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Drosophila Apc1 and Apc2 regulate Wingless transduction throughout development

Yashi Ahmed^*, Ali Nouri^* and Eric Wieschaus

HHMI/Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
^* These authors contributed equally to this work

View larger version (103K): [in a new window]   Fig. 1. Ectopic embryonic Wg transduction in a hypomorphic Apc2 mutant. (A) The Apc1 and Apc2 proteins. The conserved Armadillo repeat region (blue), ß-catenin-binding sites (purple) and Axin binding sites (green) are indicated. The position of the stop codon in Apc1Q8 and Apc2d40, as well as the epitope to which the anti-Apc2 sera was raised (black bar) are indicated. (B-E) Immunostaining of wild-type (B,D,E) and Apc2d40 maternal/zygotic mutant embryos (C) with the anti-Apc2 sera. (B,C) Embryos at similar stages obtained using the same confocal microscope settings. A dramatic reduction in Apc2 staining is found in the Apc2d40 mutant embryos. (F-H) Dark field images of cuticles of homozygous Apc2 mutant embryos from Apc2d40/Apc2d40 mothers and wild-type fathers (F), Apc2d40/Apc2d40 mothers and Apc2d40 /+ fathers (G), and Apc2d40/Df (w6) mothers and Df (w6)/+ fathers (H). Introducing a deficiency for Apc2 in the mothers and fathers results in an embryo that nearly completely lacks ventral epidermal denticles, and thus reveals the hypomorphic nature of the Apc2d40 mutation. (I-L) Immunostaining of heterozygous Apc2 embryos from Apc2d40/Apc2d40 mothers and wild-type fathers (I,K) or homozygous Apc2 mutant embryos from Apc2d40/Apc2d40 mothers and Apc2d40/+ fathers (J,L) with anti-Engrailed (red, I,J) or anti-Arm (green, K,L) antibodies. Apc2d40 maternal/zygotic mutants have an expansion of naked cuticle, and an expansion of Engrailed stripes, as well as a reduction in the striped accumulation of Arm protein, all consistent with the ectopic activation of Wg transduction.

View larger version (91K): [in a new window]   Fig. 2. Ectopic Wg transduction in the wing, leg and eye upon simultaneous reduction of Apc1 and Apc2. (A-C) Constitutive activation of Wg transduction in the adult wing upon simultaneous reduction of Apc activity in Apc1Q8 Apc2d40 double mutant clones that are marked by a mutation in the yellow gene. Apc1Q8 Apc2d40 clones in anterior wing margin (arrowhead in A) form bristles (higher magnification shown in B), whereas in the posterior margin (arrow in A) form thin tapered hairs (higher magnification shown in C). Adult wing with mutant clones homozygous for AxinS04423 (D,F) or zw3M11-1 (E,G) show the same cell fate transformations, but only the Axin mutant clones are similar in size to those produced by Apc1Q8 Apc2d40. In the adult legs (H,I), marked Apc1Q8 Apc2d40 mutant clones are associated with outgrowths (arrow in H) and incomplete duplications (arrowhead in H) in regions containing dorsal and lateral pattern elements. In I, there is a duplication of a dorsal structure, the pre-apical bristle (arrow), and an incomplete distal duplication (double arrowhead), as well as a mutant clone of cells that includes the normally formed apical bristle, which is a ventral structure (single arrowhead). Constitutive activation of Wg transduction in the adult eye in an Apc1Q8 Apc2d40 mutant clone (J) or an Axin mutant clone (K) transforms ommatidial cells within the clone to cuticular fates.

View larger version (98K): [in a new window]   Fig. 3. Apc1 is ubiquitously expressed. (A-F) Whole-mount immunostaining of Apc1Q8/Apc1Q8 (A-C) and Apc1Q8/+ (D-F) embryos with an anti-Apc1 sera. Whole embryos (A,D), epidermis (B,E) and gut (C,F) are shown. For each paired set of images, the same confocal microscope settings have been used. Although Apc1 staining is most prominent in the nervous system, there is a low, but consistent increase in the intensity of staining for Apc1 in all cells in Apc1Q8 heterozygous embryos when compared with their homozygous Apc1Q8 mutant siblings.

