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. 2017 Aug 15;6(8):1165-1173.
doi: 10.1242/bio.025221.

The Protein Phosphatase 4 Complex Promotes the Notch Pathway and wingless Transcription

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Free PMC article

The Protein Phosphatase 4 Complex Promotes the Notch Pathway and wingless Transcription

Eric T Hall et al. Biol Open. .
Free PMC article

Abstract

The Wnt/Wingless (Wg) pathway controls cell fate specification, tissue differentiation and organ development across organisms. Using an in vivo RNAi screen to identify novel kinase and phosphatase regulators of the Wg pathway, we identified subunits of the serine threonine phosphatase Protein Phosphatase 4 (PP4). Knockdown of the catalytic and regulatory subunits of PP4 cause reductions in the Wg pathway targets Senseless and Distal-less. We find that PP4 regulates the Wg pathway by controlling Notch-driven wg transcription. Genetic interaction experiments identified that PP4 likely promotes Notch signaling within the nucleus of the Notch-receiving cell. Although the PP4 complex is implicated in various cellular processes, its role in the regulation of Wg and Notch pathways was previously uncharacterized. Our study identifies a novel role of PP4 in regulating Notch pathway, resulting in aberrations in Notch-mediated transcriptional regulation of the Wingless ligand. Furthermore, we show that PP4 regulates proliferation independent of its interaction with Notch.

