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 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/Worm. Larvae. jpg
4 Stages of Vulval Development 1) Late L 1 , the VPCs are born 2) Early L 3 cell – cell interactions specify the fates of VPCs signaling events specify P 5. p, P 6. p & P 7. p to adopt vulval fates P 3. p, P 4. p & P 8. 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 L 1 P 3 NB P 4 P 5 P 6 P 7 P 8 NB NB NB L 2 L 3 SS SS L L T N T T NL L T SS
Egg Laying Neurons HSNR VC 6 VC 3 VC 5 VC 4 HSNL wormatlas. org Vulval Cell Neurons (VC) L 1 born L 3 send out processes (late L 3) L 4 branch in the region of the vulva HSN’s are born in the embryo and migrate near gonadal primordium (mid-body) prior to hatching L 2 & L 3 axon outgrowth L 3 & L 4 synapse formation. veryfunnypics. com/ pics/food/images/eggs. jpg
wormatlas. org HSN In Wild Type Animals HSNL forms en passant synapses onto the VC 4, 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(ky 652) 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 MUV’s SYG-1 localizes to each of the 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 other animals and syg-1(ky 652) lin-11(n 566) is associated with cell lineage defects syg-2(ky 673) in the 1° & 2° vulval cell lineage 1° vulval cells are generated but fail to function normally lin-11(n 566) syg-2(ky 671) ↓ 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 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& syg 2 Mutants Scanning EM; 150μm region centered on the vulva 1 WT L 4; 1 L 4 syg-1(ky 652); L 4 syg-2(ky 673); L 4 syg-2(ky 671) 2 1 Wild Type syg-1(ky 652) syg-2(ky 673) syg-2(ky 671) triangles represent active zone; diamonds represent # of synaptic vesicles
SYG-2 Encodes an Immunoglobulin Superfamily Protein 7 Ig Domains Type. II PDZ Fibronectin type. III syg-2(ky 671) G to A mutation at SL 1 splice acceptor site at the first exon of syg-2 gene LOF mutation in the syg-2 gene syg-2(ky 673) 32 kb deletion (affects 4 predicted ORF)
SYG-1 SYG-2 4 Drosophila a role in Drosophila play muscle function NEPH 1 and nephrin are the mammalian orthologs which play a role in kidney development
SYG-2 Expression Across Development SYG-1: : GFP Mid L 3 SYG-1: : GFP clusters at the same time that SYG-2: : GFP expression begins Mid L 4 SYG-2: : GFP Mid L 3 SYG-2: : GFP expressed in 1° epithelial cells Mid L 4 ↑ 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 6 hrs 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(ky 671) syg-2(ky 673) & Wild Type syg-2(ky 673) SYG-1 was diffusely distributed on the HSNL axon, syg-2(ky 671) 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(ky 673) 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 cell aggregation assay with SYG-1 & SYG-2 S 2 cells were cotransfected SYG-1 c. DNA & GFP SYG-2 c. DNA & ds. RED 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 60 min 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 HSNL ts ntac co cont acts SYG-1 VC axons & vulval muscles SYG-2 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells ? syg-1 & syg-2 Mutants HSNL ts ntac co cont acts VC axons & vulval muscles 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells ? ? SYG-1 SYG-2
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