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pygopus encodes a nuclear protein essential for Wingless/Wnt signaling

Tatyana Y. Belenkaya^*, Chun Han^*, Henrietta J. Standley^*, Xinda Lin, Douglas W. Houston, Janet Heasman and Xinhua Lin

Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA
^* These authors contributed equally to this work

View larger version (51K): [in a new window]   Fig. 1. Identification of pygo as a new segment polarity gene required for Wg signaling. Wings are oriented proximal towards the left and anterior upwards (A-C). A wild-type wing is shown in A. A wing with somatic clones of pygoF66 is shown in B. The enlarged parts of the wing in B are shown in C. Clones of pygoF66 (B) cause wing notching and the formation of ectopic bristles in nearby tissues. The wing phenotypes associated with pygoF66 are fully penetrant. Virtually identical results were obtained from other pygo alleles. The cuticle phenotypes of wild-type (D) and pygo mutant embryos (E-H) are shown. All embryos are oriented anterior towards the left and dorsal upwards. The pygo mutant embryos in E-H were derived from homozygous pygo mutant germline clones (see Materials and Methods). The wild-type embryo (D) forms a segmented larval cuticle decorated with denticles spaced by naked cuticle. pygo mutant embryos (E-H) form unsegmented cuticles that produce ‘lawn’ of denticle hairs. Homozygous mutant embryos derived from germline clones are shown for pygoF15 (E), pygoF66 (F) and pygoF107(H). A pygo mutant embryo shown in G was derived from a homozygous pygoF15 mutant germline clone and paternally mutant for pygoF66.

View larger version (80K): [in a new window]   Fig. 2. Pygo is required for Wg signaling in various embryonic developmental processes. Wild-type (wt) and pygo mutant embryos are shown for expression of En (A,B), Wg (C,D), Labial (E,F) and Eve (G-J). All the pygo mutant embryos were derived from homozygous pygoF107 mutant germline clones and paternally mutant for pygoF66. En is normally expressed in stripes in the ectoderm of the thorax and abdomen during stage 10 (A). Wg is also expressed in stripes in the ectoderm of the thorax and abdomen (C) during stage 10. Both Wg and En stripes are strikingly reduced in pygo mutant embryos at stage 10 (B,D). The endoderm is normally subdivided into discrete domains by constrictions imposed by the visceral mesoderm and Labial is expressed in one of these domains (E). Labial expression is diminished in a pygo mutant embryo (F). Eve is normally expressed in specific subsets of cells derived from the somatic mesoderm that will form the heart (G) and also expressed in specific neurons in the central nervous system (CNS), including the RP2 neurons (arrow) (I). (H,J) In pygo mutant embryos, the expression of Eve-positive cells is absent in the somatic mesoderm cells (H) and in the RP2 neurons (J), respectively.

View larger version (90K): [in a new window]   Fig. 3. Pygo is required for Wg signaling in imaginal disc development. In these images, anterior is towards the left, dorsal is upwards. All the discs were derived from third instar larvae. Somatic clones mutant for pygoF15 are marked by the absence of GFP shown in green. A complex pattern of Achaete (Ac) expression is shown in a wing disc from a wild-type third instar larva (A). Wg acts at short range to induce the expression of Ac at the DV boundary of anterior wing pouch (A). In a mosaic clone mutant for pygoF15, which is marked by absence of GFP, the expression of Ac protein is abolished (B,B'). Note that a patch of Ac-positive cells within the clone is Wg independent. Therefore the expression of Ac in these cells is not diminished. In response to Wg signaling, Dll is expressed in a graded manner with highest expression in DV boundary in a wild-type wing disc (C). Dll expression is diminished autonomously in a pygoF15 mosaic clone (D,D'). In a wild-type leg disc, Dpp-lacZ expression is repressed by Wg signaling in the ventral anterior quadrant (E). In a pygoF15 mosaic clone of the leg disc, Dpp-lacZ expression is de-repressed (F,F'). In a wild-type wing disc, Dpp-lacZ expression is induced by Hh signaling at the AP boundary. The expression of Dpp-lacZ is not altered in mosaic clones mutant for pygoF15 (H,H').