Keywords: Flfl; Notch signaling; PP4; Wingless signaling.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Reduction of PP4 subunits inhibits Wg pathway activation without inducing cell death. (A-B) Normal expression pattern of hh-Gal4 (A) in the posterior domain of the developing wing disc, shown with wild-type expression of Wg target gene Dll-lacZ (A′), as well as cleaved caspase 3 (A″) and stabilized Arm in bands flanking the D/V boundary (B, arrow). (C-D) The knockdown of PP4-19C with RNAi in the posterior domain causes a reduction in Dll expression (C,C′, arrowhead), but did not significantly increase C. Casp3 levels (C″), while inducing a marked reduction in stabilized Arm (D). (E-F) Knockdown of PPP4R2 caused a minor reduction in Dll-lacZ (E’, arrowhead) and Arm (F), but did not affect C. Casp3 levels (E″). (G-H) Flfl knockdown reduced Dll expression (G,G′, arrowhead) without increasing C. Casp3 activity (G″), and caused a reduction in stabilized Arm (H). Scale bar: 50 µm.
Fig. 2.
Fig. 2.
PP4 promotes Wg signaling through Notch pathway activation. (A) Wild-type pattern of Wg protein. (B) Using hh-Gal4, expressed in the posterior domain of the wing disc (right of the dotted line), to express flfl-RNAi caused a reduction in total Wg protein levels. (C,C′) Somatic clones of the hypomorphic flfl795 allele in the posterior domain (marked by the absence of Ci) also showed reduced Wg protein. (D-I) Wild-type pattern of wg transcription (D), Cut protein (F), and Dl protein (H). Expression of flfl-RNAi in cells in the posterior domain (right of dotted line), caused a reduction in wg transcription (E), and loss of Cut (G) and Dl (I) in the developing wing disc. Scale bar: 50 µm.
Fig. 3.
Fig. 3.
PP4 promotes Notch signaling in the Notch-signal receiving cells. (A-D) Wild-type expression pattern of Dll-lacZ (A), Wg (B), Cut (C) and NICD (D) in the developing wing disc. NICD is enriched along the dorsal/ventral (D/V) boundary (D, arrowhead) and suppressed in the adjacent cells. (E) Expression pattern of C5-Gal4 driving GFP in the D/V boundary flanking cells of the wing pouch. (F-I) The knockdown of flfl with RNAi in the D/V boundary flanking cells does not affect Dll-lacZ (F), Wg (G), Cut (H) or NICD (I). (J) Expression pattern of wg-Gal4 driving GFP along the D/V boundary. (K-N) Knockdown of flfl in the D/V boundary cells causes a loss of Dll-lacZ (K), strong reduction of Wg (L), loss of Cut (M) and a failure of NICD enrichment along the D/V boundary (N, arrow). Scale bar: 50 µm.
Fig. 4.
Fig. 4.
PP4 likely functions within the nucleus to promote Notch. (A-C) Adult wild-type wing and margin (A, inset). Overexpression of Flfl (B), or Flfl-cyto (C) throughout the entire wing does not induce any noticeable phenotype. (D-F) Knockdown of Flfl induces ectopic veins and thickening of veins, as well as a reduced wing size (D). This effect can be primarily rescued by reintroduction of a full length Flfl transgene (E), but not by Flfl-cyto (F). (G-L′) Overexpression of Nnucl induces a loss of wing veins (G) and wing margin bristles (G′). Knockdown of flfl induces mild rescue of the Nnucl loss of vein phenotype, and still reduces the overall wing size (H,H′). The overexpression of Flfl (I,I′) or Flfl-cyto (J,J′) did not disrupt the Nnucl phenotype. (K-L′) Expression of wg-RNAi in the wing induced sporadic loss of margin bristles (K,K’, arrow). Nnucl with wg-RNAi is able to maintain several margin bristles (L,L′, arrowhead).
Fig. 5.
Fig. 5.
Flfl is required for proliferation and overall tissue size independent of Notch signaling. (A) Box plots representing total wing area of the genotypes shown in Fig. 4 (n=8-13). Overexpression of Flfl (b) or Flfl-cyto (c) did not affect wing size compared to wild type (a). flfl-RNAi caused a significant reduction in wing size (d). The flfl-RNAi size defect could be fully rescued by reintroduction of full length Flfl (e), but no effect was seen with Flfl-cyto (f). Wings expressing Nnucl (g) are slightly smaller than wild-type wings. Nnucl and flfl-RNAi wings (h) have a significant size reduction compared to wild type (a), equivalent to that of flfl-RNAi alone (d). Data are presented as box plot 25-75 percentile, whiskers 10-90 percentile, (−) median, (+) mean and (•) outliers, with letters above representing significance from corresponding genotypes (P<0.01), generated from one-way ANOVA. (B-F) The normal expression pattern of en-Gal4 marked by GFP (B) in the posterior domain of the developing wing disc, shown with mitotic cell marker PH3 (B’) and represented as a ratio of posterior domain versus the anterior control (n=8) (E,F). Overexpression of Flfl in the posterior domain had no effect on area (C,E) or proliferation rate (n=7) (C′F). The knockdown of Flfl with RNAi in the posterior domain induced a significant reduction in area (D,E), and a significantly higher number of PH3-positive cells (n=9) (D′F). Data are presented as mean±s.d.; *P<0.05, **P<0.01 generated from one-way ANOVA. Scale bar: 50 µm.

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References

    1. Axelrod J. D. (2010). Delivering the lateral inhibition punchline: it's all about the timing. Sci. Signal. 3, pe38 10.1126/scisignal.3145pe38 - DOI - PubMed
    1. Baonza A. and Garcia-Bellido A. (2000). Notch signaling directly controls cell proliferation in the Drosophila wing disc. Proc. Natl. Acad. Sci. 97, 2609-2614. 10.1073/pnas.040576497 - DOI - PMC - PubMed
    1. Bray S. J. (2016). Notch signalling in context. Nat. Rev. Mol. Cell Biol. 17, 722-735. 10.1038/nrm.2016.94 - DOI - PubMed
    1. Clevers H. and Nusse R. (2012). Wnt/β-catenin signaling and disease. Cell 149, 1192-1205. 10.1016/j.cell.2012.05.012 - DOI - PubMed
    1. Cohen P. T. W., Philp A. and Vázquez-Martin C. (2005). Protein phosphatase 4 – from obscurity to vital functions. FEBS Lett. 579, 3278-3286. 10.1016/j.febslet.2005.04.070 - DOI - PubMed

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