Download 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