Figure 1.
Wing phenotype of sl2 mutants compared to ORR (Oregon R) controls.
Only male flies were used in this and subsequent figures unless otherwise noted. (A) Wild type wing. (A') shows a higher magnification of boxed area in (A), illustrating the wing region used to determine cell density. (B) sl2 mutant wing. (B') shows an amplification of boxed area in (B), used to determine cell density. (C) Histogram showing the reduced mutant sl2 wings area. n = 100. (D) Histogram showing an increase in cell density in sl mutant wings. n = 100. (E) Histogram showing the averages of total number of cells of complete wing surfaces. n = 5. sl2 and ORR do not differ significantly. *p<0.001; error bars represent SEM.
Figure 2.
Reduced gene dosage of MAPK and insulin pathway genes on sl mutant wings.
Heterozygous mutant conditions for EGF/MAPK (A) and insulin (B) signaling genes, and genes downstream of Sl (C) change the sl2 mutant wing size. Histogram in (D) shows the effects on sl9 mutant wing size of heterozygosity for genes of the insulin and MAPK pathways and of the IP3R. n = 100 in all cases, * p<0.001; error bars represent SEM. In this and subsequent figures showing genetic interactions, tests were done with hemizygous sl2 or sl9 males and heterozygous or wild type for the genes tested unless otherwise noted.
Figure 3.
Sl affects eye size. Images in (A, B) are SEMs of control (A; Canton S) and (B) sl9 homozygote female flies.
Notice mild roughness of sl9 mutant eye; red bar in A is 100 µm. (C) Average areas of whole eyes of control (Canton S) and sl9 homozygotes. Areas are expressed in µm2; n = 18 for Canton S and 16 for sl9. (D) Average number of ommatidia in eyes of Canton S and sl9 homozygotes (n = 13 for both Canton S and sl9). Digitized images of eyes were used for measurements in (C) and (D), and in both cases differences are significant at p<0.002 (C) and p<0.0001 (D). (E) Plastic section through an eye containing a y w sl9 homozygous clone, marked by the absence of red pigment surrounding the ommatidia; a black line indicates the approximate edge of the mutant clone. Three ommatidia showing the extra R7 photoreceptors characteristic of sl mutants are indicated by arrows. (F) A comparison of sl+ and sl mutant tissue in nine heads (each pair of bars represents data from an individual head; dark grey bars represent wild-type cells, light grey bars represent cells in mutant patches). The area of R1–R6 rhabdomeres was determined in three to fifteen pairs of nearby ommatidia in each head, each pair consisting of one sl+ (w+) ommatidium and one sl9 homozygous (w-) ommatidium.
Figure 4.
Reduced gene dosage of MAPK and insulin pathway genes on sl2ectopic wing veins.
Shown are effects of heterozygous mutant conditions for EGF/MAPK (A) and insulin (B) pathway genes on the sl2 ectopic vein phenotype. (C, D) show wings of heteroallelic mutant InRE19/3T5 (C) and PKB1/3 (D) flies with small ectopic vein-like patches. (C') and (D') show close-ups of boxed areas in (C) and (D), respectively. Arrows point to vein-like material present. As heterozygotes, neither InR nor PKB show ectopic wing vein-like material. (E) Effects of heterozygosity for IP3R B4, PKC53EEY14093, Rack1EE, rhove-1 and S2 on the extent of sl2 ectopic wing veins. n = 100. *p<0.001; error bars represent SEM.
Figure 5.
Reduced gene dosage of MAPK pathway genes on sl2 R7 phenotype.
Tangential sections of the distal part of eyes from sl2 (A) and sl2 heterozygous for Drke0A (B) flies, stained with toluidine blue. The arrows indicate extra R7 cells. (C) Histogram showing the effect of heterozygosity for mutations in genes of the MAPK pathway on extra R7 cells in sl2 mutants. n = 5 eyes each with ≤150 ommatidia per eye. *p<0.001; error bars represent SEM.
Figure 6.
Reduced gene dosage of insulin pathway and downstream components on the sl2 extra R7 phenotype.
Tangential sections of the distal part of eyes from sl2 (A) and sl2 heterozygous for InRE19 (B) flies, stained with toluidine blue. Arrows indicate extra R7 cells. (C) Histogram showing the effect of heterozygosity for mutations in genes of the insulin pathway on the percentage of ommatidia with extra R7 cells in sl2 mutants. n = 5 eyes each with ≤150 ommatidia per eye. *p<0.001; error bars represent SEM. Tangential sections of the distal part of eyes from sl2 (D) and sl2 heterozygous for Ip3RB4(E) flies, stained with toluidine blue. Arrow indicates ectopic R7 cells. (F) Histogram showing the effect of heterozygosity for Ip3R B4, PKC53EEY14093, Rack1EE and rhove-1 on the percentage of ommatidia with extra R7 cells in sl2 mutants. n = 5 eyes each with ≤150 ommatidia per eye. *p<0.001; error bars represent SEM.
Figure 7.
Expression of wild type (X10) and mutant sl constructs in a mutant sl2 background.
(A) Schematic representation of mutations in the SH2 domains of PLC-γ. (B) Histogram showing the effect of expression of sl constructs on sl2 wing size. n = 100. *p<0.001; error bars represent SEM. (C) Histogram showing the effect of expression of sl constructs on ectopic wing veins of sl2 mutant flies. n = 100. *p<0.001; error bars represent SEM. (D) Histogram showing the effect of expression of sl constructs on extra R7 cells in sl2 mutant eyes. n = 5 eyes each with ≤150 ommatidia per eye. *p<0.001; error bars represent SEM.
Figure 8.
Sl modes of action in growth and differentiation.
Panel (A) shows Sl, activated by the insulin pathway, acting as a liaison regulating MAPK pathway ligand processing, to foster MAPK activation to a level promoting growth (red inhibitory interaction). (B) Conversely, for differentiation, reduced insulin receptor signaling leads to lower levels of Sl activation and augmented Spi processing (different from A, gray inhibitory interaction; possibly other targets from those in A), and this, in turn, allows MAPK activation in a manner consistent with promotion of differentiation.