NIP/DuoxA is essential for Drosophila embryonic development and regulates oxidative stress response

Abstract

NIP/DuoxA, originally cloned as a protein capable of binding to the cell fate determinant Numb in Drosophila, was recently identified as a modulator of reactive oxygen species (ROS) production in mammalian systems. Despite biochemical and cellular studies that link NIP/DuoxA to the generation of ROS through the dual oxidase (Duox) enzyme, the in vivo function of NIP/DuoxA has not been characterized to date. Here we report a genetic and functional characterization of nip in Drosophila melanogaster. We show that nip is essential for Drosophila development as nip null mutants die at the 1(st) larval instar. Expression of UAS-nip, but not UAS-Duox, rescued the lethality. To understand the function of nip beyond the early larval stage, we generated GAL4 inducible UAS-RNAi transgenes. da(G32)-GAL4 driven, ubiquitous RNAi-mediated silencing of nip led to profound abnormality in pre-adult development, crinkled wing and markedly reduced lifespan at 29 degrees C. Compared to wild type flies, da-GAL4 induced nip-RNAi transgenic flies exhibited significantly reduced ability to survive under oxidative stress and displayed impaired mitochondrial aconitase function. Our work provides in vivo evidence for a critical role for nip in the development and oxidative stress response in Drosophila.

Keywords: Numb Interacting protein; dual oxidase maturation factor; embryonic development; oxidative stress..

Figure 1

Expression of nip transcript and localization of NIP protein in Drosophila embryos. (A) Northern blot analysis of nip and numb transcripts in Drosoplila embryos and larvae. Embryos were collected at different time points after egg laying (AEL). Larvae at stages L1, L2 and L3 were collected. The blot was probed with P³²-labeled nip, numb and rp49 cDNA fragments, respectively. The position of 18S rRNA (1.98 kb) is indicated with an arrow. Rp49, ribosomal protein 49. (B) Maternal NIP expression and localization during oogenesis. N, nurse cell; O, oocyte. Arrows indicate where NIP immunostain was prominent. (C) localization of NIP protein in embyos of stages 2 to 4. NIP is in red; nuclei (stained with DAPI) in blue.

Figure 2

Maternal NIP is not essential for embryogenesis. (A-C) Maternal NIP expression is lost in the pBac germline clone flies. Total RNA was isolated from eggs laid by w¹¹¹⁸ (WT), pBac/CyO (pBac), pBac GLC (pBac-G) females at one hour AEL. The nip and numb cDNA probes used were: (A) nip-RA n176-472, which is upstream of the pBac insertion site; (B) nip-RA n602-1117, which is downstream of pBac insertion site; (C) numb n286-816. The full-length nip mRNA (arrow) is detected in wt and pBac eggs, while truncated nip mRNA (arrow head) is detected in pBac and pBac-GLC eggs. The asterisk (*) shows the position of Drosophila 18S rRNA (1.98 kb). (D&D') Staining of a pBac-GLC (D) and a wild type (D') egg chamber with an anti-NIP antibody. No NIP protein was detected in the oocyte (O) or nurse cells (N) in the pBac-GLC egg chamber. (E&E') Staining of eggs laid by pBac-GLC females (E) and wt females (E'). The developmental stages are distinguishable by the nucleus staining (blue): the right eggs are at post-cellularization stages and left eggs at the pre-blastoderm stage. No NIP protein is detected in the pBac-GLC embryo at the pre-blastoderm stage. (F) Hatch ratios of embryos from the nip mutant and the germline flies.

Figure 3

Lack of NIP expression leads to growth and developmental arrest. (A) Comparison in body size between wt (right) and nip^-/- pBac homozygous (left) larvae at different time points after larval hatching. (B) Graphical representation of body lengths of pBac homozygus and wt larvae at different time points after hatch. (C) Cuticles of wt and nip mutant larvae. Anterior is left and dorsal is down. Larvae are not actual size. (D) Mallory staining pattern of wt and mutant L1 larvae showing defects in inner organ development for the nip mutant. (E) G32-Gal4 induced GFP expression to visualize morphology of wt and nip mutant larvae. Note the gross developmental defects for the mutant fly, in particular in the digestive track.

Figure 4

Developmental defects of nip-RNAi flies. Compared to wt (A), nip-RNAi flies show vestigial wing (B) and eclosion defect (C) at 29^oC. Moreover, Nip-RNAi/G32 flies exhibited dramatic preadult lethality at 29^oC.

Figure 5

nip-RNAi/G32 flies have a temperature-dependent reduction in lifespan. (A) Drastically reduced lifespan observed for two lines of nip-RNAi/G32 flies compared to the controls. (B) Effect of temperature shift from 25°C to 29°C or from 29°C to 25°C on the lifespan of nip-RNAi/G32 and wt flies. The survival percentage was determined for 20 flies/vial x 5 vials on standard cornmeal food and transferred with a 2-3 day interval.

Figure 6

(A) nip-RNAi flies are hypersensitive to paraquat induced oxidative stress compared to the wild types. Female adults reared at 29°C were scored as 5 vial X 20 flies/vial after 5 hr eclosion. The flies were allowed to recover overnight from CO₂ anesthesia and then exposed to 10 mM paraqut in 1 % sucrose. The survival rate was determined at 24 hr after exposure. Data shown are representative of two independent sets of experiments. (B) nip-RNAi flies exhibit a reduced ratio of mitochondrial/cytosolic aconitase activity. Aconitase activity was measured for flies in the 3rd instar and compared to the wild type control. Cyto, cytosolic; mito, mitochondrial. (C) A graphical representation of the mitochondrial/cytosolic aconitase activity ratio. Data shown are averages from three independent experiments.

Figure S1

A DNA map of the nip mutant PBac{RB}mole02670 and rescue of pBac homozygous flie (A) A piggyBac{RB} element is inserted in an intron of the nip gene. The arrows show the location of primers used for PCR to identify pBac insertion in B&C. (B&C) PCR products obtained from genomic DNA of y w (lane 1), pBac/CyO (lane 2) and pBac/pBac;UAS-nip/TubP-Gal4 (lane 3 in B) or pBac/pBac;UAS-nip^NN/AA/TubP-Gal4 (lane 3 in C) flies. The short band in lane 2 of (B) was too weak to detect in the lane 2 of (C).

Figure S2

Expression of NIP under an HS-GAL4 driver completely rescued the pBac/pBac mutant phenotype.

Figure S3

Phenotypes of flies with ectopic expression of Numb or NIP in the wing disc. The ap-Gal4 was used to induce the ectopic expression of Numb (A) and NIP (B) in the wing disc. Ectopic expression of Numb resulted in a bald notum (A) whereas over-expression of NIP did not result in a significant defect. The absence of a few hairs in the ap-Gal4/UAS-nip flies (B) was most likely due to Gal4 instead of NIP because it was also observed when ap-Gal4 was crossed with wild type flies (data not shown).

Figure S4

Nip-RNAi flies display hatching and eclosion defects at 29°C compared to wt flies.