Insulin receptor-mediated signaling via phospholipase C-γ regulates growth and differentiation in Drosophila

Abstract

Coordination between growth and patterning/differentiation is critical if appropriate final organ structure and size is to be achieved. Understanding how these two processes are regulated is therefore a fundamental and as yet incompletely answered question. Here we show through genetic analysis that the phospholipase C-γ (PLC-γ) encoded by small wing (sl) acts as such a link between growth and patterning/differentiation by modulating some MAPK outputs once activated by the insulin pathway; particularly, sl promotes growth and suppresses ectopic differentiation in the developing eye and wing, allowing cells to attain a normal size and differentiate properly. sl mutants have previously been shown to have a combination of both growth and patterning/differentiation phenotypes: small wings, ectopic wing veins, and extra R7 photoreceptor cells. We show here that PLC-γ activated by the insulin pathway participates broadly and positively during cell growth modulating EGF pathway activity, whereas in cell differentiation PLC-γ activated by the insulin receptor negatively regulates the EGF pathway. These roles require different SH2 domains of PLC-γ, and act via classic PLC-γ signaling and EGF ligand processing. By means of PLC-γ, the insulin receptor therefore modulates differentiation as well as growth. Overall, our results provide evidence that PLC-γ acts during development at a time when growth ends and differentiation begins, and is important for proper coordination of these two processes.

Figure 1. Wing phenotype of sl ² 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) sl ² mutant wing. (B') shows an amplification of boxed area in (B), used to determine cell density. (C) Histogram showing the reduced mutant sl ² 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. sl ² 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 sl ² mutant wing size. Histogram in (D) shows the effects on sl ⁹ 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 sl ² or sl ⁹ 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 sl ⁹ 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 sl ² ectopic vein phenotype. (C, D) show wings of heteroallelic mutant InR ^E19/3T5 (C) and PKB ^1/3 (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, PKC53E ^EY14093, Rack1 ^EE, rho ^ve-1 and S ² 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 sl ² R7 phenotype.

Tangential sections of the distal part of eyes from sl ² (A) and sl ² heterozygous for Drk ^e0A (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 sl ² 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 sl ² (A) and sl ² heterozygous for InR ^E19 (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 sl ² 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 sl ² (D) and sl ² heterozygous for Ip₃R ^B4(E) flies, stained with toluidine blue. Arrow indicates ectopic R7 cells. (F) Histogram showing the effect of heterozygosity for Ip₃R ^B4, PKC53E^EY14093, Rack1^EE and rho ^ve-1 on the percentage of ommatidia with extra R7 cells in sl ² 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 sl ² background.

(A) Schematic representation of mutations in the SH2 domains of PLC-γ. (B) Histogram showing the effect of expression of sl constructs on sl ² 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 sl ² 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 sl ² 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.