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Development and aging in Caenorhabditis elegans: Gene structure and expression of daf-12 and a longitudinal study of neurons in long-lived individuals

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Development and aging in Caenorhabditis elegans: Gene structure and expression of daf-12 and a longitudinal study of neurons in long-lived individuals

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DEVELOPMENT AND AGING IN Caenorhabditis elegans: GENE STRUCTURE
AND EXPRESSION OF daf-12 AND A LONGITUDINAL STUDY
OF NEURONS IN LONG-LIVED INDIVIDUALS
by
Mark Irvin Snow
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
in Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
December 2000
Copyright 2000 Mark Irvin Snow
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UMI Number: 3041526
Copyright 2000 by
Snow, Mark Irvin
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UNIVERSITY OF SOUTHERN CALIFORNIA
1MB GRADUATE S C HO O L
UM VERSm rFAM
L OB ANOELBS. CAL I F ORNI A 9 0 0 0 7
This dissertation, written try
Mark Irvin Snow
under the direction of h ..il — Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School in partial fulfillment of re-
quirements for the degree of
DOCTOR OF PHILOSOPHY
• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • « • • • • • • • • • • • • • • • • • • » • • • • • • • • • • • • • • « • • « •
Qe m o f G nd**te Studies
D ate .......
ATION COl
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Mark Irvin Snow Norman Amheim
ABSTRACT
DEVELOPMENT AND AGING IN Caenorhabditis elegans: GENE STRUCTURE
AND EXPRESSION OF daf-12 AND A LONGITUDINAL STUDY
OF NEURONS IN LONG-LIVED INDIVIDUALS
Environmental conditions promote a cascade of signaling molecules in C. elegans
early larval development to direct growth to the reproductive adult or to arrest as a dauer
larva. Molecular characterization o f daf-12 was performed because it is one of the most
downstream genes in the dauer formation genetic pathway and may be directly regulating
dauer development. The three transcripts made from the daf-12 gene by differential
splicing are produced throughout development and expression increases during the
preparation for and execution of dauer formation. The deduced protein isoforms are
similar to both the DNA and ligand binding domains of nuclear hormone receptors. The
isoform ratios of daf-12 steady-state mRNA are not changed by reduction of function in
the genetically upstream TGF-B and insulin-like signaling components of the dauer
pathway. The daf-12 promoter directs expression of the Green Florescent Protein (GFP)
in the pharynx, daf-12 is a putative ligand activated transcription factor acting
throughout development and functions under a variety of environmental conditions.
Neuronally controlled behaviors such as movement progressively diminish as a
function o f age. To determine if there is a relationship between the age-associated
decline in these behaviors and the status of neurons, longitudinal analysis of adult
neurons was performed. The GABA and dopmainergic neurons in individual
hermaphrodites were followed from young adulthood to just before death. The majority
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of animals with a wild-type life span showed no change in the number o f neurons by GFP
expression. In the few individuals with loss o f expression, specific neurons were
undetectable in the aged animals. Absence of GFP expression may indicate functional
loss because the gfp tagged genes encode enzymes necessary for neurotransmitter
biosynthesis. In animals with extended life spans, as a result of a mutation in the daf-2
gene, GFP expression patterns were not altered. In conclusion, it appears the age-
associated decline in movement is not correlated to loss of GFP expression in
GABAergic or dopaminergic neurons and daf-2 mutations are neural protective.
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ii
Acknowledgements
I would like to thank the following people:
My advisor, Pam Larsen for her patience and direction; my dissertation committee
members Norm Amheim, Steve Finkel, Kevin Moses, John Tower, Gary Trump and Rahul
Warrior for their advice and guidance; Patricia Herd for assistance with cloning and
sequencing; former lab members Hui Yu. Daniela Bota, Patrick Leong and Marian Tom for
all their help; Paul Sternberg and members of his laboratory for suggestions and technical
help; Bill Trusten for his administrative assistance; Yishi Jin and Robyn Lints for providing
strains; my father, Robert and my mother Florence for their support, and Orady
Souksamlane for patience and support.
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Table of Contents
Acknowledgements..................................................................................................................... ii
List of Tables..............................................................................................................................iv
List of Figures..............................................................................................................................v
Introduction...................................................................................................................................1
Chapter 1...................................................................................................................................... 7
Introduction..............................................................................................................................7
Methods.................................................................................................................................. 15
Results.................................................................................................................................... 21
Discussion..............................................................................................................................39
Chapter 2 .................................................................................................................................... 48
Introduction............................................................................................................................48
Methods.................................................................................................................................. 52
Results.................................................................................................................................... 57
Discussion..............................................................................................................................97
Bibliography............................................................................................................................ 106
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List of Tables
Table I: him-5; nisi 18 individuals with changes in the number of neurons expressing
G FP................................................................................................................................... 72
Table 2: Adult life span statistics for juls7 and juls7; daf-2 strains............................87
Table 3: Adult life span statistics for him-5; nisi 18 and daf-2; nisi 18 strains...... 90
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List of Figures
Figure 1: Life cycle of C. elegans........................................................................2
Figure 2: Genetic pathway for dauer formation............................................................ 4
Figure 3: Model for reproductive growth conditions.......................................................10
Figure 4: Model for dauer-inducing conditions...........................................................12
Figure 5: Genomic organization of the daf-12 gene.......................................................24
Figure 6: Alignment of the DAF-12 A1 sequence........................................................ 28
Figure 7: Developmental Northern analysis............................................................. 32
Figure 8: Analysis of steady-state daf-12 isoform ratios................................................... 35
Figure 9: Photomicrographs illustrating daf-I2::GFP expression....................................37
Figure 10: Photomicrographs of GFP neurons........................................................... 59
Figure 11: GFP expression in GABA neurons in moribund individuals.............................61
Figure 12: Individual expression profile forjuls7 ..............................................................63
Figure 13: Individual expression profile forjuls7; daf-2(m4l).........................................65
Figure 14: Individual expression profile for juls7; daf-2(eI370)......................................66
Figure 15: Cumulative number of animals dying over time for juls7 and juls7; daf-2
strains................................................................................................................................ 67
Figure 16: GFP expression with age in dopamine neurons................................................69
Figure 17: Individual expression profile for him-5; nisi 18............................................... 71
Figure 18: Individual expression profile for daf-2(m4l); nisi 18......................................74
Figure 19: Individual expression profile of daf-2(el370); nisi 1 8 ....................................75
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Figure 20: Cumulative number of animals dying over time for him-5; nisi 18 and daf-2;
nisi 18 strains................................................................................................................... 76
Figure 21: Movement as an indicator of fitness for juls7............................................78
Figure 22: Movement as an indicator of fitness for juls7; daf-2(m41).....................79
Figure 23: Movement as an indicator of fitness for juls7; daf-2(eI370)...................80
Figure 24: Movement as an indicator of fitness for him-5; nisi 1 8 ........................... 82
Figure 25: Movement as an indicator of fitness for daf-2(m4l); nisi 18...................83
Figure 26: Movement as an indicator of fitness for daf-2(el370); nisi 18................84
Figure 27: Survival curves comparing manipulated animals to non-manipulated animals
for juls7 and juls7; daf-2................................................................................................ 86
Figure 28: Survival curves comparing manipulated animals to non-manipulated animals
for him-5; nisi 18 and daf-2(m4I); nisi 18.................................................................... 89
Figure 29: Survival curves comparing different genotypes of non-manipulated animals
for N2 vs.juls7 and him-5; nisi 18................................................................................ 92
Figure 30: Survival curves comparing different genotypes of non-manipulated animals
for daf-2 (m4l) \s.juls7; daf-2(m41) and daf-2(m4l); nisi 18.................................94
Figure 31: Survival curves comparing different genotypes of non-manipulated animals,
for daf-2 (e!370) vs ,juls7; daf-2(eI370) and daf-2(el370); nisi 18 .........................95
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1
Introduction
The free-living soil nematode, Caenorhabditis elegans, is an excellent model
system for addressing questions pertaining to the molecular genetics o f development and
aging. The advantages of C. elegans as a model system for developmental biology
studies are a rapid life cycle of three days, a nearly completely sequenced genome, the
ease of obtaining large populations of genetically identical organisms and a nearly
invariant cell lineage. C. elegans is also an advantageous model system to study the
neurobiology o f aging. The hermaphrodite cell lineage has been determined and the 959
ceils, of which 302 are neurons, is relatively small in number compared to other
biological systems (Sulston and Horvitz, 1977). All o f the neurons in the animal have
been identified and the connections between the neurons have been mapped (White et al.,
1986). The transparency of the organism and the standard use of transgenic animals
expressing specific genes fused to the Green Florescent Protein gene allows for the
visualization of specific cells in the individuals (Chalfie et al., 1994). C. elegans also has
a short mean life span o f approximately 12-18 days at 20°C (Kenyon, 1997).
