Embed Size (px)
Citation preview
A Nematode Neuron Identity Crisis
Bruce WightmanMuhlenberg 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
Aequorea victoria
GFPNematodeneuron-specificpromoter
The AVK axons can be visualized with an flp-1::gfp transgene
AVKL
GFPflp-1
(Courtesy of C. Li)
The fax-1 gene is requiredfor AVK axon pathfinding
AVKL
Wild Type
AVKL
fax-1 (gm83)
fax-1 is also required for normallevels of flp-1::gfp expression
Wild-Type fax-1(gm83)
AVKLAVKR
AVKLAVKR
The flp-1 gene encodes a protein precursor for FMRFamide-like
peptide neurotransmitter
N C
SADPNFLRFSQPNFLRF
ASGDPNFLRFSDPNFLRF
AAADPNFLRFPNFLRF
AGSDPNFLRF Post-synapticneuron
Pre-synapticneuron
flp-1
FMRFamide is reduced in AVK neurons of fax-1 mutants
anti-FMRFamide immunostaining
fax-1 functions in both axon pathfinding and neuro-transmitter expression
FAX-1
FMRFamide
axon pathfinding
fax-1 encodes a protein that is a member of the nuclear hormone receptor family
N CDBD LBD
downstreamgene
NHR’s regulate transcriptionand bind lipophilic hormones
The FAX-1 DBD containstwo zinc- fingers
Zn2+
C C
CC
C C
CCZn2+
(J. Much)
FAX-1 is a member of a subclass of NHR’s that function in
nervous system development
Estrogen ReceptorRetinoic Acid Receptor
NHR-111TLX*
NHR-67
TLL*
FAX-1*
PNR*C. elegansDrosophilaH. sapiens
* functionsin nervoussystem
Mutations in human PNR cause defects in photoreceptor identity
Normal retina Enhanced S-conesyndrome retina
PNR is also known as NR2E3(J. Tanis)
fax-1 may regulate transcription of genes involved in speci-
fication of neuron identity
pathfindinggene
flp-1
Other neuron-specific gene
Examining FAX-1 expression using antibodies and immunofluroescence
staining
Anti-FAX-1 antibodies Immunofluorescencestaining
(N. Carmean)(B. Ebert)
fax-1 is expressed in 18 neurons including the AVKs
mid-stage embryo
adult
AVKL
AVKR
AVKAIY
RICDVA
Fax-1 Phenotypes:Unc, Che, Fab, Wandering
AVB AVAAVE MI
RICR AVBRAVARAVER
fax-1 may regulate transcription of genes involved in speci-
fication of AVK neuron identity
AVK path-finding gene
flp-1
Other AVK-specific gene
ncs-1::gfp expression in AVK requires fax-1
neuron-specific calcium sensor
wild-type ncs-1::gfp fax-1 (gm83) ncs-1::gfp
AVKL
AVKR
(Gomez, et al., 2001)
Summary of gene expression requirements
AVK
Requirefax-1
flp-1ncs-1
Cells:
What is the function of fax-1 in neurons other than the AVK’s?
DVA
AVKAIYRIC
AVBAVAAVEMI
The RIC neurons have normal anatomy and expression of tbh-1 in
fax-1 mutants
tyrosine -hydroxylase (for octopamine biosynthesis)
wild-type and fax-1 (gm83)
(M. Alkema and H.R. Horvitz, unpublished)
RIC
Expression of NMDA glutamate receptor subunits in AVA and AVE
requires fax-1
AVB
fax-1 (gm83) nmr-1::gfp
RIM
AVA/EAVB
RIM
AVG
AVBRIM
AVG
wild-type nmr-1::gfp
AVBRIMAVA
AVE
Both nmr-1 and nmr-2 show the same pattern
Expression of non-NMDA glutamate receptor subunits
does not require fax-1
AVA and AVE axons appear normal in fax-1 mutants:fax-1 not required for axon pathfinding in AVA/E
wild-type glr-1::gfp fax-1 (gm83) glr-1::gfp
AVA/EAVA/E
Summary of gene expression requirements
AVK AVA/E
Requirefax-1
flp-1ncs-1
nmr-1nmr-2
Cells:
Which genes may functionupstream of fax-1?
The homeodomain gene unc-42 may function upstream of fax-1
fax-1
UNC-42
AVK
• 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)
fax-1 is not expressed in some neurons of unc-42 mutants
Wild-type
unc-42 (e419)
FAX-1 is absent in AVB and two anterior neurons and reduced in AVK
DVA
AVKAIYRIC
AVBAVAAVEMI
AVA
AVERIC
AVK
wild-type FAX-1 unc-42(e419) FAX-1
AVA/E AVA/E
AVBRIC
AVKAVK
RIC
DVA
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
Requirefax-1
Requireunc-42
flp-1ncs-1
fax-1flp-1
nmr-1nmr-2
Cells:
Expression of ncs-1 also depends on unc-42
wild-type ncs-1::gfp unc-42(e419) ncs-1::gfp
AVKL
AVKR
Summary of gene expression requirements
AVK AVA/E
Requirefax-1
Requireunc-42
flp-1ncs-1
fax-1flp-1ncs-1
nmr-1nmr-2
Cells:
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
(Baran et al., 1999; Brockie et al., 2001)
wild-type unc-42(e419) fax-1(gm83)
nmr-1 + + -nmr-2 + + -glr-1 + - +glr-4 + - NDglr-5 + - +
Axon pathfinding by AVA and/or AVE appears defectivein unc-42 mutants
Summary of gene expression requirements
AVK AVA/E
Requirefax-1
Requireunc-42
flp-1ncs-1
fax-1flp-1ncs-1
nmr-1nmr-2
glr-1glr-4glr-5
Cells:
Models for the specification of neuron identity by unc-42 and fax-1
UNC-42
FAX-1
flp-1
Pathfindinggenes
ncs-1
AVK AVA/E
UNC-42 FAX-1
Pathfindinggenes
glr-1nmr-1
nmr-2glr-4
glr-5
Approaches to identification of FAX-1 downstream targets
(R. Haviland, R. Lombel, E. Murphy, K. Reinert, E. Smith, Y. Vidgop)
Other neuron-specific genes
Is expression of FAX-1 sufficient to change gene expression?
heat-shock promoter fax-1
(N. Bianco, T. Sundaresan)
anti-FAX-1 hs::fax-1; fax-1(gm83)
Participantsfax-1Sepi BazelNick BiancoNicole CarmeanSara CarrBryan EbertDavid GarbeRebecca HavilandChirag KalolaRebecca LombelJason MuchElissa MurphyGwen SarverDennis SladeEric SmithTilak SundaresanKristy Reinert Jessica TanisYelena Vidgop
unc-20Peter AlffJeff DotoBrook KohrtSon NguyenSuchi PandeyDoug PrechtelMatt Stein
Other genesDavid BrightbillAndrea Cerrone
Kelly KlampertSheila Mathieson
Mark Alkema, Cori Bargmann, Edouard DeCastro, Gian Garriga, Oliver Hobert, Christine Li, A.V. Maricq, Ruth Yu
