Enhanced Locomotion Caused by Loss of the Drosophila DEG/ENaC Protein Pickpocket1

AbstractCoordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms [1]. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output [2]. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain [3]. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders

A transgenic mouse line for collecting ribosome-bound mRNA using the tetracycline transactivator system

Acquiring the gene expression profiles of specific neuronal cell-types is important for understanding their molecular identities. Genome-wide gene expression profiles of genetically defined cell-types can be acquired by collecting and sequencing mRNA that is bound to epitope-tagged ribosomes (TRAP; Translating Ribosome Affinity Purification). Here, we introduce a transgenic mouse model that combines the TRAP technique with the tetracycline transactivator (tTA) system by expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under control of the tetracycline response element (tetO-TRAP). This allows both spatial control of EGFP-L10a expression through cell-type specific tTA expression, as well as temporal regulation by inhibiting transgene expression through the administration of doxycycline. We show that crossing tetO-TRAP mice with transgenic mice expressing tTA under the Camk2a promoter (Camk2a-tTA) results in offspring with cell-type specific expression of EGFP-L10a in CA1 pyramidal neurons and medium spiny neurons in the striatum. Co-immunoprecipitation confirmed that EGFP-L10a integrates into a functional ribosomal complex. In addition, collection of ribosome-bound mRNA from the hippocampus yielded the expected enrichment of genes expressed in CA1 pyramidal neurons, as well as a depletion of genes expressed in other hippocampal cell-types. Finally, we show that crossing tetO-TRAP mice with transgenic Fos-tTA mice enables the expression of EGFP-L10a in CA1 pyramidal neurons that are activated during a fear conditioning trial. The tetO-TRAP mouse can be combined with other tTA mouse lines to enable gene expression profiling of a variety of different cell-types

Behavioral Responses to Hypoxia in Drosophila Larvae Are Mediated by Atypical Soluble Guanylyl Cyclases

The three Drosophila atypical soluble guanylyl cyclases, Gyc-89Da, Gyc-89Db, and Gyc-88E, have been proposed to act as oxygen detectors mediating behavioral responses to hypoxia. Drosophila larvae mutant in any of these subunits were defective in their hypoxia escape response—a rapid cessation of feeding and withdrawal from their food. This response required cGMP and the cyclic nucleotide-gated ion channel, cng, but did not appear to be dependent on either of the cGMP-dependent protein kinases, dg1 and dg2. Specific activation of the Gyc-89Da neurons using channel rhodopsin showed that activation of these neurons was sufficient to trigger the escape behavior. The hypoxia escape response was restored by reintroducing either Gyc-89Da or Gyc-89Db into either Gyc-89Da or Gyc-89Db neurons in either mutation. This suggests that neurons that co-express both Gyc-89Da and Gyc-89Db subunits are primarily responsible for activating this behavior. These include sensory neurons that innervate the terminal sensory cones. Although the roles of Gyc-89Da and Gyc-89Db in the hypoxia escape behavior appeared to be identical, we also showed that changes in larval crawling behavior in response to either hypoxia or hyperoxia differed in their requirements for these two atypical sGCs, with responses to 15% oxygen requiring Gyc-89Da and responses to 19 and 25% requiring Gyc-89Db. For this behavior, the identity of the neurons appeared to be critical in determining the ability to respond appropriately

Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis

<div><p>Abstract</p><p>Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a <i>Drosophila</i> cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes <i>Tdc</i>2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity.</p></div

A transgenic mouse line for collecting ribosome-bound mRNA using the tetracycline transactivator system. Frontiers in molecular neuroscience

Acquiring the gene expression profiles of specific neuronal cell-types is important for understanding their molecular identities. Genome-wide gene expression profiles of genetically defined cell-types can be acquired by collecting and sequencing mRNA that is bound to epitope-tagged ribosomes (TRAP; translating ribosome affinity purification). Here, we introduce a transgenic mouse model that combines the TRAP technique with the tetracycline transactivator (tTA) system by expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under control of the tetracycline response element (tetO-TRAP). This allows both spatial control of EGFP-L10a expression through cell-type specific tTA expression, as well as temporal regulation by inhibiting transgene expression through the administration of doxycycline. We show that crossing tetO-TRAP mice with transgenic mice expressing tTA under the Camk2a promoter (Camk2a-tTA) results in offspring with cell-type specific expression of EGFP-L10a in CA1 pyramidal neurons and medium spiny neurons in the striatum. Co-immunoprecipitation confirmed that EGFP-L10a integrates into a functional ribosomal complex. In addition, collection of ribosome-bound mRNA from the hippocampus yielded the expected enrichment of genes expressed in CA1 pyramidal neurons, as well as a depletion of genes expressed in other hippocampal cell-types. Finally, we show that crossing tetO-TRAP mice with transgenic Fos-tTA mice enables the expression of EGFP-L10a in CA1 pyramidal neurons that are activated during a fear conditioning trial. The tetO-TRAP mouse can be combined with other tTA mouse lines to enable gene expression profiling of a variety of different cell-types

Mutation of the <i>Tdc2</i> gene results in decreased activity and circadian arrhythmicity for adult locomotor activity.