In C. elegans, development proceeds through four successive larval stages from
egg to adulthood (Figure 1). After embryogenesis, the first larval stage (LI) develops in
one of two ways depending upon the environmental conditions (Golden and Riddle,
1984). These environmental factors include the concentration of food, the concentration
of a constitutively secreted pheromone and the temperature (Golden and Riddle, 1984).
If the larvae are in an environment of high food concentration and low pheromone
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egg -►LI
/
\
L2d
L2 L3
\
I
/
adult
Figure 1: Life cycle of C. elegans. At the first larval stage (LI) animals can molt
(represented by arrows) to the L2, when environmental conditions are favorable or the
L2d stage when conditions are unfavorable. L2 animals reassess the suitability of the
environment for growth; if suitable the L2ds molt to the L3, if unsuitable, then L2ds will
proceed to enter the dauer larvae stage.
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3
concentration, the animal will develop through the remaining larval stages to become an
adult. If the environmental conditions are more severe and the concentration of food is
low and the concentration o f pheromone is high, the LI animal will molt to the L2d stage.
L2d animals reassess the environmental conditions and if they have improved, animals
will molt to the L3 stage and proceed to the adult stage. If environmental conditions do
not improve or become worse, the L2d animal molts to the dauer stage (Cassada and
Russell. 1975). Animals in the dauer stage do not feed (Cassada and Russell, 1975), have
an altered metabolism (O'Riordan and Burnell, 1989) and remodel certain tissues
including constriction of the pharynx (Vowels and Thomas. 1992), thickening of the
cuticle (Cassada and Russell, 1975) and condensing of the intestinal lumen (Popham and
Webster. 1979). Dauer larvae can survive several months until environmental conditions
improve which promotes recovery from this stage and development to adulthood (Golden
and Riddle. 1984).
Genetic screens have been performed to identify genes that function in dauer
formation. Animals with mutations in these genes display either the dauer constitutive
(Daf-c) or the dauer defective (Daf-d) phenotypes. Daf-c animals form dauer larvae
inappropriately while Daf-d animals are unable to form dauer larvae. Epistasis analysis
has resulted in a complex pathway for dauer formation (Figure 2) including two parallel
pathways and a third interacting, branched pathway (Gottlieb and Ruvkun, 1994; Larsen
et al., 1995; Riddle et al., 1981; Thomas et al., 1993; Vowels and Thomas, 1992).
Electron microscopy studies suggest the early genes (cilium structure genes) in this
pathway may act in reception of environmental signals (Perkins et al., 1986). Several of
the genes in the middle of the pathway have been molecularly cloned and they encode
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4
daf-11
daf-21
Daf-c
cilium
structure
genes
Daf-d
daf-22
Daf-c
daf-6
Daf-d
\
cilium
structure
genes
Daf-d
daf-1
daf-4
daf-7
daf-8
daf-14
Daf-c
/
daf-12
Daf-d — ► dauer
/ /
‘ i
i
daf-3 i
daf-S 1 i
Daf-c 7 '
i i
‘ i
i i
i /
daf-2
akt-1 _
daf-16
age-1
..........►
T
akt-2
Daf-d
Daf-c
I
daf-18
Daf-d
Figure 2: Genetic pathway for dauer formation based on epistasis analysis. Daf-C =
dauer formation constitutive, Daf-D = dauer formation defective. Wild-type gene
products activate (represented by arrows) or inhibit (represented by T bars) downstream
gene functions in the pathway.
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proteins that are similar in sequence to the TGF-B and SMAD families, (Estevez et al.,
1993; Georgi et al., 1990; Inoue and Thomas, 2000; Patterson et al., 1998; Ren et al.,
1996) two classes of molecules that are known to function in signal transduction. Loss of
function mutations in the daf-12 gene render the animals unable to form dauer larvae;
thus, the normal DAF-12 function is required to promote dauer development (Riddle et
al., 1981). The genetic analysis has placed daf-12, similar to the human Vitamin D
receptor (Yeh. 1991), at a late step in the pathway of dauer development (Larsen et al.,
1995) and it may be part of a negative regulatory loop with the daf-2 gene that is similar
to human insulin receptor gene (Kimura et al., 1997).
Analysis of life span showed that animals with a mutation in the daf-12 gene have
slightly shorter life spans than wild-type (Larsen et al., 1995). Animals with a mutation
in the daf-2 gene have doubled life spans and certain double mutant combinations of daf-
21 daf-12 live four times longer than wild type (Kenyon et al., 1993; Larsen et al., 1995).
One conclusion from the studies was that the daf-2 gene functions to promote growth and
limit life span, while the daf-12 gene modulates this function. The molecular basis of the
observed traits in these mutants in adult longevity remains to be determined.
In this dissertation, two major projects are presented. To better understand the
function of the daf-12 gene in the process of dauer formation, molecular characterization
was performed and is described in Chapter 1. Presented are the mRNA isoform
structures of the gene, the locations of three molecular lesions, the mRNA expression
throughout development, and the identification o f specific cells that express a
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6
daf-12::GFP construct. Understanding daf-12 at the molecular level contributes
mechanistic aspects to the model of how the flow of information, initiated with an
external environmental signal, evokes an internal response to result in specific
developmental programs.
To determine if there is a relationship between the age-associated decline in
behaviors and the status of neurons, it is necessary to monitor neurons in individuals as
they age. Previous neuronal studies in other model organisms have used a cross sectional
design and the correlative conclusions drawn from these experiments are intriguing, but
limited because the results are obtained from one distinct point in an individual life span.
To overcome this limitation, a protocol was designed and implemented, as described in
Chapter 2, which allowed for a longitudinal analysis of adult neurons in C. elegans.