View larger version (45K): [in a new window]   Fig. 4. Reduction in Apc1 enhances ectopic Wg transduction in the Apc2 mutant. (A-C) Cuticles of embryos from Apc2d40/Apc2d40 mothers and Apc2d40 fathers (A); Apc1Q8Apc2d40/Apc2d40 mothers and Apc2d40 fathers (B); Apc2d40/Apc2d40 mothers and Apc1Q8 Apc2d40 fathers (C). Most of the denticles that remain in Apc2d40 mutants are eliminated by reducing the maternal or zygotic dose of Apc1 by half. (D-I) Embryos from Apc1Q8 Apc2d40 germline clones, which lack maternally provided wild-type Apc1 and Apc2. Cuticles (D-F) and Engrailed stripes (G-I) in embryos from Apc1Q8 Apc2d40 germline clones with a wild-type zygotic allele of Apc1 and Apc2 (D,G), Apc1Q8 Apc2d40 germline clone embryo homozygous for only Apc2d40 (E,H) or homozygous for only Apc1Q8 (F,I). The ectopic Wg activation caused by simultaneous homozygous reduction of Apc1 and Apc2 maternally is made more severe by elimination of either zygotic wild-type Apc1 or zygotic wild-type Apc2. (J) A quantitative analysis of embryonic cuticular patterning defects that result from the Apc1Q8 and Apc2d40 mutations.

View larger version (101K): [in a new window]   Fig. 5. Rescue of retinal neuronal apoptosis in the Apc1Q8 mutant by overexpression of Apc2. Genotypes shown are as follows: (A) P[Apc2]/+; +/+; (B) Apc1Q8/Apc1Q8; (C) P[Apc2]/Y; Apc1Q8/Apc1Q8. (A) Tangential section through the eye of a fly with one extra copy of an Apc2 transgene. As in wild-type eyes, in each ommatidium there are eight photoreceptor neurons located in a highly ordered pattern. Seven neurons are seen in the plane of focus. Each group of photoreceptor cells is surrounded by a lattice of pigment cells, identified by the small, darkly stained pigment granules they contain. (B) In the homozygous Apc1Q8 mutant, there is apoptotic death of all retinal neurons in all ommatidia. The pigment cell lattice remains intact. (C) One extra copy of the Apc2 gene is sufficient to partially prevent the neuronal cell death that is induced by Apc1 loss. The degree to which the apoptosis is prevented is greater in Apc1 mutant males than in females. As the Apc2 transgene is inserted on the X chromosome, we infer that this difference is the result of dose compensation (Kelley and Kuroda, 1995).

View larger version (111K): [in a new window]   Fig. 6. Stabilization and nuclear accumulation of Arm in the Apc1Q8 Apc2d40 double mutant. Embryos from Apc1Q8 Apc2d40 germ cells that are wild type for both Apc1 and Apc2 zygotically (A), or mutant for Apc1Q8 Apc2d40 zygotically (B), stained with anti-Arm antibody. (A,B) Same confocal microscope settings. There is a dramatic increase in the intensity of Arm staining in the combined maternal and zygotic Apc1 Apc2 double mutant embryos. (C-H) Embryos from homozygous Apc1Q8 Apc2d40 germ cells that are wild-type for both Apc1 and Apc2 zygotically (C-E), or mutant for Apc1Q8 Apc2d40 zygotically (F-H) stained with anti-Arm antibody in green (C,F), or with Hoechst dye to detect nuclei in red (D,G) or both (E,H). Staining of the amnioserosa (arrows in C,F) of a stage 9 mutant embryo reveals a nuclear accumulation of Arm that persists throughout embryogenesis only in the Apc1Q8 Apc2d40 maternal/zygotic double mutant.