Transcript A Nematode Neuron Identity Crisis

A Nematode Neuron
Identity Crisis
Bruce Wightman
Muhlenberg College
The nervous
system is
comprised of
many different
types of
neurons
Homeodomain transcription
factors regulate neuron identity
in the vertebrate spinal cord
The nematode C. elegans
• DNA Sequence known
• Rich genetics
• Simple nervous system
• Defined anatomy
The C. elegans nervous system
Axon anatomy can be
examined by neuron-specific
promoter :: GFP fusions
Nematode
neuron-specific
promoter
GFP
Aequorea victoria
The AVK axons can be visualized
with an flp-1::gfp transgene
AVKL
flp-1
GFP
(Courtesy of C. Li)
The fax-1 gene is required
for AVK axon pathfinding
Wild Type
AVKL
fax-1 (gm83)
AVKL
fax-1 is also required for normal
levels of flp-1::gfp expression
fax-1(gm83)
Wild-Type
AVKL
AVKR
AVKL
AVKR
The flp-1 gene encodes a protein
precursor for FMRFamide-like
peptide neurotransmitter
flp-1
N
SADPNFLRF
SQPNFLRF
ASGDPNFLRF
SDPNFLRF
AAADPNFLRF
PNFLRF
AGSDPNFLRF
C
Pre-synaptic
neuron
Post-synaptic
neuron
FMRFamide is reduced in AVK
neurons of fax-1 mutants
anti-FMRFamide immunostaining
fax-1 functions in both axon
pathfinding and neurotransmitter expression
FMRFamide
FAX-1
axon pathfinding
fax-1 encodes a protein that is a
member of the nuclear
hormone receptor family
N
DBD
The FAX-1 DBD contains
two zinc- fingers
C C
Zn2+
C C
C C
Zn2+
C C
LBD
C
NHR’s regulate transcription
and bind lipophilic hormones
downstream
gene
(J. Much)
FAX-1 is a member of a
subclass of NHR’s that function in
nervous system development
FAX-1*
PNR*
NHR-67
TLL*
TLX*
NHR-111
C. elegans
Drosophila
H. sapiens
* functions
in nervous
system
Estrogen Receptor
Retinoic Acid Receptor
Mutations in human PNR cause
defects in photoreceptor identity
Normal retina
Enhanced S-cone
syndrome retina
PNR is also known as NR2E3
(J. Tanis)
fax-1 may regulate transcription
of genes involved in specification of neuron identity
pathfinding
gene
Other neuronspecific gene
flp-1
Examining FAX-1 expression
using antibodies and
immunofluroescence staining
Anti-FAX-1 antibodies
(N. Carmean)
(B. Ebert)
Immunofluorescence
staining
fax-1 is expressed in 18
neurons including the AVKs
mid-stage
embryo
adult
AVKR
RICR AVBR
AVAR
AVER
AVKL
AVB
RIC
AIY
AVK
DVA
Fax-1 Phenotypes:
Unc, Che, Fab, Wandering
AVA
AVE MI
fax-1 may regulate transcription
of genes involved in specification of AVK neuron identity
AVK pathfinding gene
Other AVKspecific gene
flp-1
ncs-1::gfp expression
in AVK requires fax-1
wild-type
ncs-1::gfp
fax-1 (gm83)
ncs-1::gfp
AVKL
AVKR
neuron-specific calcium sensor
(Gomez, et al., 2001)
Summary of gene
expression requirements
Cells:
Require
fax-1
AVK
flp-1
ncs-1
What is the function of fax-1 in
neurons other than the AVK’s?
AVA
MI AVE
AVB
RICAIY
AVK
DVA
The RIC neurons have normal
anatomy and expression of tbh-1
in fax-1 mutants
wild-type and fax-1 (gm83)
RIC
tyrosine b-hydroxylase (for octopamine biosynthesis)
(M. Alkema and H.R. Horvitz, unpublished)
Expression of NMDA glutamate
receptor subunits in AVA and
AVE requires fax-1
nmr-1::gfp
wild-type
AVA
AVE
AVA/E
AVB
RIM
fax-1 (gm83)
nmr-1::gfp
AVB
RIM
AVB
RIM
AVB
RIM
AVG
AVG
Both nmr-1 and nmr-2 show the same pattern
Expression of non-NMDA
glutamate receptor subunits
does not require fax-1
glr-1::gfp
wild-type
AVA/E
fax-1 (gm83)
glr-1::gfp
AVA/E
AVA and AVE axons appear normal in fax-1 mutants:
fax-1 not required for axon pathfinding in AVA/E
Summary of gene
expression requirements
Cells:
Require
fax-1
AVK
AVA/E
flp-1
ncs-1
nmr-1
nmr-2
Which genes may function
upstream of fax-1?