Presented are experiments in which individual transgenic animals expressing GFP in
GABA and dopamine neurons were followed over time, as well as movement as an
indicator of fitness, to determine what happens as they age. Neuron expression was
followed in individuals with a wild-type background and in animals carrying a mutation
in the daf-2 gene. This approach could potentially identify cellular alterations that are
predictive of death in live animals. For the work presented here, it was found that the
cellular alterations do not correlate with death and thus suggest GABA and dopamine
neurotransmitter synthesis is not life limiting in C. elegans.
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7
Chapter 1
Molecular Characterization of a Nuclear Hormone Receptor
Gene Involved in Development, daf-12
Introduction
External stimuli can alter the developmental fate of the nematode Caenorhabditis
elegans (Cassada and Russell, 1975). Conditions of low food, high temperature and high
population density cause a remodeling of certain tissues to a specialized third larval stage
called the dauer larva (Riddle and Albert, 1997). Morphology of the pharynx, cuticle and
intestinal lumen are altered in this developmentally arrested diapause stage. The dauer
state can be maintained for several months until the environmental conditions improve,
and in response the animals will re-initiate development to adulthood (Cassada and
Russell, 1975). Many of the genes involved in this process have been identified through
genetic screens and epistasis analysis has resulted in a complex pathway for genes
involved in dauer formation.
The daf-12 gene lies at the convergence of two signaling pathways in the dauer
formation genetic pathway. Several of these genes have been cloned and found to be
homologous to genes in mammalian signaling pathways. The TGF beta signaling
component o f dauer formation includes homologs of the TGF-B ligand daf-7 (Ren et al.,
1996), type II and type I TGF-B receptors daf-l (Georgi et al., 1990) and daf-4 (Estevez
et al., 1993), and Smads daf-2 (Patterson et al., 1998), daf-8 (Riddle and Albert, 1997)
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8
and daf-l4 (Inoue and Thomas, 2000). In reproductive growth conditions, daf-7::GFP is
expressed in the ASI neuron (Ren et al., 1996; Schackwitz et al., 1997) and the putative
TGF-B molecule is speculated to activate the serine/threonine kinase receptors which
phosphorylate the downstream Smads. The daf-3 gene encodes a homolog o f a DPC4-
like Smad and is suggested to be antagonized by DAF-7 (Patterson et al., 1998). These
Smads may then be localized to the nucleus where they interact with other reproductive
growth genes to promote transcription.
The second signaling pathway that converges on daf-12 is involved in
chemosensory signal transduction. Genetic evidence suggests daf-l I and daf-21 function
in sensory endings and are important in dauer formation (Vowels and Thomas, 1992).
daf-l 1 shows homology to a transmembrane guanylate cyclase, which is important in
retinal phototransduction and hormonal signal transduction (Bimby et al., 2000). A third
major signaling pathway is involved in dauer formation and differs from the other two
pathways in that it is not suppressed by mutation of the daf-12 gene. Genetic interaction
between daf-12 and the third pathway have been reported (Gottlieb and Ruvkun, 1994;
Larsen et al., 1995). Genes in this pathway include daf-2 a homolog of the insulin
receptor/insulin growth factor I (Kimura et al., 1997), age-1 a phosphatidylinositol-3-OH
(PI3) kinase (Morris et al., 1996), akt-l and akt-2 (Paradis and Ruvkun, 1998), d a f -18 a
PTEN (Gil et al., 1999; Mihaylova et al., 1999; Ogg and Ruvkun, 1998), pdk-1 (Paradis
et al., 1999) and daf-16 a Forkhead transcription factor (Lin et al., 1997; Ogg et al.,
1997). This cascade is activated in reproductive growth conditions and the DAF-2
tyrosine kinase receptor likely recruits the AGE-1 PI3 kinase ultimately resulting in
DAF-16 inactivation and down regulation of dauer specific genes, daf-16 mutant alleles
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9
display a partial dauer phenotype in which animals induced with dauer pheromone are
not completely dauer defective because dauer phenotypes are observed in some tissues
(Gottlieb and Ruvkun, 1994; Vowels and Thomas, 1992). A model of the genes involved
in reproductive growth and dauer larvae formation from the insulin-like and TGF-B
signaling pathways is depicted in Figures 3 & 4.
The daf-12 locus affects multiple processes in C. elegans development as revealed
by the phenotypes resulting from mutations in daf-12 (Antebi et al., 1997; Riddle et al.,
1981). The first phenotype of daf-12 described by Riddle, et al. (1981) was defective
dauer formation. These dauer formation defective (Daf-d) animals are unable to form
dauer larvae under any environmental conditions. Larsen, et al. (1995) subjected several
daf-12 alleles (m20. m25, m l 16, and m383) to dauer formation and life span epistasis
analysis with mutations in other genes in the dauer formation genetic pathway. This
work implicated daf-12 in efficient life maintenance, since certain allele specific
interactions between daf-2; daf-12 double mutants resulted in a synergistic increase in
life span of nearly four-fold. Mutations in the gene mig-7 were isolated in screens for
defects in distal tip cell migration and subsequent mapping showed that the mig-7
mutations were novel alleles of daf-12 (Antebi et al., 1998). Some of these daf-12 alleles
display the phenotype o f abnormal cell migrations (Mig) and abnormal cell lineage (Lin).
The Mig mutants have abnormally migrating linker and distal tip cells and the Lin
mutants display a phenotype in which hypodermal and intestinal cells repeat their second
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10
Figure 3: Model for reproductive growth conditions (black boxed genes are down-
regulated). The TGF-beta molecule activates the receptors and the Smads are
phosphorylated and move into the nucleus and may effect the DAF-12 nuclear hormone
receptor. The insulin-like signaling pathway is activated and ultimately results in down-
regulation of DAF-16. In these conditions, DAF-12 activates genes for reproductive
growth and down-regulates dauer-specific genes.
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1 1
Transforming Growth
Factor-ll signal
DAF-7 (TGF-p like)
DAF-4 DAF-1 (Type I/ll receptors)
DAF-8
DAF-14
(Smads)
DAF-3 (Smad)
Insulin-like signal
DAF-2 (lnsulin/IGF-1 like receptor)
AGE-1 (PI3 kinase)
(PTEN) D A F -18
phosphoinositides [PI(3,4)P2, PI(3,4,5)P3 ]
PDK-1 (PDK)
AKT-1 AKT-2 (AKT/PKB)
A1 A2 B
(NHR)
D A F -16 (Forkhead TF)
reproductive
growth genes
distal tip cell migration (Mig)
hypodermal seam cell lineage (Lin)
tr.insi n p t i o n of
11.-inot spt-cific
' j P r l t . - S
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12
Figure 4: Model for dauer-inducing conditions. The TGF-beta is lacking (black boxed
genes are down-regulated), the Smads are not phosphorylated, but DAF-3 may still move
into the nucleus to affect the DAF-12 nuclear hormone receptor. The insulin-like
signaling is also lacking, resulting in relief of repression o f DAF-16 that acts in parallel
with DAF-12 to promote dauer formation.
