Parental allele-specific protein expression in single cells In vivo.

Allelic expression from each parent-of-origin is important as a backup and to ensure that enough protein products of a gene are produced. Thus far, it is not known how each cell throughout a tissue differs in parental allele expression at the level of protein synthesis. Here, we measure the expression of the Ribosomal protein L13a (Rpl13a) from both parental alleles simultaneously in single cells in the living animal. We use genome-edited Drosophila that have a quantitative reporter of protein synthesis inserted into the endogenous Rpl13a locus. We find that individual cells can have large (>10-fold) differences in protein expression between the two parental alleles. Cells can produce protein from only one allele oftentimes, and time-lapse imaging of protein production from each parental allele in each cell showed that the imbalance in expression from one parental allele over the other can invert over time. We also identify the histone methyltransferase EHMT to be involved in the protein synthesis dynamics within cells.

GeneDig: a web application for accessing genomic and bioinformatics knowledge.

BACKGROUND: With the exponential increase and widespread availability of genomic, transcriptomic, and proteomic data, accessing these '-omics' data is becoming increasingly difficult. The current resources for accessing and analyzing these data have been created to perform highly specific functions intended for specialists, and thus typically emphasize functionality over user experience. RESULTS: We have developed a web-based application, GeneDig.org, that allows any general user access to genomic information with ease and efficiency. GeneDig allows for searching and browsing genes and genomes, while a dynamic navigator displays genomic, RNA, and protein information simultaneously for co-navigation. We demonstrate that our application allows more than five times faster and efficient access to genomic information than any currently available methods. CONCLUSION: We have developed GeneDig as a platform for bioinformatics integration focused on usability as its central design. This platform will introduce genomic navigation to broader audiences while aiding the bioinformatics analyses performed in everyday biology research.

Autism-linked mutations of CTTNBP2 reduce social interaction and impair dendritic spine formation via diverse mechanisms.

Abnormal synaptic formation and signaling is one of the key molecular features of autism spectrum disorders (ASD). Cortactin binding protein 2 (CTTNBP2), an ASD-linked gene, is known to regulate the subcellular distribution of synaptic proteins, such as cortactin, thereby controlling dendritic spine formation and maintenance. However, it remains unclear how ASD-linked mutations of CTTNBP2 influence its function. Here, using cultured hippocampal neurons and knockin mouse models, we screen seven ASD-linked mutations in the short form of the Cttnbp2 gene and identify that M120I, R533* and D570Y mutations impair CTTNBP2 protein-protein interactions via divergent mechanisms to reduce dendritic spine density in neurons. R533* mutation impairs CTTNBP2 interaction with cortactin due to lack of the C-terminal proline-rich domain. Through an N-C terminal interaction, M120I mutation at the N-terminal region of CTTNBP2 also negatively influences cortactin interaction. D570Y mutation increases the association of CTTNBP2 with microtubule, resulting in a dendritic localization of CTTNBP2, consequently reducing the distribution of CTTNBP2 in dendritic spines and impairing the synaptic function of CTTNBP2. Finally, we generated heterozygous M120I knockin mice to mimic the genetic variation of patients and found they exhibit reduced social interaction. Our study elucidates that different ASD-linked mutations of CTTNBP2 result in diverse molecular deficits, but all have the similar consequence of synaptic impairment.

Achilles-Mediated and Sex-Specific Regulation of Circadian mRNA Rhythms in Drosophila.

The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain using fluorescence in situ hybridization (FISH), and legacy data suggest it is not expressed in core clock neurons. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous studies, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.

TIE: A Method to Electroporate Long DNA Templates into Preimplantation Embryos for CRISPR-Cas9 Gene Editing.

Precise genome editing using CRISPR typically requires delivery of guide RNAs, Cas9 endonuclease, and DNA repair templates. Both microinjection and electroporation effectively deliver these components into mouse zygotes provided the DNA template is an oligonucleotide of only a few hundred base pairs. However, electroporation completely fails with longer double-stranded DNAs leaving microinjection as the only delivery option. Here, we overcome this limitation by first injecting all CRISPR components, including long plasmid-sized DNA templates, into the sub-zona pellucida space. There they are retained, supporting subsequent electroporation. We show that this simple and well-tolerated method achieves intracellular reagent concentrations sufficient to effect precise gene edits.

Neural circuitry coordinating male copulation.

Copulation is the goal of the courtship process, crucial to reproductive success and evolutionary fitness. Identifying the circuitry underlying copulation is a necessary step towards understanding universal principles of circuit operation, and how circuit elements are recruited into the production of ordered action sequences. Here, we identify key sex-specific neurons that mediate copulation in Drosophila, and define a sexually dimorphic motor circuit in the male abdominal ganglion that mediates the action sequence of initiating and terminating copulation. This sexually dimorphic circuit composed of three neuronal classes - motor neurons, interneurons and mechanosensory neurons - controls the mechanics of copulation. By correlating the connectivity, function and activity of these neurons we have determined the logic for how this circuitry is coordinated to generate this male-specific behavior, and sets the stage for a circuit-level dissection of active sensing and modulation of copulatory behavior.

Structural and functional analysis of single neurons to correlate synaptic connectivity with grooming behavior.

