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First published online 27 February 2008
doi: 10.1242/dev.015982

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Dual function of Src in the maintenance of adherens junctions during tracheal epithelial morphogenesis

Masayo Shindo^1,2, Housei Wada¹, Masako Kaido¹, Minoru Tateno¹, Toshiro Aigaki³, Leo Tsuda^1,* and Shigeo Hayashi^1,2,4,

¹ Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku Kobe 650-0047, Japan.
² National Institute of Genetics and the Graduate School for Advanced Studies, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan.
³ Department of Biology, Tokyo Metropolitan University, Minami-Ohsawa 1-1, Hachioji, Tokyo 192-0397, Japan.
⁴ Department of Life Science, Kobe University Graduate School of Sciences and Technology, Kobe 657-8501, Japan.

View larger version (77K): [in this window] [in a new window]   Fig. 1. Expression of phosphorylated Src42A (pSrc) in the adherens junctions of epithelial tissues. (A) Schematic diagrams of Src42A and Src64B. Three functional domains (SH3, SH2 and the kinase domain), the myristoylation (M) site at glycine 2 (G2), the catalytic lysine in the kinase domain, and the sites of activating (Y400/434) and inhibitory (Y511/547) tyrosine phosphorylation are indicated. (B-D) Staining pattern of anti-phospho Src (pSrc) antibody during tracheal development. (B) Embryos were stained for pSrc (gray scale), and tracheal cells were labeled green with trh-lacZ. pSrc was localized to the apical cell-cell junction of the ectoderm and in invaginating tracheal primordia in stage 11. (B') y-z section shows that tracheal cells (arrow) express a higher level of pSrc than ectoderm (arrowhead). (C) pSrc and E-cadherin are co-localized in the AJs of stage 11 tracheal primordia (btl>gfp-moe, green). (D) Src expression in a control (Src42A26-1/+) embryo at stage 14, showing broad cell membrane staining by anti-Src42A antibody and highly restricted AJ staining by anti-pSrc (arrow indicates trachea; arrowhead, epidermis). (E) Both signals were greatly reduced in Src42A26-1 mutant embryos. (F) Src42A overexpression in the trachea (btl>Src42AGS) greatly increased the pSrc signal (arrow). (G) Src64B overexpression in the trachea (btl> Src64BGS) did not increase the pSrc signal. (H) Src activation in the parasegmental furrow. Optical section of the embryonic ectoderm at stage 9 labeled with pSrc (green) and Arm (magenta). Nuclei were visualized by DAPI (blue). Grayscale images of the two channels are shown below. Scale bars: in C, 10 µm for B,C; in H, 100 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 2. Requirement for Src functions in tracheal morphogenesis. (A-D) Snap-shot images from a time-lapse series of tracheae labeled with -catenin-GFP (see also Movies 1-4 in the supplementary material). Genotypes are: control (A), Src42Amyri (B), btl>Src42ARNAi; Src42A26-1/+ (C) and Src42Amyri; Src64BP1 (D). Arrows in magenta indicate the position of a dorsal trunk `bend', which is likely to be due to an excessive pulling force applied by the shortened dorsal branch. Green arrowheads indicate the position of the leading edge of dorsal epidermis. Inset shows an enlarged view of AJs in the dorsal branch. The images represent more than three independent embryos of each genotype.

View larger version (73K): [in this window] [in a new window]   Fig. 3. Src antagonizes E-cadherin during tracheal branching. (A-D) Dorsal branch morphologies in embryos with various levels of E-cadherin. Markers and genotypes are indicated on the left. (A) Control embryos carrying btl>GFP-moesin. Arrowhead indicates the lumen. L, length of dorsal branch (DB) measured and plotted in I and H. (B) Control embryos carrying btl>-catenin-GFP that labels the AJ as a black line in each dorsal branch. (C) In shgIH/shgE17B mutants, AJs became diffuse and discontinuous. (D) E-cadherin-GFP-expressing tracheae undergo slightly delayed, but morphologically normal, branching. Inset shows double lines of AJs remaining in the late stage of DB elongation. (E-H) Effect of activated Src42A on dorsal branch morphology and AJs. (E) In embryos expressing activated Src42A, tracheal cell contact persisted but became loose, and the lumen became discontinuous. (F) Src42AACT loosened AJs. Dotted circles indicate transiently detached cells at the tip of the DB. (G) This phenotype was further enhanced in the shgIH/+ background. Most cells have now rounded up and lost accumulation of -catenin-GFP. (H) E-cadherin-GFP partially restored the extent of elongation of the DB and its lumen (compare with E). (I) Plot of the length of DBs (L, see A). The onset of DT fusion was set as time 0 for each measurement. When compared with DBs labeled with GFP-moesin, E-cadherin-GFP delayed DB elongation in an otherwise wild-type background. (J) E-cadherin-GFP partially restores elongation of DBs expressing Src42AACT. Original movies for A, C, D, E and H are presented as Movies 5-7 in the supplementary material. Scale bar in H: 25 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 4. FRAP analyses of -catenin-GFP. (A) Dorsal branch of btl>-catenin-GFP embryos with or without Src42AACT were photo bleached at the bracketed regions and fluorescence recovery was recorded. (B) Schematic plot of FRAP analysis. Photo bleaching reduces the fluorescence level from pre bleach (Fpre) to post bleach (Fpost). Fast recovery of fluorescence reaches an approximate plateau level (Fend) that is usually lower than Fpre. Recovery to Fpre required a longer time and was not analyzed. Mobile fraction [Mf=100x(Fend-Fpost)/(Fpre-Fpost)] and half-time of recovery (t) were estimated from the plots. (C) Representative plots of fluorescence recovery in dorsal branches of control (black), Src42A; Src64B double mutant (red) and Src42AACT-expressing (blue) embryos. (D) t and Mf values under various conditions of Src activities. Error bar indicates standard error. Values that deviate from controls are indicated (*P<0.05, **P<0.005).