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13
Transforming Growth
Factor-& signal
DAF-7 (TGF-p like)
D A F-4 DAF-1
(Type I/ll receptors)
DAF-8
E H B E
(Smads)
DAF-3 (Smad)
Insulin-like signal
DAF-2 (lnsulin/IGF-1 like receptor)
(PI3 kinase)
(PTEN) DAF-18
phosphoinositides [PI(3 4)P. PI(3 4 5)P
PDK-1 (PDK)
3 3 H E 3 E (AKT/PKB)
DAF-16 (Forkhead TF)
A1 A2 B
DAF-12 (NHR)
i t - p r o d u c t i ' A ?
]! ( )',7th f h ’-llt ‘S
l i i s t c l l tip- i ] t ‘ 1 1 !?) |l j[ , it li It] M u )
tV/f'iOl j f H U i i St :, it ] ] . f l l i i t l f . j r j t ; ! I
transcription of
dauer-specific
genes
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14
stage (L2) divisions (Antebi et al., 1998). Five of the new alleles displayed a weak dauer
constitutive phenotype and formed partial dauer larvae. From the analysis, six
phenotypic classes at this locus were defined based on distal tip cell migration,
hypodermal seam cell lineage, and dauer formation traits (Antebi et al., 1998).
Refined genetic mapping and creation of a Tel induced allele (m545) was used to
clone a portion of a cDNA which encodes 292 putative amino acids homologous to the
DNA binding domain of steroid hormone receptors (Yeh, 1991). The molecular identity
of daf-12 as a steroid hormone receptor suggests that it functions as a hormonally
regulated transcription factor mediating development, entry into the dauer stage and adult
life span. To further understand the role of daf-12 in dauer entry and aging the complete
cDNA structures of the three daf-12 transcripts have been determined with the largest
encoding a putative 753 amino acid protein. The deduced proteins show multiple
hallmarks of nuclear hormone receptors. These daf-12 transcripts are present throughout
development and mRNA levels increase prior to the dauer stage. The steady-state mRNA
isoform ratios of daf-12 do not change under the dauer-inducing or reproductive growth
conditions tested, daf-12 may provide a hormonal mechanism to repress and activate
transcription depending on the environmental conditions and internal status (tissue and
developmental stage) of the animal.
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15
Methods
Genetics
Standard methods (Brenner, 1974) were used in the handling and genetic
manipulation o f all animals, excepted where noted. Animals were cultured on 6 cm
NGM agar plates seeded with Escherichia coli OP50 or in liquid culture as described
below. Mutations used in this study were LGI: daf-l6(m26)\ LGIII: daf-7(el372)\ LG X:
daf-12(m20, m l 16 & m583), and daf-3(eI376).
Cosmid Transformation Rescue
Cosmid FI 1 Al was injected at 100 ng/pl along with plasmid pRF4 (rol-6
(su!006)) at a concentration of 100 ng/pl into the double mutant strain daf-7; daf-12. FI
animals were scored for the Rol phenotype and stable lines were established. The
animals were assayed for dauer formation at 25.5°C.
RT-PCR
N2 bristol mixed stage nematodes were ground with a mortar and pestle in liquid
nitrogen and sonicated 3 x 90 second bursts. Total RNA was extracted using the mRNA
Isolation Kit (Gibco-BRL). mRNA was isolated by passing total RNA over oligo- (dT)
Cellulose Columns (Gibco-BRL) using the method o f Ausubel, et al. (1997).
Approximately 500 ng of mRNA was reverse transcribed using an oligo-dT-adapter
primer and the SUPERSCRIPT™ Preamplification System (Gibco-BRL) for first strand
cDNA synthesis. One tenth of the volume o f the cDNA synthesis reaction was
subsequently used for PCR. The genome consortium used the GENEFINDER program
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16
to predict putative open reading frames of the FI 1A1 cosmid sequence (The C. elegans
Sequencing Consortium, 1998). 21 to 25-mer oligonucleotides complementary to the
predicted coding regions were designed for PCR amplification. An SLl primer 5’-
ATTCCGCGGTTTAATTACCCAAGTTTG-3, and a gene specific primer 5’-
AAAATTCTCCTGGCAGCTCTTCGG-3, were used with the Expand High Fidelity
PCR System (Boehringer Mannheim) in the first round of PCR. The first round reaction
was diluted 1:10 or 1:100 and second round PCR using the SLI primer and a nested gene
specific primer 5’-ACAGTTCCCATGAACAGCATTCCAG-3’. For the 3’ end, RACE
(Frohman et al., 1988) was performed using the 3’ end primer 5'-
CGGATTCCAAAAGCACTGGGATTAC-3, and the external adapter 5’-
TAACCCGGGTCTACAAAGTG-3'. The first round PCR reaction was diluted 1:10 or
1:100 and second round PCR was performed using 5'-
CGGATTCCAAAAGCACTGGGATTAC-3’ and the internal adapter 5’-
AT ACT GCGT AACTGACTAT A-3. ’
Cloning
PCR products were either fractionated on 1% agarose, gel purified (Qiagen), or
passed through a Sephadex G-50 spin column (Boehringer Mannheim) to remove
oligonucleotides smaller than 72 base pairs in length. The vector pUC 19 was either Smal
or EcoR V digested and incubated in 1XPCR buffer with dTTP and Taq polymerase for 2
hours at 72“ C. The ligation was performed with T4 DNA ligase or with pCR2.1-TOPO
vector (Invitrogen). The pUC19 ligations were phenol/chloroform extracted, ethanol
precipitated and transformed into XL-1 blue cells (Stratagene) by electroporation. The
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pC R 2.l-T0P0 ligations were transformed into TOPO TF’ cells (Invitrogen) by heat
shock. The cells were plated on LB-ampicillin plates streaked with XGAL/IPTG for the
XL-1 blue transformations or XGAL only for the TOPO TF’ transformations. White
colonies were streaked to a grid and grown at 37“ C overnight. Nitrocellulose colony lifts
were prepared, hybridized and exposed to identify putative clones (Sambrook et al.,
1989). For confirmation, positive clones were subjected to PCR by picking bacteria into
IX PCR buffer (Perkin-Elmer Cetus) and incubating 10 minutes at 95 “C. One fifth of
this reaction was added to a PCR cocktail (IX PCR buffer, MgCl?, Taq polymerase, and
appropriate primers) and cycled at 95°C for 30 sec, 59°C for 30 sec, 72°C for 1 minute
(30 cycles). Resulting reactions were fractionated on 1% agarose gel. DNA (Qiagen)
was prepared from the clones that gave positive PCR results. The DNA was digested with
restriction enzymes to further confirm that the plasmid inserts were correct. Plasmid
DNA was used as a template for sequencing and a Perkin Elmer ABI machine was used
for separation and reading of the reaction products. Sequences for each of the three
isoforms were entered into the GenBank March 22, 1999 and are currently accessible.
Mutant DNA sequences
Animals were grown on NGM plates and the protocol of Sulston and Hodgkin
(Sulston and Hodgkin, 1988) was used to extract genomic DNA. PCR was performed
with gene specific primers and resultant products were cloned into the TOPO TA vector
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18
(Invitrogen). Isolated PCR clones were picked to an overnight culture and mini-prepped
(Qiagen). Plasmid DNA was used as double stranded template for sequencing on a
Perkin-Elmer ABI machine. Plasmid clones were obtained from three independent sets
of PCR reactions. Both DNA strands were sequenced.