We describe a protocol to image the complex axonal branching structure of identified mechanosensory neurons in Drosophila, combined with a behavioral assay to evaluate the functional output of the neuron. The stimulation of identified mechanosensory neurons in live animals produces a stereotyped grooming reflex. The mechanosensory axonal arbor within the CNS is subsequently labeled with a lipophilic fluorescent dye and imaged by fluorescence microscopy. The behavioral output can therefore be correlated to the axonal morphology of the stimulated neuron in the same animal. Combining this protocol with genetic analysis provides a powerful tool for identifying the roles of molecules involved in different aspects of hard-wired neural circuit formation underlying an innate behavior. From behavioral analysis to axonal imaging, the protocol takes 4 d.

Overexpression of Down syndrome cell adhesion molecule impairs precise synaptic targeting.

Fragile X syndrome is caused by the loss of Fragile X mental retardation protein (FMRP), an RNA-binding protein that suppresses protein translation. We found that FMRP binds to Down syndrome cell adhesion molecule (Dscam) RNA, a molecule that is involved in neural development and has been implicated in Down syndrome. Elevated Dscam protein levels in FMRP null Drosophila and in flies with three copies of the Dscam gene both produced specific and similar synaptic targeting errors in a hard-wired neural circuit, which impaired the flies' sensory perception. Reducing Dscam levels in FMRP null flies reduced synaptic targeting errors and rescued behavioral responses. Our results indicate that excess Dscam protein may be a common molecular mechanism underlying altered neural wiring in intellectual disabilities such as Fragile X and Down syndromes.

Opposing roles of PlexinA and PlexinB in axonal branch and varicosity formation.

Establishing precise synaptic connectivity during development is crucial for neural circuit function. However, very few molecules have been identified that are involved in determining where and how many synapses form. The Plexin cell-surface molecules are a conserved family of axon guidance receptors that mediate axon fasciculation and repulsion during neural development, and later in development PlexinA receptors are involved in eliminating axonal branches and synapse numbers. Here we investigate the roles of PlexinA and PlexinB receptors in axonal branch and varicosity formation in Drosophila. We knocked down PlexinA or PlexinB expression using RNAi in identified mechanosensory neurons and analyzed axonal branching patterns and varicosity formations. Reducing PlexinA expression increased the axonal arbor complexity by increasing the number of branches and varicosities along the axon. In contrast, knocking down PlexinB expression decreased morphological complexity by decreasing the number of branches and the overall size of the axonal arbor, but did not reduce the number of varicosities. Our results demonstrate opposing roles for PlexinA and PlexinB in local wiring within a target region, where PlexinA functions to suppress excessive axonal branches and synapses and PlexinB facilitates axonal growth.

Long-term, high-resolution imaging in the neocortex in vivo.

INTRODUCTIONIn the neocortex, elucidating the mechanisms of structural plasticity is essential to an understanding of the emergent network properties and fundamental cognitive phenomena, such as memory formation. Time-lapse imaging microscopy has revealed a remarkable array of dynamic activities in dendritic structures in developing cortical tissue in vitro, the developing cortex in vivo, and even the adult neocortex. Chronic high-resolution in vivo imaging of the structure of neurons in the cortex became possible with the invention of two-photon laser scanning microscopy (2PLSM). This technique has key advantages over conventional, single-photon excitation techniques, such as confocal microscopy. Two-photon excitation is the near-simultaneous (within femtoseconds) absorption of two photons coinciding on a fluorophore. The absorption rate depends quadratically on the illumination intensity and is therefore confined to a small volume around the focal point. Scattered excitation light is too weak to generate fluorescence. Thus, the signal is generated exclusively in a tiny focal volume, and all emitted fluorescence photons constitute useful signals. A related advantage is that the longer wavelengths used to generate two-photon excitation penetrate scattering tissue more efficiently than the shorter wavelengths used to generate single-photon excitation of the same fluorophores. The upper layers (1-4) of neocortex are ideal for high-resolution 2PLSM imaging. This protocol describes experimental procedures for in vivo imaging in the neocortex.

The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila.

Alternative splicing of Dscam generates an enormous molecular diversity with maximally 38,016 different receptors. Whether this large diversity is required in vivo is currently unclear. We examined the role of Dscam in neuron-target recognition of single mechanosensory neurons, which connect with different target cells through multiple axonal branches. Analysis of Dscam null neurons demonstrated an essential role of Dscam for growth and directed extension of axon branches. Expression of randomly chosen single isoforms could not rescue connectivity but did restore basic axonal extension and rudimentary branching. Moreover, two Dscam alleles were generated that each reduced the maximally possible Dscam diversity to 22,176 isoforms. Reduction of Dscam diversity resulted in specific connectivity defects of mechanosensory neurons. Furthermore, the observed allele-specific phenotypes suggest functional differences among isoforms. Our findings provide evidence that a very large number of structurally unique receptor isoforms is required to ensure fidelity and precision of neuronal connectivity.

Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.

Do new synapses form in the adult cortex to support experience-dependent plasticity? To address this question, we repeatedly imaged individual pyramidal neurons in the mouse barrel cortex over periods of weeks. We found that, although dendritic structure is stable, some spines appear and disappear. Spine lifetimes vary greatly: stable spines, about 50% of the population, persist for at least a month, whereas the remainder are present for a few days or less. Serial-section electron microscopy of imaged dendritic segments revealed retrospectively that spine sprouting and retraction are associated with synapse formation and elimination. Experience-dependent plasticity of cortical receptive fields was accompanied by increased synapse turnover. Our measurements suggest that sensory experience drives the formation and elimination of synapses and that these changes might underlie adaptive remodelling of neural circuits.