siRNA-Mediated Gene Targeting in Aedes aegypti Embryos Reveals That Frazzled Regulates Vector Mosquito CNS Development

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.


Introduction
Completion of the Aedes aegypti and Anopheles gambiae genome projects uncovered orthologues of many known developmental regulatory genes in these two important mosquito vectors of dengue and malaria, respectively [1,2]. Although characterization of the function of these genes could provide insight into the evolution of insect development or potentially reveal novel strategies for vector control, extremely little is known about the genetic regulation of mosquito development [3,4]. Excellent descriptive analyses of Ae. aegypti embryogenesis were completed in the 1970's [5,6], and additional developmental analyses in this species were recently published [7,8]. Still, expression of only a handful of mosquito embryonic genes has been described in Ae. aegypti or other vector mosquitoes [9,10,11,12,13,14,15,16]. This is likely a result of the technical challenges historically encountered by those performing developmental analyses in mosquitoes. In fact, Christophers [17], author of the most comprehensive text on the biology of Ae. aegypti, indicated that the eggs of this species are not the most suitable form on which to study mosquito embryology.
Given the many known advantages of studying the biology of Ae. aegypti [3,18], we recently published a series of protocols for the study of its development [19,20,21,22,23]. These methodologies, in addition to those published previously [9,11], will promote analysis of mosquito developmental genetics. We are presently employing these techniques to examine mosquito nervous system development. Analysis of mosquito neural development will lead to a better understanding of the developmental basis of motor function, sensory processing, and behavior, key aspects of mosquito host location.
During Drosophila melanogaster nervous system development, midline cells secrete guidance molecules such as Netrin (Net) proteins that regulate the growth of commissural axons [24,25,26]. The Dr. melanogaster Net proteins are expressed at the midline and are required for proper commissural axon guidance in the embryonic ventral nerve cord. Frazzled (Fra), the Drosophila homolog of the vertebrate Deleted in Colorectal Cancer (DCC) Net receptor, guides axons in response to Net signaling [27] and also controls Net distribution in flies [28]. Previous studies indicated that deletion of netA and B or fra results in defective guidance of commissural axons in Drosophila [27,29,30]. More recent data suggest that Drosophila Nets function as short-range guidance cues that promote midline crossing [31].
Although data support the homology of axon-guiding midline cells [16,32,33,34,35,36], homology of midline cells, which form differently in various arthropod species (discussed in [32]) has been debated. To address whether common molecular mechanisms regulate nerve cord formation during arthropod nervous system development, we recently analyzed patterns of axon tract formation and the putative homology of midline cells in distantly related arthropods. These comparative analyses were aided by a cross-reactive antibody generated against the Netrin (Net) protein, a midline cell marker and regulator of axonogenesis [16]. Despite divergent mechanisms of midline cell formation and nerve cord development in arthropods, detection of conserved Net accumulation patterns suggests that Net-Fra signaling plays a conserved role in the regulation of ventral nerve cord development of Tetraconata [16]. Here, we continue to examine this hypothesis through examination of the expression of the Net receptor frazzled in both Ae. aegypti and An. gambiae. Moreover, for the first time, we use siRNA-mediated knockdown to functionally test this hypothesis in Ae. aegypti.

Development of the mosquito embryonic ventral nerve cord
A scaffold of axon pathways develop in Dr. melanogaster and give rise to the embryonic ventral nerve cord, which has a ladder-like appearance (Fig. 1D). Within each segment of the developing fruit fly embryo, a pair of bilaterally symmetrical longitudinal axon tracts are pioneered separately on either side of the midline in each segment. A number of early growth cones project only on their own side, but most CNS interneurons will project their axons across the midline in either the anterior or posterior commissural axon tracts before extending rostrally or caudally in the developing longitudinals ( [24,25]; Fig 1D). Nerve cord development was assessed during mosquito embryogenesis with an anti-acetylated tubulin antibody ( Fig. 1A-C). Acetylated tubulin is first detected in Ae. aegypti at 52 hrs. after egg laying (AEL) when the longitudinal axon tracts have begun to form and the commissural axon tracts are initiating (Fig. 1A). During the next several hours, the axon tracts thicken as additional neurons project their axons (Fig. 1B). Anterior and posterior commissures are initially fused (not shown), as observed in Dr. melanogaster [37]. At 56 hrs. AEL, the commissures have separated, and the mature ventral embryonic nerve cord of Ae. aegypti (Fig. 1B) resembles that of An. gambiae (33 hrs. AEL shown in Fig. 1C) and a St. 16 Drosophila embryo (Fig. 1D).