<p>(A) Quantification of average activity level, average rhythmicity index (RI), and percent of rhythmic flies in wild-type and <i>Tdc2^RO54</i> populations. <i>n</i> = 25 for control; <i>n</i> = 29 for <i>Tdc2^RO54</i>. Error bars represent SEM. *<i>p</i><0.0001. (B) Representative actograms, mean activity, and correlograms for control flies and the <i>Tdc2^RO54</i> mutant.</p

Expression of EGFP-L10a and assays of function in clock cells.

<p>(A–C) Expression of EGFP-L10a in a large neurosecretory cell. Nu, nucleolus; N, Nucleus; C, Cytoplasm. Staining for a nuclear protein called LARK (red signal) is used to identify the nucleus. (D) Schematic representation of the structure of the nucleolus. FC, Fibrillar Center; DFC, Dense Fibrillar Components; GC, Granular Components. GC is the location of ribosome assembly. (E) Expression pattern of EGFP-L10a in the brain and ventral ganglion using the <i>elav-Gal4</i> pan-neuronal driver. (F) Expression of EGFP-L10a in all clock cells driven by <i>tim-Gal4</i>. (G) Restricted expression of EGFP-L10a to clock neuron but not glia using a combination of <i>tim-Gal4</i> and <i>repo-Gal80</i>. (H) Expression of EGFP-L10a in clock cells does not disrupt normal circadian behavior. Left panels shows representative free-running actograms of control flies and flies expressing EGFP-L10a in either PDF neurons (using <i>pdf-Gal4</i>) or all clock cells (using <i>tim-Gal4</i>). Right panels show the corresponding correlograms. (I) TRAP is capable of detecting changes in mRNA translation, as assayed by changes in the translational status of Ferritin 1 Heavy Chain Homolog (Fer1HCH) mRNA in response to overexpression of the Iron Regulatory Protein (IRP). Control, <i>w¹¹¹⁸; act5C-Gal4/tub-Gal80^ts; UAS-EGFP-L10a/+</i>. IRP overexpression, <i>w¹¹¹⁸; act5C-Gal4/tub-Gal80^ts; UAS-EGFP-L10a/UAS-IRP</i>. (J) Circadian changes in the translation of period (<i>per</i>) and timeless (<i>tim</i>) mRNAs. Genotype of the flies assayed, <i>elav-Gal4; UAS-EGFP-L10a/+</i>. Error bar represents standard error of the mean (SEM). *<i>p</i><0.01; **<i>p</i><0.001 (Student's <i>t</i> test).</p

Biological processes represented by the rhythmically translated mRNAs.

<p>(A) Pie chart showing different represented processes. The number of mRNAs belonging to each category is shown next to each slice of the pie. (B) Translational profile of thioredoxin system mRNAs.</p

TDC2 protein shows circadian changes in the PDF-positive large ventral lateral neurons (l-LNvs) and dorsal lateral neurons (LNds).

<p>(A–B) Translational profile of <i>Tdc2</i> revealed by RNA sequencing (A) and Q-PCR (B). In the Q-PCR graph, the level of mRNA expression for the first time point (CT0) serves as a reference, and is thus designated a value of 1. RNA expression levels at other time points are plotted relative to the value at CT0. Negative and positive error bars show the range of possible relative values calculated based on the SEM of the Ct values obtained in the Q-PCR experiments. <i>n</i>≥4 for all time points. (C) Abundance of TDC2 protein in the l-LNvs and LNds at two different times of the circadian cycle, using immunohistochemical methods. (D) Sample images showing differential expression of TDC2 in l-LNvs (red channel) at ZT1 and ZT9. Quantification of average pixel intensities is described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001703#s5" target="_blank">Materials and Methods</a> section. For LNvs, 10 pairs of brain hemispheres were compared between ZT1 and ZT9. For LNds, nine pairs of brain hemispheres were compared between ZT1 and ZT9. *<i>p</i><0.01; **<i>p</i><1.5E-05 based on paired Student's <i>t</i> test.</p

Knockdown of <i>Tdc2</i> in clock neurons results in circadian behavioral arrhythmicity.

<p>(A–E) Representative actograms showing free-running locomotor activity of flies with a Tdc2 knockdown in PDF neurons (A) or all clock neurons (B), as well as relevant control files (C–E). (F) Quantification of the average rhythmicity index (RI) for various genotypes. Number of flies tested is indicated on the histograms. *<i>p</i><1.4E-30 for comparison with the control groups based on Student's <i>t</i> test.</p