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

Semaphorin-1a Is Required for Aedes aegypti Embryonic Nerve Cord Development

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

Pupal ORN targeting defects correlate with <i>sema1a</i> knockdown.

<p>Severe ORN targeting defects (dye fills in B1; compare to control-fed animal in A1) correlate with loss of Sema1a protein expression at 24 hours APF (detected through lack of antibody staining in B2; compare to control-fed in A2). Overlays are shown in A3 and B3.</p

<i>sema1a</i> knockdown disrupts pupal antennal ORN targeting.

<p>In wildtype (A1–A4) and control-fed (B1–B4) pupae at 24 hours APF, ORNs innervate the antennal lobe and target specific glomeruli within the lobe (dye fills in A1, B1; mAb nc82 staining in A3, B3). Distinct glomerular structures are fully formed by this time point (asterisks in A, B). <i>sema1a</i> knockdown animals show defects in ORN targeting and glomerular structure (dye fills in C1, mAb nc82 label in C3). Levels of both serotonergic neuronal (C2) and synaptic neuropil markers (C3) were decreased in <i>sema1a</i> knockdown animals as compared to wildtype (A2, A3) and control-fed (B2, B3) larvae. Levels of mAb nc82 staining were increased slightly in C3 so that morphological phenotypes are viewable. Dorsal is up in all panels, and overlays of all three labels are depicted at right. Comparable sections of the antennal lobe are shown in panels A, B, and C. Panels A4, B4 & C4 represent the merged images from their respective first three columns. Scale bar = 25 µm.</p

Chitosan/siRNA nanoparticles elicit knockdown in the developing olfactory system of <i>Ae.</i> aegypti.

<p>Knockdown of <i>sema1a</i> was assessed in mosquitoes fed with nanoparticles containing a mixture of <i>sema1a</i>-targeting siRNA¹¹⁹⁸ and siRNA⁸⁹⁰ (A, C, E). qRT-PCR experiments (A) demonstrated that <i>sema1a</i> levels were significantly reduced in whole L4 knockdown (KD) animals as compared to control-fed animals (p<0.01, N = 5, where N is the number of biological replicates). Nearly complete loss of <i>sema1a</i> transcript expression is observed in the brain of an L4 animal fed with chitosan/siRNA nanoparticles targeting <i>sema1a</i> (C; compare to control-fed animal in B). Arrowheads mark the antennal lobes in B and C, and brains are oriented dorsal up in both panels. Knockdown could also be detected through anti-Sema1a antibody staining 24 hours APF (E1). In control-fed pupae, Sema1a protein (D1) is detected in a wild-type lateral to medial gradient expression pattern 24 hours APF. The nearly complete loss of Sema1a expression observed in an animal fed with chitosan/siRNA nanoparticles targeting <i>sema1a</i> results in glomeruli malformation (visualized with mAb nc82 staining in E2, levels are increased to view glomerular structures; compare to control-fed animals in D2). Merge images are shown in D3 and E3. Images in D and E are a compilation of 5 confocal sections so as to ensure that the gradient is not an artifact of natural tissue curvature. Dorsal is up in all panels. Scale bar = 25 µm.</p

Larval antennal lobe defects in <i>sema1a</i> knockdown animals.

<p>In wildtype (A) and control-fed (B) L4 larvae, antennal sensory neurons innervate the antennal lobe (marked by dotted yellow circle throughout the figure), which is labeled by mAb nc82 staining (A3, B3). Serotonergic projection neurons are marked by 5HT staining in the antennal lobes of these animals (A2, B2; overlays of all three labels are shown in A4 and B4). <i>sema1a</i> knockdown animals (C–E) show a reduction in the number of antennal neurons (C1, D1, E1) targeting the antennal lobe, which is marked by reduced mAb nc82 synaptic neuropil staining (C3, D3, E3; weak levels are slightly increased in these panels so that the antennal lobe staining can be viewed). Reduced anti-serotonin 5HT staining of projection neurons is also observed in knockdown animals (C2, D2, E2; overlays of all three labels are shown in C4, D4, and E4). Comparable antennal lobe <i>sema1a</i> knockdown phenotypes are observed in animals fed with siRNA¹¹⁹⁸ (D), siRNA⁸⁹⁰ (E), or a combination of the two (<i>sema1a</i> KD in C). Dorsal is up in all panels. Scale bar = 25 µm.</p

<i>sema1a</i> transcript is expressed in the antennae of larvae and pupae.

