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Synaptic Specificity is Synaptic Specificity is Generated by the Generated by the
Synaptic Guidepost Synaptic Guidepost Protein SYG-2 and it’s Protein SYG-2 and it’s
Receptor, SYG-1Receptor, SYG-1
Kang Shen, Richard D. Fetter and Kang Shen, Richard D. Fetter and Cornelia I. BargmannCornelia I. Bargmann
Cell, Vol. 116, 869 – 881, March 19, 2004Cell, Vol. 116, 869 – 881, March 19, 2004
Caenorhabditis elegansCaenorhabditis elegansUnderstand the cellular and molecular mechanisms that underlie synapse Understand the cellular and molecular mechanisms that underlie synapse
formation formation in vivoin vivo
Studied en passant synapses formed by HSNL near the Studied en passant synapses formed by HSNL near the C. elegansC. elegans vulva vulva
wormatlas.org
C. elegans Well Characterized Nervous System En Passant Synapses
Presynaptic Specializations Postsynaptic Specializations
C. e
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Postembryonic Postembryonic DevelopmentDevelopment
Figure adapted fr www.eb.tuebingen.mpg.de/.../wurm_entw.html
devgen.com/.../productoffering/c_elegans.htm
l
biol.unt.edu/~ppadilla/WormLarvae.jpg
4 Stages of Vulval 4 Stages of Vulval DevelopmentDevelopment1)1) Late L1 Late L1 , the VPCs are born, the VPCs are born
2)2) Early L3 Early L3 cell – cell interactions specify the fates of VPCs cell – cell interactions specify the fates of VPCs
signaling events specifysignaling events specify P5.p, P6.p & P7.pP5.p, P6.p & P7.p to adopt vulval fatesP3.p, P4.p & P8.pP3.p, P4.p & P8.p to adopt non-vulval fates
3)3) VPCs execute the specified fatesVPCs execute the specified fates (generate # & types of vulval cells)
4)4) Vulval cells undergo cell movements, cell fusion and eversion to form Vulval cells undergo cell movements, cell fusion and eversion to form mature vulvamature vulva
P3P3 P4P4 P5P5 P6P6 P7P7 P8P8
TTTT TT TT LL LL TTNNLL LL TT NNSS SS SS SS
NBNB
SSSS
NBNBNBNBNBNBNBNBNBNB
L1L1
L2L2
L3L3
Vulval Cell Neurons (VC)Vulval Cell Neurons (VC)L1 bornL3 send out processes (late L3)L4 branch in the region of the vulva
VC3VC3
VC4VC4
VC6VC6
VC5VC5
HSN’s are born in the embryo and migrate near gonadal HSN’s are born in the embryo and migrate near gonadal primordium (mid-body) prior to hatchingprimordium (mid-body) prior to hatching
L2 & L3 axon outgrowthL3 & L4 synapse formation
HSNRHSNR
HSNLHSNL
Egg Laying Neurons Egg Laying Neurons
wormatlas.org
.veryfunnypics.com/ pics/food/images/eggs.jpg
HSNHSN
In Wild Type AnimalsIn 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
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The Ig Superfamily Protein SYG-1 The Ig Superfamily Protein SYG-1 Determines the Location of Specific Determines the Location of Specific
Synapses in Synapses in C. elegansC. elegans (Cell, Vol. 112, 619-630, March 7, 2003)(Cell, Vol. 112, 619-630, March 7, 2003)
SYG-1 protein is localized to SYG-1 protein is localized to the site of future synapses. the site of future synapses.
