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First published online 19 October 2005
doi: 10.1242/dev.02092

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Developmental analysis and squamous morphogenesis of the peripodial epithelium in Drosophila imaginal discs

University of Washington, Department of Biology, Seattle, WA 98195, USA

View larger version (90K): [in a new window]   Fig. 1. Cell morphologies in wing discs. Pseudo-colorized confocal sections of a wing disc (110 hours AED) stained with -spectrin illustrates cell morphologies in the peripodial epithelium (PE) and disc proper (DP). Disc oriented with anterior leftwards, dorsal upwards. Broken white lines mark the orientations of optical cross-section and longitudinal section. Red, squamous peripodial epithelium (sPE); yellow, cuboidal cells of the margin in the peripodial epithelium (mPE) and disc proper (mDP); blue, columnar cells of the disc proper (cDP). (A) Confocal image of the PE; (B) longitudinal section; (C) confocal section in plane of the DP (5 µm below PE); (D) cross-section. Scale bar: 50 µm.

View larger version (70K): [in a new window]   Fig. 2. Cell shape changes in the PE of different imaginal discs. Temporal sequence of epithelial morphogenesis in wing (A-E), leg T2 (F-H) and eye (I-M) discs at different larval stages. -Spectrin outlines cell morphology (A-M). Each panel displays an image of the PE (left) and longitudinal section (right) taken through the center of the disc (indicated by arrows only in A,F,I). In longitudinal sections, the PE is oriented left of the disc lumen. White arrowheads indicate squamous PE cells, grey arrowheads mark margin cells. Bolwig's nerve is indicated by red asterisks. Broken yellow lines mark the AP boundary, anterior is towards the left. MF, morphogenetic furrow.

View larger version (69K): [in a new window]   Fig. 3. Compartment boundaries in the peripodial epithelium. (A-C) Expression of en-lacZ in the wing PE and DP during larval development. (A) 55 hours; (B) 72 hours; (C) 110 hours AED. An anterior shift of en-expression in the PE relative to the en-expression in the DP coincides with the formation of squamous PE cells. Scale bar in A: 50 µm (A-F,M-O). (D-F) The en-expression domain in the wing PE defines a clonal restriction. (D) A GFP-expressing PE cell clone straddles (arrowhead) but does not cross the en-expression domain (F) in the PE. (E) Overlay of D and F. (G-I) Large M+ PE cell clones respect the AP compartment boundary in the wing PE. (G) A +/+ PE cell clone, generated in a Minute mutant background, is distinguished by two copies of GFP (outlined in red) and grows along but does not cross the anterior Ci expression domain in the PE (arrowhead) in I. (H) Overlay of G and I. (J-L) PE cells in the wing disc belong to the V compartment. (J) Random GFP-expressing PE clones grow adjacent to without crossing the ap-lacZ expression domains (L) in the wing PE or DP. (L) ap-lacZ expression (red asterisk) is limited to 40+ margin cells in the P compartment of the PE. (K) Overlay of J and L. (J'-L') Enlargements of the boxed region in K. (J') A PE cell clone (outlined in red) that grows next to but does not cross the ap-lacZ (asterisk in L') expression region in the PE (L'). (K') Overlay of J' and L'. A yellow line marks the DV compartment boundary in the DP (lower layer), asterisk indicates ap-lacZ in PE. .The PE cell clone in J-L' traverses the DV compartment in the DP. Scale bars: 50 µm. (M-O) M+ cell clone in the eye PE reveals that the Bolwig's nerve defines a DV lineage restriction. (M) Heterozygous M/+ cells are marked by Arm-lacZ. A +/+ PE cell clone is distinguished by the absence of Arm-lacZ (M) and two copies of GFP (O) and defines a straight line left of the Bolwig's nerve (arrowheads and outlined in red). (N) Overlay of M and O.

View larger version (103K): [in a new window]   Fig. 4. hh and dpp activity are required for squamous morphogenesis in the wing PE. In each panel, confocal sections from top to bottom are of the PE (A-F) and disc cross-section (A'-F'). Red arrows in A-F indicate cross-section levels in A'-F'. Red lines indicate region of squamous cells in A'-C',F'. AP compartment boundary (yellow lines) in the PE (A-F). (A,A') Control disc from larvae heterozygous for wgIL (wgIL/CyO y+). (B,B') Discs that lack wg activity (wgIL/wgIL) are small yet form squamous PE cells and columnar DP cells (B') (n=22 discs). (C,C') Control disc from larvae heterozygous for hhts2 (hhts2/TM6B). (D,D') In the absence of hh function (hhts2/hhts2), discs lack squamous PE cells, while DP cells become columnar (D') (n=46 discs). (E,E') Discs from hhts2 larvae that overexpress cyclin-D-Cdk4 are larger than discs from hhts2/hhts2 larvae yet squamous cells do not form (E') (n=26 discs). (F,F') Discs from hhts2 larvae that overexpress dpp restores squamous morphogenesis in the PE (F') (n=34 discs). (G,G',H,H',I-K) Ubx>dad wing discs have aberrant squamous morphogenesis. Panels from top to bottom are confocal sections in plane of DP and disc cross-section. Red arrows in G,H indicate cross-section levels in G',H'. Red lines indicate region of squamous cells in H'. (G') A PE that consists entirely of cuboidal-shaped cells and (H') a PE with limited squamous cells apposed to clefted folds in the DP. (I-K) Cross-sections of Ubx<dad wing discs, shows that the AP compartment boundary in the PE (red and yellow arrowheads) fails to shift in the absence of squamous morphogenesis (compare with Fig. 3C). (I) -Spectrin staining. (K) En expression. (J) Overlap of I,K. Scale bar: 50 µm.

View larger version (94K): [in a new window]   Fig. 5. Dpp signaling pathway is activated during squamous morphogenesis in the wing disc. Color images show p-MAD (red) and -spectrin (green). Black and white images (A'-D') show p-MAD and (D'') -spectrin alone. Wing discs are from larvae at 67 hours AED (A,A'), 72 hours AED (B,B') and 96 hours AED (C,C'). (A,A') Prior to squamous morphogenesis, p-MAD is present in a ventral wedge in the PE. (B,B') Early in squamous morphogenesis, a stripe of p-MAD expression appears in the PE (yellow arrowhead). (C,C') During the third instar, p-MAD expression broadens and is observed in the majority of squamous cells of the posterior compartment in the PE. (D-D'') Enlargement of region boxed in C. (D-D'') p-MAD is observed only in squamous cells of the posterior compartment and is absent from margin cells (C,D') in both A and P compartments of the PE. (E) hh-lacZ expression in the wing PE from a wandering third instar larva. Scale bar in A: 50 µm.

View larger version (18K): [in a new window]   Fig. 6. Estimated doubling times of squamous PE and columnar DP cells in wing, eye and leg (T2) imaginal discs during larval development. (A) Cell clones were induced (act5C<Gal4, UAS GFP) by heat-shocking larvae for 10-20 minutes at 37°C at the following times: first (42 hours AED), second (48, 52, 60 hours AED) and third instar (72, 84, 90, 96 hours AED). Below the timeline are eight horizontal lines, the left end of the horizontal lines indicate the time of clone induction the right end indicate time of fixation for clone analysis of each experimental set. W (wing), E (eye) and L (leg) indicate the discs analyzed for each clone induction. A vertical mark in each horizontal line represents the mid-point between clone induction and fixation (=average clone age). Estimated cell doubling times are plotted at this mid-point. At each time interval more than 100 clones were scored in over 50 discs. Data represent median cell doubling times±s.d. Cell doubling times of squamous PE cells (blue circles) and columnar DP cells (red squares) in the wing (B), leg (C) and eye (D) imaginal discs.

View larger version (77K): [in a new window]   Fig. 7. Clonal relationship between the wing disc epithelial layers. PE-specific cell clones induced during the second larval instar (48-72 hours AED) using UAS-flp-EBD, Ubx-Gal4 and act5C>stop>nuclacZ. Analysis of clone distribution and frequency was performed in 110 hours AED larvae. (A) Clones with 1-3 cells are present and also a clone with 30+ cells limited to the sPE (yellow asterisks). Cuboidal cell clones are also present at the disc margins in either the PE or DP (arrows), as is a clone that includes squamous and margin cells of both epithelial layers (yellow arrowhead). Scale bar: 50 µm. (B-B'') A z-series of confocal images of the boxed region in A. Teashirt labels squamous PE cells and margin cells in both PE and DP, ß-gal labels clones. Images in the PE (B,B') and the DP (B'') showing a clone that consists of both squamous PE cells and margin cells present in both the disc epithelia. (C) Distribution and frequency of clones born in the wing disc PE during the second (48-72 hours AED) and third (72-96 hours AED) larval instars. sPE, clones of squamous cells in the PE; mPE, clones of margin cells in the PE; mDP, clones of margin cells in the DP; sPE+mPE+mDP clones of squamous PE cells and margin cells in both the PE and DP.

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