JCS044271 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Analysis of control and tkv^a12 bsk⁻ clones. (A) Clones of cells homozygous mutant for bsk⁻ induced in the second instar larval stage and stained 60 hours later for Fasciclin III (FasIII, red), E-cadherin (blue), viking-GFP (green) and DAPI (white). Clones (asterisks) are marked by the absence of GFP (green). Cross-section view (xz). bsk⁻ cells have an apparently normal shape. (B,C) Clones of cells homozygous mutant for tkv^a12 bsk⁻ marked by the presence of CD8-GFP staining (green) expressing tkv from a transgene were induced at the second instar larval stage and analyzed 48 hours later for F-actin (red) and E-cadherin (blue). x-y views (B) and x-z cross-sectional views (C) are shown. DAPI staining (white) is shown in (C). Expression of tkv results in a high frequency of clone recovery within the wing disc pouch. Cells have a normal morphology (absence of epithelial invagination). (D-F) Clones of tkv^a12 bsk⁻ cells, marked by the presence of CD8-GFP, were induced at the second instar larval stage and stained as indicated 24 hours later. xz views. (D) F-actin staining is increased at the apical side of tkv^a12 bsk⁻ cells and adjacent control cells (arrow) compared with non-adjacent control cells (arrowhead). Apical-basal length of tkv^a12 bsk⁻ cells is reduced compared with control cells (double-sided arrows). (E) Rho1 staining intensity is not obviously changed in tkv^a12 bsk⁻ cells (arrow) compared with control cells (arrowhead). (F). Moesin is decreased at the apical side of tkv^a12 bsk⁻ cells and adjacent control cells (arrow) compared with most non-adjacent control cells (arrowhead). Control cells within a normal epithelial fold display decreased Moesin levels (asterisk). (G-K) Clones of tkv^a12 bsk⁻ cells induced at the second instar larval stage and stained 48 to 60 hours later as indicated. (G) x-z view. tkv^a12 bsk⁻ cells are marked by the absence of GFP (green). PSβ-integrin is increased at the clonal border (arrows). (H) x-z view. tkv^a12 bsk⁻ cells are marked by the presence of CD8-GFP (green). Dlg is present in clones forming an invagination. (I) x-z view. White line outlines clone. Arrow points to high F-actin staining at the apicolateral side of mutant cells, where E-cadherin is highly reduced. (J) x-y view. Asterisk points to a normal epithelial fold where F-actin staining and E-cadherin are high. Arrow points to the apicolateral side of mutant cells, where F-actin is strong, but E-cadherin is highly reduced. (K) x-z view. Dlg is detected at the apical side of control cells (arrowhead), but undetectable in tkv^a12 bsk⁻ cells extruded from the epithelium (labeled with CD8-GFP, arrow). (L,O) Clones of tkv^a12 bsk^- cells labeled with CD8-GFP (green) induced at the second instar larval stage and stained 60 hours later as indicated. x-z (L) and x-y (M-O) views are shown. (L-M) Extruded mutant cells display actin-rich extensions (arrows). (N) Twist is detected in muscle precursor cells (asterisk) but not in tkv^a12 bsk⁻ cells (arrow). (O) A magnified view of the area boxed in N. Scale bars: 10 µm (A,C-M,O); 50 µm (B,N).

• Supplemental Figure S2 -

  Fig. S2. Consequences of Dad overexpression on P-Mad, epithelial morphology, and cell identity. (A-D) Clones of cells coexpressing Dad and p35 (Act5C>Gal4 UAS-dad UAS-p35) marked by the presence of p35 (green) and stained for F-actin (red). (A) Cross section x-z view of a disc carrying a few, separate clones (data not shown). Clonal cells are part of an epithelial invagination. (B-D) A wing disc carrying many Dad and p35 coexpressing clones. (B) Low magnification x-y view. (C) High magnification view of the wing discs shown in B. The control cells (asterisks) are surrounded by Dad and p35 coexpressing cells. (D) Cross section view of the x-y section shown in C. DAPI staining (white) is shown in addition. Note that in these wing discs, which display many, and large, clones of cells coexpressing Dad and p35, the control cells (asterisks) were part of epithelial invaginations. This indicates that the degree of percolation of control cells, and cells displaying reduced Dpp signaling, has an influence on the epithelial architecture. (E) Cross section view of a wing disc coexpressing Dad and p35 in the dorsal compartment (ap-GAL4 tubP-gal80^ts UAS-dad UASp35) 24 hours after temperature shift to inducing conditions stained for F-actin (red), P-Mad (green) and DAPI (white). The dorsal compartment is to the right. P-Mad staining is highly reduced in dorsal cells. (F,G) x-z sections of wing discs coexpressing Dad and p35 in the dorsal compartment (ap-GAL4 tubP-gal80^ts UAS-dad UASp35) (F) 0 hours, or (G) 56 hours, after temperature shift to inducing conditions stained for F-actin (red), Wingless (green), p35 (blue), and DAPI (white). The arrowheads indicate Wingless protein expressed at the dorsal-ventral compartment boundary and the arrows indicate Wingless protein in epithelial fold 2, which limits the pouch region. (G) Shortened cells expressing Dad and p35 are part of the wing pouch region, as delineated by Wingless (arrow) and epithelial fold 2. The x-z sections are taken approximately through the center of the wing disc perpendicular to the dorsal-ventral interface. Numbers denote folds in the wing disc (compare to Fig. 1E). The dorsal compartment is shown to the right. Scale bars: 10 µm (A,C,D,E); 20 µm (F,G).