Anterior displacement of the vulvaAnterior 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 Elimination of vulval epithelial cells results in diffuse cells results in diffuse
localization of SYG-1 on HSNL localization of SYG-1 on HSNL axon axon
Isolated the Mutant Isolated the Mutant syg-1(ky652)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
HypothesisHypothesis
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 PaperGoal of this Paperwormatlas.org
Mutations in Mutations in syg-2syg-2 disrupt disrupt synaptic vesicle clustering synaptic vesicle clustering
in HSNLin HSNLSNB-1 (synaptobrevin) SNB-1 (synaptobrevin)
synaptic vesicle protein that synaptic vesicle protein that labels synaptic vesicle clusters labels synaptic vesicle clusters in in C. elegansC. elegans and other animals and other animals
WT animalsWT animals
syg-1(ky652)
syg-2(ky673)
syg-2(ky671)
↓ ↓ accumulation of SNB-accumulation of SNB-1::YFP near the vulva1::YFP near the vulva
lin-11(n566)lin-11(n566) is associated with cell is associated with cell lineage defects in the 1° & 2° vulval lineage defects in the 1° & 2° vulval
cell lineage 1° vulval cells are cell lineage 1° vulval cells are generated but fail to function generated but fail to function
normallynormally lin-11(n566)
SNB-1::YFP clusters shifted to SNB-1::YFP clusters shifted to ectopic anterior positionsectopic anterior positionslin-11lin-11 mutants have defects in mutants have defects in
synaptic localization similar to those synaptic localization similar to those of syg-1 and syg-2 mutantsof syg-1 and syg-2 mutants
(Pre/Post)synapse(Pre/Post)synapse
in some cases see thickening of post synaptic membrane (difficult to visualize)
electron dense presynaptic electron dense presynaptic specializationsspecializations
vesiclevesicle
dense core dense core vesiclevesicle
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Synapse Characterization in Synapse Characterization in syg-1syg-1 & & syg2syg2 Mutants MutantsScanning EM; 150Scanning EM; 150μμm region centered on the vulva m region centered on the vulva
1 WT L4; 1 L4 1 WT L4; 1 L4 syg-1(ky652);syg-1(ky652); 2 L4 2 L4 syg-2(ky673);syg-2(ky673); 1 L4 1 L4 syg-2(ky671)syg-2(ky671)
syg-1(ky652)syg-1(ky652) syg-2(ky673)syg-2(ky673) syg-2(ky671)syg-2(ky671)Wild TypeWild Type
triangles represent active zone; diamonds represent # of synaptic vesiclestriangles represent active zone; diamonds represent # of synaptic vesicles
SYG-2 Encodes an SYG-2 Encodes an Immunoglobulin Immunoglobulin
Superfamily ProteinSuperfamily 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 genesyg-2(ky673) 32kb deletion (affects 4 predicted ORF)
7 Ig Domains
Fibronectin typeIII
TypeII PDZ
NEPH1 and nephrin are the NEPH1 and nephrin are the mammalian orthologs which play mammalian orthologs which play
a role in kidney developmenta role in kidney development
SYG-1SYG-1
SYG-2SYG-2
4 4 Drosophila Drosophila play a role in play a role in DrosophilaDrosophilamuscle functionmuscle function
SYG-2SYG-2 Expression Across Expression Across DevelopmentDevelopment
SYG-2::GFPSYG-2::GFP
Mid L3Mid L3
SYG-2::GFP expressed in 1° epithelial cellsSYG-2::GFP expressed in 1° epithelial cells
Mid L4Mid L4
↑ ↑ SYG-2::GFP expression in SYG-2::GFP expression in 1° vulval epithelial cells1° vulval epithelial cells
YADYAD
SYG-2::GFP expression disappeared SYG-2::GFP expression disappeared within the first 6hrs of adulthoodwithin the first 6hrs of adulthood
SYG-1::GFPSYG-1::GFP
Mid L4Mid L4
Mid L3Mid L3
SYG-1::GFP clusters at the same SYG-1::GFP clusters at the same time that SYG-2::GFP expression time that SYG-2::GFP expression
begins begins
YADYAD
Disappearance of SYG-2::GFP Disappearance of SYG-2::GFP correlates to more diffuse SYG-correlates to more diffuse SYG-
1::GFP distribution1::GFP distribution
SYG-2 required for early synaptic specification, not for maintenance of synaptic functionSYG-2 required for early synaptic specification, not for maintenance of synaptic function
SYG-2 Expression is necessary to SYG-2 Expression is necessary to localize SYG-1localize SYG-1Synapses 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?Does the SYG-2 protein correspond to this vulval signal?
