Fig. 3. Reducing Cindr triggered basal delamination and lateral cell movement. (A-B) Overlays of the apical 1.23 ?m (green), and lower 1.23 ?m zones (red) of a ptc>Dcr-2, GFP, lacZ larval wing pouch. Apical and lower zones are indicated in B; please refer to Fig. S4 for helpful illustration of the wing. In an orthogonal cross section of this wing (B) we observe some ‘bulging’; of GFP-cells beneath the surface of the A/P adherens boundary (indicated). (C-E) The apical 1.23 ?m (green), and lower 1.23 ?m zones (red) of a ptc>Dcr-2, GFP, cindrRNAi2 wing. In two orthogonal cross sections (D,E) we observe substantial movement of Concanamycin A
across the A/P adherens boundary. Position of these cross-sections indicated in C. (F-G) The apical zones (only) of control lacZ and cindrRNAi2-expressing tissue. Actin (red) and Ecadherin (blue, white in F? and G?) are also shown. Red boxes indicate tissue presented at higher magnification in H and I. A/P boundaries are indicated with white lines. (H-I) Higher magnification images of a small region of tissue straddling the A/P adherens boundary (marked by white lines). In control tissue, GMA (GFP), Actin and Ecad accumulate apically marking the ZA. Higher levels of Ecadherin are detected at the smaller circumferences of cindrRNAi2-cells. Cells arrange into rosettes (pink) and numerous diminutive cells (green) are also observed. Green arrows indicates a large oval cell migrating apically, enriched in actin. (J-K) The lower 1.23 ?m zones of lacZ and cindrRNAi2-expressing tissue. Actin (red) and Ecadherin (blue, white in J? and G?) are homogenously localized about the periphery of cells in control wings (J?), marking the lateral membranes. A marked decrease in Ecadherin and increase in Actin is observed in most basal cindrRNAi2-cells (blue bracket, K?, white arrow (K??) indicates an F-actin punctum), except in several ‘healthier’; migrating cells that retain peripheral Ecadherin (inset in K?-K??).Figure optionsDownload full-size imageDownload high-quality image (2637 K)Download as PowerPoint slide
To understand the morphology of displaced cindrRNAi2-cells better, we examined the effect of reducing Cindr in cuboidal epithelial cells of the pupal wing. At 14 h after puparium formation (h APF), these cells
are four to five times larger than cells of the larval wing epithelium. At 14 h APF the wing is rather like an oval water balloon: the single-layered cuboidal epithelium encloses a fluid-filled lumen. We peeled back the chitinous pupal-case to expose the dorsal surface of the wing for live-imaging (Fig. 2F). Using this strategy we observed GFP-labeled cells projecting dynamic extensions and confirmed that in ptc>GFP wings, cells remained in stable positions, shifted only by occasional cell division ( Fig. 2G, data not shown). In contrast, reducing Cindr released small groups and individual cells from the epithelium (Fig. 2H-K). These cells delaminated into the underlying lumen and we expect were later swept away in the fluid hemolymph. Whilst delaminating, some cells adopted morphologies reminiscent of migrating mesenchymal cells (Fig. 2I). These observations support the hypothesis that in the absence of Cindr, epithelial cells delaminate and acquire migratory potential.
3.2. Features associated with cell delamination and migration accompanied loss of Cindr
The apical circumferences of many cindrRNAi2-cells still within the ‘intact’; ptc-domain (defined above) were mildly reduced (compare Fig. 3F? and G?, H-b and I-b, quantified in Fig. S3), indicating apical constriction which often portents cell delamination ( Katoh and Fujita, 2012 and Eisenhoffer and Rosenblatt, 2013). Consistent with this suggestion, we observed several small cindrRNAi2-cells distributed through the ptc domain (examples are colored green in Fig. 3I-c) as well as groups of cells arranged in rosettes (examples are colored pink in Fig. 3I-c) - we expect that a cell originally lay in the center of these rosettes, but had delaminated. These observations are consistent with those of delamination in other systems (for eg. Marinari et al. (2012) and Muliyil et al. 2011)) and described by T2 transition models (Fletcher et al., 2014). Interestingly, the apical circumferences of several rows of <img src="http://farm6.static.flick