Abl Regulates Planar Polarized Junctional Dynamics through β-Catenin Tyrosine Phosphorylation  Masako Tamada, Dene L. Farrell, Jennifer A. Zallen  Developmental Cell  Volume 22, Issue 2, Pages 309-319 (February 2012) DOI: 10.1016/j.devcel.2011.12.025 Copyright © 2012 Elsevier Inc. Terms and Conditions

Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Tyrosine Kinase Signaling Is Planar Polarized in Intercalating Cells (A–D) Antibodies to phosphotyrosine (pY) (green, A and C) and phospho-Src (pY416) (green, B) detect increased staining at AP junctions, in addition to cytoplasmic vesicles. (D) Quantitation reveals an enrichment of pY at edges oriented at 90° relative to the AP axis (AP edges) (n = 9 embryos, 97–224 edges/embryo). (E–I) Antibodies to Abl (green, E and G) and GFP (green, F and H) in stage 7 wild-type (WT, E and G) or Abl:GFP embryos (F and H). Anterior left, dorsal up. Cross sections shown in C, G, and H. Cells were costained with Baz (red in A, C, E, and F) or α-catenin (red in B).(I) Quantitation reveals an enrichment of Abl:GFP at AP edges (n = 8 embryos, 88–247 edges/embryo). Each bar represents the mean intensity of edges in a 15° angular range. Error bars indicate the SEM between embryos. Values were normalized to the mean intensity of edges parallel to the AP axis (0°–15°). Scale bars represent 10 μm. See also Figure S1. Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Abl and β-Catenin Are Tyrosine Phosphorylated in Elongating Embryos (A) Lysates from 2–4 hr and 16–18 hr WT embryos were incubated with anti-β-catenin (β-cat) or control mouse antibody. Eluted immunocomplexes and lysates were analyzed by anti-pY or anti-β-cat immunoblot, 110 kDa (arrows) and 80 kDa (arrowhead) bands are indicated. (B) Lysates from 2–4 hr embryos were incubated with anti-pY or anti-pY416 antibody. Eluted immunocomplexes and lysates were analyzed by anti-pY or pY416 immunoblot. (C) Lysates from 2–4 hr WT or embryos with β-cat:GFP inserted on chromosome III (lane 2) or II (lane 3) were incubated with anti-β-cat antibody. Eluted immunocomplexes were analyzed as in (A). (D) Lysates from 2–4 hr WT or Abl:GFP embryos were incubated with anti-GFP antibody. Equivalent amounts of eluted immunocomplexes were analyzed by anti-pY or anti-GFP immunoblot. (E) Lysate from 2–4 hr Abl:GFP embryos was incubated with anti-GFP, anti-pY416, or control rabbit antibody. Equivalent amounts of eluted immunocomplexes were analyzed by anti-GFP immunoblot. Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 abl Mutants Are Defective for Axis Elongation and Rosette Formation (A–H) Cell behavior in time-lapse movies of WT and abl mutant embryos expressing β-catenin:GFP (n = 5 WT and 8 abl movies). (A) Germband elongation was reduced in abl mutants (p = 0.001). Germband length was normalized to the length at the onset of elongation (t = 0), defined as the time in early stage 7 when the derivative of the elongation curve intersects zero. (B and C) The frequency of neighbor exchange in abl mutants was similar to WT (p = 0.12), but the frequency of rosette formation was significantly reduced (p = 0.0013). Only cells that lost a neighbor through neighbor exchange or rosette formation were counted as having participated in the event. (D) The average lifetime of vertices where four or more cells meet was 6.86 ± 0.25 min (mean ± SEM) in WT (n = 324 vertices in three embryos) and 4.21 ± 0.27 min in abl mutants (n = 238 vertices in eight embryos, p < 0.0001) (black circles/lines show the mean/standard deviation). (E and G) Stills from bright field movies of WT and abl embryos. Arrowheads indicate the posterior end of the germband, t = 0 was the onset of dorsal pole cell displacement. (F and H) Stills from confocal movies of WT and abl embryos expressing β-catenin:GFP, time in min. (F) Linked AP edges (marked by the black line) contract to form a rosette in WT. (H) Linked AP edges in an abl mutant did not noticeably contract during the period of observation. Anterior left, dorsal up. Scale bar represents 5 μm. See also Movies S1 and S2. Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Abl Is Not Required for Planar Polarized Actomyosin Localization and Contractility (A–J) Localization of myosin II (red) and Baz/Par-3 (green) in WT (A,I) and abl mutant (E and J) embryos at stage 7. Quantitation of planar polarity in WT (B–D) and abl (F–H). Values were normalized to the mean intensity of edges oriented at 0°–15° (for myosin II and F-actin) or at 75°–90° (for Baz), where 0° is parallel to the AP axis. n = 7–12 embryos, 77–247 edges/embryo. Planar polarity is significantly decreased in abl mutants for Baz (p = 0.00017), and is only slightly increased for myosin (p = 0.036) and F-actin (p = 0.33). (K) Peak retraction velocities after ablation in WT (n = 10 AP, 6 DV edge ablations) and abl (n = 6 AP, 7 DV edge ablations). (L and M) Stills from time lapse movies in WT and abl knockdown (KD) embryos. E-cadherin:GFP (green), Myo:mCherry (red). Time in min. Anterior left, dorsal up. Cross sections shown in (I and J). Scale bars represent 10 μm (A–J); 5 μm (L and M). See also Figure S2 and Movies S3 and S4. Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Abl Is Required for the Planar Polarized Localization and Dynamics of β-Catenin (A–H) β-catenin localization in WT (A and B), abl mutant (C and D), and abl KD embryos (E and F). Values were normalized to the mean intensity of edges perpendicular (75°–90°) to the AP axis. β-catenin planar polarity was significantly reduced in abl mutant (p = 0.0002) and abl KD embryos (p = 0.000017) (n = 7 WT, 12 abl mutant, 3 abl KD embryos, 76–169 edges/embryo). Anterior left, dorsal up. Cross sections shown in (G) and (H). (I and J) The percentage of prebleach fluorescence observed after bleaching β-catenin:GFP at AP or DV junctions. (K) In WT, β-catenin displayed a higher mobile fraction at AP junctions (63 ± 6%, n=19 edges) (mean ± SEM) than at DV junctions (47 ± 4%, n = 25) (p = 0.023). This difference was abolished in abl mutants (48 ± 3% at AP junctions, n = 25, 46 ± 4% at DV junctions, n = 20) (p = 0.317). (L) The time to recover half maximal fluorescence (t½) was similar for all edges. Scale bars represent 10 μm. See also Figure S3. Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Abl Promotes the Phosphorylation of β-Catenin on Tyrosine 667 (A) Schematic of β-catenin transgenes. β-catΔC is a deletion of aa 681–843, equivalent to the armXM19 allele. β-catΔCS10/150A and β-catΔCS12 have an additional deletion of aa 35–87 or 549–606, respectively. All tyrosine (Y) residues in β-catΔC and Y to phenylalanine (F) or alanine (A) mutations are indicated. Asterisks indicate conserved tyrosines. (B) S2R+ cells were transfected with Venus-tagged β-catenin plasmids with or without Abl:HA. Asterisk indicates β-catΔC667F. (C) S2R+ cells were transfected with Venus-tagged β-catΔC, β-catΔC667F, or β-catΔC667E plasmids with or without Abl:HA. Cell lysates were incubated with anti-GFP antibody to detect Venus, and eluted immunocomplexes were analyzed by anti-pY or anti-GFP immunoblot. Top shows protein levels. (D and E) Lysates from 3–6 hr embryos expressing HA-tagged β-catΔC, β-catΔC667F, or β-catΔC667E transgenes at 20°C were immunoprecipitated with anti-E-cadherin (D) or anti-HA (E) antibody and eluted immunocomplexes were analyzed by anti-β-catenin, anti-E-cadherin, or anti-α-catenin immunoblot. See also Figure S4. Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Phosphorylation of β-Catenin on Tyrosine 667 Regulates β-Catenin Dynamics and Rosette Formation (A and B) The percentage of prebleach fluorescence after bleaching AP junctions in embryos expressing Venus-tagged β-catΔC, β-catΔC667E, or β-catΔC667F. (C) β-catΔC667E had increased mobility compared to β-catΔC (p = 0.0009) and β-catΔC667F (p = 0.0034). There was no difference in the mobility of β-catΔC and β-catΔC667F (p = 0.5132) (n = 21 β-catΔC, 18 β-catΔC667E, 23 β-catΔC667F). (D) The t½ was similar for all transgenes. (E and F) Stills from movies of β-catenin KD embryos expressing HA:β-catΔC667E or HA:β-catΔC667F. Cells labeled with Spider:GFP. Linked AP edges (black line) contract to form a rosette in an embryo expressing β-catΔC667E (E) (new contacts, yellow line at 14 min). Little contraction occurs in an embryo expressing β-catΔC667F (F). (G) Percentage of linked AP edges that joined a rosette of five or more cells in WT, abl, and β-cat KD embryos expressing the indicated β-catΔC transgenes. Fewer linked AP edges formed rosettes in abl (p = 0.0421 versus WT) and β-catΔC667F (p = 0.0233 versus β-catΔC and p = 0.0149 versus β-catΔC667E) (38–168 edges scored in three to eight embryos/genotype). Error bars indicate the SEM between embryos. (H) Percentage of fully extended embryos. Ninety-seven percent of Gal4 control embryos completed elongation (n = 147 embryos) versus 37% of abl KD embryos (n = 527) (p < 0.0001). Sixty percent of abl KD embryos expressing full-length HA:β-catenin667E completed elongation (n = 469) (p < 0.0001 compared to abl KD), versus 35% of abl KD embryos expressing full-length HA:β-catenin (n = 239) (p = 0.57) and 33% of abl KD embryos expressing full-length HA:β-catenin667F (n = 511) (p = 0.17). Scale bar represents 5 μm. See also Figures S4 and S5. Developmental Cell 2012 22, 309-319DOI: (10.1016/j.devcel.2011.12.025) Copyright © 2012 Elsevier Inc. Terms and Conditions