Growth of Animals for Northern Analysis
For developmental samples, cultures of gravid N2 adults were washed in M9
buffer and treated with alkaline hypochlorite solution to obtain eggs (Sulston and
Hodgkin, 1988). These were collected before hatching and frozen in liquid nitrogen for
the egg sample. For the other developmental stages, the collected eggs were allowed to
hatch overnight without food to obtain synchronous L 1 larvae. These larvae were fed
and grown to the desired stage which was verified by scoring gonadal or vulva lineage
cells with DIC optics (Kimble and Hirsh, 1979; Sulston and Horvitz, 1977). The staged
animals were collected by centrifugation, separated from bacteria and debris by sucrose
flotation twice, rinsed in M9 buffer for 30 minutes and frozen by dripping into liquid
nitrogen. Dauer larvae were purified by treating animals from a 2 week old starved
culture with 1% SDS. PD1 animals were from feeding purified dauer larvae for 8 hours
at 20“ C. PD2 animals were from fed dauer larvae that had recovered and progressed to
the mid-L4 stage.
Dauer-inducing conditions for isolation of L2d larvae were created by filter
sterilizing media from 3-4 week old N2 cultures and mixing 3 volumes of old media with
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19
one volume of fresh media. A small amount of OP50 was given to the starved
synchronous LI larvae. The cultures were checked every 6-8 hours, fed if necessary,
until the gonads of most animals had 12 cells and a few had 16 cells.
Northern Analysis
Total RNA was isolated from approximately 0.25 grams of frozen packed worms
using RNA isolator (Genosys). The total RNA pellet was resuspended in DEPC treated
water, and poly-A+ mRNA selected by passing total RNA over oligo- (dT) Cellulose
Columns (Gibco-BRL) (Ausubel et al., 1997). Approximately Ipg of poly A+ mRNA
was denatured in formaldehyde, formamide, and running buffer (Sambrook et al., 1989)
and loaded onto a 1% agarose-formaldehyde gel submerged in IX RNA running buffer.
3 pg o f RNA MW markers (Gibco-BRL) were subjected to the same treatment. The gel
was run at 80 volts with the buffer re-circulating, until the dye migrated 8 cm from the
wells. The lanes containing the MW markers were cut away from the gel and stained in
ethidum bromide and de-stained in water. The gel containing the RNA samples was
rinsed in DEPC-water for one minute. Magna charge nylon (MSI) was pre-wetted in
water and soaked in 2X SSC. The RNA was transferred to the nylon in 20X SSC
overnight (Sambrook et al., 1989). The membrane was dried at room temperature for one
hour and then baked at 80’C for one hour. Church-Gilbert hybridization solution was
used (Church and Gilbert, 1984). A cDNA clone that encodes ubiquitin/ribosomal
protein s27A, YK104e5 (a gift of Yuji Kohara), was used to assess for equal loading of
mRNA in all stages. The daf-12 antisense-RNA probe was transcribed from Xbal
digested clone PL# 114 using T7 RNA Polymerase (Boehringer Mannheim) and 32P-UTP.
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20
The reaction contents were passed through a Sephadex-G50 spin column (Boehringer
Mannheim) to remove unincorporated radionucleotides. The pre-hybridization and
hybridization buffers were incubated at 57° C. The membranes were washed three times
in 0.1 X SSC. 1% SDS for 30-40 minutes at 65° C. The membranes were exposed to a
phosphoimager screen for 1 to 24 hours.
Promoter Analysis
Genomic DNA was extracted from mixed stage animals and used as a template
for Expand High Fidelity PCR (Boehringer Mannheim). A 4kb portion of the genomic
sequence o f that precedes daf-12 was amplified and it included the first two exons and
first intron of the gene. The PCR product was amplified with oligonucleotides containing
BamHI and Xbal restriction sites and these were used to clone into a TOPO TA vector
and transformed into TOPO TF’ cells. The promoter fragment was subcloned into GFP
gene containing vector pPD95.73 (a gift of A. Fire). For injection of the resulting clone,
plasmid DNA was isolated (Qiagen), precipitated with potassium acetate and
resuspended into injection buffer. Transgenic strains were created by the gonad
syncytium injection method of Mello and Fire (1995). GFP DNA constructs were
injected into N2 hermaphrodite at a concentration of 200 ng/pl along with 50 ng/pl of
plasmid pRF4, encoding rol-6(su!006), a semi-dominant mutation of the collagen gene.
FI animals were scored for the roller phenotype and several F2 transmitting lines were
established. For GFP analysis, adult animals were treated with basic hypochlorite to
establish a population of eggs. Animals were allowed to develop to various stages and
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21
were analyzed for expression. For visualization of the stage and GFP, a Leica inverted
microscope equipped with Differential Image Contrast optics, a GFP filter (Chroma) and
a fluorescent light source was used at a magnification of 1000 X.
Results
Cosmid transformation rescue
The C. elegans Genome Sequencing Consortium sequenced cosmid FI 1 Al and
the Genefinder program predicted an open reading frame FI 1A 1.3 with sequence
homology to a nuclear hormone receptor (The C. elegans Sequencing Consortium, 1998).
This position coincided with the flanking genomic sequence of the previously identified
transposon insertion mutation, daf-l2(m524) (Yeh, 1991). Before proceeding with
molecular characterization of this locus, extrachromosomal transgenic lines were created
to determine whether cosmid FI 1 Al was sufficient to rescue the Daf-d phenotype of daf-
12 mutant animals. DNA from cosmid FI 1 Al was prepared and co-injected with
plasmid pRF4 into the gonad syncytium o f daf-7(e!372); daf-l2(m20) double mutant
animals. The double mutant animals used in this experiment are dauer defective (Daf-d)
at 25°C and do not form dauer larvae due to the daf-l2(m20) mutation suppressing the
dauer constitutive phenotype of daf-7(el372). Dauer constitutive (Daf-c) animals form
dauer larvae inappropriately in ample food and low pheromone concentrations. If the
FI 1A1 cosmid, carrying the daf-12 gene, rescued the Daf-d phenotype of this double
mutant strain, then the Daf-c phenotype would be observed. Three stable transgenic
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22
strains were obtained and scored for dauer formation at 25.5°C. For two of the daf-7;
daf-12; Ex [FI lAl+pRF4] strains 99% of Rol animals were dauer constitutive (n=90 and
n=202). For the third daf-7; daf-12; Ex [FI 1 Al+pRF4] strain 59% of the transgenic
animals were dauer larva (n=4l). These results confirm that the functional daf-12 gene is
contained within the FI I Al cosmid. The above work was conducted by Dr. Pamela
Larsen.
cDNA clones for the daf-12 gene
Previously, a partial cDNA clone was obtained from a cDNA library using the
genomic region flanking the transposon (Yeh, 1991). This work sought to clone the full-
length cDNA by a RT-PCR strategy. Overlapping DNA clones were obtained from three
independent samples o f mixed stage N2 (wild-type) animals using two methods. Nearly
full-length clones were obtained by performing PCR with primers complementary to the
C. elegans splice-leader sequence (SL1) and gene specific primers in the last predicted
exon (exon 17) o f the gene. A second method was used to obtain the remaining portion
of the gene located 3' of exon 17, including the untranslated region. Rapid Amplification
of cDNA Ends (Frohman et al., 1988) was performed using adapter primers (see
materials and methods) at the 3' end and gene specific primers from exon 12 at the 5' end.
