Transcript syg-2
Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1 Kang Shen, Richard D. Fetter and Cornelia I. Bargmann Cell, Vol. 116, 869 – 881, March 19, 2004 Caenorhabditis elegans Understand the cellular and molecular mechanisms that underlie synapse formation in vivo C. elegans C. elegans II, pg 613 wormatlas.org Well Characterized Nervous System En Passant Synapses Presynaptic Specializations Postsynaptic Specializations Studied en passant synapses formed by HSNL near the C. elegans vulva Postembryonic Development devgen.com/.../productoffering/c_elegans.html Figure adapted fr www.eb.tuebingen.mpg.de/.../wurm_entw.html biol.unt.edu/~ppadilla/WormLarvae.jpg 4 Stages of Vulval Development 1) Late L1 , the VPCs are born 2) Early L3 cell – cell interactions specify the fates of VPCs signaling events specify P5.p, P6.p & P7.p to adopt vulval fates P3.p, P4.p & P8.p to adopt non-vulval fates 3) VPCs execute the specified fates (generate # & types of vulval cells) 4) Vulval cells undergo cell movements, cell fusion and eversion to form mature vulva L1 P3 NB P4 P5 P6 P7 P8 NB NB NB NB NB L2 L3 SS SS L L T N T T T T NL L T SS Egg Laying Neurons HSNR VC6 VC3 VC5 VC4 HSNL wormatlas.org Vulval Cell Neurons (VC) L1 born L3 send out processes (late L3) L4 branch in the region of the vulva HSN’s are born in the embryo and migrate near gonadal primordium (mid-body) prior to hatching L2 & L3 axon outgrowth L3 & L4 synapse formation .veryfunnypics.com/ pics/food/images/eggs.jpg wormatlas.org HSN In Wild Type Animals HSNL forms en passant synapses onto the VC4, 5 motor neurons and the vulval muscles near the vulva region synaptic vesicle clustering in HSNL is not dependent on postsynaptic neurons and muscles something is contacting HSNL and acting as synaptic guidepost to directing HSNL presynaptic vesicles to adjacent regions The Ig Superfamily Protein SYG-1 Determines the Location of Specific Synapses in C. elegans (Cell, Vol. 112, 619-630, March 7, 2003) Isolated the Mutant syg-1(ky652) Encodes an Ig superfamily protein that acts in the presynaptic HSNL axon HSNL synaptic vesicles fail to accumulate at normal synaptic locations and form ectopic anterior clusters Defects in synapse formation similar to animals lacking vulval epithelial cells SYG-1 protein is localized to the site of future synapses. Elimination of vulval epithelial cells results in diffuse localization of SYG-1 on HSNL axon Anterior displacement of the vulva SYG-1 localization is displaced anteriorly matching the position of the ectopic vulva Hypothesis Synapses made by the C. elegans HSNL neuron are initiated by the guidepost role of the primary vulval epithelial cells, acting through the TM Ig superfamily protein SYG-1 on HSNL Goal of this Paper Suggest a SYG-1/SYG-2 interaction stimulates the formation of appropriate synapses while suppressing inappropriate synapses syg-2 encodes the guidepost signal in the vulval epithelial cells wormatlas.org Mutations in syg-2 disrupt synaptic vesicle clustering in HSNL WT animals SNB-1 (synaptobrevin) synaptic vesicle protein that labels synaptic vesicle clusters in C. elegans and other animals syg-1(ky652) lin-11(n566) is associated with cell lineage defects syg-2(ky673) in the 1° & 2° vulval cell lineage 1° vulval cells are generated but fail to function normally lin-11(n566) syg-2(ky671) ↓ accumulation of SNB-1::YFP near the vulva SNB-1::YFP clusters shifted to ectopic anterior positions lin-11 mutants have defects in synaptic localization similar to those of syg-1 and syg-2 mutants (Pre/Post)synapse dense core vesicle vesicle electron dense presynaptic specializations C. elegans II, pg 614 in some cases see thickening of post synaptic membrane (difficult to visualize) Synapse Characterization in syg-1 & syg2 Mutants Scanning EM; 150μm region centered on the vulva 1 WT L4; 1 L4 syg-1(ky652); 2 L4 syg-2(ky673); 1 L4 syg-2(ky671) Wild Type syg-1(ky652) syg-2(ky673) syg-2(ky671) triangles represent active zone; diamonds represent # of synaptic vesicles SYG-2 Encodes an Immunoglobulin Superfamily Protein 7 Ig Domains TypeII PDZ Fibronectin typeIII syg-2(ky671) G to A mutation at SL1 splice acceptor site at the first exon of syg-2 gene LOF mutation in the syg-2 gene syg-2(ky673) 32kb deletion (affects 4 predicted ORF) SYG-1 SYG-2 4 Drosophila play a role in Drosophila muscle function NEPH1 and nephrin are the mammalian orthologs which play a role in kidney development SYG-2 Expression Across Development SYG-1::GFP Mid L3 SYG-1::GFP clusters at the same time that SYG-2::GFP expression begins Mid L4 SYG-2::GFP Mid L3 SYG-2::GFP expressed in 1° epithelial cells Mid L4 ↑ SYG-2::GFP expression in 1° vulval epithelial cells YAD Disappearance of SYG-2::GFP correlates to more diffuse SYG-1::GFP distribution YAD SYG-2::GFP expression disappeared within the first 6hrs of adulthood SYG-2 required for early synaptic specification, not for maintenance of synaptic function SYG-2 Expression is necessary to localize SYG-1 Synapses made by the C. elegans HSNL neuron are initiated by the guidepost role of the primary vulval epithelial cells, acting through the TM Ig superfamily protein SYG-1 on HSNL Does the SYG-2 protein correspond to this vulval signal? Examined synaptic localization of SYG-1 in a syg-2 Mutant WT animals SYG-1 was localized to the segment of the axon contacting the primary vulval epithelial cells syg-2(ky671) & syg-2(ky673) Wild Type syg-2(ky673) SYG-1 was diffusely distributed on the HSNL axon, syg-2(ky671) Suggests that SYG-2 is essential for SYG-1 localization at synaptic sites. note unc-86 promoter expressed only in HSN neurons SYG-2 in secondary vulval epithelial cells relocalizes SYG-1 & SNB-1 labelled vesicles Does SYG-2 directly specify the location of SYG-1 & synaptic vesicles? Expressed SYG-2 in 2° vulval epithelial cells using an egl-17 promoter in syg-2(ky673) Wild Type SYG-2 expression in the 2° vulval epithelial cells is sufficient to attract SYG-1 protein from HSNL axon SYG-1::GFP Wild Type The interaction of SYG-1 & SYG-2 is sufficient to trigger the clustering of SNB-1 vesicles SNB-1::GFP egl-17 is expressed in a subset of vulval epithelial cells Do SYG-1 & SYG-2 Interact Directly? Drosophila S2 cell aggregation assay with SYG-1 & SYG-2 S2 cells were cotransfected SYG-1 cDNA & GFP SYG-2 cDNA & dsRED transfected cells tested for aggregation (homo or hetero) homophilic interactions of SYG-1 & SYG-2 are weak SYG-1 transfected cells specifically aggregated with SYG-2 transfected cells complete aggregation within 60min of incubation Summary Synapses made by the C. elegans HSNL neuron are initiated by the guidepost role of the primary vulval epithelial cells, acting through the TM Ig superfamily protein SYG-1 on HSNL Ig superfamily protein SYG-2 is the molecular guidpost cue on the epithelial cells SYG-2 SYG-1 direct interaction specifies the location of synapses drives the selection of appropriate postsynaptic targets excludes inappropriate HSNL targets synaptic specificity Summary ? Wild-Type Animals SYG-1 VC axons & vulval muscles SYG-2 HSNL 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells ? syg-1 & syg-2 Mutants VC axons & vulval muscles HSNL 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells ? ? SYG-1 SYG-2