synaptic specificity is generated by the synaptic guidepost protein syg-2 and it’s receptor, syg-1

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Synaptic Specificity Synaptic Specificity is Generated by the is Generated by the Synaptic Guidepost Synaptic Guidepost Protein SYG-2 and Protein SYG-2 and it’s Receptor, SYG-1 it’s Receptor, SYG-1 Kang Shen, Richard D. Fetter Kang Shen, Richard D. Fetter and Cornelia I. Bargmann and Cornelia I. Bargmann Cell, Vol. 116, 869 – 881, March 19, Cell, Vol. 116, 869 – 881, March 19, 2004 2004

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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. C. elegans II, pg 613. wormatlas.org. Caenorhabditis elegans. - PowerPoint PPT Presentation

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Page 1: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 2: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

leg

an

s II, p

g 6

13

Page 3: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 4: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 5: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 6: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

worm

atl

as.

org

Page 7: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 8: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 9: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 10: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

(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

C. e

leg

an

s II, p

g 6

14

Page 11: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 12: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 13: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 14: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 15: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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)

Page 16: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 17: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 18: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

Page 19: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1

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

?

?

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

?

?

Page 20: Synaptic Specificity is Generated by the Synaptic Guidepost Protein SYG-2 and it’s Receptor, SYG-1