Edited by Matthew P. Scott, Stanford University, Howard Hughes Medical Institute, Stanford, CA, and approved April 25, 2013 (received for review July 25, 2012)
Distinct neoplastic propensities of lgl− clones in proximal and distal wing imaginal disc epithelium. Cartoons of (A) an adult wing and (B) third-instar larval wing primordium. Notum and hinge (green) are parts of the proximal wing; wing pouch (gray) represents the distal wing blade. P→D designates proximal-distal axis. (C) Cartoon of lateral view of the wing imaginal disc epithelium, as seen in an optical section, displaying its proximal (green) and distal (gray) domains. (D–G) Images and data from mosaic discs where lgl− M+ clones (absence of β-gal) are surrounded by cell competition compromised M−/M+ heterozygous cells. (D) Note that lgl−M+ clones in the distal domain (blue stars) do not transform neoplastically as revealed by their intact cytoarchitecture (actin, gray). Proximally (yellow stars), however, their altered cytoarchitecture reveals their neoplastic transformation. (D′) An x–z optical section along the yellow dotted line in D to reveal intact and altered cytoarchitecture of distal (blue star) and proximally neoplastic clones (yellow star), respectively. (E) Proximal clones (yellow star) shows expression of matrix metalloproteinase-1 (MMP1, red) and breakdown of the basement membrane, marked by collagen-IV-GFP (Coll-IV, green) while the distal clone (blue star) does not; absence of β-gal (gray) marks clone boundary. Higher magnifications of boxed area of E is shown at the far right column to reveal correspondence between disrupted basement membrane (Coll-IV, arrowhead) with MMP1 expression (red) within the clonal area (absence of β-gal, gray). (F–F′′) In older (day 6) mosaic discs, distal (blue star) lgl−M+ clones (absence of β-gal, green) were not seen in the apical plane (F), while in the basal plane (F′) these displayed extensive cell death (caspase, red). Optical section along the x–z plane (F′′) through the yellow dotted line shown in F and F′ reveals basal extrusion of these distal (blue star), caspase-expressing (red) clones. Note the abundant neoplastic transformation of proximal clones (yellow stars) in all of the three optical planes. A←B indicates the direction of of apical-basal polarity. (G) Plot of total and transformed clonal area of proximal and distal lgl−M+ clones in mosaic wing imaginal discs through successive days of clonal growth. (H) lgl− clones marked by GFP (green) and generated in a cell death compromised H99/+ genetic background. Distal clones (blue star) display intact cytoarchitecture (actin, gray) but proximally (yellow stars) these are neoplastically transformed. (I) Cartoon representation of lgl− clones in mosaic wing disc epithelium in different genetic contexts. Clonal age is shown as days after clone induction. (Scale bars, 100 µm.)
Recruitment of multiple signaling pathways during proximal wing neoplasia. (A–D) Transcriptional status of Hippo (A), Wg (B), TGF-β (C), and EGFR (D) signaling pathways and their functional cooperation for lgl− neoplasia. (A, i–D, i) GSEA plots display enrichment of these pathways at P < 0.05; y axis displays enrichment score (ES) and x axis displays ranked order of genes in descending order of their fold-changes in lgl− vs. Wild-type; (A, ii–D, ii) Quantitative real-time PCR of representative members of the pathways show their up-regulation in lgl− mosaics. Error bars represent SEM. (A, iii–D, iii) Functional cooperation of individual signaling pathways for lgl− neoplasia; n represents the number of mosaic discs examined in each case. (A, iii) Functional cooperation of Hippo signaling: mosaic wing discs bearing ft− or lgl− ft− clones (loss of GFP). Distal (blue star) and proximal (yellow star) twin spots in boxed areas are shown at higher magnification in the Center; the far right panel shows their cartoon representation. Note that proximal and distal ft− clones outgrow their wild-type twins (dark green, red line). In contrast, distal lgl− ft− clones are conspicuous by their absence (question mark, blue star), but their wild-type twin-spot grew as expected. Proximal lgl− ft− clones display neoplastic transformation (actin, gray) and outgrow their wild-type twins (dark green). lgl− clones (GFP, green) overexpressing UAS-dsh (B, iii), UAS-tkvQD (C, iii), and UAS-EGFRλtop (D, iii). Note that proximally all these clones (yellow stars) are transformed (actin) but distally (blue stars) these are rarely seen in the apical optical plane; however, basally these were invariably seen to be extruded. Mosaic disc in B, iii is marked for cell death (arrows, caspase, red), which are seen in the basal plane. (E) Selective up-regulation of Expanded, Ex (red) and (F) Wg (red) in proximally transformed lgl− clones (absence of green, yellow stars). Note that distally, where lgl− clones are not neoplastically transformed, Wg expression too was not altered in both 4- and 6-d-old clones (blue arrowheads). [Scale bar (applies to all panels), 100 µm.]
