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Figures
- Fig. 1.
sal/SALL4 induces cell invasion in the larval body and wing disc. (A) GFP signal driven by the dpp-Gal4 was expressed in a stripe in the anterior wing disc. A indicates the anterior compartment and P is posterior compartment. Dashed lines in A-D contour the rough dpp-Gal4 region. In this and subsequent figures, wing imaginal discs were oriented anterior left and dorsal up. The developmental stages were late third-instar and the x-y images were focused on the middle section of the wing pouch and hinge region, unless indicated elsewhere. (B–D) Cells expressing salm (B), salr (C), or SALL4 (D) in the dpp-Gal4 domain invaded into both A and P compartments. In most cases, there was a groove in the pouch region due to sal discontinuity regulated cell sorting. The red arrowheads indicate the single migrating cells and the yellow arrowheads indicate the cell mass in B–G. (E–G) GFP-labeled clone cells. Compared with the control (E), cells overexpressing salm (F) or SALL4 (G) tended to disperse into the single cell level (red arrowheads). The yellow arrowheads represent the hyperproliferative tumor cells. (H) Control clones that expressing the membrane CD8-GFP. (I) The filopodia-like structure appeared in the moving cells shown by CD8-GFP. I′ was the magnification of the box in I. The arrowhead shows the membrane protrusion. Scale bars: 50 µm.
- Fig. 2.
The apico-basal polarity is disrupted in sal/SALL4-overexpressing wing discs. (A) α-integrin was specifically concentrated at the basement membrane. Wing discs as shown in Fig. 1 were sectioned along the x-z axis and images here showed the side view. In all x-z scans apical cells were up and anterior cells were left. (B) Expressing salr induced cell extrusion and ECM degradation. Arrowheads show the degradation of integrin in extrusion cells. (C) DE-cad was rearranged in cells overexpressing salr. The apical DE-cad was comparable in salr-overexpressing and non-overexpressing cells, but the lateral localization was increased in salr-overexpressing cells (GFP expressing regions). Dashed lines in C-E mark the boundary of GFP-expressing and non-expressing cells. (D) The lateral DE-cad was increased in cells overexpressing salm. (E) The profile of DE-cad fluorescence intensity. (F–H) The EMT marker DN-cad occurred in salr/SALL4-overexpressing cells. Arrowheads indicate the ectopic DN-cad. Scale bars: 50 µm.
- Fig. 3.
sal/SALL4 promotes cell invasion through the JNK signaling. (A) Wild-type cells had no obvious JNK activation as indicated by the Mmp1 staining. (B,C) The Mmp1 level was upregulated in salr/SALL4-overexpressing wing discs. Arrowheads in B-H indicate the increased JNK signaling. (D) Mmp1 was activated in clone cells overexpressing salr. (E) pJNK expression was slightly activated in the central stripe of wild-type wing discs. (F) Overexpression of salr promoted JNK phosphorylation. (G) puc was not activated in the control wing disc. (H) puc was activated in the salm-overexpressing cells. Arrowheads show the autonomously increased JNK signaling and non-autonomous increase in the surrounding cells. (I,J) Co-expression of salm and puc suppressed salm-induced cell invasion as well as the Mmp1 level. (K–M) Cell invasion induced by salr/SALL4 was significantly inhibited by bskDN. (N,O) Co-expression of salr and Timp suppressed salr-induced cell invasion. (P) Co-expression of salr and Timp suppressed salr-induced cell extrusion. (Q) Quantification of the area of invading cells into the P compartment. Each genotype was quantified for 30 wing discs. *** represents P<0.001 (two-tailed one-way ANOVA tests for each genetic interaction with salm, salr and SALL4 overexpression). Error bars indicate s.e.m. Scale bars are the same except in P. Scale bars: 50 µm.
- Fig. 4.
sal/SALL4 inhibits dMyc expression. (A) dMyc was expressed in the wing discs. (B,C) dMyc was downregulated in salr/SALL4-overexpressing cells. Arrowheads in B′ and C′ indicate the areas that dMyc was obviously repressed. (D–F) dMyc was reduced in salr/SALL4-overexpressing clone cells. The arrowheads mark the clone cells. E″ and F″ are higher resolution images for box areas in E and F. Scale bars: 50 µm except in the higher resolution images where scale bars are 25 µm.
- Fig. 5.
sal/SALL4-induced cell invasion depends on dMyc expression. (A) Expressing dMyc showed subtle migration phenotype. The outline of GFP at the A/P compartment boundary was not as smooth as that in previous dpp>GFP controls. (B,C) Overexpression of dMyc greatly repressed salr/SALL4-induced cell invasion. (D) Mmp1 was not activated in the wing discs co-expressing salr and dMyc. (E) Mmp1 level was not increased in the wing discs co-expressing SALL4 and dMyc. (F) Downregulation of dMyc alone induces cell migration. Arrowheads indicate single cell migration into the P compartment. (G) The Mmp1 level was upregulated in dMyc-knockdown wing discs. Arrowheads show the high Mmp1 expression in the dMyc-knockdown cells. (H) Co-expression of salr and dMyc-RNAi (dMyc-i) exacerbated salr-induced cell invasion. (I) Quantification of invading cell areas. Each genotype was quantified for 30 wing discs. *** represents P<0.001 (two-tailed pairwise comparison of t-tests). Error bars indicate s.e.m. Scale bars are the same except in G. Scale bars: 50 µm.
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