View larger version (107K): [in a new window]   Fig. 4. Pygo acts downstream or in parallel with Arm to regulate nuclear Arm activity. An embryo lacking both maternal and zygotic axin exhibits the characteristic naked cuticle phenotype associated with constitutive Wg signaling (A). In a axin-pygoF15 double null embryo, the naked cuticle phenotype is reversed and exhibits a ‘lawn’ of denticles phenotype (B). A wing bearing somatic clones of axin produced by vg Q1206-Gal4/UAS flipase, is shown in C. Numerous bristles were produced by clones mutant for axin (C). A wing with axin-pygoF15 somatic clones (D) exhibits no bristle within the wing and produces notching and the formation of ectopic bristles in nearby tissues, which is similar to the wing bearing pygoF15 clones (see Fig. 1B,C). A axin somatic clone in the wing disc produces autonomously the expression of Ac (E,E'). In a axin-pygoF15 somatic clone, no induction of Ac expression is observed (F,F'). In a wild-type embryo, Arm protein levels are upregulated in a segmentally repeated fashion in the ventral ectoderm (G). The expression of Arm protein remains in a segmentally repeated fashion in embryos mutant for pygo (H). In a wing disc, Arm protein is strikingly upregulated in a clone mutant for axin (I,I',I''). This upregulated Arm protein is not diminished in clones mutant for axin-pygo (J,J',J''). There is no difference in the subcellular localization of Arm protein in clones of axin and axin-pygo (compare I,I',I'' with J,J',J'').

View larger version (58K): [in a new window]   Fig. 5. pygo encodes a novel putative nuclear protein with a PHD finger domain. The amino acid sequence of Drosophila Pygo is shown in (A). A putative nuclear localization signal (NLS) is found in the N-terminal region (blue) and a PHD finger is found in C-terminal region (red). Three pygo mutants were sequenced and the mutations (green bars) are shown in B. Homologs of Pygo are also found in Xenopus and Mus musculus (B). They all have a NLS (blue box) at the N terminus and a PHD domain (red box) at the C terminus. The C-terminal regions of the four proteins are very conserved, and the alignment is show in C, which includes the PHD domains and the flanking sequences. The identical residues among four proteins are shown in pink and the similar residues are boxed (C).

View larger version (25K): [in a new window]   Fig. 6. Pygo protein forms a complex with Arm in vivo and contains a transactivation domain(s). (A) Co-immunoprecipitation of Pygo and Arm. 293T cells were transfected with plasmids expressing either Myc-tagged Pygo (amino acid 105 to 815) or HA-tagged Arm (amino acid 128 to 844) alone and together as indicated. Whole-cell lysates were prepared 36 hours after transfection. Lysates were immunoprecipitated with mouse monoclonal anti-Myc antibody. Immunoprecipitated materials and a fraction of each lysate were resolved by SDS-PAGE and analyzed by western blotting with antibodies as shown. IP, immunoprecipitation; IB, immunoblot. (B) Analysis of the transcription activation domain(s) in Pygo. Cells were transfected with the pG5E1b-luciferase reporter construct (Hsu et al., 1994) and with vectors expressing GAL4 DNA-binding domain alone (pM1) (Sadowski et al., 1992) or with GAL4-Pygo fusion protein. A GAL4-Jun AC-containing Jun activation domain (amino acids 5 to 89) fused with GAL4 was used as a positive control. Luciferase acitivities are expressed as relative activities compared with cells transfected with the plasmid containing the GAL4 DNA-binding domain alone.

View larger version (29K): [in a new window]   Fig. 7. Xpygo depletion results in a ventralized phenotype. (A) Maternal Xenopus pygo (Xpygo) mRNA transcripts are depleted in a dose-dependent manner upon injection of an antisense Xpygo oligo into oocytes. mRNA levels were assayed 24 hours after oligo injection. (B) Uninjected control embryo (above; blue) at stage 36, and embryos derived from sibling oocytes injected with antisense Xpygo (below; mauve). Xpygo– embryos are ventralized. (C) Relative expression of markers normalized to ODC in wild type and Xpygo– embryos at late blastula (9.5) and gastrula (10.5) stages. Expression of dorsal markers is reduced in Xpygo– embryos.