An overlap of 831 bases between the two sets of clones was found. At least three clones
from each independent set o f animals were sequenced to reveal the presence o f three SL1
spliced transcripts, which have been designated the A l, A2 and B isoforms (Figure 5).
Comparison of the cDNA sequence and the Genefinder predicted open reading frames
revealed several discrepancies. The Genefinder program did not predict exon 1 of the Al
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23
iso form. Two exons, one located between exon 1 and exon 2 and the other between exon
2 and exon 3, were predicted but were not found in our Al sequence. A discrepancy was
also found at the boundary of exons 13 and 14. Exon 13 contains more nucleotides at the
3' end than what was predicted by the Genefinder program. The GenBank accession
numbers for the isoforms are AF136238 (Al), AF136239 (A2), and AF136240 (B).
These accession numbers were cited in a report that describes a daf-12 A3 iso form that
has a 16 amino acid difference from the Al described here (Antebi et al., 2000).
The Al iso form transcript contains 17 exons and spans 3,683 bases. Al has a
putative initiating methionine in the first exon located 35 nucleotides 3' of the start of the
exon. The Al methionine is immediately preceded by the sequence TTAA. This
sequence is similar to the C. elegans consensus translation initiation sequence AAAA. A
14kb intron is located between exon 2 and exon 3 which is unusually long for C. elegans
(Blumenthal and Steward, 1997). The other daf-12 introns range in size from 46 to 693
nucleotides. Genefinder predicted a putative protein of 219 amino acids on the
complementary strand between exon 3 and exon 2 located in the large I4kb intron. The
A2 isoform transcript lacks exon 1 and exon 2 and is 203 bases shorter than the Al
iso form. A2 has a putative methionine initiator codon located 8 nucleotides into exon 3,
which is the first exon for this isoform. The sequence TCAA immediately precedes the
ATG sequence and is similar to the consensus translation initiation site. The B isoform
transcript consists of 2,192 nucleotides spanning 5 exons (numbers 13 through 17) at the
3’ end of the gene. The B iso form does not include the DNA binding domain. The
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24
Figure 5: Genomic organization o f the daf-12 gene. The daf-12 gene produces three
transcripts. Exons are represented by rectangles, introns by lines. The genomic region is
depicted across the top and the putative domain structure based on homology to other
genes in the steroid/thyroid hormone receptor superfamily is depicted along the bottom.
The Al isoform of the daf-12 gene contains 17 exons, the A2 isoform lacks the first two
exons, and the B isoform consists of a small portion of the hinge region and the entire
ligand binding domain.
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CM
C s |
□ H
C M
<
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H ypervariable binding H inge Ligand binding
26
putative initiating methionine is the first amino acid o f exon 13. It is common for the
splice leader (SL) in C. elegans to be spliced close to the initiating methionine and the SL
may therefore play a role in the initiation of translation (Blumenthal and Steward, 1997).
The A l, A2 and B isoforms share 2,192 bases and the 3’ UTR extends 1,388 bases
beyond exon 17. The above work was conducted with the assistance of Patricia Herd.
Sequence homology and putative functional domains
The Al isoform deduced protein is 753 amino acids long (Figure 6) and this
predicted protein sequence was used in a BLAST homology sequence search (Altschul et
al., 1997). The most homologous sequence to that of DAF-12 is a steroid/thyroid
hormone receptor gene from S. stercoralis. a human parasitic nematode. This protein is
homologous throughout the entire daf-12 protein, specifically 96% (73/76) identical in
the DNA binding domain and 40% (96/238) identical in the ligand binding domain.
DAF-12 is similar to the D. melanogaster hormone receptor 96 (DHR96), 68% (52/76)
identical in the DNA binding domain and 26% (62/238) identical in the ligand binding
domain. DAF-12 is 64% (49/76) identical in the DNA binding domain and 18% (42/238)
identical in the ligand binding domain to C. elegans NHR-8. DAF-12 is 53% (40/76)
identical in the DNA binding domain and 5% (12/238) identical in the ligand binding
domain to H. sapiens vitamin D Receptor.
The predicted protein product o f the daf-12 Al isoform has a modular structure similar to
hormone receptors, including an N terminal hypervariable region, zinc finger DNA
binding domain, hinge region, and ligand binding domain. The hypervariable region
contains the amino acids 32KRVT35, a potential phosphorylation site preferred by
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27
cGMP-dependent protein kinases (Fremisco et al., 1980; Glass et al., 1986; Glass and
Smith, 1983). The DNA binding domain, approximately 75 amino acids, consists of two
C4 type zinc finger regions and contains a total of 9 cysteine residues in conserved
positions. The two zinc fingers are encoded across different exons, similar to other
nuclear hormone receptors (Huckaby et al., 1987). The first zinc finger contains the P-
box sequence CESCKA. The PSORTII program identified an 18 amino acid bipartite
nuclear localization signal at the end of the DNA binding domain (residues 184-201).
This sequence has the hallmarks of the bipartite pattern with 2 basic residues followed by
a spacer of 10 amino acids and ending with a portion in which at least 3 of the 5 residues
are basic (Robbins et al., 1991). The hinge region that spans approximately 400
nucleotides contains several potential N-myristylation sites. A portion of the ligand
binding domain o f DAF-12 (amino acids 565-588) is identical with some residues in the
t, region o f group II hormone receptors. The x, region, along with the dimerization
region, are two subdomains within the ligand binding domain in group II receptors
(Forman and Samuels, 1990). A tyrosine kinase phosphorylation site lies in the ligand
binding domain at residues 654-661.
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28
Figure 6: Alignment o f the DAF-12 Al sequence with members of the nuclear hormone
receptor superfamily. The Al isoform was translated with MacVector © and the amino
acid sequence subjected to BLAST analysis. Black boxes indicate identical amino acids
and gray boxes indicate similar amino acids. The locations of sequenced mutations in
DAF-12 are depicted by dot (•) with the allele number. Domains are depicted above
amino acid sequence: hypervariable (— ), DNA binding (////), hinge (****), and ligand
binding (\ \ \ \ ). At the end of each row the amino acid numbers are given for the
particular protein. Abbreviations: D12=C. elegans DAF-12; SGR=5’ . stercoralis
nuclear hormone receptor-like; N48= C. elegans NHR-48; D96= D. melanogaster
hormone receptor 96; N8= C. elegans NHR-8; VDR= H. sapiens vitamin D receptor.
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29
------------------------------------------------------------------------------------------------- »m583-----------------
D12MGTNGGVIA£QSMEXETNENPDKVEEPWRRKRVTRRRHRRIHSKNN|l'EPpH | dD P H | B ^ H H I 73
SGR 44
N48 I 3 H ^ n I 63
--------------------------------------------------------------------- --------------------/ / / / / / / / / / / / / / / / / / / / / / / / « m ll6
12 l | A A P ( 4 N G Y H | s | v | L E | s | G § C S | F | D E S i H n ^ | ^ ^ n ^ ^ H ^ H H ^ B ^ R 4 6
« « i i
. . . 1 9J L D U J B 1 B V H P 54
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/ / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / • m 2 0 / / / » ~ ......................................
