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First published online 27 April 2005
doi: 10.1242/dev.01850

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Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila

¹ Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
² UPBSB, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (134K): [in a new window]   Fig. 1. Subcellular localization of Src42A. Boxed regions are enlarged in insets or separate panels. (A-F2) Ovaries stained for Src42A (green) and E-cad (magenta). Asterisks indicate oocytes; arrowheads indicate polar or invading border cells. Some cystocytes in region 2a/b exhibited transient but strong Src42A expression (A). In subsequent stages, relatively strong Src42A signals associated with E-cad signals became evident along the follicle/germline-cell boundary (D2). E-cad signals are shown only in enlarged panels. Nurse cell membrane E-cad signals surround ring-canal-Src42A signals (C1,C2). During oogenesis stages 6-9, oocytes incorporated Src42A into the cytoplasm (B,D,E) and began accumulating it on the oocyte surface (D2,D3). (F1,2) Membrane localization of E-cad-free Src42A was clearly seen at stage 10b, when centripetal cells strongly expressed Src42A and E-cad (arrow indicates oocyte membrane). (G1-V) Embryos stained for Src42A and E-cad. Src42A and E-cad signals are colored in green and magenta, respectively (except in U). Stages are shown in the bottom left-hand corner. In stage 2 embryos, membranous Src42A (I) signals scarcely associated with E-cad. (G1-3) Panels showing three phases of membrane extension in stage 5 embryos. Arrowheads indicate extending membrane tips marked with Src42A. (J) At stage 7, apical signals for Src42A and E-cad in furrow-forming or invaginating cells (H2,K) became stronger than in others (H1). At mid-late-stages, E-cad-free Src42A expression occurred between ectodermal and mesodermal cell-layers (see arrows in L). Src42A and E-cad signals colocalized at the apical tips of mesectoderm cells (M1,M2) and the apical surface of ectodermal tubular structures such as stomodeum (N), salivary gland (P), hindgut (Q,R) and Malpighian tubules (R). Malpighian tubules are hindgut protrusions (Skaer, 1993) and, unlike hindgut enclosed with the visceral mesoderm (inset in R), are not associated with visceral mesoderm. Arrows in R show that basal Src42A signals abruptly have disappeared with Malpighian-tubule protrusion. (O) Src42A and E-cad signals at stage-13 leading edge. (S) E-cad/Src42A colocalization at fusion points of dorsal trunk of trachea (see arrowheads). Arrows, Src42A signals in the tendon, rich in integrin (Martin-Bermudo and Brown, 1996). (T,U) E-cad and Src42A expression in CNS. E-cad signals were detected only in midline cells (see arrowheads). Fas2 expression (magenta) (U) shows Src42A expressed in axons, optic lobe and Bolwig's organs. (V) Src42A signals in gonad. hg, hindgut; np, neuropile; pc, pole cells; pm, posterior-midgut primordium; pr, proctodeum primordium; st, stomodeum; tr, trachea; vm, visceral mesoderm; cl, clypeus; ec, ectoderm; me, mesoderm; sg, salivary gland; mt, Malpighian tubule; m, muscle; bo, Bolwig's organ; ol, optic lobe.

View larger version (118K): [in a new window]   Fig. 2. Phenotypes of Src42A26-1 mutants. (A1) Size and location of the Src42A26-1 deletion are shown by the filled box. Triangle indicates P insertion; red boxes indicate exons; H, HindIII; S, SalI. (A2,A3)Anti-Src42A antibody staining of wild-type (A2) and Src42A26-1 (A3) embryos, indicating Src42A26-1 a protein-null allele. (B1-B3) Src42A26-1 embryos stained for Fas3 (B1,B2) or Engrailed (B3). These embryos were associated with slightly irregular midline (B1,B2; bottom). Staining for Engrailed (magenta) and E-cad (white) indicated cell-cell matching failure along the midline in these embryos (B3). Bottom panel provides an interpretation (magenta, Engrailed-expressing cells). (B4-F4) Functional redundancy of Src42A and Src64 found in dorsal closure/germ-band retraction (B4,B5), CNS (C1-C4), trachea (D1-D4), visual system (E1-E3) and ovary (F1-F4). Anterior is towards the left. (B4) Dorsal view (top) and enlargement of the dorsal midline region (bottom) in Src42A26-1;Src64P1/+ embryos. (B5) Lateral view of Src42A26-1;Src64P1 embryos. No germband retraction was observed. (C) CNS stained for Fas2 (top) or Elav (bottom). In Src double mutants, longitudinal axons were misrouted but neurons appeared not significantly reduced in number. (D) Embryos stained for Trachealess (Trh, top) and lumen (2A12, bottom). (E) Head regions stained for Fas2. Bolwig's organ (bo) fails to separate from the optic lobe (ol). (F) Ovaries stained with Rhodamine-phalloidin (magenta) and SYTOX (green).

View larger version (151K): [in a new window]   Fig. 3. Genetic interactions between Src42A and shg/arm. Anterior is leftwards in A-J. (A-C) Enhancement of the rough eye phenotype of sev-Src42A[KR]/+ flies (A) by shgE(7A-1) (B) and shgR64a (C). (D,E) Lateral views of stage 13 embryos of wild-type (D) and shgE(7A-1) (E) stained for E-cad, showing significant E-cad-signal reduction in shgE(7A-1). (F-J) Dorsal views of stage 15 embryos stained for Fas3, indicating dorsal open phenotype in double mutants. (K-N) Thorax-cleft phenotypes. Classification except for class 4 is based on Tateno et al. (Tateno et al., 2000). (O) Numerical data show that thorax-cleft phenotypes of Src42A6-1 are enhanced by the introduction of a shg mutation, shgg317.

