chitosaninterfering-rna-nanoparticle-mediated-gene-silencing-disease

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

University of Notre Dame

Statutory recognition of the right to die: the California Natural Death Act.

Scientific dispute resolution: first use of provision 404 of the Food and Drug Administration Modernization Act of 1997.

Protocols for both generation of mosaic clones and wholemount in situ hybridization detection of mRNA have led to an advanced understanding of Drosophila development. A simple means of combining these techniques for staining of imaginal discs would be useful, as it would allow for analysis of mRNA transcripts in marked mosaic clones. However, few researchers attempt such experiments due to the technical difficulty of simultaneously detecting clones and mRNA transcripts. Furthermore, maintaining the ability to use GFP-marked clones is desirable. However, typical Drosophila in situ hybridization protocols result in loss of GFP fluorescence. The method for double labeling of imaginal discs described here maintains the ability to identify mosaic clones, including those marked by GFP, while simultaneously allowing the detection of mRNA transcripts. The methodology described is technically manageable, robust, rapid, relatively inexpensive and of interest to many Drosophila researchers.

Whole-mount in situ hybridization detection of mRNA in GFP-marked Drosophila imaginal disc mosaic clones.

Although neurite outgrowth has been linked to axon guidance regulators, the effects of guidance molecules on cellular growth are not well understood. Use of the Drosophila wing imaginal disc, an epithelial tissue and a well-characterized system for analysis of cellular growth regulation, permits analysis of the impacts of guidance molecules on cellular growth in a setting in which axon guidance is not a confounding factor. In this investigation, the impacts of Netrin A (NetA) and Semaphorin-1a (Sema1a) signaling on cellular growth are examined during wing development. Levels of these genes were modulated in somatic clones in the developing wing disc, and clone areas, as well as individual sizes of clonal cells were assessed. NetA and Sema1a signaling were found to induce cellular growth in these assays. Furthermore, immunohistochemical analyses indicated that NetA and Sema1a signaling induce expression of several growth regulators, including myc, cycD, cdk4, PCNA, and MapK in the wing disc. These data illustrate that NetA and Sema1a can specifically promote growth through induction of key cellular growth regulators. The abilities of NetA and Sema1a to regulate cellular growth are likely critical to their functions in both nervous system development and oncogenesis.

Induction of cellular growth by the axon guidance regulators netrin A and semaphorin-1a.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

Aedes aegypti: an emerging model for vector mosquito development.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol describes a method for fixation and dissection of Ae. aegypti embryos, larvae, and pupae. Tissue processed in this manner can be used subsequently for in situ hybridization detection of mRNA or immunohistochemical analysis of protein expression.

Fixation and preparation of developing tissues from Aedes aegypti.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. This protocol for whole-mount in situ hybridization can be used to analyze gene expression in Ae. aegypti embryos and larvae, a critical aspect of understanding developmental gene function in this vector mosquito.

Whole-mount in situ hybridization for analysis of gene expression during Aedes aegypti development.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and gene products that regulate development are of particular interest. This protocol for immunohistochemical analysis of protein expression can be used to analyze expression of developmental proteins of interest in Ae. aegypti embryos, larvae, and pupae, which will be critical for the development of markers for particular developing tissues.

Immunohistochemical analysis of protein expression during Aedes aegypti development.

Loss of heterozygosity at 18q, which includes the Deleted in Colorectal Cancer (DCC) gene, has been linked to many human cancers. However, it is unclear if loss of DCC is the specific underlying cause of these cancers. The Drosophila imaginal discs are excellent systems in which to study DCC function, as it is possible to model human tumors through the generation of somatic clones of cells bearing multiple genetic lesions. Here, these attributes of the fly system were utilized to investigate the potential tumor suppressing functions of the Drosophila DCC homologue frazzled (fra) during eye-antennal disc development.

The Drosophila Netrin receptor frazzled/DCC functions as an invasive tumor suppressor.

