The EGF Receptor Defines Domains of Cell Cycle Progression and Survival to Regulate Cell Number in the Developing Drosophila Eye  Nicholas E. Baker, Sung-Yun Yu  Cell  Volume 104, Issue 5, Pages 699-708 (March 2001) DOI: 10.1016/S0092-8674(01)00266-5

Figure 7 Summary of EGFR Activity during the Second Mitotic Wave The cartoon illustrates how EGFR functions summarized in Figure 1G regulate the number of cells that will differentiate in the retina. (A)–(C) represent normal eye development. (D) and (E) illustrates the consequences of a mistake in ommatidim spacing. Shading reflects EGFR activity. (A) Cartoon of the eye disc epithelium around column 1. R8 cells (<) activate EGFR to arrest and recruit 4 other precluster cells (shaded). Other cells lack EGFR activity, reenter the cell cycle and perform S phase (unshaded). (B) Column 3. Cells of each precluster are tightly associated in a stereotypic morphology. All other cells are in the SMW. Precluster contact activates EGFR in most cells (shaded), allowing progression into mitosis. Some cells are not activated and remain in G2 (unshaded). On average 7.7 divided and 2.0 remained in G2 per ommatidium (Figure 1F). (C) Column 10. After the SMW there are an average of 17.4 unspecified cells per precluster in either G1 or G2 (2+ [7.7 × 2]), an excess of 3.4 over the number required to complete each ommatidium. EGFR activity (shaded) protects most G1 progenitor cells from cell death. EGFR activity also protects some remaining G2 cells without causing their mitosis (light shading). 0.3 cells died per ommatidium (star; Figure 5G and data not shown). More deaths occur ∼30 hr after puparium formation (2.5 cells per ommatidium; Wolff and Ready, 1991; Miller and Cagan, 1998; our unpublished data). These individual measurements tally well with the 19 progenitor cells that finally construct each ommatidium. (D) Cartoon of an eye disc around column 3 where a precluster is missing. Precluster contact activates EGFR in neighboring cells (shaded). Many cells lack precluster contacts and arrest in G2 (unshaded). (E) Cartoon of an eye disc around column 10 where a precluster is missing. Reduced mitosis and survival adjust the number of progenitor cells towards the number necessary to complete the extant ommatidia. Symbols as in (C) Cell 2001 104, 699-708DOI: (10.1016/S0092-8674(01)00266-5)

Figure 1 The Second Mitotic Wave (A–E) show eye discs with anterior to the left. In all figures a vertical arrowhead indicates column 0 in the morphogenetic furrow, a vertical arrow column 4, the center of SMW mitosis. Panels F,G summarize the data and ommatidial development. (A) Eye disc labelled for BrdU incorporation in red and the senseless protein (Nolo et al., 2000) in green. (B and D) Eye discs labelled for mitotic cells in red, and specified ommatidial cells in green (mAbBP104 and mAbsca1 combined). (C and E) Eye discs labelled for cyclin B expression (dark HRP reaction product). (F) The number of G2 cells per ommatidium plotted against position from column 3 to column 11. Cells were counted with nuclear cyclin B immediately after ectopic STG expression (see Figure 6B for an example). (G) Accurate summary of the SMW in relation to ommatidium differentiation (see Figure 7 also). A timeline of multiple EGFR functions is shown by the pink bars underneath. “MF” indicates extent of the morphogenetic furrow. In columns 0 and 1 the precluster cells R8, R2, R3, R4, and R5, are first visible as arcs of cells (outlined). Surrounding cells all incorporate BrdU during SMW S phase (shown by the gold bar) and accumulate cyclin B (green). Mitosis (red) is not completed before the first waves of cell fate specification and onset of survival signalling. Eleven of the nineteen precursor cells have been recruited by the end of the diagram at column 10 Cell 2001 104, 699-708DOI: (10.1016/S0092-8674(01)00266-5)

Figure 5 Ommatidia Provide a Survival Signal Independent of Mitosis (A) and (B) show segments of eye discs from Elp/Elp; GMRp21 (2X) individuals. (A) Cyclin B labelling reduced to background posterior to the the morphogenetic furrow verifying arrest prior to the SMW. (B) Basic fuchsin staining. Apoptosis still occurred (compare Figure 6H). (C–I) Green: cyclin B. Red: caspase activation (CM1 antibody). (C) Elp/Elp. (D) The labelled cells progressively adopt an apoptotic morphology. CM1 labelling disappears towards the posterior of the disc as cells are lost. (E and F) Elp/Elp; GMRp35. Morphology of the CM1 labelled cells is indistinguishable from normal interommatidial cells. CM1 labelling persists to the posterior of the eye disc. (F) shows examples of cells labeled by CM1 but not for cyclin B, and vice versa (arrows). (G) wild type. Some cells die between columns 7–15. (H and I) GMRp35. Protected cells can be in G2 (cyclin B: green) or postmitotic (no green) Cell 2001 104, 699-708DOI: (10.1016/S0092-8674(01)00266-5)

