1. 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

2. Caenorhabditis elegans Understand the cellular and molecular mechanisms that underlie synapse formation in vivo Studied en passant synapses formed by HSNL near the C. elegans vulva wormatlas.org C. elegans  Well Characterized Nervous System  En Passant Synapses  Presynaptic Specializations  Postsynaptic Specializations C. elegans II, pg 613

3. Postembryonic Development Figure adapted fr www.eb.tuebingen.mpg.de/.../wurm_entw.html devgen.com/.../productoffering/c_elegans.html biol.unt.edu/~ppadilla/WormLarvae.jpg

4. 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 P5.p, P6.p & P7.p  to adopt vulval fates P3.p, P4.p & P8.p P3.p, P4.p & P8.p  to adopt non-vulval fates 3) VPCs execute the specified 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 mature vulva P3P4P5P6P7P8 TTTTLLT N LLTN SSSS NB SS NBNBNBNBNB L1 L2 L3

5. Vulval Cell Neurons (VC) L1  born L3  send out processes (late L3) L4  branch in the region of the vulva VC3 VC4 VC6 VC5 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 HSNR HSNL Egg Laying Neurons wormatlas.org.veryfunnypics.com/ pics/food/images/eggs.jpg

6. HSN In Wild Type Animals synaptic vesicle clustering in HSNL is not dependent on postsynaptic neurons and muscles HSNL forms en passant synapses onto the VC4, 5 motor neurons and the vulval muscles near the vulva region something is contacting HSNL and acting as synaptic guidepost to directing HSNL presynaptic vesicles to adjacent regions wormatlas.org

7. The Ig Superfamily Protein SYG-1 Determines the Location of Specific Synapses in C. elegans (Cell, Vol. 112, 619-630, March 7, 2003) SYG-1 protein is localized to the site of future synapses. Anterior displacement of the vulva  Anterior displacement of the vulva  SYG-1 localization is displaced anteriorly matching the position of the ectopic vulva MUV’s  MUV’s  SYG-1 localizes to each of the vulva Elimination of vulval epithelial cells results in diffuse localization of SYG-1 on HSNL axon 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

8. 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 syg-2 encodes the guidepost signal in the vulval epithelial cells Suggest a SYG-1/SYG-2 interaction stimulates the formation of appropriate synapses while suppressing inappropriate synapses Goal of this Paper wormatlas.org

9. Mutations in syg-2 disrupt synaptic vesicle clustering in HSNL SNB-1 (synaptobrevin)  synaptic vesicle protein that labels synaptic vesicle clusters in C. elegans and other animals WT animals syg-1(ky652) syg-2(ky673) syg-2(ky671) ↓ accumulation of SNB-1::YFP near the vulva lin-11(n566) is associated with cell lineage defects in the 1° & 2° vulval cell lineage 1° vulval cells are generated but fail to function normally lin-11(n566) 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

10. (Pre/Post)synapse in some cases see thickening of post synaptic membrane (difficult to visualize) electron dense presynaptic specializations vesicle dense core vesicle C. elegans II, pg 614

11. 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) syg-1(ky652) syg-2(ky673)syg-2(ky671) Wild Type triangles represent active zone; diamonds represent # of synaptic vesicles

12. SYG-2 Encodes an Immunoglobulin Superfamily Protein 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) 7 Ig Domains Fibronectin typeIII TypeII PDZ

13. NEPH1 and nephrin are the mammalian orthologs which play a role in kidney development SYG-1 SYG-2 4 Drosophila play a role in Drosophila muscle function

14. SYG-2 Expression Across Development 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 SYG-2::GFP expression disappeared within the first 6hrs of adulthood SYG-1::GFP Mid L4 Mid L3 SYG-1::GFP clusters at the same time that SYG-2::GFP expression begins YAD Disappearance of SYG-2::GFP correlates to more diffuse SYG-1::GFP distribution SYG-2 required for early synaptic specification, not for maintenance of synaptic function

15. 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 Suggests that SYG-2 is essential for SYG-1 localization at synaptic sites. note unc-86 promoter expressed only in HSN neurons WT animals SYG-1 was localized to the segment of the axon contacting the primary vulval epithelial cells syg-2(ky671) & syg-2(ky673) SYG-1 was diffusely distributed on the HSNL axon, Wild Type syg-2(ky671) syg-2(ky673)

16. 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) egl-17 is expressed in a subset of vulval epithelial cells egl-17 is expressed in a subset of vulval epithelial cells Wild Type SYG-1::GFP SNB-1::GFP SYG-2 expression in the 2° vulval epithelial cells is sufficient to attract SYG-1 protein from HSNL axon The interaction of SYG-1 & SYG-2 is sufficient to trigger the clustering of SNB-1 vesicles

17. Do SYG-1 & SYG-2 Interact Directly? Drosophila S2 cell aggregation assay with SYG-1 & SYG-2 GFP S2 cells were cotransfected  SYG-1 cDNA & GFP dsRED  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

18. 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 directinteraction specifies the location of synapses drives the selection of appropriate postsynaptic targets excludes inappropriate HSNL targets synapticspecificity

19. Summary HSNL Wild-Type Animals contacts VC axons & vulval muscles 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells contacts SYG-1 SYG-2 SYG-1 SYG-2 ? ? syg-1 & syg-2 Mutants HSNL contacts VC axons & vulval muscles 1° & 2° vulval epithelial cells PVQL, PVPR, AVKL axons body wall muscles hypodermal cells contacts ? ?