Although mosquito genome projects have uncovered orthologues of many known developmental regulatory genes, extremely little is known about mosquito development. In this study, the role of semaphorin-1a (sema1a) was investigated during vector mosquito embryonic ventral nerve cord development. Expression of sema1a and the plexin A (plexA) receptor are detected in the embryonic ventral nerve cords of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector), suggesting that Sema1a signaling may regulate mosquito nervous system development. Analysis of sema1a function was investigated through siRNA-mediated knockdown in A. aegypti embryos. Knockdown of sema1a during A. aegypti development results in a number of nerve cord phenotypes, including thinning, breakage, and occasional fusion of the longitudinal connectives, thin or absent commissures, and general distortion of the nerve cord. Although analysis of Drosophila melanogaster sema1a loss-of-function mutants uncovered many similar phenotypes, aspects of the longitudinal phenotypes differed between D. melanogaster and A. aegypti. The results of this investigation suggest that Sema1a is required for development of the insect ventral nerve cord, but that the developmental roles of this guidance molecule have diverged in dipteran insects.

Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development.

Genome sequencing projects have presented the opportunity for analysis of developmental genes in three vector mosquito species: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A comparative genomic analysis of developmental genes in Drosophila melanogaster and these three important vectors of human disease was performed in this investigation. While the study was comprehensive, special emphasis centered on genes that 1) are components of developmental signaling pathways, 2) regulate fundamental developmental processes, 3) are critical for the development of tissues of vector importance, 4) function in developmental processes known to have diverged within insects, and 5) encode microRNAs (miRNAs) that regulate developmental transcripts in Drosophila. While most fruit fly developmental genes are conserved in the three vector mosquito species, several genes known to be critical for Drosophila development were not identified in one or more mosquito genomes. In other cases, mosquito lineage-specific gene gains with respect to D. melanogaster were noted. Sequence analyses also revealed that numerous repetitive sequences are a common structural feature of Drosophila and mosquito developmental genes. Finally, analysis of predicted miRNA binding sites in fruit fly and mosquito developmental genes suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. The results of this study provide insight into the evolution of developmental genes and processes in dipterans and other arthropods, serve as a resource for those pursuing analysis of mosquito development, and will promote the design and refinement of functional analysis experiments.

Comparative genomic analysis of Drosophila melanogaster and vector mosquito developmental genes.

Despite the devastating impact of mosquito-borne illnesses on human health, surprisingly little is known about mosquito developmental biology, including development of the olfactory system, a tissue of vector importance. Analysis of mosquito olfactory developmental genetics has been hindered by a lack of means to target specific genes during the development of this sensory system. In this investigation, chitosan/siRNA nanoparticles were used to target semaphorin-1a (sema1a) during olfactory system development in the dengue and yellow fever vector mosquito Aedes aegypti. Immunohistochemical analyses and anterograde tracing of antennal sensory neurons, which were used to track the progression of olfactory development in this species, revealed antennal lobe defects in sema1a knockdown fourth instar larvae. These findings, which correlated with a larval odorant tracking behavioral phenotype, identified previously unreported roles for Sema1a in the developing insect larval olfactory system. Analysis of sema1a knockdown pupae also revealed a number of olfactory phenotypes, including olfactory receptor neuron targeting and projection neuron defects coincident with a collapse in the structure and shape of the antennal lobe and individual glomeruli. This study, which is to our knowledge the first functional genetic analysis of insect olfactory development outside of D. melanogaster, identified critical roles for Sema1a during Ae. aegypti larval and pupal olfactory development and advocates the use of chitosan/siRNA nanoparticles as an effective means of targeting genes during post-embryonic Ae. aegypti development. Use of siRNA nanoparticle methodology to understand sensory developmental genetics in mosquitoes will provide insight into the evolutionary conservation and divergence of key developmental genes which could be exploited in the development of both common and species-specific means for intervention.

Disruption of Aedes aegypti olfactory system development through chitosan/siRNA nanoparticle targeting of semaphorin-1a.