Figure 2 Role of SPI and EGFR in Cell Cycle Progression (A–C) spi clones. Mutant cells lack β-galactosidase (red). A. Green: mitotic figures. Posterior to the furrow most mitotic cells within spi clones were adjacent to wild type cells (blue arrows). B. Green: Cyclin B. C. Green: BrdU incorporation. (D and E) egfr mutant clones. Mutant cells lack β-galactosidase (red). (D) Green: cyclin B. In egfr mutant regions cyclin B was maintained cell autonomously up to the clone boundaries. (E) Green: BrdU incorporation Cell 2001 104, 699-708DOI: (10.1016/S0092-8674(01)00266-5)

Figure 3 Differentiating Cells Are the Source of Mitotic Signals (A and B) ato clones. Mutant cells lack β-galactosidase (red). (A) Green: cyclin B. Small ato clones arrest in the SMW (red arrow). Cells near the boundaries of large clones arrest in G2 but clone interiors do not enter the SMW (blue arrow). (B) Green: BrdU. ato clones affect S phase nonautonomously. (C and D) Cyclin B labelling of genotypes lacking ommatidia. (C) Cells accumulated in G2 in the region lacking preclusters (red bracket) when differentiation was transiently blocked by activation of Notch. The disc is shown 9 hr after 40 min heat shock induction of Notch intracellular domain. (D) In Elp/Elp most cells remain in G2; only the few ommatidia lack cyclin B. (E) Elp/Elp. Blue: mitotic figures. Red: Neuroglian, labelling differentiating ommatidial cells at the apical microvilli the rest of cell surface more faintly. (F and G) Elp/Elp disks. Red: differentiating ommatidia (neuroglian protein). Blue: mitosis. Green: cyclin B. (F) Elp/Elp. (G) Elevated EGFR expression by GMRGal4, UASG-EGFR (Type 1) restored the SMW in Elp/Elp. Almost all cells divide. Some extra cells begin differentiation more posteriorly Cell 2001 104, 699-708DOI: (10.1016/S0092-8674(01)00266-5)

Figure 4 Role of EGFR in Survival (A–D) egfr (A-C) or spi (D) mutant clones induced in the GMRp35 background. Mutant cells lack β-galactosidase (blue). (A) Green: differentiating neurons (ELAV). Red: BOSS (specific for R8 cells). At the nuclear plane, BOSS protein is detectable in the perinuclear ER, permitting precise correlation with the nuclear ELAV marker (Kramer et al., 1991). (B) Adult section, egfr mutant clones, GMRp35 background. Some cells differentiate small rhabdomeres (arrowheads). (C and D) Red: caspase activation (CM1 antibody). (C) egfr mutant cells autonomously activate caspases posterior to column 7. Note the morphologically distinct apoptotic figures ahead of the furrow which are not protected by p35. (D) In spi clones CM1 labelling does not correspond autonomously to the boundary of the clone. (E–H) egfrts eye discs. Green: differentiating neurons (ELAV). Red: caspase activation (CM1 antibody). (E) Control at the permissive temperature. (F) 2 hr at restrictive temperature. Interommatidial cells over a wide area label with CM1 and adopt typical apoptotic morphology. (G) 6 hr at restrictive temperature. (H) 8 hr at restrictive temperature. As the morphogenetic furrow progresses apoptotic cells disappear from posterior regions but new labelling arises between columns 6–10 Cell 2001 104, 699-708DOI: (10.1016/S0092-8674(01)00266-5)

Figure 6 Ectopic STG Expression (A) STG protein in wild type. Apical mitotic nuclei label for STG protein just before metaphase (horizontal arrow). (B–E) Effects of ectopic STG expression. Cyclin B labelling except for (D). (B) Cyclin B entered the nuclei of G2 cells immediately after a 30 min heat shock induction of the hs-stg transgene. These nuclei lie basally within the disc epithelium. (C) 60 min later the nuclei are large, apical and mitotic. (D) The mitotic marker phosphorylated histone H3 became detectable simultaneously. (E) 120 min after induction. Cyclin B has been degraded by all cells posterior to column 5, in contrast with the many such labelled cells at the start of the experiment (B). A few apical mitotic nuclei are still seen (horizontal arrows). (F) Ectopic STG expression in egfr mutant cells (lacking red marker). Green: phosphorylated histone H3. Many mutant cells were mitotic (blue arrows; 50 min after 30 min heat shock). (G–J) Basic fuchsin labelling. (G) Wild type, basic fuchsin stains mitotic figures posterior to the morphogenetic furrow (blue arrows). (H–J) Effects of ectopic STG on Elp mutants. (H) In Elp mitotic figures are absent. Red arrows exemplify apoptotic bodies. (I) 55 min after a 40 min heat shock, most cells posterior to the furrow are mitotic (blue arrows). These mitotic figures remain for 2–3 hr. (J) 120 min after heatshock, mitotic figures have been replaced by apoptotic bodies. Extensive cell loss often fissured the epithelium Cell 2001 104, 699-708DOI: (10.1016/S0092-8674(01)00266-5)