View larger version (37K): [in this window] [in a new window]   Fig. 5. Regulation of E-cadherin-catenin complex by Src42A. (A) Complex formation by E-cadherin, Arm and Src42A. Expression constructs of E-cadherin-GFP and Src42A were transfected into S2 cells and the E-cadherin-Arm complex was analyzed by immunoprecipitation. Blue and red boxes mark the lanes of whole cell extract and immunoprecipitates of anti-Arm, respectively. The expression of E-cadherin-GFP stabilized Arm in apparent high and low molecular weight forms, indicated by H and L. Although both Src42AACT and Src42ADN in whole cell extract were phosphorylated at Y400, Src42ADN in complex with Arm was under phosphorylated. (B) Src42A downregulates the cell surface level of E-cadherin. Surface biotinylation was applied to S2 cells expressing E-cadherin-GFP and Src42A. Western blot analyses were performed on whole cell extracts (whole) and proteins were recovered by avidin beads (surface). Septin and avidin-reacting proteins were used as loading controls for each blot. The positions of marker proteins are shown in the avidin panel. (C) Downregulation of E-cadherin-GFP by Src42AACT. Transfected S2 cells were stained with a method to detect extracellular E-cadherin (surface, magenta). GFP signal in the intracellular domain (intra, green) is shown. Src42AACT reduced both signals. (D) The amount of E-cadherin was reduced in Src mutant embryos. Control and Src42Amyr; Src64BP1 embryos (src-/-) were collected and analyzed for E-cadherin and pSrc expression by western blotting. Protein extracts were analyzed in the same gels and blots. Relative amount is shown above each band.

View larger version (116K): [in this window] [in a new window]   Fig. 6. Src activates E-cadherin transcription through Arm. (A-C) Control; (D-F) btl>Src42AACT; (G,H) Src42Amyr; (I) btl>TCFN; (J,K) btl>TCFN+Src42AACT; (L,M) btl>wg. Tracheal cells are labeled with btl>GFP-moesin (green). (A,D,G) Arm (grayscale) is localized to the apical cell-cell junction in control embryos (A). In Src42AACT embryos, the Arm signal increased to fill the entire tracheal cell (D). Arm staining was reduced in Src42A mutants (G). (B,E,H,J,L) Esg expression (magenta) was localized to fusion cells in control embryos (B), and the number of Esg-expressing cells was greatly increased in embryos overexpressing Src42AACT or wg (E,L). In Src42Amyr or Src42AACT, TCFN embryos, Esg was almost undetectable (H,J). (C,F,I,K,M) shg-lacZ (nuclear signal, magenta) was broadly expressed, with elevated levels in tracheal fusion cells (C, asterisk in C'). (C'-M') Framed area in C-M enlarged and shown in grayscale. The number of strong shg-lacZ-expressing cells increased in embryos overexpressing Src42AACT or wg (F,M). In Src42Amyr or Src42AACT, TCFN embryos, shg-lacZ expression was reduced in tracheal cells (I,K; compare the levels in the trachea and ectoderm). Scale bar in A: 20 µm for A-M. (N) RNA quantification. Src42AACT, Src42AGS or wg was expressed under the control of the ubiquitous da-Gal4 driver, and the levels of E-cadherin, arm and rac1 mRNAs were measured by quantitative RT-PCR. da-Gal4 embryos were used as a control.

View larger version (110K): [in this window] [in a new window]   Fig. 7. Essential role of Src-induced Arm/TCF activity in tracheal cell adhesion. (A-F) Time-lapse observations of btl>GFP-moesin (A-D,F) or btl>Cat-GFP (E) in control (B), btl>TCFN (C), btl>Src42AACT (A,D), btl>Src42AACT; shgIH (E) and btl>Src42AACT, TCFN (F) embryos. (A) Metastasis-like behavior of tracheal cells expressing Src42AACT. Cells numbered 1 and 2 detached from one branch and reattached to an adjacent branch. (B-E) Src42AACT and TCFN synergistically caused a strong cell dissociation phenotype. Asterisk in E indicates the autofluorescence of yolk. Original movies for A,B,D and F are presented as Movies 8, 9 in the supplementary material. (G) Model of the dual function of Src42A on E-cadherin. Scale bar in B: 50 µm for B-D,F; 25 µm for E; 10 µm for A.

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