Nitric Oxide Synthase Regulates Growth Coordination During Drosophila melanogaster Imaginal Disc Regeneration

Abstract

Mechanisms that coordinate growth during development are essential for producing animals with proper organ proportion. Here we describe a pathway through which tissues communicate to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint through expression of Dilp8. This both produces a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. Here we demonstrate that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland coordinates tissue growth through regulation of endocrine signals.

Keywords: allometry; ecdysone; growth control; nitric oxide; regeneration.

Figure 1

NOS is required for imaginal disc growth coordination during the regeneration checkpoint. (A) Growth reduction of undamaged eye imaginal discs in larvae with targeted tissue damage in the wings (Bx > eiger) and control larvae (Bx > GFP). Eyes were isolated at 104 hr AED and stained with rhodamine-labeled phalloidin. Bar, 100 μm. (B) Dilp8 is required for coordinating imaginal tissue growth during targeted wing damage. Eye imaginal disc size measured at 104 hr AED following targeted wing expression of eiger (Bx > eiger) or control (Bx > LacZ) in larvae wild type for dilp8 or homozygous dilp8^−/−. (C) Dilp8 is required for coordinating imaginal tissue growth during irradiation damage. Measurement of undamaged eye imaginal disc size following targeted irradiation (shielded, 25 Gy) compared to unirradiated control (0 Gy) in wild type (w¹¹¹⁸) and homozygous dilp8^−/− larvae. Posterior tissues were exposed to 25 Gy ionizing irradiation at 80 hr AED while anterior tissues were shielded using lead tape (see Materials and Methods and Figure S1B for more detail). Eye imaginal disc size measured at 104 hr AED. (D) NOS is required for coordinating imaginal tissue growth during the regeneration checkpoint. Coordination of growth during targeted irradiation is lost in larvae mutant for NOS. Measurement of undamaged eye imaginal disc size following targeted irradiation compared to unirradiated control in wild type (w¹¹¹⁸) and larva heterozygous or homozygous for NOS mutant (NOS¹). Posterior tissues were exposed to irradiation at 80 hr AED, and eye imaginal disc size was measured at 104 hr AED. (E) Dilp8 growth restriction requires NOS. Eye imaginal disc growth restriction during dilp8 overexpression in the wing (Bx > dilp8) is lost in larvae mutant for NOS (NOS^{1 −/−}). Larvae were raised at 29°, and eye imaginal disc size was measured at 100 hr AED. Statistical analysis: mean ± SD. *P < 0.05, ****P < 0.001 calculated by two-tailed Student’s t-test.

Figure 2

NOS is required in the PG to coordinate imaginal tissue growth during the regeneration checkpoint. (A) A systemic pulse of NOS expression early during the larval feeding period restricts imaginal disc growth throughout the rest of larval development. NOS was systemically expressed by heat shock (∆) at 76 hr AED, and eye imaginal disc size was measured in populations of larva at subsequent time points. NOS overexpression at 76 hr AED extends larval development. Measurement of pupariation timing (marked by eversion of anterior spiracles) following systemic expression of NOS (hs > NOS) is depicted on the right. (B) NOS overexpression in the PG (phm > NOS) restricts imaginal disc growth and extends larval development. phm > GFP- and phm > NOS-expressing larvae were raised at 21° (see Figure S2D). (C) Both targeted tissue damage (Bx > eiger) and systemic dilp8 expression (Tub > dilp8) increase NO production in the PG. Measurement of NO production by the fluorescent reporter DAF2-DA. Brain complexes with the PG attached were isolated and stained with DAPI and DAF2-DA at 93 hr AED. n for Bx > LacZ = 36, Bx > eiger = 23, Tub > LacZ = 20, and Tub > dilp8 = 23. (D) NOS is required in the PG for regeneration checkpoint growth coordination. Measurement of undamaged eye imaginal disc size following shielded irradiation (25 Gy) compared to unirradiated control (0 Gy) in control (phm > LacZ) or NOS-targeted RNAi expressed in the PG (phm > NOS^IR-X or phm > NOS^{Ri BL28792}). Posterior tissues were exposed to 25 Gy ionizing irradiation at 80 hr AED and anterior tissues, including the eye discs, were shielded using lead tape. Eye imaginal disc size was measured at 104 hr AED. Statistical analysis: (A) Differing letters denote statistical significance calculated by one-way ANOVA with Tukey’s post-test. (B) Mean ± SD. Time in A and B is the mean of triplicate experiments ± SEM. (C) Mean of triplicate experiments. (D) Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001 calculated by two-tailed Student’s t-test.

