Distributions of Nkd and Dsh in embryos. (A) Heat-fixed wild-type stage 11 embryo stained for endogenous Nkd. Arrowheads mark positions of parasegmental boundaries. Note segmental variation in Nkd abundance (brackets, also in B). (B–B″) Heat fixed wild-type embryo stained for endogenous Nkd (green, B) and Dsh (red, B′); B″ is a merged image. Insets in B–B″: each protein is cytoplasmic and weakly nuclear (large arrowhead) with some cytoplasmic and/or plasma membrane colocalization (yellow arrows). (C) Formalin-fixed stage 11 cas-Dsh^myc/UAS-Nkd^GFPC;nkd^7H16da-Gal4/nkd^7H16 embryo stained with α-myc and α-GFP (only the latter is shown). Note uniform, predominantly cytoplasmic but low-level nuclear distribution of Nkd^GFPC. (D–D″) Medium power; same genotype as C. Note uniform distributions of both proteins in cytoplasm and nucleus (arrowhead in insets). Punctate areas with predominantly Nkd (green arrow), Dsh (red arrow), or both (yellow arrow) can be identified in magnified insets. (E) Western blot of extracts from 0- to 5-hr embryos expressing GFP (left) or Nkd^GFPC (right) and probed with α-GFP antibody highlighting specific GFP (arrow), Nkd^GFPC (large arrowhead), and nonspecific bands (small arrowheads). (F) Western blot of extracts from 0 to 5 hr. cas-Dsh^myc embryos probed with α-myc. Note doublet in left lane that collapses to a single band following phosphatase (CIP) treatment. (G) High power; same genotype as C, with DNA (blue) marking nuclei in G′ and G″. Note punctate cytoplasmic Nkd (green arrow) and Dsh (red arrow) staining with rare cytoplasmic (yellow arrow) and nuclear (arrowhead) colocalization.

Nkd^GFPC and Dsh^myc do not undergo rapid CRM-1-dependent nuclear export. Each panel shows low- and high-power views of representative stage 11 embryos expressing the indicated construct via da-Gal4 (except cas-Dsh^myc, which expresses Dsh^myc at appropriate levels via the dsh promoter) stained with the appropriate antibody to reveal subcellular distributions ±30 min treatment with the CRM-1-dependent nuclear export inhibitor LMB. The positive control (A and B) is Lines^myc, which is nuclear in Wg-receiving cells and cytoplasmic in Hh-receiving cells of the dorsal ectoderm (Hatini et al. 2000). LMB treatment drives Lines^myc into the nucleus of most dorsal ectoderm and head (arrow) cells. Asterisks designate positions of nuclei. The negative control (C and D) is the myristoylated, N-terminal 89 amino acids of D. melanogaster Src (Simon et al. 1985) fused to GFP. No change in the membrane-associated Src^GFP distribution is seen after LMB treatment. Nkd^GFPC (E) is cytoplasmic and nuclear, whereas Dsh^myc (G) is predominantly cytoplasmic in the absence of LMB, and these distributions remain unchanged in LMB-treated embryos (F and H).

Properties of Nkd proteins with mutant Dsh-binding regions. (A) Dsh (purple) has DIX, PDZ, and DEP domains. Nkd (yellow) has EFX domain. Red bars indicate regions that mediate Nkd/Dsh association (Rousset et al. 2002). Nkd constructs lacking Dsh-binding regions (designated by spliced-out regions) are schematized at left, with representative images from activity assay (stage 11 α-En and larval cuticle) and α-GFP localization in stage 11 embryo, depicted from left to right. The top two constructs, lacking the EFX domain, each narrowed the En stripe to two to three cells and restored cuticle pattern, while the bottom two constructs, lacking the Zn²⁺-binding domain, partially narrowed the En stripe but also restored most of the cuticle pattern. Note that EFX deletion increased the ratio of nuclear-to-cytoplasmic GFP, with the exception of the two Nkd^ΔR1S/GFPC-expressing cells in the center of the field with condensed chromatin indicative of late telophase. ND, not done. (B) Distribution of wild-type and nkd cuticles in rescue cross for indicated Nkd construct. For crosses in which the UAS-Nkd insertion is lethal, the presence of the CyO balancer in the rescue cross results in ∼12.5% of cuticles remaining unrescued and hence “strong” (indicated by the black-topped bars). Note that constructs lacking Dsh-binding regions have a reduced ability to rescue relative to wild-type Nkd, as evidenced by a reduced proportion of wild-type cuticles and increased weak and moderate cuticles. (C) Y2H of strains expressing Nkd bait constructs (left) and Dsh (top) or Dsh^bPDZ (middle) prey assayed for growth under double-dropout (2D) or quadruple-dropout (4D) media. Bar graphs on right show ONPG units for strains harboring indicated constructs in quantitative Y2H assay. (Bottom) Western blot of yeast extracts expressing indicated Nkd-Gal4 DNA-binding domain (DBD) fusion proteins. Porin is loading control. (D) High power of stage 11 UAS-Nkd^ΔR1S/GFPC/cas-Dsh^myc;nkd^7H16da-Gal4/nkd^7H16 epidermis stained for α-GFP (D, green), α-myc (D′, red), and DNA (D″, blue); D‴ is a merged image. Although the majority of Nkd^ΔR1S/GFPC is nuclear, rare cytoplasmic Nkd/Dsh colocalization (yellow arrows) can be identified.

