of full-length Pez, ΔFERM-Pez (only B), or PezPD (only B). The presence of UAS-Pez full-length alone, without any Gal4 driver, does not rescue (A) or rescues only partially (B). Error bars indicate the standard deviations.
(C) Flies of the indicated genotypes were mock treated or fed with 25 μg/ml Bleomycin. pH3-positive cells per midgut were quantified after 2 days of feeding. Statistical analyses as in Figure 2C (n ≥ 16; p = 0.45; p = ); error bars indicate the standard deviations.
(D and E) The overabundance of pH3-positive cells (D; statistical analyses as in Figure 2C; n = 7; p = ; p = ) and the induction of vn (E) in Pez mutant adult midguts are partially rescued by Myo1Ats-driven knockdown of yki or Myo1Ats-driven overexpression of hpo, respectively. Error bars indicate the standard deviations.
(F) The overabundance of pH3-positive cells in Pez mutant adult midguts is partially rescued by Dlts-driven overexpression of hpo or Pez but not by knockdown of yki. Statistical analyses as in Figure 2C (n ≥ 13; p = 2.3E-05; p = 0.30; p = ); error bars indicate the standard deviations.
(G and H) Surface (G, G′) and cross-section (H, H′) of adult midgut epithelium stained for DAPI (blue) and expressing the GFP-tagged Pez genomic rescue construct (stained with GFP antibody; green) and esg-lacZ (red, only G′). The scale bars represent 50 μm.
(I) Domain organization of Pez, ΔFERM-Pez, ΔPTP-Pez, PezPro, PezLink, and Kibra as well as location of amino acid replacements in PezPD, PezPPPA PPSGA, and KibraWW mutant.
(J) Wild-type Kibra coimmunoprecipitates with full-length Pez, ΔFERM-Pez, ΔPTP-Pez, PezPPPA PPSGA (all J), and PezPro (J′) but not PezLink (J′) in S2 cells. KibraWW mutant does not coimmunoprecipitate with full-length Pez in S2 cells (J). Kibra-FLAG or KibraWW mutant-FLAG and Pez-HA, ΔFERM-Pez-HA, ΔPTP-Pez-HA, PezPPPA PPSGA-HA, PezPro-HA, or PezLink-HA were cotransfected into S2 cells. HA immunoprecipitates were blotted for FLAG-tagged proteins. HA-GFP was used as a negative control.
(K and L) Surface (K–K″) and cross-section (L–L″) of adult midgut epithelium stained for DAPI (blue) and expressing the GFP-tagged Pez genomic rescue construct (stained with GFP antibody; green) as well as UAS-Kibra18 under the control of Myo1A-Gal4 (red). The scale bars represent 50 μm.
See also Figure S4.
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