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Subdivision of the Drosophila wing imaginal disc by EGFR-mediated signaling

Department of Genetics and Development, Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA

View larger version (73K): [in a new window]   Fig. 1. EGFR/Ras signaling is required for notum development. (A,B) Organization of the mature wing imaginal disc. (A) Subdivision of a wild-type wing imaginal disc into AP compartments (as marked by en-Gal4 driven GFP expression in posterior cells; green), DV compartments (as marked by the expression of Ap in dorsal cells; blue) and notum-wing primordium (as marked by elevated expression of mirr-lacZ in prospective notum cells; red). The notum-wing boundary is marked by an arrowhead; mirr-lacZ is also expressed in prospective pleural cells in the mature wing disc (to the right and below the arrowhead). (B) mirr-lacZ expression (red) relative to Wg expression (blue) in a wild-type wing imaginal disc. Overlapping expression appears in pink. Note that mirr-lacZ expression is generally restricted to the prospective lateral notum (region III), which is demarcated dorsally by a stripe of Wg expression (arrows in B-E) and ventrally by a characteristic deep fold (arrowheads in A-E). However, as noted in the text, the Iro-C genes are also expressed in additional domains in mature, third instar wing discs, including in a stripe of cells extending ventrally along the edge of the disc. The prospective medial notum (region IV) is located dorsal to this stripe of Wg expression, and the prospective wing blade (region I) is encircled by two closely associated rings of Wg expression (only one ring is visible here, asterisk), distinguishing it from the surrounding prospective wing hinge (region II). Wg is also expressed along the DV compartment boundary, bisecting the wing blade primordium into D and V halves. (C-F) Egfr– (C) and ras– (D-F) clones marked by the absence of either GFP expression (C-E, green) or CD2 expression (F, green). Wg expression is shown in blue (C-E) and faint green (F); tsh-lacZ expression, a marker for prospective wing hinge, is shown in blue (F). (C) Distribution of Egfr– clones induced during late second/early third larval instar using the Minute technique. Note that clones are present throughout the disc, except in an area corresponding to the prospective lateral notum (between the arrow and arrowhead). (D) Distribution of ras– clones generated in a non-Minute background during first larval instar. ras+ twin spots are marked by bright GFP expression. ras– clones generally failed to survive in the prospective lateral notum (see also Fig. 2B). Note the presence of a large ras– clone in the prospective medial notum (asterisk), which abuts the prospective lateral notum (defined by the stripe of Wg expression), which straddles the boundary between the two domains. (E) Disc with at least two large ras– clones generated during first larval instar using the Minute technique. Note that the mutant clones populate most of the prospective wing blade and wing hinge and show a normal pattern of Wg expression; however, the clones do not contribute to the notum, which also appears to develop normally and shows normal Wg expression. (F) Disc with large ras– clones generated during first larval instar using the Minute technique. The notum is ablated and the disc is composed largely of prospective wing hinge tissue (marked by high levels of tsh-lacZ expression; blue) and wing blade tissue (encircled by a thin stripe of Wg expression and bisected by an additional stripe of Wg expression along the DV compartment boundary (faint green).

View larger version (26K): [in a new window]   Fig. 2. Survival of ras– clones in the prospective wing blade, wing hinge, lateral notum and medial notum. (A) Subdomains of the wing imaginal disc: (I) presumptive wing blade, (II) presumptive wing hinge, (III) presumptive lateral notum and (IV) presumptive medial notum (see also Fig. 1B). (B) Histogram showing the survival rates of ras– clones in each of the four territories in the mature third instar wing disc, following clone induction during the first larval instar (see Materials and Methods). ras– clones generally survive less well than their ras+ twin clones. However, survival in the presumptive lateral notum was severely reduced relative to survival in other regions of the wing disc. Bars represent the percentage of ras+ clones associated with a ras– twin clone within a given disc area. n, total number of ras+ clones (with or without a paired ras– clone). Clones were induced at 24-48 hours after egg laying (AEL).

View larger version (61K): [in a new window]   Fig. 3. EGFR/Ras signaling is required for Iro-C expression. (A-E) ras– (A-B) and Egfrts (C-E) clones, marked by the absence of GFP (green), fail autonomously to express mirr-lacZ (A,C,D, red) and autonomously upregulate expression of tsh-lacZ (B, red) or endogenous Tsh protein (E, red). Wing blade specific vgQ-lacZ expression is also seen (C,D, red). Egfrts clones were generated using the Minute technique. Note the round shape of mutant clones (A-C,E) located within the prospective lateral notum when compared with the irregular shape of the clones located elsewhere in the disc. (A,B) ras– clones generated during first larval instar. (C) Egfrts clones generated during second larval instar and raised at the severely restrictive temperature of 31°C. (D-E) Egfrts clones generated during first larval instar and raised at intermediate restrictive temperature of 28°C.

View larger version (144K): [in a new window]   Fig. 4. EGFR/Ras signaling is sufficient to activate Iro-C expression. (A-D) Ectopic mirr-lacZ and Ara/Caup expression (red; arrows) associated with clones ectopically expressing either EGFR (A,B) or RasV12 (C,D). Clones are marked either by loss (A,B) or gain (C,D) of GFP expression. Cells co-expressing GFP and mirr-lacZ appear yellow. EGFR-expressing clones are associated with ectopic mirr-lacZ and Ara/Caup expression in some regions of the prospective wing hinge, but not others (A; see also Fig. 5); they can also induce mirr-lacZ expression in the prospective wing blade (B). RasV12-expressing clones activate mirr-lacZ (C) and Ara/Caup (D) expression in a more penetrant and strictly autonomous fashion within the wing hinge and blade (but not within the medial notum), as indicated by the exact correspondence between the clone marker (GFP, green) and mirr-lacZ or Ara/Caup (red) expression (overlapped expression appears in yellow). Note that Ara/Caup is normally expressed in localized patches of anterior compartment cells within the prospective wing blade; these appear red instead of yellow in D (left-hand panel).