A 30-aa motif is necessary for Nkd activity and nuclear localization. (A) Data from indicated UAS-Nkd constructs as described in Figure 6A. Note that constructs with the 30-aa motif (light blue box) narrowed the En stripe, restored cuticle pattern, and were detected in nuclei, whereas constructs lacking the 30-aa motif did not narrow the En stripe, did not restore or only partially restored cuticle pattern, and (with the exception of Nkd^7H/GFPC) were excluded from nuclei. (B) Quantitation of cuticle phenotypes. Note that constructs with a 30-aa motif (blue bars) efficiently rescued, while those lacking the 30-aa motif (white bars) partially rescued or did not rescue. (C) Y2H of Nkd or Nkd^Δ30aa binding to Dsh or Dsh^bPDZ. (Right) Western blot of yeast extracts expressing Nkd or Nkd^Δ30aa. Note that deletion of the 30-aa motif did not affect Dsh binding.

Nkd alignment and protein family. (A) ClustalW alignment of predicted An. gambiae (Ag, GenBank BK005845) and D. melanogaster (Dm, GenBank AF213376) Nkd proteins. Identities are in a black background, and similarities are shaded. EFX is designated by a red line and the 30-aa motif by light blue shading. Arrowheads designate the position of nonsense mutations in each nkd allele shown in Figure 9A as well as the C-terminal extent of constructs Nkd^NIN3/GFPC and Nkd^NIN4/GFPC (each named for the position of nkd intron 3 and 4, respectively). (B) Nkd protein family showing region of EF-hand similarity between Recoverin (Rec), consisting of four EF hands, and Nkd proteins. Vertical lines indicate sequence homology, whereas dashed lines indicate preserved motif order, but not sequence conservation, between recoverin, fly and mosquito Nkd, and Mus musculus (Mm) and Homo sapiens (Hs) Nkd1 (orange) and Nkd2 (green). Thirty-amino-acid motifs (light blue in insect, dark blue in mammal) are each predicted to be an amphipathic α-helix, part of which is depicted in helical wheel format above the fly sequence (beginning with Nkd residue 552). Diagonal line separates charged (+ or −) residues on one face of the predicted helix from hydrophobic residues (H) that mostly lie on the opposite face. Residues: pink, acidic; blue, basic; orange, nonpolar; green, polar uncharged.

Fly 30-aa motif increases mouse Nkd1 activity and confers nuclear localization. Schematic of mouse Nkd1^GFPC (left) and chimeric construct with substitution of fly 30-aa domain (light blue, right). (A and B) Representative B119-Gal4 wg/UAS-mNkd1^GFPC (A) or B119-Gal4 wg/UAS-mNkd1^f30aa/GFPC (B) female abdomens showing segments with reduced numbers of sternite bristles (arrows). mNkd1^f30aa/GFPC reduced sternite bristle counts approximately twofold relative to mNkd1^GFPC (the mean ±SD of bristles for 10 flies from the indicated number of lines for each construct is shown). (C and D) Epidermal cells of stage 11 UAS-mNkd1^GFPC;nkd^7H16da-Gal4/nkd^7H16 (C) or UAS-mNkd1^f30aa/GFPC;nkd^7H16da-Gal4/nkd^7H16 embryos (D) stained with α-GFP (green). Magnified insets show low-level nuclear GFP in mNkd1^f30aa/GFPC but not mNkd1^GFPC (asterisks mark center of nuclei). C and D were stained in parallel and imaged under identical confocal settings. (E and F) Salivary gland expressing mNkd1 constructs driven by da-Gal4. Note absence of nuclear GFP in mNkd1^GFPC-producing salivary gland cells (E) but abundant nuclear GFP (arrow) in mNkd1^f30aa/GFPC-producing cells (F). DNA is purple.