Expression of fra in the developing mosquito CNS
Net accumulation data have indicated that Net-Fra signaling may play conserved roles during insect ventral nerve cord development [16,36]. However, in insects, fra expression has not been examined outside of Drosophila, where it is expressed on developing axons of the commissural and longitudinal axon pathways, including the earliest commissural axons [27]. Expression of Ae. aegypti fra (Aae fra) and An. gambiae fra (Aga fra) were therefore analyzed through whole-mount in situ hybridization at the onset of nerve cord development in both species. Aae fra expression initiates in developing neurons, including the earliest commissural axons, just prior to establishment of the axonal scaffold and is maintained during ventral nerve cord formation ( Fig. 2B-D). Comparable fra expression patterns are detected in the developing nervous system of An. gambiae ( Fig. 2A). These data are consistent with the hypothesis that Fra functions to regulate growth of commissural axons in mosquitoes.
si-RNA mediated knockdown of fra during Ae. aegypti development Analysis of fra expression (Fig. 2) suggested that this gene may regulate ventral nerve cord development in mosquitoes. Functional testing of this hypothesis required the development of a strategy to selectively inhibit gene function during mosquito development. RNA interference (RNAi) technology, which has emerged as an effective method for inhibiting gene function in many organisms, was therefore combined with previously described Ae. aegypti microinjection techniques [38,39] to knockdown fra during Ae. aegypti development. Two separate siRNAs corresponding to siRNAs were injected pre-cellular blastoderm, and knockdown was assessed through both quantitative real-time PCR (qRT-PCR) and whole-mount in situ hybridization. Multiple qRT-PCR replicates at three different time points, including 24, 48 (not shown), and 72 hrs. (Fig. 3), confirmed knockdown of fra that was maintained through the end of embryogenesis. At 72 hrs., the time point that was typically assayed once injection protocols and knockdown strategies had been optimized, fra transcript levels were reduced by 80% on average (Fig. 3, p,0.0001), and a maximum of 90% knockdown was achieved in one replicate. Knockdown in the developing CNS was verified through in situ hybridization, which confirmed reduced levels of fra transcripts in the embryonic CNS at levels comparable to those detected by qRT-PCR, and which revealed nearly complete knockdown in the developing nervous systems of embryos bearing strong phenotypes (Fig. 4C). These studies suggest that siRNA methodology can be used for targeted disruption of embryonic gene function in Ae aegypti.

Ae. aegypti fra knockdown CNS phenotypes
The impact of fra knockdown on Ae. aegypti embryonic nerve cord development was assessed through anti-acetylated tubulin staining at 54 hrs. AEL. In embryos injected with fra siRNA-A, 71% of anterior commissures and 80% of posterior commissures are thin or absent (Fig. 4B, C, Table 1). As observed in Drosophila [27], the posterior commissure is more severely disrupted than the anterior, with 51% of the embryos displaying a severe phenotype in the posterior commissure and 36% of embryos displaying a severe anterior commissure phenotype (Table 1). Occasional breaks in the longitudinal tracts were also noted in fra knockdown embryos. Injection of either fra siRNA-A (Fig. 4B,C) or siRNA-B (Fig. 4D), which correspond to two separate Aae fra sequences, produced similar phenotypes. This result indicates that the knockdown phenotypes described are due to loss of fra and are not the result of off-site targeting. Injection of the scrambled control siRNA did not disrupt nerve cord development (Fig. 4A, Table 1).
It should be noted that the penetrance and severity of the Aae fra knockdown phenotype are higher than that reported for the Drosophila fra null, in which only 12% of the anterior commissures and 43% of the posterior commissures are reportedly thin or absent [27]. In fact, in embryos in which CNS transcripts are nearly depleted, the Aae fra knockdown phenotype (Fig. 4B,C) bears strong resemblance to the Drosophila commissureless phenotype, in which commissure formation is entirely blocked [40]. These results suggest that Net-Fra signaling may play a more critical role in formation of the Ae. aegypti ventral nerve cord, and that the guidance cues postulated to compensate for loss of Net-Fra signaling in Dr. melanogaster [29] may not be present in mosquitoes.  These observations suggest that further analysis of embryonic nerve cord development in mosquitoes may uncover underlying differences between Dr. melanogaster and mosquito nervous system development. In support of this concept, our ongoing analysis of semaphorin knockdown in Ae. aegypti suggests that the function of this gene in nerve cord development has evolved in insects (data not shown).