Distal neoplasia of lgl− clones under conditions that override their tissue microenvironmental surveillance and cell death. (A) lgl− clones (absence of β-gal, green) in a genetic context compromising both cell competition (M−/M+) and cell death (H99/+) display neoplastic transformation (actin, red) in both proximal (yellow star) and distal (blue star) domains. (B and C) lgl− UAS-yki clones (GFP, green) induced by a short pulse of heat shock (4 min). On day 3, only proximal clones are transformed (yellow star #1), but distal clone (blue star #2) display only early signs of actin reorganization (actin, red) (B). On day 4, proximal clones displayed neoplasia (yellows star) but distally one of the two large clones is neoplastically transformed (blue star), while other is not (arrow head) (C); optical sections along the x–z plane through the dotted line across transformed (blue) and nontransformed (pink) clones are shown at the bottom right panels to reveal altered F-Actin cytoarchitecture in only the former. (D) Cartoon representation of transformed lgl− clones in both distal and proximal territories under conditions of reduced tissue surveillance and cell death. n = number of discs examined. Scores for distal (d) or proximal (p) clones showing neoplasia are displayed in the panel. A←B indicates apical-basal polarity. [Scale bar (applies to all panels), 100 µm.]
Loss of cell fate during distal neoplasia of lgl− clones. (A–C) Yki-expressing (UAS-yki, green) clones do not alter distal expression of Vg (A), Dll (B), or Nub (C) but (D–F) their lgl− UAS-yki counterparts (GFP, green) do (blue stars). Distal lgl− UAS-yki clones shown in D display intact cytoarchitecture (actin), whereas those shown in E and F are neoplastically transformed (actin). (G and H) Expression of Vg in lgl−M+ clones (loss of GFP, green) induced in a cell competition (M−/M+) and cell death (H99/+) compromised context. Boxed area in a mosaic distal wing imaginal disc, dissected day 5 after clone induction highlights wiggly clonal borders (G); Vg (red) expression in these clones, however, is not affected (shown at higher magnification). At a later stage of their growth, such clones display both neoplastically transformed (blue star) and nontransformed (yellow arrows) territories (H). Yellow boxed area is shown at a higher magnification at far right column to reveal clonal territory (yellow dotted lines), which are not transformed yet (actin, gray) but show down-regulation of Vg (arrowhead) and smooth clone border. Blue boxed area shown at a higher magnification (shown at the far right panel) display neoplasia (Actin, gray), loss of Vg (blue star), and smooth clone border (dotted blue line). Scores of distal (d) clones are also shown. (Scale bars, 100 µm.)
Distal cell fate is incompatible with neoplastic cell state. (A) Wild-type third-instar wing imaginal disc displaying expressions of a N target, Ct (red) along the dorsal-ventral wing margin, and Vg (green) in distal wing (pouch domain); expression of Hth (purple) marks the proximal wing. (B) In a neoplastically transformed (actin, gray, blue star) distal lgl− UAS-yki clones (green) expression of Ct (red) is extinguished. Boxed area marks a nontransformed distal clone (actin, gray), which is shown at a higher magnification on the right to reveal early signs of loss of Ct (between the arrowheads). (C and D) Clones displaying gain of Wg signaling (UAS-dsh, green) activate Vg in distal (blue arrowheads) and in select proximal (yellow arrowhead) clones (C). In contrast, lgl− UAS-dsh (green) clones are not seen in the distal wing (marked by Vg, red, D); proximally (outside the domain of Vg, red, D) these are neoplastically transformed, but fail to display gain of Vg, unlike their UAS-dsh counterparts (C). (E–H) Gain of Vg (UAS-vg, green) does not affect growth or survival in distal (blue star) or proximal (yellow star) domains of the wing primordium (E). However, Vg-expressing lgl− clones (lgl− UAS-vg, green), are distally eliminated because of cell death (caspase, red, F), but those that displayed proximal survival were not transformed (G). Higher magnification of boxed area in F and G is shown at the right and x–z sections over the dotted line are shown at the far right. Apical-Basal (A←B) orientation in the x–z sections is shown. Note the dispersed distribution of the lgl− UAS-vg clones (GFP, green) in the periphery of this proximal clone (marked by yellow arrowhead, G) and their basal extrusion as revealed in the x–z section. Nonextruded lgl− UAS-vg cells at the center of the clone are not transformed, as revealed by their intact cytoarchitecture (actin). (H) lgl UAS-vg; UAS-yki (green) clones display cell death (caspase, red) both distally (blue star) and proximally (yellow star). Higher magnifications of proximal clones (marked by blue box) are shown at the bottom panel to reveal densely packed cells and their intact cyto-architecture (actin). (I and J) Schematic presentation of the outcome of gain of Wg signaling (I) or the Vg transcription factor (J) in lgl− clones. Note that distally, where Wg signaling induces Vg expression (see C, above), lgl− UAS-dsh clones are eliminated while those that are proximally transformed do not activate Vg (see D, above). Gain of Vg in lgl− clones, however, inevitably induces distal elimination while proximally these remain growth-compromised and die extensively (see F–H). Scores for distal (d) or proximal (p) clones showing a given phenotype (loss or gain of a marker or neoplasia) are displayed in respective panels. [Scale bar (A, B, and E–J), 100 μm, (C and D), 200 μm.]