Examined synaptic localization of SYG-1 in a Examined synaptic localization of SYG-1 in a syg-2 syg-2 MutantMutant
Suggests that SYG-2 is essential Suggests that SYG-2 is essential for SYG-1 localization at synaptic for SYG-1 localization at synaptic
sites.sites.
note note unc-86unc-86 promoter expressed only in HSN neurons promoter expressed only in HSN neurons
WT animalsWT animalsSYG-1 was localized to the segment of the axon contacting the primary vulval epithelial cells
syg-2(ky671)syg-2(ky671) & & syg-2(ky673)syg-2(ky673) SYG-1 was diffusely distributed on the HSNL axon,
Wild TypeWild Type
syg-2(ky671)syg-2(ky671)
syg-2(ky673)syg-2(ky673)
SYG-2 in secondary vulval epithelial cells SYG-2 in secondary vulval epithelial cells relocalizes SYG-1 & SNB-1 labelled relocalizes SYG-1 & SNB-1 labelled
vesiclesvesiclesDoes SYG-2 directly specify the location of SYG-1 & synaptic 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)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 cellsegl-17 is expressed in a subset of vulval epithelial cells
Wild TypeWild Type
Wild TypeWild Type
SYG-1::GFPSYG-1::GFP
SNB-1::GFPSNB-1::GFP
SYG-2 expression in SYG-2 expression in the 2° vulval the 2° vulval
epithelial cells is epithelial cells is sufficient to attract sufficient to attract SYG-1 protein from SYG-1 protein from
HSNL axon HSNL axon
The interaction of The interaction of SYG-1 & SYG-2 is SYG-1 & SYG-2 is
sufficient to trigger sufficient to trigger the clustering of the clustering of SNB-1 vesiclesSNB-1 vesicles
Do SYG-1 & SYG-2 Interact Do SYG-1 & SYG-2 Interact Directly?Directly?DrosophilaDrosophila S2 cell aggregation assay with SYG-1 & SYG-2 S2 cell aggregation assay with SYG-1 & SYG-2
S2 cells were cotransfected SYG-1 cDNA & GFPGFP SYG-2 cDNA & dsREDdsRED
transfected cells tested for
aggregation (homo or hetero)
homophilic homophilic interactions of SYG-1 interactions of SYG-1
& SYG-2 are weak& SYG-2 are weak SYG-1 SYG-1 transfected cells transfected cells
specifically specifically aggregated with aggregated with
SYG-2 SYG-2 transfected cellstransfected cells
complete complete aggregatioaggregatio
n within n within 60min of 60min of
incubationincubation
SummarySummarySynapses 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 HSNLIg superfamily protein SYG-2 is the molecular guidpost cue on the epithelial cells
SYG-2 SYG-1
direct direct interactioninteraction
specifies the location of synapsesspecifies the location of synapses
drives the selection of appropriate postsynaptic targetsdrives the selection of appropriate postsynaptic targets
excludes inappropriate HSNL targetsexcludes inappropriate HSNL targets
synapticsynapticspecificityspecificity
SummarySummary
HSNL
Wild-Type AnimalsWild-Type Animals
contactscontactsVC axons & vulval musclesVC axons & vulval muscles
1° & 2° vulval epithelial cells1° & 2° vulval epithelial cellsPVQL, PVPR, AVKL axonsPVQL, PVPR, AVKL axonsbody wall musclesbody wall muscleshypodermal cellshypodermal cells
contactscontacts
SYG-1
SYG-2
SYG-1
SYG-2
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syg-1syg-1 & & syg-2 syg-2 MutantsMutants
HSNLcontactscontactsVC axons & vulval musclesVC axons & vulval muscles
1° & 2° vulval epithelial cells1° & 2° vulval epithelial cellsPVQL, PVPR, AVKL axonsPVQL, PVPR, AVKL axonsbody wall musclesbody wall muscleshypodermal cellshypodermal cells
contactscontacts
?
?