Nkd antagonizes Arm/β-catenin accumulation. (A) Schematic of ectodermal gene expression in wild type (wt, left) and nkd (right) embryos. Stages 6–7: Wg (blue) and Hh/En (red) initiate in adjacent cell rows. Stage 9: In wild type, Wg protein distributes in a gradient (blue triangle) and maintains Hh/En in approximately two cell rows while, in nkd, Hh/En is expressed in all competent cells (red shading). Wg expression-competent cells are in blue shading. Stage 11: In wild type, the Wg gradient is biased to the anterior while, in nkd, Wg is ectopically produced by some anterior Wg-competent cells, causing boundary duplications with reversed polarity (arrows). Parasegmental (PS) and segmental (S) boundaries are designated. “hatch”: Denticle rows 1–6 are produced in a trapezoidal band, two belts of which are depicted. In nkd, ectopic Wg promotes cell death (X) and suppresses denticle synthesis. (B and C) Five segmental anlagen of stage 10 wild type (B) or nkd^7H16 (C) embryos stained for Arm (green) above schematic. (D) Mean pixel intensity across five segmental anlagen from Arm-stained stage 10 wingless (blue), wild-type (green), and naked (red) mutants. (E) Stage 10 UAS-Nkd^GFPC;nkd^7H16prd-Gal4/nkd^7H16 embryo stained for Arm (green) and GFP (purple). Note reduced Arm in Nkd^GFPC-expressing segments (asterisks).

Nkd rescues nkd mutants to adulthood. (A–C) Stage 11 embryos of indicated genotypes stained for En and viewed at low power (left) and high power showing two segments (middle). (Right) Representative cuticle. Wt (A) has a two- to three-cell En stripe (arrows and bar), while nkd^7H16/nkd^7E89 (B) has a wide En stripe and develops a strong nkd phenotype with a severe head involution defect (arrowhead) and widely split posterior spiracles (arrow). (C) UAS-Nkd^GFPC;nkd^7E89da-Gal4/nkd^7H16 has a narrow En stripe and a wild-type cuticle pattern. (D–M) Eye (D–H) or ventral abdomen (I–M) of wild type (wt) (D and I) or B119-Gal4/UAS-Nkd female (E–H and J–M). Weaker UAS-Nkd lines Nkd^mycC#9 and Nkd^GFPC#3 produce a teardrop-shaped ventral eye (arrowheads, E and F) relative to wild type (D), whereas the stronger lines Nkd^mycC#3-2 and Nkd^GFPC#1 cause ventral eye reduction (G and H). Wild-type female abdomen has ∼90 sternite bristles (arrowhead) in the ventral aspect of segments A2–A6 (I). Weaker UAS-Nkd lines (J and K) caused bristle loss and lateral bristle displacement with indicated mean ±SD bristle numbers, while stronger lines (L and M) resulted in near total bristle loss. (N) Distribution of wild-type and nkd cuticle phenotypes (weak, moderate, strong) in rescue cross (UAS-X; nkd^7H16/TM3 × da-Gal4,nkd^7E89/TM3, where X is indicated construct) for each Nkd construct. UAS-lacZ (light blue) is the negative control, giving rise to an ∼3:1 Mendelian ratio (75:25%) of wild-type-to-strong nkd mutants. w− is viability control, with 100% wild type. Magenta, untagged Nkd; blue, Nkd^mycC; green, Nkd^GFPC. Note that each UAS-Nkd line rescues nearly all cuticles to wild type. (O) Percentage of nkd embryos that hatched into crawling first instar larvae as a function of UAS-Nkd rescue construct. w− and UAS-lacZ are controls. Above each bar is the number of embryos cultured. (P) Percentage of rescued nkd crawling first instar larvae that survived to indicated stage. w− is the control for culture conditions. Key shows the number of larvae of each genotype that were cultured. Note that a lower percentage of Nkd^GFPC than Nkd^mycC-rescued embryos hatch and survive to later stages. (Q) UAS-Nkd^mycC;nkd^7E89da-Gal4/nkd^7H16 hatched adult with wing-to-notum transformation (arrowhead). (R) Wild-type anterior wing margin with evenly spaced stout bristles (blue arrowhead) and slender bristles (red arrowheads), each of the latter interspersed by four wing trichomes. (S and T) Wing margins from rescued nkd adult showing displaced stout bristle and decreased spacing between slender bristles (S) and ectopic sensory bristles near wing margin (T).

