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First published online 8 December 2005
doi: 10.1242/dev.02198

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Drosophila Dok is required for embryonic dorsal closure

¹ Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
² Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.

View larger version (46K): [in a new window]   Fig. 1. Drosophila Dok sequences and their relationship to the mammalian Dok proteins. (A) Alignment of the Ddok PH domain with mammalian Dok1, Dok2 and Dok3. Identical amino acids are indicated by shading. (B) Alignment of the Ddok PTB domain with the PTB domains of mammalian Dok1-Dok3. (C) Schematic representations of Ddok and Dok1-Dok3, showing C-terminal tyrosines predicted to be phosphorylated by NetPhos. The line joining diamonds in Ddok shows the region of overlap of the Ddok clones obtained in the two-hybrid screen. (D) A phylogenetic tree of Ddok and the mammalian Dok nucleotide sequences compiled using the Clustal W method within the Lasergene Navigator program.

View larger version (53K): [in a new window]   Fig. 2. Interaction of Shark and Ddok in yeast and S2 cells. (A) Dependence of the interaction between Shark and Ddok on tyrosine phosphorylation. LexA, the DNA-binding domain in the pGilda vector was fused to Shark in pGilda without activated Src (Lex A-Shark), to Shark in pGilda with activated Src (LexA-Shark/Src) or, as a control, to nothing (LexA). AD, the activation domain in the pB42AD vector was fused to either Ddok (AD-Ddok), DdokYF mutations (AD-Ddok Y---F) or nothing (AD). Transformed yeast were plated on replica plates containing X-gal for screening ß-galactosidase-positive blue colonies (upper panel) and Leu- plates for screening Leu-positive colonies (lower panel). (B) Y427, Y499, Y515 and Y537 are involved in the tyrosine phosphorylation-dependent interaction of Ddok with Shark. The constructs used in A were used to perform the more sensitive ß-galactosidase assays of yeast cell lysates (±s.e.m.; n=3; *P<0.05, Student's t-test, significantly different from LexA-Shark/Src+AD-Ddok). (C) Ddok antibody detects a single protein in a western blot of Drosophila embryo extract. (D) Endogenous Shark and Ddok are associated in S2 cells. (E) Dependence of Shark and Ddok association on Ddok tyrosine phosphorylation in co-transfected S2 cells. S2 cells were transfected with pMT vector alone, Myc-Shark alone, Ddok-Flag alone, both Myc-Shark and Ddok-Flag, or Myc-Shark with each of the following Ddok-Flag mutants individually: Ddok-M1 (Ddok Y499F,Y515F), Ddok-M2 (Ddok Y499F,Y515F,Y427F), or Ddok-M3 (Ddok Y499F,Y515F,Y427F,Y537F). Cell lysates were immunoprecipitated with Flag antibody and subjected to SDS-PAGE and western blotting with the indicated antibodies. (F) Inhibition of Ddok tyrosine phosphorylation in S2 cells by the Src inhibitor, PP2. Ddok-Flag or Ddok-Flag+Shark transfected cells were incubated in medium containing 10 µm PP2 or solvent (DMSO) for 1 hour prior to the immunoprecipitation of cell lysates with Flag antibody. (G) Ddok and Shark are colocalized at the cell cortex. S2 cells were transfected with Myc-Shark alone (a,b), Ddok-Flag alone (c,d), or co-transfected with Myc-Shark and Ddok-Flag (e-h), immunofluorescently stained for Myc (red; b,g,h) and Flag (green; d,f,h), and examined by confocal microscopy. The merged panel (h) shows the colocalization (yellow).

View larger version (90K): [in a new window]   Fig. 3. Ddok gene organization and the phenotypes of DdokPG155/Y male `escapers'. (A) Ddok gene organization, showing the position of the P-element insertion in the DdokPG155 line. (B) RT-PCR with Ddok and RpS3 (control) primers. Lane 1, 100 bp DNA markers; lane 2, wild-type (WT) flies, with expected 223 bp product; lane 3, DdokPG155/Y flies, no detectable product; lane 4, no reverse transcriptase (-RT). (C) The wings of the male escapers are shriveled (b,c) compared with wild-type wings (a). (D) Defects (arrowed) in thorax (b), head (d) and eye (f) of the male escapers compared to wild-type thorax (a), head (c) and eye (e). Male escapers exhibit a split thorax phenotype (b) with loss of bristles at the dorsal midline (arrow). Occasionally, the escapers also show loss of the anterior orbital bristle in the head (d; asterisk indicates the normal developmental process) and irregularly placed bristles (f) in the eyes (arrows).

View larger version (80K): [in a new window]   Fig. 4. Characterization of DdokPG155 mutant embryos. (A) Bright-field photographs of cuticle preparations of wild-type and DdokPG155(GLC) embryos. DdokPG155(GLC) cuticles exhibit a severe dorsal open phenotype with large anterior holes (arrow). (B) Ddok immunofluorescent antibody staining of lateral epidermal cells of stage 13 embryos. Arrow and hatched line indicate LE. (C) Anti-Fasciclin III antibody staining of epidermal cells of stage 12 embryos. (D) In situ hybridization to dpp mRNA in DdokPG155 GLC reveals a loss of dpp mRNA specifically from the LE cells (lateral stripes, arrows) in lateral views of stage 11 (a,b), and in dorsal views of stage 13 (c,d), embryos. dpp mRNA staining in other structures of the stage 11 and stage 13 mutant embryos is indistinguishable from that in wild-type embryos.

View larger version (160K): [in a new window]   Fig. 5. Phalloidin staining of F-actin. Phalloidin staining of F-actin in (A) wild-type, (B) DdokPG155/Y and (C) DdokPG155(GLC) embryos at 10 and 12 hours after egg laying. Arrows indicate leading edge.

View larger version (80K): [in a new window]   Fig. 6. Epistatic analysis of Ddok and components of the JNK signaling pathway. DdokPG155 GLC flies were crossed to males bearing JNK pathway transgenes or mutations. Embryos bearing heat-shock transgenes were heat shocked for 30 minutes at 37°C at stage 9-11, and then kept at 25°C. Cuticle preparations of (A) wild-type embryos and DdokPG155 mutant (Ddok*) embryos, and (B-F) DdokPG155 mutant embryos (right), (B) expressing a constitutively activated form of Jun (hs-SEjunasp), (C) heterozygous for pucE69, (D) overexpressing Shark (hs-shark-10), (E) overexpressing kinase-dead Shark (hs-shark-10-K698R) and (F) overexpressing Src42A (hs-Src42A22.3), together with cuticles of control, DC-defective, DdokPG155/Y embryos (left). +hs, heat shocked; -hs, no heat shock. Arrows indicate partial rescue of the dorsal open phenotype.

View larger version (138K): [in a new window]   Fig. 7. Expression of tyrosine phosphorylated Shark at the periphery of epidermal cells is decreased in Ddok mutant embryos, during DC. (A,B) Embryo extract western blotted with (A) anti-pY927 and anti-phosphotyrosine (anti-P-Tyr-100) antibodies, and (B) anti-pY927, before (lane 1) and after (lane 2) phosphatase (CIAP) treatment of membrane, and with anti-Shark antiserum (lane 3). (C,D) Staining of wild-type and DdokPG155 mutant embryos with anti-pY927 Shark peptide antibody. Arrowheads indicate the LE.

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