Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis - PubMed (original) (raw)

Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis

Mirka Uhlirova et al. Proc Natl Acad Sci U S A. 2003.

Abstract

The transcription factor Broad-Complex (BR-C) is required for differentiation of adult structures as well as for the programmed death of obsolete larval organs during metamorphosis of the fruit fly Drosophila melanogaster. Whether BR-C has a similar role in other holometabolous insects could not be proven without a loss-of-function genetic test, performed in a non-drosophilid species. Here we use a recombinant Sindbis virus as a tool to silence BR-C expression in the silkmoth Bombyx mori. The virus expressing a BR-C antisense RNA fragment reduced endogenous BR-C mRNA levels in infected tissues (adult wing and leg primordia) via RNA interference (RNAi). The RNAi knock-down of BR-C resulted in the failure of animals to complete the larval-pupal transition or in later morphogenetic defects, including differentiation of adult compound eyes, legs, and wings from their larval progenitors. BR-C RNAi also perturbed the programmed cell death of larval silk glands. These developmental defects correspond to loss-of-function phenotypes of BR-C Drosophila mutants in both the morphogenetic and degenerative aspects, suggesting that the critical role of BR-C in metamorphosis is evolutionarily conserved. We also demonstrate that the Sindbis virus is a useful vehicle for silencing of developmental genes in new insect models.

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Figures

Fig. 1.

Fig. 1.

Amino acid identity between the BR-C Z4 isoform of M. sexta (top, AF0326761) and the protein sequence deduced from the B. mori BR-C cDNA fragment is 88%. The highly conserved C-terminal part of the BTB domain and the N-terminal portion of the Z4 zinc finger are indicated by arrows; between them is a variable part of the core, not conserved in Drosophila. The dashed line indicates which portion of the Bombyx cDNA was used for cloning into TE 3′2J-based SINV vectors (Fig. 2) and as a probe for hybridizations in Fig. 7.

Fig. 7.

Fig. 7.

SINV mediates specific BR-C silencing via RNAi. (A) Northern blot hybridization shows that infection with TE 3′2J-Bras reduced the levels of BR-C mRNAs. Total RNA was isolated from wing discs (Left) and legs (Right) of fifth-instar larvae, either uninfected or 6 days after virus injection. The _Bombyx BR-C_-specific probe detected three transcripts. To show equal RNA loading, the membranes were rehybridized with a probe for a constitutive ribosomal protein RpL3. Hybridization with a probe for the Sindbis E1 structural protein (for wing discs) showed the full-length viral genomic RNA (arrow) and the first subgenomic viral RNA before (black arrowhead) and after (open arrowhead) deletion, which probably removed RNA at the 3′ end. N, uninfected larvae; C, control TE 3′2J virus; Bras, TE3′2J-Bras virus. (B) Hybridization of small RNAs, isolated from mixed tissues of fifth-instar larvae 6 days after infection with TE 3′2J-Bras (right lane) but not the control TE 3′2J virus (left lane) shows _BR-C_-specific siRNAs of the indicated size range.

Fig. 2.

Fig. 2.

Design and replication of recombinant SINV. Recombinant TE 3′2J-based viruses contain a full Sindbis viral genome (13.8 kb in length), represented by single-stranded positive sense RNA with a second subgenomic promoter added at the 3′ end (17). Parts of the construct are not to scale in this scheme. (A) The 705-bp BR-C cDNA fragment was cloned in antisense orientation downstream of the second subgenomic promoter, thus generating TE 3′2J-Bras (top). On infection, a negative sense RNA copy of the genome (–RNA) is made by viral RNA-dependent RNA polymerase from a signaling sequence (*) in the 3′ noncoding region of the viral genome. The–RNA serves as a template for first and second subgenomic RNAs produced from two internal promoters and for new full-length positive sense RNA. (B) The chimeric TE 3′2J-EGFP-Bras virus contains a fusion of the EGFP coding region and the antisense Bombyx BR-C cDNA. The TE 3′2J-EGFP and TE 3′2J (bottom) viruses without the BR-C insert were used for controls.

Fig. 3.

Fig. 3.

Various tissues of the silkmoth are SINV targets. The TE 3′2J-EGFP virus was injected into fourth-instar larvae. Expression of EGFP serves as a marker of successful viral replication and shows the temporal spreading of the infection. (A) The middle part of the silk gland in a fifth-instar larva. (B) Strong EGFP signals in wing discs (arrows), imaginal leg primordia (arrowhead), and eye primordia in the head (arrow) are visible through the cuticle of a fifth-instar larva. Asterisk shows a leg apparently not infected with the virus. (C and D) Infected animals survive metamorphosis and show the viral EGFP expression in adult organs such as the compound eye (C) and forewing (D), both derived from infected imaginal progenitors.

Fig. 4.

Fig. 4.

Differentiation of compound eyes is disrupted by BR-C RNAi. Shown are scanning electron micrographs of eyes from adults infected as day 0 fifth-instar larvae with the control TE 3′2J virus (A and B) and with TE 3′2J-Bras (C and D). Compared to controls, animals carrying TE 3′2J-Bras show cone-shaped eyes with invaginations and folds. The bars correspond to 500 μm in A and C and to 200 μm in B and D.

Fig. 5.

Fig. 5.

BR-C function is required for pupation, elongation, and differentiation of adult legs and wings. Most larvae infected on day 0 of the fifth instar undergo the larval–pupal transition. Compared to a control pupa carrying the TE 3′2J virus (A), a pupa infected with TE 3′2J-Bras displays short forewings and short malformed legs (B). The arrowhead in B indicates where the wings should extend and meet normally. A mesothoracic leg of a control TE 3′2J infected adult (C) shows the normal number and size of segments, whereas infection with TE 3′2J-Bras leads to deletions of segments and overall leg malformation (D). Brackets show the normal (C) and shortened (D) tarsi; arrow in D points to an undeveloped mesothoracic leg. (E and F) Although a majority of animals injected as day 2 fourth-instar larvae with control viruses form normal pupae (E), most of those infected with TE 3′2J-Bras die when trying to ecdyse (F).

Fig. 6.

Fig. 6.

BR-C plays a role in the programmed cell death of larval silk glands. Animals infected as day 2 fourth-instar larvae were dissected 12 h after pupation. Silk glands found in control TE 3′2J-infected pupae displayed late phase of histolysis in the anterior and middle parts (A). In contrast, a middle gland dissected from a TE 3′2J-Bras-infected animal showed no signs of degeneration (B). In a day 1 pupa infected with TE 3′2J-EGFP-Bras, only one gland from the pair failed to degenerate (C); shown is the posterior part where the EGFP fluorescence indicates viral infection. The middle parts of silk glands were still visible in a day 3 pupa infected with TE 3′2J-Bras (D) but not in control pupae (not shown).

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