View larger version (102K): [in a new window]   Fig. 5. EGFR signaling controls cell-affinity. (A-C) Wild-type wing imaginal discs containing clones of cells ectopically expressing either EGFR (A), Rho (B) or Spi* (C), monitored for mirr-lacZ expression (red). Clones were induced during the first larval instar and are marked either by loss (A,B) or gain (C) of GFP expression (green). (A) EGFR-expressing clones located within prospective notum (arrows) intermix freely with surrounding cells, as indicated by their ‘wiggly’ borders. Some clones within the hinge ectopically express mirr-lacZ and these tend to adopt a circular shape and sort out from surrounding wild-type cells (black arrowhead). Other clones do not express mirr-lacZ and these intermix with surrounding cells (asterisk). Finally, the sorting out of high mirr-lacZ-expressing from neighboring cells can occur within the same clone (white arrowhead). (B,C) Clones of Rho- or Spi*-expressing cells located within the prospective wing hinge form ‘wiggly’ borders and induce circular patches of mirr-lacZ-expressing cells that tend to sort out from surrounding cells that do not express mirr-lacZ. The inset in the left panel of B shows the overlay between GFP and mirr-lacZ expression in the vicinity of the clone.

View larger version (30K): [in a new window]   Fig. 6. Distribution, size and frequency of Egfr mutant clones in the D and V compartments. (A) Frequency of Egfrts clones that populate the D (black) or V (white) compartment, or both compartments (D/V, gray) as a function of temperature. Clones were induced during the first larval instar using the Minute technique, and larvae were kept at the indicated restrictive temperatures thereafter. n, total number of clones scored for each temperature condition. The percentage of Egfrts clones in the D compartment declines progressively as the temperature increases; conversely, the percentage of these clones rises in the V compartment. More clones are found within the D compartment than in the V compartment at the permissive temperature, reflecting the larger pool of cells from which the D compartment will arise. All clones within the wing disc were scored in this experiment. (B) Frequency of Egfr– clones that populate the D (black) or V (white) compartment, or both compartments (D/V, gray), as a function of the time of clone induction [indicated by hours (h) AEL]. The Minute technique was used; n, total number of clones scored for each time interval. Egfr– clones generated before the DV compartments are established during the second larval instar (approximately 48-72 hours AEL) preferentially populate the ventral compartment; clones induced thereafter frequent both compartments equally. Only clones populating the presumptive wing blade were scored in this experiment. (C) Relative sizes of Egfr– clones shown in B (see Materials and Methods for quantitation of size); for clones that populated both compartments, the sizes of the mutant territories in the D and V compartments were scored separately and are designated, respectively as D/v (dark gray bars) and d/V (light gray bars). Egfr– clones induced before the DV compartments contribute poorly to the D compartment, probably because most of the cells fail to activate ap and sort into the V compartment or out of the disc epithelium; clones induced thereafter contribute equally well to each compartment.

View larger version (125K): [in a new window]   Fig. 7. Early EGFR/Ras-mediated signaling is required for establishing the D compartment. (A,B) Examples of large Egfr– clones that preferentially populate the ventral compartment. The clones were generated during first larval instar using the Minute technique and are marked by the absence of GFP (green). Ap protein is shown in red (A), Wg protein in blue (B,C). Black arrows in A indicate small mutant clones located within the dorsal compartment. The white arrow in B indicates a small cluster of mutant cells that appear to have been left behind in the D compartment when the remainder of the clone sorted into the V compartment. Note that cells within this cluster do not express Wg, indicating that they are of D, rather than V type. (C,D) Example (C) and schematic representation (D) of a disc containing a dorsally situated clone of Egfr– cells that has developed as an ectopic V compartment. The wing blade primordium (all cells located within the outer ring of Wg expression) is shown in color: V compartment cells are represented in yellow, and D compartment cells in blue. Egfr+ cells are indicated by hatching. The dorsoventral compartment boundary, which correlates with the inner ring of Wg expression, is outlined in red.

View larger version (79K): [in a new window]   Fig. 8. Early activation of the EGFR/Ras transduction pathway is sufficient to generate an ectopic D compartment. (A-C) Wing imaginal discs containing clones of cells ectopically expressing either EGFR (A), RasV12 (B) or Rho (C), monitored for ap-lacZ (A, red), Ap (B,C, red), Wg (A, blue) or mirr-lacZ (C, blue) expression. Clones were induced during first (A,B) or late second/early third larval instar (C), and are marked either by absence (A,C) or presence (B) of GFP expression. Cells expressing both Ap (or ap-lacZ) and GFP appear yellow. (A) EGFR-expressing cells can form an ectopic D compartment within the V compartment. Note that only some cells within the clone express ap-lacZ and form the ectopic D compartment, and that the ectopic D compartment is encircled by a stripe of Wg-expressing cells that flank the ectopic DV boundary. (B) Early-induced RasV12-expressing clones autonomously express ap and form an ectopic D compartment. (C) Late-induced Rho-expressing clones fail to induce ectopic Ap expression, but still retain the ability of inducing ectopic mirr-lacZ expression within the presumptive wing hinge.