Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation - PubMed (original) (raw)

Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation

L Misquitta et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of mesodermal cells known as the muscle "pioneer" or "founder" cells. These cells are thought to establish the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the embryo is associated with one or more nautilus-positive cells. However, because of the lack of known (isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. We now have utilized toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) to determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic interference upon injection of nautilus double-stranded RNA. These results define a crucial role for nautilus in embryonic muscle formation. The application of RNA interference to a variety of known Drosophila mutations as controls gave phenotypes essentially indistinguishable from the original mutation. RNA-i provides a powerful approach for the targeted disruption of a given genetic function in Drosophila.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Muscle formation and gene expression patterns in the Drosophila embryo as modulated by specific cell ablation, antisense expression, and RNA interference by the injection of dsRNA. Ablation of the nautilus_-positive muscle founder cells by ricin toxin disrupts muscle formation. (A) Ricin not induced. (B and C) Ricin induced in nautilus_-positive cells. Antisense expression of nautilus RNA disrupts muscle formation in three different UAS antisense (AS) nautilus lines (D_– F). Injection of_nautilus dsRNA blocks muscle formation (G and H) and does not depend on the bHLH domain for the disruption [dsRNA for the C terminus (I), dsRNA for the bHLH domain (J), and dsRNA for the amino terminus of_nautilus (K)]. Injection of β-galactosidase dsRNA does not disrupt the muscle pattern but eliminates normal lacZ expression (shown in M) without affecting muscle pattern (similar to A), whereas injection of nautilus dsRNA into a_nautilus lacZ line 14.1 disrupts the lacZ muscle pattern (compare M and N) and reduces lacZ expression. Injection of dsRNA for DMEF2 [uninjected (L) and injected (O)], S59 [uninjected (P) and injected (Q)],daughterless [uninjected (R) and injected (S); C, CNS; P, PNS], and white [uninjected (T) (w+) and injected (U)] results in the disruption of gene function for these genes.A_–_L and_O_–Q were stained with antimyosin;M and N were stained with 5-bromo-4-chloro-3-indolyl β-

d

-galactoside;R and S were stained with monoclonal 22C10 and horseradish peroxide for CNS and PNS. AS in the upper left-hand corner marks the nautilus antisense lines, -U indicates the uninjected phenotype for the designated gene, and -I indicates dsRNA injection for the indicated gene.

Figure 2

Figure 2

Cuticular patterns in early larvae induced by injection of twist and engrailed dsRNAs. (A) Wild-type cuticular pattern for early larva. (B) Twist phenotype of the known_twist_ mutation twi_1096. (C) Embryos injected with twist dsRNA show the same phenotype as in B. (D) Fused cuticular band phenotype seen for enIIB86 null mutants. (E) Embryos injected with the engrailed dsRNA show the same_en null phenotype as in D.

Similar articles

Cited by

References

    1. Rushton E, Drysdale R, Abmayr S M, Michelson A M, Bate M. Development. 1995;121:1979–1988. - PubMed
    1. Bate M. Development. 1990;110:791–804. - PubMed
    1. Ho R K, Ball E E, Goodman C S. Nature (London) 1983;301:66–69. - PubMed
    1. Keller C A, Grill M A, Abmayr S M. Dev Biol. 1998;202:153–156. - PubMed
    1. Abmayr S M, Erickson M S, Bour B A. Trends Genet. 1995;11:153–159. - PubMed

MeSH terms

Substances

LinkOut - more resources