Nkd reverses Dsh- but not Dsh^ΔbPDZ-induced eye phenotype. (A) Wild-type eye. (B) wg^Glazed eye. (C) GMR-Gal4/UAS-Dsh;UAS-lacZ eye. (D) GMR-Gal4/UAS-Dsh;UAS-Nkd eye showing strong suppression of Dsh-induced phenotype. (E) GMR-Gal4/UAS-Dsh^ΔbPDZ;UAS-lacZ eye (left) is identical to GMR-Gal4/UAS-Dsh^ΔbPDZ;UAS-Nkd eye (right), with no ommatidia or bristles.

Nkd's Dsh-binding regions are important for blocking Dsh-induced eye phenotype. (A–D) SEMs of representative wild type (A), GMR-Gal4/UAS-Dsh;UAS-lacZ (B), GMR-Gal4/UAS-Dsh;UAS-Nkd^ΔR1S/GFPC (C), or GMR-Gal4/UAS-Dsh;UAS-Nkd^R1S/mycC (D) eyes. Note that Nkd^ΔR1S only weakly rescues the Dsh-induced phenotype, while Nkd^R1S, consisting of only Dsh-binding sequences, restored ommatidia and bristles.

Phenotypes and molecular lesions of nkd alleles. (A) Predicted truncated Nkd protein based on sequencing of indicated nkd allele, relative to full-length Nkd (top). Note that each predicted protein is truncated N-terminally of the 30-aa motif. Stage 11 mutant embryos have a widened En stripe and a variable nkd phenotype. Right column shows DNA sequence trace from a heterozygote, indicating the codon number with mutation (arrow). (B) Mean number of complete (black bars) and partial (shaded bars) abdominal denticle belts (A1–A8) in each nkd allele (n = 25 mutant cuticles scored for each allele). The molecular lesion of nkd^7H16 was reported in Zeng et al. (2000).

The 30-aa motif is required for Nkd to reduce Arm/β-catenin levels. Each panel shows four segmental anlagen of a representative stage 10 UAS-X;nkd^7H16prd-Gal4/nkd^7H16 embryo, where X is each indicated Nkd construct, stained for Arm (green) and GFP (purple). Colored arrowheads indicate position of nkd mutant (green) and rescued (purple) segments. Right column shows mean grayscale intensity (ordinate range 0–200) of Arm staining as a function of position along the anterior–posterior axis. To quantitate the extent of Arm reduction by each Nkd construct, we defined γ as the ratio of peak grayscale intensity in nkd mutant segments to that in adjacent segments expressing each construct. When GFP is used in the rescue assay, γ = 1.0 (i.e., no rescue), while for full-length Nkd, γ > 1.8. Both constructs that lacked the 30-aa motif had γ = 1.2, indicating a reduced ability to decrease Arm levels.

A heterologous NLS partially restores Nkd^Δ30aa activity. (A–C) Representative stage 11 nkd^7H16 mutant embryos expressing Nkd^GFPC (A), Nkd^Δ30aa/GFPC (B), or Nkd^{Δ30aaNLS/GFPC} (C) stained for GFP (green) and En (red). The NLS promotes Nkd nuclear localization and narrows the En stripe of most segments (arrowheads). (D) Quantitation of nkd cuticle rescue. Note increased proportions of wild type and reduced moderate and weak nkd cuticles with rescue by Nkd^{Δ30aaNLS/GFPC} (red bars) as compared to Nkd^Δ30aa/GFPC (purple). (E) UAS-Nkd1^{Δ30aaNLS/GFPC};nkd^7H16da-Gal4/nkd^7H16 adult. (F) Anterior wing margin of animal in E with marked bristle disarray, including absent (x) and ectopic (blue arrowhead) stout bristles and mispatterned and ectopic slender bristles (red arrowhead).