D 12H H |^ B B H B H B H ^ ^ ^H |H H H ^ H |^ H H ^ H j^ B lT G ?C N K R SQ P G N Q Q S2 1
N 48 § I ^ H H h s h iss
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g| g
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429
D12TATG|GDAAEEM YKRM t4FYENCXQSALDHENQ|PKPQEABPKEEYM THHGE(^QSBYQVKABlig|511
s g r T i Til m ■ i l iB Ip 5 1 2
D96 I ggi 476
\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \_\\\\\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ U \ \ \ \ \ \ \ \ \ \ \
0 1 2 H H | ^ n n r n f r i f c ^ n i n p F A K R A N A Q A Q K A K c R H i * f lR ii 6 ^ E s i
i t r j i ■ _ m E 656
s r S n W f 1 d F 1. . 1 A t t s ;
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\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \
D12Q
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\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \
Di2,E rN # n cii 753
SGR E l 3 [ I H 749
D96 I i r i l I 721
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30
Sequence o f mutant alleles
The zinc finger domains of three different dauer defective daf-12 alleles (m583,
m20, and m l 16) were sequenced to determine whether or not their molecular lesions
altered this region. This domain was chosen because the site of the Tel insertion was at
the end o f an exon just outside the DNA binding domain (Yeh, 1991). The lesion in the
m583 allele is a thymine rather than a cytosine at base 176 of the Al isoform that results
in a glutamine (CAA) to stop codon (TAA) at amino acid 59. Allele m l 16 has a change
from a guanine to adenine at base 428 of the A 1 iso form resulting in an arginine (AGG)
to lysine change (AAG) at amino acid 143. The m20 mutation has a change from a
guanine to adenine at base 561 of the Al isoform resulting in a tryptophan (TGG) to stop
codon (TGA) at amino acid 187. The m583 mutation is predicted to effect only the Al
isoform while the m20 and m l 16 mutations effect the Al and A2 isoforms, but not the B
isoform, so these 3 alleles are not molecular nulls.
Northern analysis
Poly A+ RNA was extracted from animals at ten different stages, from egg to adult, for
analysis of daf-12 expression during development. A daf-12 B anti-sense RNA probe
was used to detect the three isoforms to be able to compare relative levels o f expression
between the isoforms. The developmental Northern shows an upper band of
approximately 3.8 kb, which is consistent with the size of the Al and A2 isoforms, and a
lower band at 2.2 kb which corresponds to the B transcript. The cDNA sequence of the
Al and A2 isoforms indicates that they differ by 203 bases and mRNAs of this size
would not be expected to resolve in a formaldehyde gel system, daf-12 mRNA is
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31
detected at all stages of development (Figure 7A). A cDNA clone that encodes
ubiquitin/ribosomal protein s27A was used to assess loading of mRNA in each stage
(Figure 7B). The relative levels of mRNA were quantified and after normalization to the
loading control, the highest levels were observed in egg and L2d samples (Figure 1C).
An approximate two-fold increase in daf-12 expression is observed in L2d pre-dauer
compared to the level detected in eggs. LI and L2 levels are lower than egg levels.
Although very low, daf-12 mRNA is detected in the post dauer-1 (PD1) stage, in which
animals recovering from the dauer stage resume normal pharyngeal pumping and resume
growth. The daf-12 mRNA is more easily detected in samples of post dauer-2 (PD2)
larvae that are morphologically similar to an L4 animal (Liu and Ambros, 1991). The
daf-12 mRNA is detected at approximately equivalent levels for L3, L4 and adult stages,
although they are lower than the early developmental stages. Transcription of many
genes has been shown to decrease in the dauer stage with the notable exception being
hsp90 that showed a very strong signal in dauer larvae (Dailey and Golomb, 1992). A
strong hsp90 signal was found in dauer larvae samples, but no daf-12 signal could be
detected.
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32
Figure 7: Developmental Northern analysis. A. I pg of the twice selected poly-A+
RNA was fractionated on a 1% formaldehyde containing agarose gel, transferred to a
nylon membrane and was probed with j2P labeled anti-sense RNA from a daf-12 B
isoform clone template. The lanes are labeled with the developmental stage o f the
animals from which RNA was isolated. B. To assess loading, the membrane was probed
with a cDNA clone, ykl04e5, which encodes ribosomal protein 27 fused to ubiquitin.
Numbers depicted on the right side of figure indicate size of RNA in kilobases derived
from molecular weight markers. C. The daf-12 and ykl04e5 expression levels were
quantified with a phosphoimager. The Y axis represents the normalized volume o f pixels
detected by the phosphoimager.
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33
Al/A*
daf-12
B yk!04e5
c.
e
z
8
7
■ A isoforms
□ K Lsoform
6
5
4
3
1
0
EGG LI L2 L2D PDl PD2 L3 L4 ADULT
Stage
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34
To determine whether daf-12 isoform levels are regulated by genes in the dauer
formation genetic pathway, mRNA samples were prepared from animals with mutations
in daf-7, daf-3, daf-12 and daf-16 that had been grown under dauer pheromone inducing
conditions. Northern analysis was performed with a daf-12 B antisense RNA probe. The
A1/A2 and B isoform bands were detected in the mRNA from each mutant strain (Figure
8A). Quantification of the daf-12 bands within each strain shows the relative ratio of
mRNA for the A isoforms is approximately equal to the mRNA levels of the B isoform
(Figure 8B). This is even the case for the daf-l2(m20) strain that has a nonsense
mutation in the Zn-finger domain, again suggesting that the allele is not a molecular null
due to an unaffected B isoform.
Analysis of GFP expression under the control of the daf-12 promoter
A frequently used marker to determine when and in which tissue a promoter
drives expression is GFP (Ogg et al., 1997; Paradis et al., 1999; Paradis and Ruvkun,
1998; Patterson et al., 1998; Ren et al., 1996; Schackwitz et al., 1997). It is assumed that
the GFP pattern resembles the normal cellular distribution, although over-expression of a
reporter gene may not represent endogenous expression exactly. The putative promoter
region and first two exons of the daf-12 gene were cloned into a GFP vector for C.
elegans. Transgenic lines were isolated and GFP fluorescence was detected in the cells
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35
Figure 8: Analysis of steady-state daf-12 isoform ratios in various genetic backgrounds.
A. For each lane, 1 jig o f the twice selected poly-A+ RNA was loaded from mutant
strains grown from eggs under dauer-inducing conditions. The alleles used were daf-
3(el376), daf-7 (el372), daf-16(m26), daf-12(m20) and wild type. The membrane was
probed with 32P labeled anti-sense RNA daf-12 B isoform. The bands in the daf-3 sample
are shifted slightly lower due to a gel artifact. B. Ratio of the volume o f pixels detected
for the A isoforms to the volume of pixels detected for the B isoform.
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36
A1/A2
B .
1.2
1.0
0.8
a
^ 0.6
w
3 0 L 4
X
0.2
0.0
llllll
daf-3 daf-7 daf-16 daf-7; daf-12 N2
daf-12
Strain
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37
Figure 9: Photomicrographs illustrating daf-12.-.QFP expression.
Expression is detected in animals carrying extrachromosomal array construct. DIC (1)
images are depicted on the left and the corresponding fluorescent images (2) are on the
right. A and B are the anterior and posterior view, respectively, of the pharynx of the
same adult. C is an L3 larvae. D is an LI larvae.
