siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development

Abstract

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.

Figure 1. Development of the Ae. aegypti embryonic ventral nerve cord.

Anti-acetylated tubulin staining (A–C) marks the developing axon tracts in 52 hr. (A) and 56 hr. (B) Ae. aegypti embryos. By 56 hrs. (C), the Ae. aegypti nerve cord resembles that of a 33 hr. An. gambiae embryo and a St. 16 Dr. melanogaster nerve cord (BP102 staining is shown in D). These time points in the three respective species correspond to germ-band retracted embryos in which segmentation is obvious and organogenesis has initiated. Filleted nerve cords are oriented anterior up in all panels. The anterior commissure is marked by a black arrowhead, and a white arrowhead marks the posterior commissure.

Figure 2. Expression of fra in the developing mosquito CNS.

Comparable fra expression patterns are detected in lateral views of the developing nervous systems (arrows) of An. gambiae (33 hrs., A) and Ae. aegypti (52 hrs., B). Ventral views of Aae fra expression in 52 hr. (C, segments T3–A5) and 54 hr. (D; segments A2–A6) Ae. aegypti embryos are shown. Anterior is oriented left in A and B and up in C and D.

Figure 3. Confirmation of fra knockdown in Ae. aegypti.

qRT-PCR was used to assess fra levels following microinjection of fra siRNA-A. A scrambled version of fra siRNA-A was injected as a control. At 72 hrs. post injection, levels of fra were 80% less than that of the control-injected group (N = 3, p<0.0001).

Figure 4. Ae. aegypti fra knockdown CNS phenotypes.

Anti-acetylated tubulin staining (reddish brown) marks the axons of the ventral nerve cords of scrambled control (A) and fra siRNA injected embryos (B–D). Knockdown phenotypes characterized by thinning or loss of commissural axons were observed at 54 hrs. (B–D). Comparable results were obtained with two different siRNAs (fra siRNA-A in B,C; fra siRNA-B in D). Knockdown of fra was confirmed by double-labeling to detect fra mRNA expression (dark blue in C). Nerve cords are oriented anterior up in each panel. The anterior commissure is marked by a black arrowhead, and a white arrowhead marks the posterior commissure.