<p>Antennal ORNs, a subset of which express <i>sema1a</i> transcripts (B), are marked by <i>orco/OR7</i> transcript expression (A) in the antennae of fourth instar larvae. <i>sema1a</i> transcript expression is maintained in the wildtype pupal antenna at 24 hours APF (E). In contrast to L4 animals in which <i>sema1a</i> transcript expression is not altered following feeding with control siRNA/chitosan nanoparticles (C), no <i>sema1a</i> transcript expression is detected in L4 ORNs of an animal fed with nanoparticles containing a mixture of <i>sema1a</i>-targeting siRNA¹¹⁹⁸ and siRNA⁸⁹⁰ (D). The proximal ends of antennae are oriented up in A–D and right in E.</p

Larval antennal neuron targeting defects in <i>sema1a</i> knockdown animals.

<p>In the larval antennal lobe, antennal gustatory neurons (red arrowhead) target the antennal lobe and project ventrally (oriented down in all panels) toward the subesophageal ganglion. These neurons target properly in <i>sema1a</i> knockdown animals (C–J). In wildtype (A) and control-fed (B) L4 larvae, a subset of antennal sensory neurons innervating the antennal lobe send projection neurons dorsally (oriented up in all panels) to a target region (marked by blue arrows) in the SuEG. These neurons do not target properly to the SuEG in <i>sema1a</i> knockdown animals (C–J). Phenotypic categories observed include: antennal neurons failing to innervate outside of the antennal lobe (C, D; 38% of phenotypes; n = 38), neurons (yellow arrowheads) stopping short of the SuEG target (E, F; 19% of phenotypes), extension beyond the target region (blue arrows) within the SuEG (G, H; 24% of phenotypes), and extraneous branching within the SuEG (I, J; 19% of phenotypes). All four phenotypes were detected in animals fed with siRNA⁸⁹⁰ alone (H, J), siRNA¹¹⁹⁸ alone (D, F), or a combination of the two siRNAs (referred to as <i>sema1a</i> KD in C, E, G, and I), although only a subset of these results are included here. These phenotypes were never observed in wildtype larvae (A) or those that were fed with control siRNA nanoparticles (B). Scale bar = 25 µm.</p

Levels of <i>sema1a</i> correlate with performance in yeast behavioral assays.

<p>The above set of data represents a compiled summary of results obtained from four replicate experiments in which control-fed vs. <i>sema1a</i> knockdown (KD) animals were tested in a yeast odorant attractant assay. The total number of animals (n) indicates the number of individuals that were assessed in these assays. The number of individuals (# Animals) that were attracted (left; animals that touched the yeast pellet and received a score of 1) or not attracted (right; animals that did not touch the yeast pellet and received a score of 0) under each condition (Control or KD) are indicated, and the percentages of total animals are reported after the raw numbers. The levels of <i>sema1a</i> were assessed in the brains and antennae of animals attracted (left) or not attracted (right) to the yeast. The raw number/percentage of # animals with Normal (wildtype <i>sema1a</i>), Null (no detectable <i>sema1a</i> transcript), or Moderate (reduced but not wildtype <i>sema1a</i>) levels are indicated. Loss of <i>sema1a</i> expression correlated well with a lack of attraction to the yeast.</p

Lung Spatial Profiling Reveals a T Cell Signature in COPD Patients with Fatal SARS-CoV-2 Infection

People with pre-existing lung diseases such as chronic obstructive pulmonary disease (COPD) are more likely to get very sick from SARS-CoV-2 disease 2019 (COVID-19). Still, an interrogation of the immune response to COVID-19 infection, spatially throughout the lung structure, is lacking in patients with COPD. For this study, we characterized the immune microenvironment of the lung parenchyma, airways, and vessels of never-and ever-smokers with or without COPD, all of whom died of COVID-19, using spatial transcriptomic and proteomic profiling. The parenchyma, airways, and vessels of COPD patients, compared to control lungs had (1) significant enrichment for lung-resident CD45RO+ memory CD4+ T cells; (2) downregulation of genes associated with T cell antigen priming and memory T cell differentiation; and (3) higher expression of proteins associated with SARS-CoV-2 entry and primary receptor ubiquitously across the ROIs and in particular the lung parenchyma, despite similar SARS-CoV-2 structural gene expression levels. In conclusion, the lung parenchyma, airways, and vessels of COPD patients have increased T-lymphocytes with a blunted memory CD4 T cell response and a more invasive SARS-CoV-2 infection pattern and may underlie the higher death toll observed with COVID-19