Developmental Genetics in Vector Mosquitoes
Although we have made great advances in understanding developmental genetics in Drosophila, comparatively little is known about the genetic basis for development in mosquitoes and other arthropods. In this investigation, we examined the role of Fra during development of two vector mosquitoes. Expression of fra in the developing ventral nerve cord was found to be conserved between the two mosquitoes and Dr. melanogaster. However, the results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, illustrate that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance may have evolved in distantly related insects. This is a somewhat unexpected finding given the many similarities in insect CNS development that have been observed (for example, see [34,41]). Given these findings in Ae. aegypti, it would also be interesting to apply the siRNA-mediated knockdown strategies utilized here to An. gambiae and to formally assess the function of Aga fra.
Characterizing the function of additional developmental genes in mosquitoes is critical. To date, expression patterns of only a handful of mosquito developmental genes [9,10,11,12,13,14,15,16] have been reported. Adelman et al. [13] showed that control sequences for one of these genes, nanos [11], demonstrated promise as part of a transposable element-based gene drive system that may be used to spread and fix antipathogen effector genes in natural populations. Their investigations illustrate the exciting potential for the application of evo-devo approaches in efforts to develop strategies for vector control. The methodologies used in this investigation, in particular the siRNAmediated knockdown strategy for functional analysis of developmental genes in Ae. aegypti embryos, will broaden and enhance these efforts.

Ethics statement
This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was approved by the University of Notre Dame Institutional Animal Care and Use Committee (Study # 11-036).

Mosquito Rearing, Egg Collection, and Fixation
The Ae. aegypti Liverpool-IB12 (LVP-1B12) strain and An. gambiae (M Form) were used in these investigations. Procedures for mosquito rearing and egg collection [22,42], which was performed at 26uC, have been described. Ae. aegypti embryos were fixed as described [20]. An. gambiae embryos were fixed using a comparable procedure, except that eggs were fixed at room temperature.

In situ hybridization
Riboprobes corresponding to Aae fra (AAEL014592) and Aga fra (AGAP006083) were synthesized according to the Patel [43] protocol. In situ hybridization was performed as previously described [23].

RNA interference
Knockdown was performed through embryonic microinjection of siRNAs targeting Aae fra. siRNA design and microinjection were performed as described [21]. The following siRNAs were synthesized by Dharmacon RNAi Technologies (Lafayette, CO): siRNA-A sense: CCA GAT GGG TAT GGG AGA T and antisense: GGT CTA CCC ATA CCC TCT A (corresponding to base pairs 2011-2032 of Aae fra) and siRNA-B sense: TCC ATA CAC CTA CGA AGG A and antisense: AGG TAT GTG GAT GCT TCCT (corresponds to base pairs 3862-3883 of Aae fra). A scrambled version of siRNA-A was used as a control: sense GAT TAG ACG AAT ACC ACT A and antisense: CTA ATC TGC TTA TGG TGA T. siRNAs were injected at a concentration of 6 ug/uL. Measurement of knockdown effectiveness was determined through in situ hybridization (see above) and through qRT-PCR. qRT-PCR was performed as previously described [44]. In short, total RNA was extracted from ,30 pooled siRNA-microinjected mosquito embryos using Trizol (Invitrogen, Carlsbad, CA). cDNA was prepared with the High Capacity RNA to cDNA Kit (Applied Biosystems, Foster City, CA), which includes a blend of random and oligo(dT) primers, according to the manufacturer's instructions. Real-time quantification was performed using the SYBR Green I PCR kit (Applied Biosystems, Foster City, CA) in conjunction with an Applied BioSystems Step One Plus Real-Time PCR System. Primer sets for Aae fra were: For 59 GCG ACC CAA CAC TCA ATA TG 39 and Rev 59 GTC GTA GGA TAC CGT GAG AT 39. Primer sets for the housekeeping gene rpS17, which was included as the internal standard for data normalization as previously described [44], were as follows: For 59 AGA CAA CTA CGT GCC GGA AG 39 and Rev 59 TTG GTG ACC TGG ACA ACG ATG 39. Three independent biological replicates were conducted, and all PCR reactions were performed in triplicate. Quantification of results was accomplished by standardizing reactions to rpS17 levels, and then using the DDCt method as described [45]. Results were expressed as fold-difference compared with the scrambled control-injected embryos. qRT-PCR data from replicate experiments were statistically analyzed with the Student T Test.