Figure 3

NOS is not required for regulation of developmental time during the regeneration checkpoint. Loss of NOS function either by (A) knockdown of NOS in the PG (phm > NOS^Ri) or (B) NOS mutant (NOS¹) does not alter developmental delay. Measurement of pupariation timing for larvae with irradiation damage (25 Gy) and control larvae (0 Gy). Mean of triplicates ± SEM. *P < 0.05 calculated by one-way ANOVA with Tukey’s post-test.

Figure 4

NOS overexpression during larval feeding inhibits ecdysone biosynthesis. (A) NOS activity in the PG reduces ecdysteroid production. The presence of ecdysteroids is reduced in larvae with NOS overexpression in the PG (phm > NOS) compared to control (phm > LacZ) larvae. Ecdysone levels were measured by ELISA assay for independent isolation triplicates. (B) NOS expression in the PG reduces ecdysone signaling. Transcription of E74B is reduced in larvae with NOS overexpression in the PG (phm > NOS) compared to control (phm > LacZ) larvae. Transcription levels measured by qRT-PCR in triplicate, normalized to control expression levels at 116 hr AED. (C) NOS activity in the PG reduces Halloween gene transcription. Relative expression of spok and dib in control (phm > LacZ) larvae and larvae with NOS overexpression in the PG (phm > NOS) are depicted. Transcription levels were measured by qRT-PCR in triplicate, normalized to control transcription levels. (D) Expression of NOS early during the larval feeding period (76 and 80 hr AED) substantially delays larval development, while NOS expression late during the wandering period (96 and 104 hr AED) does not delay development. NOS was systemically expressed by heat shock (hs > NOS) once at 76, 80, 96, or 104 hr AED, and time to pupariation was measured. (E) Expression of NOS early during the larval feeding period restricts imaginal disc growth, while NOS expression late during the wandering period does not inhibit growth. NOS was systemically expressed by heat shock (hs > NOS) at either 76 or 104 hr AED, and eye imaginal disc size was measured at 116 hr AED. All phm > LacZ- and phm > NOS-expressing larvae were raised at 21°. Statistical analysis: A and E, mean of triplicates ± SD calculated by two-tailed Student’s t-test. (B and C) Mean of triplicates ± SEM, calculated by paired one-tailed t-test. (D) Mean of triplicates ± SEM. *P < 0.05, **P < 0.01, ****P < 0.001.

Figure 5

Imaginal disc growth restriction during the regeneration checkpoint is the result of reduced ecdysone signaling. Ecdysone levels are rate-limiting for imaginal disc growth during the regeneration checkpoint. 20-Hydroxyecdysone (20E) rescues growth restriction induced by (A) targeted wing damage (Bx > eiger), (B) systemic dilp8 misexpression (Tub > dilp8), (C) systemic NOS misexpression (hs > NOS), and (D) PG NOS overexpression (phm > NOS) compared to control ethanol fed only larvae (EtOH). Statistical analysis: mean ± SD. *P < 0.05 and ****P < 0.001 calculated by two-tailed Student’s t-test.

Figure 6

Model for allometric growth regulation during the regeneration checkpoint. Growth is coordinated between regenerating and undamaged imaginal discs through the PG. During the larval growth period, Dilp8 secreted from regenerating imaginal discs activates nitric oxide synthase in the prothoracic gland, inhibiting ecdysone biosynthesis and reducing undamaged imaginal disc growth. Dilp8-dependent developmental delay is produced through a NOS-independent mechanism.