The temporal requirements for insulin signaling during development in Drosophila

Abstract

Recent studies have indicated that the insulin-signaling pathway controls body and organ size in Drosophila, and most metazoans, by signaling nutritional conditions to the growing organs. The temporal requirements for insulin signaling during development are, however, unknown. Using a temperature-sensitive insulin receptor (Inr) mutation in Drosophila, we show that the developmental requirements for Inr activity are organ specific and vary in time. Early in development, before larvae reach the "critical size" (the size at which they commit to metamorphosis and can complete development without further feeding), Inr activity influences total development time but not final body and organ size. After critical size, Inr activity no longer affects total development time but does influence final body and organ size. Final body size is affected by Inr activity from critical size until pupariation, whereas final organ size is sensitive to Inr activity from critical size until early pupal development. In addition, different organs show different sensitivities to changes in Inr activity for different periods of development, implicating the insulin pathway in the control of organ allometry. The reduction in Inr activity is accompanied by a two-fold increase in free-sugar levels, similar to the effect of reduced insulin signaling in mammals. Finally, we find that varying the magnitude of Inr activity has different effects on cell size and cell number in the fly wing, providing a potential linkage between the mode of action of insulin signaling and the distinct downstream controls of cell size and number. We present a model that incorporates the effects of the insulin-signaling pathway into the Drosophila life cycle. We hypothesize that the insulin-signaling pathway controls such diverse effects as total developmental time, total body size and organ size through its effects on the rate of cell growth, and proliferation in different organs.

Figure 1. Temperature Sensitivity in Inr^E19/Inr^GC25 Flies

Increasing the rearing temperature of Inr ^E19/Inr^GC25 females from 18 °C to 25 °C causes a reduction in wing area from approximately wild-type (Oregon-R [Ore-R]) to that of an insulin pathway mutant (chico). Wing size is expressed as percentage area of Oregon-R female wing at 25 °C. No Inr^E19/Inr^GC25 flies survived rearing at 29 °C. The standard errors are smaller than the markers.

Figure 2. Increasing the Temperature of Inr^E19/Inr^GC25 Flies Suppresses the Insulin-Signaling Pathway

The dFOXO panel shows localization of dFOXO protein in the fat body, the propidium iodide panel shows the position of the nuclei, and the merge panel clarifies the degree of dFOXO localization to the nuclei. (A) Endogenous dFOXO in the fat body of Inr^E19/Inrv^GC25 third instar larvae has weak nuclear localization at 17 °C. (B) Increase in rearing temperature causes a decrease in cytoplasmic distribution and an increase in nuclear localization of dFOXO, consistent with a decrease in the level of insulin signaling (C and D) Temperature has no detectable effect on dFOXO localization in Inr^E19/TM3 control flies. (E) GPH membrane localization reveals high levels of insulin signaling in the fat body of Inr^E19/Inr^GC25 second instar larvae reared at 15 °C. GPH is in green, DNA is stained blue. (F) This localization is lost when the larvae are moved to 25 °C for 12 h, consistent with a decrease in the level of insulin signaling. (G and H) Temperature has no detectable effect on GPH membrane localization in Inr^E19/TM3 control flies.

Figure 3. Suppression of Inr Expression in Inr^E19/Inr^GC25 Flies Affects Developmental Time and Adult Size

(A) Developmental time and adult wing size of Inr^E19/Inr^GC25 females switched from 17 °C to 24 °C increasingly late in development, expressed as percentage of developmental time and adult wing size of Inr^E19/TM3 females maintained under identical thermal conditions. Temperature-control flies were maintained at 17 °C throughout development. TSPs of female Inr^E19/Inr^GC25 for wing area and delayed eclosion can be seen as regions of the chart where switching from 17 °C to 24 °C increasingly early in development results in increasingly abnormal phenotypes (that is, where the gradient of the relationship between switch day and phenotype is non-zero). For delayed adult eclosion, the TSP of female Inr^E19/Inr^GC25 is before the ninth day of development at 17 °C. For reduced wing size, the TSP of female Inr^E19/Inr^GC25 is between the ninth and approximately the 20th day of development at 17 °C. (B) The stages of development of Inr^E19/Inr^GC25 flies at 17 °C (A, adult; E, embryo; L1, first instar; L2, second instar; L3, third instar; P, pupae). The point at which suppression of the insulin pathway changes from delaying adult development to reducing adult wing size occurs approximately 40% into the third instar (vertical gray bar) (C) Dry mass of Inr^E19/Inr^GC25 males switched from 17 °C to 24 °C at different points in development, expressed as percentage of dry mass of Inr^InrE19/TM3 males maintained under identical thermal conditions. The TSP of male Inr^E19/Inr^GC25 for reduced adult mass is after the ninth day of development but before pupariation. (D) Proportion of 17 °C Inr^E19/Inr^GC25 larvae pupariating when completely starved at different points in development. The point at which 50% of larvae pupariate in the absence of food marks the critical size. The critical size is reached approximately 40% through the third instar and coincides with the end of the TSP for delayed eclosion and the beginning of the TSP for reduced wing size and adult dry mass (vertical gray bar). All pupariating larvae successfully completed metamorphosis and eclosed as adults.

Figure 4. Developmental Delay in 24 °C Inr^E19/Inr^GC25 Flies Occurs primarily through Elongation of the Third Larval Instar

Area shows percentage of individuals (n = 20) in each developmental stage at different times in development. Time is shown in DDs to control for the effect of temperature on developmental rate.

Figure 5. Different Organs Respond Differently to Suppression of Inr Activity

Bars show organ area in Inr^E19/Inr^GC25 males as a percentage of area in Inr^E19/TM3 males, to control for temperature effects. Bars with different letters indicate organs that differ: A, B, and C are significantly different at α = 0.05 (Tukey-Kramer pairwise comparison). Mean areas of all organs given in Table S1. s.e., standard error.

Figure 6. A Reduction in Inr Activity Affects Cell Size and Cell Number Independently

(A) At 17 °C, the difference in wing area between Inr^E19/Inr^GC25 and Inr^E19/TM3 flies is due to a difference in cell size, whereas at 24 °C the difference is due to an additional difference in cell number. Bars show wing area, cell area, and cell number in Inr ^E19/Inr ^GC25 flies as a percentage of area or number in Inr^E19/TM3 flies. (B) At 17 °C the reduced Inr activity in Inr^E19/Inr^GC25 flies reduces cell area to approximately 85% the area in Inr^E19/TM3 flies, whereas at 24 °C there is no further reduction in cell area, but there is a reduction in cell number to approximately 75% of the number in Inr^E19/TM3 flies. Mean wing and cell area, and cell number are given in Table S2.

Figure 7. A Model of the Insulin-Signaling Regulation of Growth and Development

(A) Under normal conditions, imaginal discs grow to a critical size, which initiates an increase in the ecdysteroid titer. When ecdysteroid levels rise above a maximum threshold, the discs cease cell proliferation and undergo differentiation, fixing their final size. A, adult; E, embryo; L1–L3, first to third larval instar; P, pupa. (B) In Inr mutants, growth of imaginal discs to critical size is slowed, retarding development. When critical size is reached, the ecdysteroid titer again increases, rising above the maximum threshold for cell proliferation in the imaginal discs. Temporal changes in the ecdysteroid titer are unaffected by insulin signaling. Because the rate of cell proliferation is slowed, the imaginal discs are smaller when they begin to differentiate, reducing final organ size. Different discs have different thresholds of sensitivity to ecdysteroid and so cease cell proliferation at different times. Hormones other than ecdysteroids may also be involved.