Hth-independent neoplasia in lgl− clones in wing primordium. (A–C) lgl− UAS-yki clones (GFP, green) at different clonal age; Dll (A and C) and Vg (B) mark the distal domain. Note the progressive loss of these markers in distal lgl− UAS-yki clones (blue star) and gain of Hth (gray) on day 4 (B) and day 5 (C), but not on day 3 (A) of clone induction. Yellow star in C marks a proximal clone. (D) A proximal lgl− UAS-yki; UAS-hth-RNAi (GFP, green) clone (yellow star) displaying loss of Hth (gray); it is, however, neoplastically transformed such as its distal (blue star) counterpart (actin, purple). (E–G) Gain of Ras/EGFR signaling in lgl− clones represses distal cell fate and induces lgl neoplasia. lgl− UAS-rasV12 clones (GFP, green, E) on day 3 of clone induction repress Vg (red) and display neoplasia (actin purple, F); boxed areas in these images are shown at a higher magnification on their respective right panels. (G) Schematic representation of proximal and distal neoplasia in lgl clone upon gain of Ras/EGFR signaling. Scores displayed in A–C, E, and F are for distal clones while scores in D are for for distal (d) or proximal (p) clones. (Scale bars, 100 µm.)
Neoplastic cells of the eye primordium switch to an Hth-dependent progenitor-like cell state. (A) Cartoon of third-instar larval eye imaginal disc displaying three distinct stages of retinal development. The anterior-most progenitor cells express Hth (green) followed by a zone of Dac-expressing committed cells (peach) preceding the morphogenetic furrow (MF, arrowhead), which is succeeded by differentiated retinal cells (Elav, red). (B) An eye imaginal disc displaying domains of Elav and Hth expression. (C) scrib− retinal clone (loss of GFP, blue star). Boxed area in C is shown at a higher magnification on the right to reveal loss of Elav (red) in the clone and its neoplastic transformation (actin). (D and E) scrib−M+ clones (absence of GFP, blue star) generated in a M−/M+ surrounding. Boxed area in D is magnified on its right panels to display gain of Hth (red) on day 3 of clone induction (D). Expansion of clonal area on day 5 of clone induction results in near ubiquitous expression of Hth in the mosaic eye primordium. (F–J) Switch to a progenitor-like cell state drives neoplasia in the eye primordium. An UAS-hth clone (F, green) in the eye primordium; boxed area is shown on the right panels to reveal loss of Elav (red, arrow) corresponding to the domain of gain of Hth (GFP, green, arrow). scrib− UAS-hth RNAi clones (G, green, yellow arrows) do not lose Elav (red) and display characteristic ommatidial cytoarchitecture (actin, gray) and, thus, are no longer neoplastically transformed. In lgl− M+ clones (loss of GFP, green), generated in M−/M+ surrounding, endogenous domains of Elav (red, H) or Hth (red, I) are not altered. In contrast, lgl− UAS-hth clones (green, blue star, J) display loss of Elav (red) and neoplastic transformation (actin, blue). The central large and neoplastically transformed clone (blue star) has popped out of the plane of the rest of the eye disc. The clone on the margin of the eye disc (yellow star) represents neoplastic transformation of margin cells. [Scale bar (for all panels), 100 µm.]
Both lineage-committed and differentiated cells switch to primitive cell states during carcinogenesis. Cartoons of (A) wing and (B) eye discs displaying the cells which are committed to proximal (Hth), distal (Vg) wing, or retinal (Dac) cell fates or differentiated into retinal cells proper (red, Elav). Neoplastic transformations (irregular mass of cells) of these cells entail the following: first, loss of transcription factors that display their cell commitment (Vg) or differentiation (Elav) and, second, switch to a primitive/progenitor-like cell state (light green). Primitive cell state assumed by transformed lgl− clones in the wing is Hth-independent (A) while in the eye discs it is Hth-dependent (B).
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