Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi)

Abstract

Background: RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.

Results: Beltless mRNA is expressed in all developmental stages except in 0-6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2-A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.

Conclusions: We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.

Figure 1

Developmental expression of blt mRNA. The RT-PCR assay was performed with the total RNA extracted at different developmental stages. Shown are results from a typical experiment. Lane 1: embryos, 0–6 h after egg laying (AEL); lane 2: late embryos (17–18 h AEL) and 1st instar larvae; lane 3: 2nd-3rd instar larvae; lane 4: pupae; lane 5: adult heads; lane 6: adult female body; lane 7: adult male body. Upper bands, blt mRNA; lower bands, ribosomal rp46 mRNA (control). Note the absence of blt mRNA in lane 1, the early embryos.

Figure 2

Localization of blt mRNA determined by in situ RT-PCR (A, B) and in situ hybridization with a digoxigenin-labeled blt riboprobe (C-H). In stage 8–11 embryos, specific staining was observed in the neuroectoderm (nec) and in the invaginating pole cells (pc) (A). In stage 15–16 embryos, specific staining appears in the developing central nervous system (CNS) (B). In the third instar larvae, blt mRNA is localized in neuromuscular junctions (C and D; arrows), and in the ring gland (RG) (E). (F-H) In adult flies, panel F shows blt mRNA localization in the interneurons of the optic lobe (arrowheads) and in the large cells (arrows), panel G shows specific staining in the corpus allatum (CA) and panel H, in the ovary.

Figure 3

Embryonic blt RNAi induces a prominent cuticular phenotype. (A) A control embryo, injected with a human 5-lipoxygenase 22 bp dsRNA; (B-E) embryos injected with blt dsRNA. RNAi induced phenotype: Panels B, D and E show missing or interrupted denticle belts from the abdominal segments A2, A3 and A4 (arrows). Panel C shows an example of a more severe cuticular phenotype.

Figure 4

Adult blt RNAi, triggered by injections of blt dsRNA into the abdomen of adult female flies. RT-PCR assay was performed 4.5 days after injections. Lane 1: control, lane 2: blt dsRNA (100 ng/μl), lane 3: blt dsRNA 10 ng/μl. Upper bands, blt mRNA; lower bands, ribosomal rp46 mRNA. Note the absence of blt mRNA in lanes 2 and 3.

Figure 5

Adult blt RNAi-induced alterations of ovarian morphology. (A) Control ovary isolated from flies injected with a human 5-lipoxygenase 22 bp dsRNA. (B) Confocal microscopy of a control developing egg. (C) Ovary isolated from flies injected with blt 22 bp dsRNA. (D) Confocal microscopy of a blt RNAi developing egg. Note the smaller size and presence of deformities in the ovaries and eggs in C and D, respectively.

Figure 6

Df(1)RK4 embryonic cuticular phenotype. In this mutation, the denticle belt is missing from the abdominal A4 segment (A), or is interrupted (B) (arrows). (C, D) In a large number of embryos, the phenotypical changes are more severe; for example, no denticle belts are formed; panel C: dorsal view. However, even in these severe cases, the mouth hook formation is not affected (panel D, ventral view).

Figure 7

The absence of blt mRNA in the Df(1)RK4 embryos with missing belts. Shown are the RT-PCR products from the total RNA extracted from embryos with normal denticle patterns (lane 1) and embryos with missing belts (lane 2). Upper panel, blt mRNA; lower panel, corresponding rp46 mRNA.

Figure 8

The exon/intron structure of the blt gene and Northern blot analysis. (A) Exons: filled squares, introns: lines. (B) Northern blotting revealed a single band (the arrow indicates the 2.37 kb marker size), visualized with digoxigenin-labeled probe, using the total RNA extracted from heads and bodies of male and female flies. Lane 1: female body, lane 2: male body, lanes 3 and 4: their respective heads. The lower panel shows the corresponding ribosomal rp46 mRNA.

Figure 9

The putative beltless protein; analysis and multiple alignment. (A) The putative beltless (Blt) protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, including the NRF domain and the acyltransferase domain (Acyl_transf_3). Using the PROSITE motif search engine, we found a high probability for multiple potential phosphorylation sites in the putative Blt (shaded rectangles). (B) The BLAST homology search and the CLASTLOW multiple alignment tools aligned the Blt protein (Q9VXX9) with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains.