A Nematode Neuron Identity Crisis

1
A Nematode Neuron Identity Crisis

• Bruce Wightman
• Muhlenberg College

2
The nervous system is comprised of many different
types of neurons
3
Homeodomain transcription factors regulate neuron
identity in the vertebrate spinal cord
4
The nematode C. elegans

• DNA Sequence known
• Rich genetics
• Simple nervous system
• Defined anatomy

5
The C. elegans nervous system
6
Axon anatomy can be examined by neuron-specific
promoter GFP fusions
Nematode neuron-specific promoter
GFP
Aequorea victoria
7
The AVK axons can be visualized with an
flp-1gfp transgene
AVKL
flp-1
GFP
(Courtesy of C. Li)
8
The fax-1 gene is requiredfor AVK axon
pathfinding
Wild Type
AVKL
fax-1 (gm83)
AVKL
9
fax-1 is also required for normallevels of
flp-1gfp expression
Wild-Type
fax-1(gm83)
AVKL
AVKL
AVKR
AVKR
10
The flp-1 gene encodes a protein precursor for
FMRFamide-like peptide neurotransmitter
flp-1
N
C
Pre-synaptic neuron
SADPNFLRF SQPNFLRF ASGDPNFLRF SDPNFLRF AAADPNFLRF
PNFLRF AGSDPNFLRF
Post-synaptic neuron
11
FMRFamide is reduced in AVK neurons of fax-1
mutants
anti-FMRFamide immunostaining
12
fax-1 functions in both axon pathfinding and
neuro-transmitter expression
FMRFamide
FAX-1
axon pathfinding
13
fax-1 encodes a protein that is a member of the
nuclear hormone receptor family
DBD
LBD
C
N
NHRs regulate transcription and bind lipophilic
hormones
The FAX-1 DBD contains two zinc- fingers
C
C
C
C
Zn2
Zn2
downstream gene
C
C
C
C
(J. Much)
14
FAX-1 is a member of a subclass of NHRs that
function in nervous system development
FAX-1
C. elegans Drosophila H. sapiens
PNR
NHR-67
TLL
functions in nervous system
TLX
NHR-111
Estrogen Receptor
Retinoic Acid Receptor
15
Mutations in human PNR cause defects in
photoreceptor identity
Enhanced S-cone syndrome retina
Normal retina
PNR is also known as NR2E3
(J. Tanis)
16
fax-1 may regulate transcription of genes
involved in speci-fication of neuron identity
pathfinding gene
Other neuron- specific gene
flp-1
17
Examining FAX-1 expression using antibodies and
immunofluroescence staining
Anti-FAX-1 antibodies
Immunofluorescence staining
(N. Carmean) (B. Ebert)
18
fax-1 is expressed in 18 neurons including the
AVKs
mid-stage embryo
adult
RICR
AVBR
AVKR
AVAR
AVER
AVKL
AVA
AVB
RIC
AVE
DVA
MI
AIY
AVK
Fax-1 Phenotypes Unc, Che, Fab, Wandering
19
fax-1 may regulate transcription of genes
involved in speci-fication of AVK neuron identity
AVK path- finding gene
Other AVK- specific gene
flp-1
20
ncs-1gfp expression in AVK requires fax-1
fax-1 (gm83) ncs-1gfp
wild-type ncs-1gfp
AVKL
AVKR
neuron-specific calcium sensor
(Gomez, et al., 2001)
21
Summary of gene expression requirements
AVK
Cells
Require fax-1
flp-1 ncs-1
22
What is the function of fax-1 in neurons other
than the AVKs?
DVA
AVB
AVA
RIC
AVE
MI
AIY
AVK
23
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)
24
Expression of NMDA glutamate receptor subunits
in AVA and AVE requires fax-1
fax-1 (gm83) nmr-1gfp
wild-type nmr-1gfp
AVB
AVB
RIM
RIM
AVA AVE
AVB
AVB
AVA/E
RIM
RIM
AVG
AVG
Both nmr-1 and nmr-2 show the same pattern
25
Expression of non-NMDA glutamate receptor
subunits does not require fax-1
wild-type glr-1gfp
fax-1 (gm83) glr-1gfp
AVA/E
AVA/E
AVA and AVE axons appear normal in fax-1
mutants fax-1 not required for axon pathfinding
in AVA/E
26
Summary of gene expression requirements
AVK
AVA/E
Cells
Require fax-1
flp-1 ncs-1
nmr-1 nmr-2
27
Which genes may functionupstream of fax-1?
28
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 path-finding defects
  that are similar to fax-1 mutant defects

(Wightman et al, 1997 Baran et al., 1999)
29
fax-1 is not expressed in some neurons of unc-42
mutants
unc-42(e419) FAX-1
wild-type FAX-1
AVB
RIC
AVA/E
AVA/E
RIC
AVK
AVK
Wild-type
DVA
AVB
AVA
RIC
AVE
MI
AIY
AVK
unc-42 (e419)
DVA
AVA
RIC
AVE
AVK
FAX-1 is absent in AVB and two anterior neurons
and reduced in AVK
30
unc-42 is also required for flp-1gfp
expression
Percentage of AVK neurons Express
flp-1gfp fluorescence
Wild-Type 100 fax-1 (gm83) 65
unc-42 (e419) 0 unc-42 (e419)
fax-1 (gm83) 0
(S. Mathieson)
31
Summary of gene expression requirements
AVK
AVA/E
Cells
Require fax-1
flp-1 ncs-1
nmr-1 nmr-2
Require unc-42
fax-1 flp-1
32
Expression of ncs-1 also depends on unc-42
unc-42(e419) ncs-1gfp
wild-type ncs-1gfp
AVKL
AVKR
33
Summary of gene expression requirements
AVK
AVA/E
Cells
Require fax-1
flp-1 ncs-1
nmr-1 nmr-2
Require unc-42
fax-1 flp-1 ncs-1
34
What about unc-42 and fax-1 in the AVA and AVE
interneurons?
35
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 unc-42(e419) fax-1(gm83)
nmr-1 - nmr-2 - glr-1 - glr-
4 - ND glr-5 -
Axon pathfinding by AVA and/or AVE appears
defective in unc-42 mutants
(Baran et al., 1999 Brockie et al., 2001)
36
Summary of gene expression requirements
AVK
AVA/E
Cells
Require fax-1
flp-1 ncs-1
nmr-1 nmr-2
Require unc-42
fax-1 flp-1 ncs-1
glr-1 glr-4 glr-5
37
Models for the specification of neuron identity
by unc-42 and fax-1
AVK
AVA/E
FAX-1
UNC-42
UNC-42
FAX-1
glr-1
nmr-1
flp-1
glr-4
nmr-2
Pathfinding genes
glr-5
ncs-1
Pathfinding genes
38
Approaches to identification of FAX-1 downstream
targets
Other neuron- specific genes
(R. Haviland, R. Lombel, E. Murphy, K. Reinert,
E. Smith, Y. Vidgop)
39
Is expression of FAX-1 sufficient to change gene
expression?
heat-shock promoter
fax-1
hsfax-1 fax-1(gm83)
anti-FAX-1
(N. Bianco, T. Sundaresan)
40
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