We have initiated research to determine the genetic basis of a male wing polymorphism in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae). Previous studies showed that this polymorphism is controlled by a single biallelic locus, which we name aphicarus (api), on the X chromosome. Our objectives were to confirm that api segregates as a polymorphism of a single gene on the X chromosome, and to obtain molecular markers flanking api that can be used as a starting point for high-resolution genetic and physical mapping of the target region, which will ultimately allow the cloning of api. We have established an F2 population segregating for api and have generated X-linked AFLP markers. The segregation pattern of api in the F2 population shows that the male wing polymorphism segregates as a polymorphism of a single gene, or set of closely linked genes on the X chromosome. Using a subset of 78 F2 males, we have constructed a linkage map of the chromosomal region encompassing api using seven AFLP markers. The map spans 74.1 cM and we have mapped api to an interval of 10 cM. In addition, we confirmed X linkage of our AFLP markers and api by using one X-linked marker developed in an earlier study. Our study presents the first mapping of a gene with known function in aphids, and the results indicate that target gene mapping in aphids is feasible.

MARCM (mosaic analysis with a repressible cell marker) involves specific labeling of GAL80-minus and GAL4-positive homozygous cells in otherwise heterozygous tissues. Here we demonstrate how the concurrent use of two independent binary transcriptional systems may facilitate complex MARCM studies in the Drosophila nervous system. By fusing LexA with the VP16 acidic activation domain (VP16) or the GAL4 activation domain (GAD), we obtained both GAL80-insensitive and GAL80-suppressible transcriptional factors. LexA::VP16 can mediate MARCM-independent binary transgene induction in mosaic organisms. The incorporation of LexA::GAD into MARCM, which we call dual-expression-control MARCM, permits the induction of distinct transgenes in different patterns among GAL80-minus cells in mosaic tissues. Lineage analysis with dual-expression-control MARCM suggested the presence of neuroglioblasts in the developing optic lobes but did not indicate the production of glia by postembryonic mushroom body neuronal precursors. In addition, dual-expression-control MARCM with a ubiquitous LexA::GAD driver revealed many unidentified cells in the GAL4-GH146-positive projection neuron lineages.

The ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2,800 transgenic lines for use with the split-GAL4 intersectional method.

Drosophila sensory neurons form distinctive terminal branch patterns in the developing neuropile of the embryonic central nervous system. In this paper we make a genetic analysis of factors regulating arbor position. We show that mediolateral position is determined in a binary fashion by expression (chordotonal neurons) or nonexpression (multidendritic neurons) of the Robo3 receptor for the midline repellent Slit. Robo3 expression is one of a suite of chordotonal neuron properties that depend on expression of the proneural gene atonal. Different features of terminal branches are separately regulated: an arbor can be shifted mediolaterally without affecting its dorsoventral location, and the distinctive remodeling of one arbor continues as normal despite this arbor shifting to an abnormal position in the neuropile.

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of Metand gce throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.

Exogenous DNA sequences were introduced into the Drosophila germ line. A rosy transposon (ry1), constructed by inserting a chromosomal DNA fragment containing the wild-type rosy gene into a P transposable element, transformed germ line cells in 20 to 50 percent of the injected rosy mutant embryos. Transformants contained one or two copies of chromosomally integrated, intact ry1 that were stably inherited in subsequent generations. These transformed flies had wild-type eye color indicating that the visible genetic defect in the host strain could be fully and permanently corrected by the transferred gene. To demonstrate the generality of this approach, a DNA segment that does not confer a recognizable phenotype on recipients was also transferred into germ line chromosomes.

Many polyphenisms are examples of adaptive phenotypic plasticity where a single genotype produces distinct phenotypes in response to environmental cues. Such alternative phenotypes occur as winged and wingless parthenogenetic females in the pea aphid (Acyrthosiphon pisum). However, the proportion of winged females produced in response to a given environmental cue varies between clonal genotypes. Winged and wingless phenotypes also occur in males of the sexual generation. In contrast to parthenogenetic females, wing production in males is environmentally insensitive and controlled by the sex-linked, biallelic locus, aphicarus (api). Hence, environmental or genetic cues induce development of winged and wingless phenotypes at different stages of the pea aphid life cycle. We have tested whether allelic variation at the api locus explains genetic variation in the propensity to produce winged females. We assayed clones from an F2 cross that were heterozygous or homozygous for alternative api alleles for their propensity to produce winged offspring. We found that clones with different api genotypes differed in their propensity to produce winged offspring. The results indicate genetic linkage of factors controlling the female wing polyphenism and male wing polymorphism. This finding is consistent with the hypothesis that genotype by environment interaction at the api locus explains genetic variation in the environmentally cued wing polyphenism.

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

The discovery of intracellular Ca(2+) signals within astrocytes has changed our view of how these ubiquitous cells contribute to brain function. Classically thought merely to serve supportive functions, astrocytes are increasingly thought to respond to, and regulate, neurons. The use of organic Ca(2+) indicator dyes such as Fluo-4 and Fura-2 has proved instrumental in the study of astrocyte physiology. However, progress has recently been accelerated by the use of cytosolic and membrane targeted genetically encoded calcium indicators (GECIs). Herein, we review these recent findings, discuss why studying astrocyte Ca(2+) signals is important to understand brain function, and summarize work that led to the discovery of TRPA1 channel-mediated near-membrane Ca(2+) signals in astrocytes and their indirect neuromodulatory roles at inhibitory synapses in the CA1 stratum radiatum region of the hippocampus. We suggest that the use of membrane-targeted and cytosolic GECIs holds great promise to explore the diversity of Ca(2+) signals within single astrocytes and also to study diversity of function for astrocytes in different parts of the brain.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(2+)-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca(2+)]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca(2+) affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.