The homeodomain gene unc-42
may function upstream of fax-1
AVK
UNC-42
fax-1
• unc-42 is expressed in the AVK,
AVA, AVE and other neurons
• unc-42 mutants have AVK pathfinding defects that are similar to
fax-1 mutant defects
(Wightman et al, 1997;
Baran et al., 1999)
fax-1 is not expressed in some
neurons of unc-42 mutants
wild-type
AVA/E
Wild-type
AVA
MI AVE
FAX-1
AVB
RIC
AVK
AVB
RICAIY
AVK
unc-42(e419)
AVA/E
FAX-1
RIC
AVK
DVA
unc-42 (e419)
AVA
AVE
RIC
AVK
DVA
FAX-1 is absent in AVB and two anterior neurons and reduced in AVK
unc-42 is also required
for flp-1::gfp expression
Percentage of AVK neurons
Express flp-1::gfp
fluorescence
Wild-Type
100
fax-1 (gm83)
65
unc-42 (e419)
0
unc-42 (e419);
fax-1 (gm83)
0
(S. Mathieson)
Summary of gene
expression requirements
AVK
AVA/E
Require
flp-1
ncs-1
nmr-1
nmr-2
Require
fax-1
flp-1
Cells:
fax-1
unc-42
Expression of ncs-1 also
depends on unc-42
wild-type
ncs-1::gfp
AVKL
AVKR
unc-42(e419)
ncs-1::gfp
Summary of gene
expression requirements
AVK
AVA/E
Require
flp-1
ncs-1
nmr-1
nmr-2
Require
fax-1
flp-1
ncs-1
Cells:
fax-1
unc-42
What about unc-42 and fax-1
in the AVA and AVE interneurons?
•Expression of non-NMDA
glutamate receptor subunits in
AVA/E requires unc-42
•Expression of NMDA glutamate
receptor subunits in AVA/E
does NOT require unc-42
wild-type
nmr-1
nmr-2
glr-1
glr-4
glr-5
+
+
+
+
+
unc-42(e419) fax-1(gm83)
+
+
-
+
ND
+
Axon pathfinding by AVA and/or AVE appears defective
in unc-42 mutants
(Baran et al., 1999; Brockie et al., 2001)
Summary of gene
expression requirements
AVK
AVA/E
Require
flp-1
ncs-1
nmr-1
nmr-2
Require
fax-1
flp-1
ncs-1
glr-1
glr-4
glr-5
Cells:
fax-1
unc-42
Models for the specification of
neuron identity by unc-42 and fax-1
AVK
AVA/E
UNC-42
FAX-1
glr-1
flp-1
ncs-1
FAX-1
UNC-42
Pathfinding
genes
nmr-1
glr-4
glr-5
nmr-2
Pathfinding
genes
Approaches to identification of
FAX-1 downstream targets
Other neuronspecific genes
(R. Haviland, R. Lombel, E. Murphy,
K. Reinert, E. Smith, Y. Vidgop)
Is expression of FAX-1 sufficient
to change gene expression?
heat-shock
promoter
anti-FAX-1
fax-1
hs::fax-1; fax-1(gm83)
(N. Bianco, T. Sundaresan)
Participants
fax-1
Sepi Bazel
Nick Bianco
Nicole Carmean
Sara Carr
Bryan Ebert
David Garbe
Rebecca Haviland
Chirag Kalola
Rebecca Lombel
Jason Much
Elissa Murphy
Gwen Sarver
Dennis Slade
Eric Smith
Tilak Sundaresan
Kristy Reinert
Jessica Tanis
Yelena Vidgop
unc-20
Peter Alff
Jeff Doto
Brook Kohrt
Son Nguyen
Suchi Pandey
Doug Prechtel
Matt Stein
Other genes
David Brightbill
Andrea Cerrone
Kelly Klampert
Sheila Mathieson
Mark Alkema, Cori Bargmann, Edouard DeCastro, Gian Garriga,
Oliver Hobert, Christine Li, A.V. Maricq, Ruth Yu