• Supplemental Figure S3 -

  Fig. S3. Consequences of Katanin-60 and Klp10A co-overexpression on epithelial morphology. (A,B) Wing disc coexpressing Dad and p35 in the dorsal compartment (ap-GAL4 tubP-gal80^ts UAS-dad UASp35) 36 hours after temperature shift to inducing conditions stained for F-actin (red), α-tubulin (green), and p35 (blue). (A) x-y section. The dorsal compartment is to the top. (B) Cross section view (x-z) of the wing disc shown in A. The position of the x-y section is indicated in A by a dotted line. (B’) Only the α-tubulin channel is shown. The apical microtubule web is highly reduced in dorsal cells (arrow) compared with the ventral control cells (arrowhead). At this time point, cell shape in the dorsal compartment is not yet overtly changed compared with the ventral compartment. (C,D) Wing disc coexpressing Katanin-60 and Klp10A in the dorsal compartment (ap-GAL4 tubP-gal80^ts UAS-katanin-60 UAS-Klp10A) 17 hours after temperature shift to inducing conditions stained for F-actin (red), α-tubulin (green) and DAPI (white). (C) x-y section. The dorsal compartment is to the top. (D) Cross section view (x-z) of the wing disc shown in C. The position of the x-z section is indicated in C by a vertical dotted line. Dorsal is to the right. (D’) Only the α-tubulin channel is shown. Apical microtubules are reduced in columnar cells coexpressing Katanin-60 and Klp10A (arrow) compared with control ventral cells (arrowhead). Microtubules in the squamous cells, in which ap-GAL4 is not active, appear not to be affected (asterisk). (D’’) Only the F-actin channel is shown. The length of dorsal and ventral cells is similar (double-sided arrows). Note that the accumulation of F-actin, which normally labels zonula adherens, is positioned more basally in cells coexpressing Katanin-60 and Klp10A compared with control ventral cells (arrowheads). Prolonged coexpression of Katanin-60 and Klp10A resulted in apoptotic cells. Scale bars: 20 µm.

• Supplemental Figure S4 -

  Fig. S4. Consequences of reducing Moesin or Dia activity on epithelial morphology. (A-C) Wing disc coexpressing moesin^dsRNA and p35 in the dorsal compartment (ap-Gal4 tubP-gal80^ts UAS- moesin^dsRNA UASp35) (A,B) 36 hours or (C) 80 hours after temperature shift to inducing conditions stained for F-actin (red), Moesin (green), and DAPI (white). (A) x-y section. The dorsal compartment is to the top. (B,C) Cross section views (x-z); dorsal is to the right. (B) 36 hours after temperature shift, Moesin is highly reduced in dorsal cells compared with the ventral control cells. At this time point, cell shape in the dorsal compartment is not overtly changed compared with the ventral compartment (double-sided arrows). (C) 80 hours after temperature shift, a mass of nuclei, as identified by DAPI staining, is present below the basal surface of the dorsal compartment (asterisk), indicating that the epithelium of the dorsal compartment has largely disintegrated. (D,E) Wing disc expressing dia^dsRNA in the dorsal compartment (ap-GAL4 tubP-gal80^ts UAS- dia^dsRNA) 56 hours after temperature shift to inducing conditions stained for F-actin (red), Dia (green), and DAPI (white). (D) x-y section. The dorsal compartment is to the top. (E) Cross section views (x-z); dorsal is to the right. (E) Dia is highly reduced in dorsal cells compared with the ventral control cells. Apical-basal cell length in the dorsal compartment is not overtly changed compared with the ventral compartment (double-sided arrows). Prolonged expression of dia^dsRNA resulted in the disintegration of the epithelium of the dorsal compartment (data not shown). Scale bars: 20 µm.

• Supplemental Figure S5 -

  Fig. S5. Clonal loss of Mbs leads to cell shortening and epithelial invagination. (A-F) Clones of cells homozygous mutant for mbs^T666 induced (A, B) 24 hours, (C, D) 48 hours and (E, F) 72 hours before analysis in late third instar larval stage are detected by the absence of GFP staining (green). (A,C,E) Cross-sectional views and (B,D,F) x-y views of wing discs stained for F-actin (red), E-cadherin (blue) and DAPI (white) are shown. The positions of the x-y sections are indicated in the corresponding x-z sections by horizontal lines. mbs^T666 cells initially shorten and result in apical and basal invaginations of the epithelium (arrowheads). Early induced clones lead to deep epithelial invaginations resembling tkv^a12 bsk⁻ clones. Note that, unlike tkv^a12 bsk⁻ clones, mbs^T666 clones do not display a round shape in the x-y dimension, indicating that, unlike Tkv, Mbs does not effect the sorting of cells within the plane of the epithelium. Scale bars: 10 µm.

• Movie 1 -

  Movie 1. tkv^a12 bsk⁻ clones form deep epithelial invaginations. Different views of a 3D-rendered wing disc displaying a clone of cells homozygous mutant for tkv^a12 bsk⁻ stained for Fasciclin III (red), E-cadherin (blue), viking-GFP (green) and DAPI (white).

• Movie 2 -

  Movie 2. tkv^a12 bsk⁻ clones form deep epithelial invaginations. Different views of a 3D-rendered wing disc displaying a clone of cells homozygous mutant for tkv^a12 bsk⁻ labelled with CD8-GFP (green) and stained for F-actin (red), PSβ-integrin (blue) and DAPI (white). The later part of the movie shows the green channel only. Note the opening at the apical side of the clone.