Bone morphogenic protein-4 (BMP4) and fibroblast growth factor-8 (FGF8) are thought to have opposite roles in defining epithelial versus neurogenic fate in the developing olfactory/vomeronasal system. In particular, FGF8 has been implicated in specification of olfactory and gonadotropin releasing hormone-1 (GnRH) neurons, as well as in controlling olfactory stem cell survival. Using different knock-in mouse lines and Cre-lox-mediated lineage tracing, Fgf8 expression and cell lineage was analyzed in the developing nose in relation to the expression of Bmp4 and its antagonist Noggin (Nog). FGF8 is expressed by cells that acquire an epidermal, respiratory cell fate and not by stem cells that acquire neuronal olfactory or vomeronasal cell fate. Ectodermal and mesenchymal sources of BMP4 control the expression of BMP/TGFβ antagonist Nog, whereas mesenchymal sources of Nog define the neurogenic borders of the olfactory pit. Fgf8 hypomorph mouse models, Fgf8(neo/neo) and Fgf8(neo/null), displayed severe craniofacial defects together with overlapping defects in the olfactory pit including (1) lack of neuronal formation ventrally, where GnRH neurons normally form, and (2) altered expression of Bmp4 and Nog, with Nog ectopically expressed in the nasal mesenchyme and no longer defining the GnRH and vomeronasal neurogenic border. Together our data show that (1) FGF8 is not sufficient to induce ectodermal progenitors of the olfactory pit to acquire neural fate and (2) altered neurogenesis and lack of GnRH neuron specification after chronically reduced Fgf8 expression reflected dysgenesis of the nasal region and loss of a specific neurogenic permissive milieu that was defined by mesenchymal signals.

The indirect role of fibroblast growth factor-8 in defining neurogenic niches of the olfactory/GnRH systems.

Despite the devastating impact of mosquito-borne illnesses on human health, very little is known about mosquito developmental biology, including development of the mosquito visual system. Mosquitoes possess functional adult compound eyes as larvae, a trait that makes them an interesting model in which to study comparative developmental genetics. Here, we functionally characterize visual system development in the dengue and yellow fever vector mosquito Aedes aegypti, in which we use chitosan/siRNA nanoparticles to target the axon guidance gene semaphorin-1a (sema1a).

Article	Year
Statutory recognition of the right to die: the California Natural Death Act. Boston University law review. Boston University. School of Law\| PubMed ID: 11664705	1977
Scientific dispute resolution: first use of provision 404 of the Food and Drug Administration Modernization Act of 1997. Food and drug law journal\| PubMed ID: 12866554	2003
Whole-mount in situ hybridization detection of mRNA in GFP-marked Drosophila imaginal disc mosaic clones. Fly\| PubMed ID: 19029797	2008
Induction of cellular growth by the axon guidance regulators netrin A and semaphorin-1a. Developmental neurobiology\| PubMed ID: 20162636	2010
Aedes aegypti: an emerging model for vector mosquito development. Cold Spring Harbor protocols\| PubMed ID: 20889691	2010
Fixation and preparation of developing tissues from Aedes aegypti. Cold Spring Harbor protocols\| PubMed ID: 20889705	2010
Whole-mount in situ hybridization for analysis of gene expression during Aedes aegypti development. Cold Spring Harbor protocols\| PubMed ID: 20889706	2010
Immunohistochemical analysis of protein expression during Aedes aegypti development. Cold Spring Harbor protocols\| PubMed ID: 20889707	2010
The Drosophila Netrin receptor frazzled/DCC functions as an invasive tumor suppressor. BMC developmental biology\| PubMed ID: 21672235	2011
Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development. PloS one\| PubMed ID: 21738767	2011
Comparative genomic analysis of Drosophila melanogaster and vector mosquito developmental genes. PloS one\| PubMed ID: 21754989	2011
Disruption of Aedes aegypti olfactory system development through chitosan/siRNA nanoparticle targeting of semaphorin-1a. PLoS neglected tropical diseases\| PubMed ID: 23696908	2013
The indirect role of fibroblast growth factor-8 in defining neurogenic niches of the olfactory/GnRH systems. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24336726	2013
Role of semaphorin-1a in the developing visual system of the disease vector mosquito Aedes aegypti. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 25045063	2014

Ellen Flannery

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