-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS Neglected Tropical Diseases 05/2013; 7(5):e2215. · 4.69 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Despite the devastating global impact of mosquito-borne illnesses on human health, very little is known about mosquito developmental biology. In this investigation, functional genetic analysis of embryonic salivary gland development was performed in Aedes aegypti, the dengue and yellow fever vector and an emerging model for vector mosquito development. Although embryonic salivary gland development has been well studied in Drosophila melanogaster, little is known about this process in mosquitoes or other arthropods.
Mosquitoes possess orthologs of many genes that regulate Drosophila melanogaster embryonic salivary gland development. The expression patterns of a large subset of these genes were assessed during Ae. aegypti development. These studies identified a set of molecular genetic markers for the developing mosquito salivary gland. Analysis of marker expression allowed for tracking of the progression of Ae. aegypti salivary gland development in embryos. In Drosophila, the salivary glands develop from placodes located in the ventral neuroectoderm. However, in Ae. aegypti, salivary marker genes are not expressed in placode-like patterns in the ventral neuroectoderm. Instead, marker gene expression is detected in salivary gland rudiments adjacent to the proventriculus. These observations highlighted the need for functional genetic characterization of mosquito salivary gland development. An siRNA- mediated knockdown strategy was therefore employed to investigate the role of one of the marker genes, cyclic-AMP response element binding protein A (Aae crebA), during Ae. aegypti salivary gland development. These experiments revealed that Aae crebA encodes a key transcriptional regulator of the secretory pathway in the developing Ae. aegypti salivary gland.
The results of this investigation indicated that the initiation of salivary gland development in Ae. aegypti significantly differs from that of D. melanogaster. Despite these differences, some elements of salivary gland development, including the ability of CrebA to regulate secretory gene expression, are conserved between the two species. These studies underscore the need for further analysis of mosquito developmental genetics and may foster comparative studies of salivary gland development in additional insect species.
EvoDevo. 01/2013; 4(1):9.
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Most fra loss of function clones are eliminated during development. However, when mutant clone cells generated in the developing eye were rescued from death, partially differentiated eye cells were found outside of the normal eye field, and in extreme cases distant sites of the body. Characterization of these cells during development indicates that fra mutant cells display characteristics of invasive tumor cells, including increased levels of phospho-ERK, phospho-JNK, and Mmp-1, changes in cadherin expression, remodeling of the actin cytoskeleton, and loss of polarity. Mutation of fra promotes basement membrane degradation and invasion which are repressed by inhibition of Rho1 signaling. Although inhibition of JNK signaling blocks invasive phenotypes in some metastatic cancer models in flies, blocking JNK signaling inhibits fra mutant cell death, thereby enhancing the fra mutant phenotype.
The results of this investigation provide the first direct link between point mutations in fra/DCC and metastatic phenotypes in an animal model and suggest that Fra functions as an invasive tumor suppressor during Drosophila development.
BMC Developmental Biology 06/2011; 11:41. · 2.79 Impact Factor
-
[show abstract]
[hide abstract]
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.
PLoS ONE 01/2011; 6(1):e16730. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS ONE 01/2011; 6(6):e21694. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
PLoS ONE 01/2011; 6(7):e21504. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in the pathogenesis of intimal hyperplasia, the main cause of restenosis following vascular reconstruction. Here, the impact of sonic hedgehog (Shh)/Gli family zinc finger 2 (Gli2) signaling on VSMC proliferation was assessed.
Increased Shh signaling was detected in VSMCs in the neointima of vein grafts obtained from mice undergoing restenosis. Comparable results were found in primary cultured human VSMCs (hVSMCs) obtained from patients undergoing coronary bypass surgery, which were used to further assess the impacts of Shh signaling on VSMC proliferation. Inhibition of Shh signaling in hVSMCs through treatment with cyclopamine or knockdown of Gli2 results in G(1) arrest and reduced cyclin D1, cyclin E, and phosphorylated retinoblastoma (pRB) levels. In contrast, activation of Shh/Gli2 signaling in hVSMCs results in increased levels of G(1) cyclins and promotes G(1)-S transition. Stimulation of hVSMC proliferation by Shh is abolished by cyclin D1 knockdown.
Combined, these results demonstrate that Shh/Gli2 signaling stimulates VSMC proliferation via regulation of the G(1) cyclin-retinoblastoma axis and suggest that antagonists that target the Shh pathway may be therapeutically beneficial in the prevention of intimal hyperplasia.
Arteriosclerosis Thrombosis and Vascular Biology 09/2010; 30(9):1787-94. · 6.37 Impact Factor
-
Developmental Biology 08/2010; 344(1):528. · 4.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:473–484, 2010
Developmental Neurobiology 02/2010; 70(7):473 - 484. · 3.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Developmental Neurobiology 02/2010; 70(7):473-84. · 3.55 Impact Factor
-
Anthony Clemons,
Morgan Haugen,
Ellen Flannery,
Michael Tomchaney,
Kristopher Kast,
Caitlin Jacowski,
Christy Le,
Akio Mori,
Wendy Simanton Holland,
Joseph Sarro,
David W Severson, Molly Duman-Scheel
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cold Spring Harbor Protocols 01/2010; 2010(10):pdb.emo141. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 methods for culturing Ae. aegypti and includes a procedure for egg collection that can be used in conjunction with fixation, immunohistochemistry, and in situ protocols.
Cold Spring Harbor Protocols 01/2010; 2010(10):pdb.prot5507. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cold Spring Harbor Protocols 01/2010; 2010(10):pdb.prot5508. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cold Spring Harbor Protocols 01/2010; 2010(10):pdb.prot5509. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cold Spring Harbor Protocols 01/2010; 2010(10):pdb.prot5510. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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. In recent years, RNA interference (RNAi) has proven to be an effective strategy for inhibiting gene function in many organisms. This protocol describes a method for knockdown of embryonic genes in Ae. aegypti embryos by microinjection of small interfering RNA (siRNA) designed to target a specific gene of interest. The procedure includes a strategy for siRNA design, microinjection, and measurement of knockdown effectiveness.
Cold Spring Harbor Protocols 01/2010; 2010(10):pdb.prot5511. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Fly 12/2008; 2(6):323-5. · 1.30 Impact Factor
-
Developmental Biology - DEVELOP BIOL. 01/2006; 295(1):350-350.
-
[show abstract]
[hide abstract]
ABSTRACT: Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless (Wg) generates G(1) arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G(1) arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G(1)-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G(1)-S progression. Thus, G(1) arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G(1) progression, dMyc appears to regulate growth independently of the RBF/E2f pathway.
Proceedings of the National Academy of Sciences 04/2004; 101(11):3857-62. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although mutations that activate the Hedgehog (Hh) signalling pathway have been linked to several types of cancer, the molecular and cellular basis of Hh's ability to induce tumour formation is not well understood. We identified a mutation in patched (ptc), an inhibitor of Hh signalling, in a genetic screen for regulators of the Retinoblastoma (Rb) pathway in Drosophila. Here we show that Hh signalling promotes transcription of Cyclin E and Cyclin D, two inhibitors of Rb, and principal regulators of the cell cycle during development in Drosophila. Upregulation of Cyclin E expression, accomplished through binding of Cubitus interruptus (Ci) to the Cyclin E promoter, mediates the ability of Hh to induce DNA replication. Upregulation of Cyclin D expression by Hh mediates the distinct ability of Hh to promote cellular growth. The discovery of a direct connection between Hh signalling and principal cell-cycle regulators provides insight into the mechanism by which deregulated Hh signalling promotes tumour formation.
Nature 06/2002; 417(6886):299-304. · 36.28 Impact Factor
