Complementary Expression of Immunoglobulin Superfamily Ligands and Receptors in Synaptic Pairs in the Drosophila Visual System
ABSTRACT: Purpose: Information processing in the brain relies on precise patterns of synapses between neurons. The molecular mechanisms by which this specificity is achieved remains elusive. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Methods: we developed methods to purify seven neuronal cell types (R7, R8 and L1-L5 neurons) using Fluorescence Activated Cell Sorting. Results: we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA sequencing and MiMIC-based protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of Immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig superfamily proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity. Conclusions: This complexity is mirrored by the complexity of the cell surface and secreted molecules expressed by each of the R cell and lamina neurons profiled in this study. How this complexity contributes to specificity remains elusive, but the convergence of improved histological, genetic and molecular tools promises to provide important insights into the molecular recognition strategies controlling synaptic specificity. We chose 7 time points for RNA-seq analysis of R cells during pupal development corresponding to 24, 35, 40, 45, 53, 65 and 96 hrs after pupal formation (APF).
Project description:The goal of this gene expression profiling experiment was to identify the entire set of transcription factors expressed during late pupal wing development (~80h APF) when pigmentation genes are expressed We used Affymetrix microarrays to profile the expression of the transcription factors in late pupal wings of Oregon R flies. The staging of the flies was made by eye, based on the extent of wing pigmentation. RNA was extracted from batches of 40 flies and three biological replicates were analyzed.
Project description:The mammalian forebrain is a tissue of stunning complexity comprised of numerous regions each containing many distinct cell types that differ in their intrinsic and synaptic physiology, morphology and connectivity. These differences are likely conferred by differential gene expression, but the extent and nature of cell type specific gene expression is largely unknown. Here, we carried out microarray analysis of twelve major classes of fluorescently labelled neurons within the forebrain and provide the first comprehensive view of gene expression differences. The results demonstrate a profound molecular heterogeneity among neuronal subtypes, represented disproportionately by gene paralogs, and begin to reveal the genetic programs underlying the fundamental divisions between neuronal classes including that between glutamatergic and GABAergic neurons. Experiment Overall Design: Total of 36 samples from 12 different cell types were anlyzed. Three biological replicates (each sample from different animal(s)) were analyzed for each cell type.
Project description:Loh KH, Stawski PS, Draycott AS, Udeshi ND, Lehrman EK, Wilton DK, Svinkina T, Deerinck TJ, Ellisman MH, Stevens B, Carr SA, Ting AY. Cell 2016 Excitatory synapses are connections between neurons that promote the propagation of action potentials while inhibitory synapses repress them. Normal brain function relies on the careful balance of these antagonistic connections, which occur via molecularly distinct synaptic clefts. Understanding how this is
achieved relies on knowledge of their protein compositions, yet the clefts remain uncharacterized because they cannot be isolated biochemically. Here, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons, using a spatially restricted enzymatic tagging strategy. These proteomes reveal dozens of novel synaptic candidates, and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a specificity factor regulating the presynaptic neurotransmitter recruiting activity of Neuroligin-2 at inhibitory synapses.
Project description:Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6-/-Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD) Anterior and posterior lobes of PN7 mouse cerebellum were isolated separately and differentially expressed genes were identified.
Project description:The sense of hearing depends on the faithful transmission of sound information from the ear to the brain by spiral ganglion (SG) neurons. However, how SG neurons develop the connections and properties that underlie auditory processing is largely unknown. We catalogued gene expression in mouse SG neurons at six developmental stages, ranging from embryonic day 12 (E12), when SG neurons first extend projections, up until postnatal day 15 (P15), after the onset of hearing. For comparison, we also analyzed the closely-related vestibular ganglion (VG) at the same time points. To identify genes involved in SG axon guidance and branching, target selection, synaptogenesis, synaptic refinement, and synaptic function, we collected SG at E12 and E13, E16, P0, P6, and P15. We also collected VG at the same time points. For E12 and E13 time points, SG and VG were microdissected from Rnx-cre; Z/EG embryos, which express GFP in the VG. E16-P15 VG was also isolated by microdissection from Rnx-cre; Z/EG animals. E16-P15 SG neurons were isolated by FACS sorting dissociated cochlea from Mafb-GFP animals.
