A conserved juxtacrine signal regulates synaptic partner recognition in Caenorhabditis elegans

<p>Abstract</p> <p>Background</p> <p>An essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism <it>Caenorhabditis elegans</it>.</p> <p>Results</p> <p>Here, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in <it>unc-6 </it>and <it>unc-40 </it>animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in <it>unc-40 </it>mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners.</p> <p>Conclusions</p> <p>These results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization.</p

The pioneer round of translation ensures proper targeting of ER and mitochondrial proteins

The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC???SRP interaction safeguards against abnormal expression of polypeptides from a ribosome???nascent chain complex (RNC)???SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC???SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins

The loss of ammonia response localizes to the <i>Amt</i> gene.

<p>(A) Traces on the left show that the responses to a 500 ms pulse of 0.1% ammonia are similar in flies heterozygous for the <i>Amt¹</i> transposon and the <i>Df(3R)BSC471</i> deficiency that removes ∼30 kb including <i>Amt</i> and nine other genes. In contrast, <i>Amt¹</i>/<i>Df(3R)BSC471</i> flies have greatly reduced responses to ammonia (n = 9 each, p<0.0001). Averaged data are shown in the graph on the right. (B) The lack of response to 0.1% ammonia in ac1 sensilla from <i>Amt¹</i> flies is rescued by the addition of a genomic fragment containing <i>Amt</i> and the neighboring gene <i>Hsc70-4</i> (n = 8 each, p<0.0001). (C) The response to ammonia in <i>Amt¹</i> mutant flies is also rescued by transgenic expression of <i>UAS-Amt</i> under the control of an <i>Amt-Gal4</i> promoter (n = 9 each, p<0.0001).</p

The <i>Amt¹</i> defect is restricted to the ammonia response of ac1 sensilla.

<p>(A) Odor responses of ac1 sensilla in <i>Amt¹</i>. Ammonia, 2-oxovaleric acid, and pyrrolidine each activate one of the three ORNs in ac1; only the ammonia response was impaired (n = 10–11, p<0.0001). (B–D) Response profiles of other sensillar types appeared normal (n = 8–9 each).</p

An RNA-Seq Screen of the <i>Drosophila</i> Antenna Identifies a Transporter Necessary for Ammonia Detection

<div><p>Many insect vectors of disease detect their hosts through olfactory cues, and thus it is of great interest to understand better how odors are encoded. However, little is known about the molecular underpinnings that support the unique function of coeloconic sensilla, an ancient and conserved class of sensilla that detect amines and acids, including components of human odor that are cues for many insect vectors. Here, we generate antennal transcriptome databases both for wild type <i>Drosophila</i> and for a mutant that lacks coeloconic sensilla. We use these resources to identify genes whose expression is highly enriched in coeloconic sensilla, including many genes not previously implicated in olfaction. Among them, we identify an ammonium transporter gene that is essential for ammonia responses in a class of coeloconic olfactory receptor neurons (ORNs), but is not required for responses to other odorants. Surprisingly, the transporter is not expressed in ORNs, but rather in neighboring auxiliary cells. Thus, our data reveal an unexpected non-cell autonomous role for a component that is essential to the olfactory response to ammonia. The defective response observed in a <i>Drosophila</i> mutant of this gene is rescued by its <i>Anopheles</i> ortholog, and orthologs are found in virtually all insect species examined, suggesting that its role is conserved. Taken together, our results provide a quantitative analysis of gene expression in the primary olfactory organ of <i>Drosophila</i>, identify molecular components of an ancient class of olfactory sensilla, and reveal that auxiliary cells, and not simply ORNs, play an essential role in the coding of an odor that is a critical host cue for many insect vectors of human disease.</p></div

Expression of <i>Amt.</i>

<p>(A) RT-PCR analysis of <i>Amt</i> expression in CS. <i>Synaptogmin</i> was used as a positive control. (B) Whole-mount confocal image of a third antennal segment of an <i>Amt-GAL4; UAS-mCD8::GFP</i> fly. GFP expression is seen in large, amorphous auxiliary cells, but not in neurons. White arrowheads indicate the sacculus here and in panels E and F. Scale bar  = 30 µm. (C) Confocal image of an <i>in situ</i> hybridization to an antennal section from an <i>Amt-GAL4; UAS-mCD8::GFP</i> fly using antisense probes for <i>Amt</i> (red) and <i>GFP</i> (green). The two probes co-localize. (D, E, G, H), confocal images of antennal sections labeled with an antisense probe for <i>Amt</i> (red) and an antibody against GFP (green) driven by (D) <i>IR76b-Gal4</i>, (E) <i>IR8a-Gal4</i>, (G) <i>IR92a-Gal4</i>, and (H) <i>IR76a-Gal4</i>. <i>IR76b-Gal4</i> and <i>IR8a-Gal4</i> are co-receptors that label at least one ORN in each surface coeloconic sensillum type (ac1–4). <i>IR8a-Gal4</i> also labels coeloconic ORNs in the third chamber of the sacculus. <i>Amt</i> is detected in larger neighboring auxiliary cells in a subset of the coeloconic sensilla. (F) Confocal image of an <i>in situ</i> hybridization to an antennal section from a CS fly using antisense probes for <i>Amt</i> (red) and <i>Obp84a</i> (green). <i>Amt</i> is expressed in different auxiliary cells from those that express <i>Obp84a</i>, which is also expressed in coeloconic sensilla. (G) Expression of <i>Amt</i> on the antennal surface is found surrounding the IR92a ammonia receptor-expressing neurons, which are in ac1. (H) <i>Amt</i> is not detected in ac4 sensilla, which contain ORNs that express <i>IR76a</i>. (I, J) Higher magnification images of (F) and (G) respectively.</p