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. 2012 Sep-Dec;129(9-12):339-49.
doi: 10.1016/j.mod.2012.05.007. Epub 2012 Jun 15.

Regulation of Yorkie activity in Drosophila imaginal discs by the Hedgehog receptor gene patched

Affiliations

Regulation of Yorkie activity in Drosophila imaginal discs by the Hedgehog receptor gene patched

Jacob D Kagey et al. Mech Dev. 2012 Sep-Dec.

Abstract

The Hedgehog (Hh) pathway was first defined by its role in segment polarity in the Drosophila melanogaster embryonic epidermis and has since been linked to many aspects of vertebrate development and disease. In humans, mutation of the Patched1 (PTCH1) gene, which encodes an inhibitor of Hh signaling, leads to tumors of the skin and pediatric brain. Despite the high level of conservation between the vertebrate and invertebrate Hh pathways, studies in Drosophila have yet to find direct evidence that ptc limits organ size. Here we report identification of Drosophila ptc in a screen for mutations that require a synergistic apoptotic block in order to drive overgrowth. Developing imaginal discs containing clones of ptc mutant cells immortalized by the concurrent loss of the Apaf-1-related killer (Ark) gene are overgrown due, in large part, to the overgrowth of wild type portions of these discs. This phenotype correlates with overexpression of the morphogen Dpp in ptc,Ark double-mutant cells, leading to elevated phosphorylation of the Dpp pathway effector Mad (p-Mad) in cells surrounding ptc,Ark mutant clones. p-Mad functions with the Hippo pathway oncoprotein Yorkie (Yki) to induce expression of the pro-growth/anti-apoptotic microRNA bantam. Accordingly, Yki activity is elevated among wild type cells surrounding ptc,Ark clones and alleles of bantam and yki dominantly suppress the enlarged-disc phenotype produced by loss of ptc. These data suggest that ptc can regulate Yki in a non-cell autonomous manner and reveal an intercellular link between the Hh and Hippo pathways that may contribute to growth-regulatory properties of the Hh pathway in development and disease.

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Figures

Figure 1
Figure 1. A mosaic screen for conditional suppressors of growth identifies a novel allele of patched, ptcB.2.13, as a conditional suppressor of growth
Mosaic adult eyes and imaginal wing discs from L3 larvae, stained with phalloidin: (A-A′) Ark82 (mutant tissue is pigmented) (B-B′) ptcB.2.13,Ark82 (mutant tissue is pigmented) (C-C′) ptcB.2.13 (wild type tissue is pigmented);(D) Quantification of mosaic wing discs size from L3 larvae demonstrates the condional nature of the ptcB.2.13,Ark82 overgrowth; Mosaic larval eye discs stained with anti-Cleaved Caspase 3 (cCasp) shows ptcB.2.13mutant clones accumulate cCasp without Ark82(E-E″) ptcB.2.13,Ark82 (mutant tissue is GFP negative)(F-G″) ptcB.2.13(inset is amplified in G-G″)(mutant tissue is GFP negative).
Figure 2
Figure 2. ptcB.2.13,Ark82 wing discs have increased proliferation in both mutant cells and adjacent wild type cells
Imaginal mosaic wing discs dissected from wandering L3 larvae stained with the proliferation markers BrdU and phospho-histoneH3 (pH3) in the anterior of the wing disc; ptcB.2.13,Ark82 (A-A″) stained with BrdU, see arrows for examples of non-autonomous increased proliferation (B-B″) stained with pH3, see arrow for example of non-autonomous increased proliferation; Ark82 (C-C″) stained with BrdU (D-D″) stained with pH3; (E) quantification of the portion of larval wing discs composed of mutant tissue reveals a non-autonomous component of ptcB.2.13,Ark82 overgrowth. Mutant tissue is GFP negative.
Figure 3
Figure 3. mutant tissue has an autonomous increase in canonical Hedgehog signaling
ptcB.2.13,Ark82 mosaic wing discs have a strong autonomous increase in Hedgehog signaling confined to the anterior portion of wing discs as demonstrated by (A-A‴) anti-Cubitus Interruptus (Ci), (B-B‴) anti-Patched (Ptc), (C-C‴) anti-Decapentaplegic (Dpp). The phenotype is dependent on ptcB.2.13, as Ark82 mosaic wing discs have normal Hedgehog signaling as demonstrated by (D-D″) Ci, (E-E″) Ptc, (F-F″) Dpp. Mutant tissue is GFP negative.
Figure 4
Figure 4. ptcB.2.13,Ark82 clones activate Dpp signaling most robustly in wild type cells immediately adjacent to mutant clones
ptcB.2.13,Ark82 mosaic wing discs have an increase of Dpp signaling within the anterior of the wing disc, the signaling is highest in those cells immediately adjacent to mutant clones. Increases in Dpp signaling in ptcB.2.13,Ark82 mosaic discs measured by levels of phosphorylated-Mothers Against Decapentaplegic (p-Mad1/5) as marked by (A-A″) anti-p-Mad1/5 (p-Mad1/5) see arrows, (B-B″) Ptc denotes mutant clones (Figure 3B) and pMad1/5 levels create a ring round the mutant clones, see arrow; these same mutant discs have an autonomous increase in the inhibitory smad, Daughters Against Decapentaplegic (Dad) (C-C″) measured by the Dad transcriptional reporter Dad-LacZ (Dad-Z), see arrow; these phenotypes are dependent upon ptcB.2.13, as mosaic discs for Ark82 have normal pattern of (D-D″) p-Mad1/5 and (E-E″) Dad-Z. Mutant tissue is GFP negative, unless otherwise noted.
Figure 5
Figure 5. ptcB.2.13,Ark82 mutant clones result in gradient dependent activation of a subset of Yorkie target genes, which necessary for the ptcB.2.13,Ark82 overgrowth phenotype
ptcB.2.13,Ark82 larval mosaic wing discs have elevated Yki activity in the anterior portion of the wing discs, with the highest levels occurring in wild type cells immediately adjacent to mutant clones. Increases of several Yki targets were elevated within ptcB.2.13,Ark82 mutant clones and in those wild type cells adjacent to the clones (A-A″) anti-Drosophila Inhibitor of Apoptosis Protein 1 (DIAP1), see arrow, (B-B″) Ci denotes mutant clones (Figure 3B) and DIAP1 levels are most elevated just outside the mutant clones, see arrow, (C-C″) Expanded levels as measured by the reporter Expanded-lacZ (Ex-Z), (D-D″) Enlarged image of inset from 5C of Ex-Z, (E-E″) Ci denotes mutant clones and bantam levels are highest immediately adjacent to mutant clones as see by decreasing levels of the bantam reporter, ban-GFP. Not all Yki targets are altered by ptcB.2.13,Ark82 as Wingless pattern and levels are unchanged (F-F″) anti-Wingless (Wg). The alteration of Yki targets is dependent on the ptcB.2.13 mutation given that we find no alteration of Yki reporters in wing discs mosaic for Ark82, (G-G″) DIAP1, (H-H″)Ex-Z, (I-I″) Wg. Dominant suppression of the ptcB.2.13, Ark82 overgrowth phenotype by a single copy of banΔ1(J). A single copy loss of function yki allele (ykiB5) dominantly suppresses the overgrowth of larval wing discs generated from driving UAS-ptcIR in clones (K), despite the relative clone size remaining the same in both (K′) UAS-ptcIR and (K″) UAS-ptcIR, ykiB5/+.

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