Biology subjects

Potter, C. J.

Publications and source records attributed to Potter, C. J..

8 recordsLinked to original sources

bioRxiv2022

Neurogenetic identification of mosquito sensory neurons

Anopheles mosquitoes, as vectors for the malaria parasite, are a global threat to human health. To find and bite a human, they utilize neurons within their sensory appendages. However, the identity and quantification of sensory appendage neurons are lacking. Here we use a neurogenetic approach to label all neurons in Anopheles coluzzii mosquitoes. We utilize the Homology Assisted CRISPR Knock-in (HACK) approach to generate a T2A-QF2w knock-in of the synaptic gene bruchpilot. We use a membrane-targeted GFP reporter to visualize the neurons in the brain and to quantify neurons in all major chemosensory appendages (antenna, maxillary palp, labella, tarsi). By comparing labeling of brp>GFP and Orco>GFP mosquitoes, we predict the extent of neurons expressing Ionotropic Receptors or other chemosensory receptors. This work introduces a valuable genetic tool for the functional analysis of Anopheles mosquito neurobiology and initiates characterization of the sensory neurons that guide mosquito behavior. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/517370v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1ebbf69org.highwire.dtl.DTLVardef@63bcd2org.highwire.dtl.DTLVardef@1bab0org.highwire.dtl.DTLVardef@18799a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

A spatial map of antennal-expressed olfactory ionotropic receptors in the malaria mosquito

The malaria mosquito Anopheles coluzzii uses odors to guide various behaviors such as host-seeking. The detection of behaviorally relevant odors is mediated by a diverse family of receptors including the olfactory Ionotropic Receptors (IRs). Olfactory receptors are expressed on olfactory neurons, with the mosquito antennae representing the main olfactory appendage for detecting volatile chemical cues from the environment. It is currently unknown how many neurons across the antenna express a certain IR, or how these IR-expressing neurons are spatially arranged. Here, we performed whole mount fluorescent in situ hybridization of all IRs expressed in the antennae. The organization of IR-positive cells within an antennal segment (flagellomere) appeared stereotyped across multiple antennae. The spatial map of IR-expressing neurons revealed that the antenna might be organized into proximal and distal functional domains. Highly expressed tuning (odor-binding) IRs exhibit distinct co-localization patterns with cognate IR co-receptor(s) in a combinatorial fashion that might predict their functional properties. These findings reveal organizing principles of Anopheles IR-expressing neurons in the mosquito which might underlie their functional contribution to the detection of behaviorally relevant odors.

neuroscience↗

bioRxiv2021

Base editing using CRISPR/Cas9 in Drosophila

Cas9 and a guide RNA function to target specific genomic loci for generation of a double stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase to induce C>T (or G>A) mutations at targeted sites. Here, we test base editing in Drosophila by expressing a transgenic Drosophila base editor (DBE2, based on the mammalian BE2) which consists of a fusion protein of cytidine deaminase, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the Drosophila Base Editor (Actin5C-DBE2) and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C>T were found in positions 3-9 of the gRNA targeted site, with conversion reaching nearly 100% of targeted DNA is somatic tissues. The simultaneous use of two gRNA targeting a genomic region spaced [~]50 bps apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in Drosophila, and could be used to induce point mutations at select loci.

genetics↗

bioRxiv2020

Widespread Polymodal Chemosensory Receptor Expression in Drosophila Olfactory Neurons

Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila, the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs). The dozens of odorant binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a, Ir8a, and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in Drosophila (Orco, Ir8a, Ir76b, Ir25a). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. These results suggest co-expression of chemosensory receptors is common in olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.

neuroscience↗

bioRxiv2020

Dense reconstruction of elongated cell lineages: overcoming suboptimum lineage encoding and sparse cell sampling

Acquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.

developmental biology↗

bioRxiv2020

A Feedback Mechanism Regulates Odorant Receptor Expression in the Malaria Mosquito, Anopheles gambiae