View larger version (92K): [in a new window]   Fig. 4. Src-dependent temporal change in Fas3/tubulin (A1-A9) and E-cad/Arm (B1-B9) accumulation in DME-cells during dorsal closure. Embryos were collected at times indicated. Arrows indicate leading edge. (A) In wild-type, polarized expression of Fas3 and tubulin bundling and cell-elongation along the dorsoventral axis were observed at all times. (B) Leading edge was always smooth in appearance and leading edge signals of E-cad (green) and Arm (magenta) increased gradually 9-11 hours AEL. Src42A26-1;Src64P1/+ embryos exhibited almost normal cell-shape change and signal deposition at 9 hours AEL (B2), but, at later stages, leading edge frequently kinked (arrowheads in B4) and simultaneously Arm and E-cad signals reduced significantly (B4,B6,B8). In B6 and B8, cytoplasmic Arm signals were found. Src42A26-1 embryos were almost normal in cell-shape change and marker-protein deposition at the leading edge (A9,B9).

View larger version (92K): [in a new window]   Fig. 5. F-actin accumulation in DME-cells during dorsal closure. Embryos were collected at times indicated. Arrows indicate the leading edge. (A) In wild type, leading edge signals of F-actin increased gradually during 9-11 hours AEL. Src42A26-1;Src64P1/+ embryos exhibited almost normal F-actin deposition at 9 hours AEL, but, at later stages, leading edge F-actin was reduced significantly. Src42A26-1 embryos were almost normal in F-actin deposition at the leading edge (B4). In shgR64a mutants (B1-B3), thick accumulation of F-actin at leading edge disappeared at 10 hours AEL. (C1-C3) Change in dpp expression at leading edge. dpp expression, which is under the control of the JNK signaling (Goberdhan and Wilson, 1998), was monitored using nuclear enhancer trap (dpp-lacZ). In Src mutants, dpp expression has ventrally expanded as in the case of puc mutants (Martin-Blanco et al., 1997). (D1-D3) Effects of shg activity on Src42A distribution. shgR69 clone on pupal wing disc was marked by the absence of lacZ signals (green). In the shgR69 clone, Src42A signals (magenta) in plasma membrane were significantly reduced. Merged picture is on the left.

View larger version (137K): [in a new window]   Fig. 6. Effects of Src42A[KR] (B1-B3), Src42A[WT] (C1-C3) or Src42A[YF] (D1-D3) on cell-shape and Arm/E-cad distribution at stage 13 and possible release of Src-overexpressing cells from amnioserosa and dorsal epidermis (E-Q). pnr-GAL4 was used as driver for forced expression of Src42A genes. Green, Src42A; magenta, E-cad or Arm. (A1,A2) Wild type. Anterior is leftwards and dorsal upwards. Unlike Src42A[KR] (B1-B3), Src42A[WT] and Src42A[YF] expression caused significant change in morphology and increased cytoplasmic Arm and E-cad signals (B1-D3). (E-G) Arrowheads indicate Src-overexpressing isolated cells in Src42A[YF] embryos. (H-K) Cll (red) and Fas3 (green) are markers for amnioserosa (Jagla et al., 2001) and epidermis, respectively. Blue, Src42A. Most Src-overexpressing isolated cells (arrowheads in I-K) exhibited Cll (H-J) or Fas3 (K). (L-Q) Src-overexpressing isolated cells were TUNEL-stained (magenta) at stage 16 (N, arrows in Q) but not stage 13 (L,M, arrowheads in O,P). Green, Src42A. Insets in Q show enlarged TUNEL-positive isolated cells.

View larger version (67K): [in a new window]   Fig. 7. Src42A/E-cad/Arm ternary complex formation and Src-dependent Arm tyrosine-phosphorylation. (A) Arm/Src42A signals in E-cad immunoprecipitates of untransfected and Src42A/arm-transfected S2 cells. (B) Comparison of Arm/Src42A signals in cytosolic and membranous anti-Fas3 and anti-Arm immunoprecipitates of wild-type embryos. Identical amounts of membrane and cytosolic Arm were precipitated and examined. (C) Immunoprecipitation of membranous and cytosolic fractions of wild-type, Src42A[KR], Src42A[WT] and Src42A[YF] embryos. Same volumes of membrane and cytosolic fractions were analyzed. (a-d) Western blots of fractionated extracts. Tubulin is used as a control. (e-g) Arm, E-cad and Src42A blots of Anti-Arm antibody immunoprecipitates. (h) Src42A blots of anti-E-cad-antibody immunoprecipitates. (i,j) Arm blots of the supernatant of anti-Src42A antibody immunoprecipitates with (j) or without (i) anti-pTyr-antibody treatment. (D) Pull-down assay. Src42A was dissected as shown in the lower margin and expressed as MBP fusions. Y indicates the autophosphorylation site. GST-Arm-repeat signals were detected only in lanes 6 and 8, indicating Arm repeats to bind to the 14 amino acid, autophosphorylation-site-containing Src42A[Kinase] fragment. Lane 10 may indicate that the kinase domain other than the autophosphorylation peptide is unrelated to Arm/Src42A interactions. Input protein signals are shown in lower half as anti-MBP antibody signals. (E) Src dependency of Arm tyrosine phosphorylation. Western blots of extracts from S2 cells transfected with various combinations of metallothionein (MT)-GAL4 and UAS-arm, and dsRNAs indicated are shown (a-f). Signals for whole cell (d) and E-cad-free (e) tyrosine-phosphorylated Arm significantly reduced by Src42A RNAi (compare lane 2 with lane 5).