Project description:An understanding of how heterozygous loss-of-function mutations in ASD risk genes, such as TBR1, contribute to ASD remains elusive. Conditional Tbr1 deletion during late mouse gestation in cortical layer 6 neurons (Tbr1layer6 mutants) provides novel insights into its function, including dendritic patterning, synaptogenesis, and cell intrinsic physiology. These phenotypes occur in heterozygotes, providing insights into mechanisms that may underlie ASD pathophysiology. Restoring expression of Wnt7b, largely rescues the synaptic deficit in Tbr1layer6 mutant neurons. Furthermore, Tbr1layer6 heterozygotes have increased anxiety-like behavior, a phenotype seen ASD. Integrating TBR1 ChIP-Seq and RNA-Seq data from layer 6 neurons, and activity of TBR1 bound candidate enhancers, provides evidence for how TBR1 regulates layer 6 properties. Moreover, several putative TBR1 targets are ASD risk genes, placing TBR1 in a central position both for ASD risk and for regulating transcriptional circuits that control multiple steps in layer 6 development essential for the assembly of neural circuits. Overall design: Examination of TBR1 genomic binding in P2 wildtype mouse cortex
Project description:Interstitial cells of Cajal (ICC) have important functions in regulation of motor activity in the gastrointestinal tract. In murine small intestine ICC are gathered in the region of the myenteric plexus (ICC-MY) and within the deep-muscular plexus near the submucosal surface of the circular muscle layer (ICC-DMP). These two classes of ICC have different physiological functions. ICC-MY are pacemaker cells and generate the slow wave electrical rhythmicity of gastrointestinal organs. ICC-DMP form synaptic connections with the varicose nerve terminals of enteric motor neurons and are involved in reception and transduction of motor neurotransmission. In the present study we used recently developed highly selective techniques to isolate the two classes of ICC from enzymatically dispersed intestinal muscles by fluorescence-activated cell sorting. Transcriptional expression of the two functional classes was investigated using DNA microarray analysis. Experiment Overall Design: ICC-DMP and ICC-MY cells were isolated from the murine small intestinal tissues and their transcriptional expression was compared with that of the tunica muscularis tissues. Transcriptional expression profiles of ICC-DMP and ICC-MY were compared to each other also.
Project description:Recent advances in single-cell RNAseq technologies are enabling new cell type classifications. For neurons, electrophysiological properties traditionally guide cell type classification but correlating RNAseq data with electrophysiological parameters has been difficult. Here we demonstrate RNAseq of electrophysiologically and synaptically characterized individual, patched neurons in the hippocampal CA1-region and subiculum, and relate the resulting transcriptome data to their electrical and synaptic properties. In this analysis, we explored the hypothesis that precise combinatorial interactions between matching cell-adhesion and signaling molecules shape synapse specificity. In analyzing interneurons and pyramidal neurons that are synaptically connected, we identified two independent, developmentally regulated networks of interacting genes encoding cell-adhesion, exocytosis and signal-transduction molecules. In this manner, our data allow postulating a presumed cell-adhesion and signaling code, which may explain neuronal connectivity at the molecular level. Our approach enables correlating electrophysiological with molecular properties of neurons, and suggests new avenues towards understanding synaptic specificity. Overall design: These data include 15 tissue samples (including 3 independent replicas in 5 developmental stages) as well as 93 single-cell samples (including CA1 cholecystokinin, parvalbumin, and pyramidal neurons as well as subiculum burst and regular firing pyramidal neurons).
Project description:Interstitial cells of Cajal (ICC) have important functions in regulation of motor activity in the gastrointestinal tract. In murine small intestine ICC are gathered in the region of the myenteric plexus (ICC-MY) and within the deep-muscular plexus near the submucosal surface of the circular muscle layer (ICC-DMP). These two classes of ICC have different physiological functions. ICC-MY are pacemaker cells and generate the slow wave electrical rhythmicity of gastrointestinal organs. ICC-DMP form synaptic connections with the varicose nerve terminals of enteric motor neurons and are involved in reception and transduction of motor neurotransmission. In the present study we used recently developed highly selective techniques to isolate the two classes of ICC from enzymatically dispersed intestinal muscles by fluorescence-activated cell sorting. Transcriptional expression of the two functional classes was investigated using DNA microarray analysis. Keywords: comparative transcriptional profiling Overall design: ICC-DMP and ICC-MY cells were isolated from the murine small intestinal tissues and their transcriptional expression was compared with that of the tunica muscularis tissues. Transcriptional expression profiles of ICC-DMP and ICC-MY were compared to each other also.
Project description:Stem cells are highly abundant and proliferate rapidly during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. Here, we show that changes in energy metabolism induced by the steroid hormone Ecdysone initiate an irreversible cascade of events leading to cell cycle exit in Drosophila neural stem cells. We show that the timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple cell cycle progression from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Neuroblasts isolated from brain tumors fail to undergo this shrinkage process and this may explain why they are immortalized. Our findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation control in stem cells. Comparison of transcriptomes of Drosophila melanogaster central brain NBs from wild-type larval NBs, wild type pupal NBs and med27 RNAi pupal NBs.