Mosquitoes locate and approach humans ( host-seek) when specific Olfactory Neurons (ORNs) in the olfactory periphery activate a specific combination of glomeruli in the mosquito Antennal Lobe (AL). We hypothesize that dysregulating proper glomerular activation in the presence of human odor will prevent host-seeking behavior. In experiments aimed at ectopically activating most ORNs in the presence of human odor, we made a surprising finding: ectopic expression of an AgOr (AgOr2) in Anopheles gambiae ORNs dampens the activity of the expressing neuron. This contrasts studies in Drosophila melanogaster, the typical insect model of olfaction, in which ectopic expression of non-native ORs in ORNs confers ectopic neuronal responses without interfering with native olfactory physiology. To gain insight into this dysfunction in mosquitoes, RNA-seq analyses were performed comparing wild-type antennae to those ectopically expressing AgOr2 in ORNs. Remarkably, almost all Or transcripts were significantly downregulated (except for AgOr2), and additional experiments suggest that it is AgOR2 protein rather than mRNA that mediates this downregulation. Our study shows that ORNs of Anopheles mosquitoes (in contrast to Drosophila) employ a currently unexplored regulatory mechanism of OR expression, which may be adaptable as a vector-control strategy. SIGNIFICANCE STATEMENTStudies in Drosophila melanogaster suggest that insect Olfactory Receptor Neurons (ORNs) do not contain mechanisms by which Odorant Receptors (ORs) regulate OR expression. This has proved useful in studies where ectopic expression of an OR in Drosophila ORNs confers responses to the odorants that activate the newly expressed OR. In experiments in Anopheles gambiae mosquitoes, we found that ectopic expression of an OR in most Anopheles ORNs dampened the activity of the expressing neurons. RNA-seq analyses demonstrated that ectopic OR expression in Anopheles ORNs leads to downregulation of endogenous Or transcripts. Additional experiments suggest that this downregulation required ectopic expression of a functional OR protein. These findings reveal that Anopheles mosquitoes, in contrast to Drosophila, contain a feedback mechanism to regulate OR expression. Mosquito ORNs might employ regulatory mechanisms of OR expression previously thought to occur only in non-insect olfactory systems.

neuroscience↗

bioRxiv2019

Commonly used insect repellents hide human odors from Anopheles mosquitoes

The mode of action for most mosquito repellents is unknown. This is primarily due to the difficulty in monitoring how the mosquito olfactory system responds to repellent odors. Here, we used the Q-system of binary expression to enable activity-dependent Ca2+ imaging in olfactory neurons of the African malaria mosquito Anopheles coluzzii. This system allows neuronal responses to common insect repellents to be directly visualized in living mosquitoes from all olfactory organs including the antenna. The synthetic repellents DEET and IR3535 did not activate Odorant Receptor Co-Receptor (Orco) expressing olfactory receptor neurons (ORNs) at any concentration, while picaridin weakly activated ORNs only at high concentrations. In contrast, natural repellents (i.e. lemongrass oil and eugenol) strongly activated small numbers of ORNs in the mosquito antennae at low concentrations. We determined that DEET, IR3535, and picaridin decrease the response of Orco expressing ORNs when these repellents are physically mixed with activating human-derived odorants. We present evidence that synthetic repellents may primarily exert their olfactory mode of action by decreasing the amount of activating ligand reaching ORNs. These results suggest that synthetic repellents disruptively change the chemical profile of host scent signatures on the skin surface rendering humans invisible to mosquitoes.

neuroscience↗

bioRxiv2017

An olfactogenetic approach identifies olfactory neurons and brain centers directing negative oviposition decisions in Drosophila.

The sense of smell influences behaviors in animals, yet how odors are represented in the brain remains unclear. The nose contains different types of olfactory sensory neurons (OSNs), each expressing a particular odorant receptor, and OSNs expressing the same receptors converge their axons on a brain region called a glomerulus. In Drosophila, second order neurons (projection neurons) typically innervate a single glomerulus and send stereotyped axonal projections to the lateral horn. One of the greatest challenges to studying olfaction is the lack of methods allowing activation of specific types of olfactory neurons in an ethologically relevant setting. Most odorants activate many olfactory neurons, and many olfactory neurons are activated by a variety of odorants. As such, it is difficult to identify if individual types of olfactory neurons directly influence a behavior. To address this, we developed a genetic method in Drosophila called olfactogenetics in which a narrowly tuned odorant receptor, Or56a, is ectopically expressed in different olfactory neuron types. Stimulation with geosmin (the only known Or56a ligand), in an Or56a mutant background leads to specific activation of only the target olfactory neuron type. We used this approach to identify which types of olfactory neurons can directly guide oviposition decisions. We identified 5 OSN-types (Or71a, Or47b, Or49a, Or67b, and Or7a) that, when activated alone, suppress oviposition. Projection neurons partnering with these OSNs share a region of innervation in the lateral horn, suggesting that oviposition site-selection might be encoded in this brain region.\n\nSignificance StatementThe sense of smell begins by activation of olfactory neurons in the nose. These neurons express an olfactory receptor that binds odorants (volatile chemicals). How the sense of smell is encoded in the brain remains unclear. A key challenge is due to the nature of olfactory receptors themselves - most respond to a wide range of odorants - so it is often impossible to activate just a single olfactory neuron type. We describe here a novel approach in Drosophila called olfactogenetics which allows the specific experimental activation of any desired olfactory neuron. We use olfactogenetics to identify olfactory neurons and brain regions that guide egg-laying site selection. Olfactogenetics could be a valuable method to link olfactory neuron activities with circuits and behaviors.

neuroscience↗