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38
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39
of the pharynx (Figure 9). The pharynx of C. elegans displays 3-fold symmetry and
consists of 4 regions: the procorpus, metacorpus, isthmus, and terminal bulb (White,
1988). Animals expressing the daf-l2v.GFP construct display the highest levels of GFP
throughout the procorpal region in the pharyngeal muscle (pm3) cells. GFP is also
expressed throughout the metacorpus (pm4 cells) as well as the isthumus (pm5 cells) and
portions of the terminal bulb (pm6). These transgenic animals appear to be express GFP
throughout the muscles of the pharynx and the pattern o f expression is similar to
fluorescently labeled phalloidin which stains muscles in C. elegans (Waterston, 1988).
GFP florescence is detected in each developmental stage including dauer larvae.
Discussion
In this chapter, rescue of the dauer defective phenotype of daf-12 animals with the
FI 1A1 cosmid was demonstrated. There are three differentially spliced mRNAs from
this locus and each isoform shares homology with nuclear hormone receptors and is SL1
spliced. The A1 and A2 isoforms differ by 203 nucleotide bases at the 5’ end while the B
isoform lacks 1,491 bases from the 5’ end of the A 1 isoform. The three isoforms share
2,192 bases in common. The three iso forms are present throughout development and the
quantity peaks in pre-dauer animals, at the L2d stage. The sequence o f three daf-12
mutations that display a dauer defective phenotype shows each to be single base pair
point mutation in or near the DNA binding domain. Two of the alleles are nonsense
mutations and one is a missense mutation. The ratio of daf-12 A isoforms to B isoform
mRNA levels are not altered in samples from daf-12, daf-3, daf-7 and daf-16 mutants.
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40
Expression is detected in the pharynx, which is known to be remodeled in the dauer stage
(Vowels and Thomas. 1992), in transgenic animals carrying a construct containing the
daf-12 promoter region fused to the GFP gene.
The deduced protein o f DAF-12 can be assigned a modular structure based on
other hormone receptors. DAF-12 has two C4-type zinc finger domains including 9
cysteines in conserved positions which are common in the DNA binding domain of
members of the steroid and thyroid hormone receptors (Evans, 1988). DAF-12 contains a
P-box sequence, CESCKA, in the first zinc finger. This motif is important in DNA
binding specificity and the CESCKA sequence has been reported in nuclear hormone
receptors from a number of other species (Sluder et al., 1999). DAF-12A may bind to
response elements similar to those of these other nuclear hormone receptors.
Three daf-12 dauer formation defective mutants were sequenced (m20, m l 16, and
m583) and the molecular lesion for each allele is a point mutation located in or close to
the DNA binding domain. The zinc finger DNA binding domain of nuclear hormone
receptors is critical for the transcriptional regulation of target sequences (O'Malley et al.,
1991). daf-12 likely binds to target genes important in the control of dauer formation and
longevity. Furthermore, these target genes could interact antagonistically with the daf-2
pathway components and give rise to the negative regulatory loops proposed in the
genetic pathways (Gems et al., 1998; Gottlieb and Ruvkun, 1994; Larsen et al., 1995).
Mosaic analysis of daf-2 by Apfield and Keynon (1998) showed daf-2 is cell
nonautonomous and they propose that daf-2 controls the daf-12 ligand production or
activity in dauer formation, but not for adult longevity. This may be the case, or since the
daf-2 pathway has been shown to regulate fat metabolism and storage (Ogg et al., 1997),
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41
the daf-12/daf-2 pathway interaction may be cross-talk similar to one recently described
with fatty acids regulating a nuclear hormone receptor (Tobin et al., 2000). Hsin and
Kenyon demonstrated that laser ablation of germline precursor cells can lead to an
increase in life span (Hsin and Kenyon, 1999). They performed germline ablations in a
daf-12(m20) background and found daf-12 is necessary for the life span extension
phenotype o f these laser ablated animals. Taking into consideration the daf-12 isoform
structure and location of molecular lesions, it can now be further stated that function of
the DAF-12 A isoforms are necessary for the life span extension phenotype o f these laser
ablated animals. Since the daf-12(m20) mutation only affects the Al and A2 isoforms
further details of this phenotype may be uncovered by finding the target genes regulated
by Al and A2. Germline ablations in a daf-16 background showed that it is necessary for
the life span extension phenotype of these laser ablated animals (Hsin and Kenyon,
1999). Thus, the target genes may be regulated by the transcription factor DAF-16 as
well as DAF-12.
daf-12 was originally identified in a genetic screen as a gene that mediates dauer
formation and interpretation o f genetic epistasis studies placed daf-12 at the end of the
regulatory pathway (Riddle et al., 1981). Phenotypically, daf-12 mutants and some daf-
12 double mutants, in the presence of pheromone make L2d animals but do not form
dauer larvae (Vowels and Thomas, 1992). Mutations in daf-2, daf-5, and daf-6 display
the Daf-d phenotype and do not form L2d animals or dauer larvae, suggesting daf-12 is
required for the L2d to dauer transition but may not be important for the LI to L2d
transition (Vowels and Thomas, 1992). The developmental Northern analyses in Figures
7A & 7B shows an approximate two fold increase in daf-12 mRNA levels at the L2d
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42
stage. In addition, the location of the lesions of the three completely dauer defective
alleles in the DNA binding domain suggests this region is important for dauer formation.
Taken together, these results suggest that increased steady-state levels of mRNA and a
Zn-fmger domain o f wild-type sequence fulfill the requirement for daf-12 function at the
L2d stage for dauer entry, daf-12 may be regulating the genes responsible for dauer entry
as well as genes necessary for continued maintenance of the dauer state.
The promoter region and exons 1 and 2 fused to the GFP is expressed in the
pharynx throughout development. The pharynx is known to be remodeled during dauer
formation (Vowels and Thomas, 1992) and daf-12 may function in cells in the pharynx to
remodel this tissue by binding to targets in this tissue to positively regulate genes
important for morphological changes that accompany dauer entry. The daf-12 mRNA
was found to be rare, so it is conceivable that the GFP pattern observed underestimates
the cells in which daf-12 is expressed. In dauer larvae, daf-12 mRNA was not detected
but expression of GFP was observed which may be due to the stability of the GFP.
Recently, the pattern of a FI 1 Al ::GFP construct was reported to be in most tissues
(Antebi et al., 2000) which is probably due to genomic regulatory domains that are not
included in the construct studied. There may be a second promoter located between the
second and third exons in the 14 kb intron. This second promoter would be predicted to
direct expression of the A2 and B isoforms. There is precedence for such an arrangement
in the ecdysone receptor and hormone receptor 38, two nuclear receptors from
Drosophila that have been demonstrated to have two distinct promoters (Kozlova et al.,
1998; Talbot etal., 1993).
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To understand how other d a f genes affect daf-12 at the molecular level, mRNA
expression was examined from animals grown under dauer-inducing conditions. One
branch of the dauer formation pathway includes genes that show sequence similarity to
the TGF-6 signaling pathway components (e.g. daf-3, daf-7) and an interacting branch
includes genes homologous to insulin-like signaling (e.g. daf-16) components (see
Figures 3 & 4). The ratio of the daf-12 mRNA A isoforms/B isoform are not altered in
animals with mutations in daf-7 (Daf-c), daf-3 (Daf-d), daf-16 (partial Daf-d), or daf-12
(Daf-d). The Al and A2 isoforms may be differentially regulated in these mutant
backgrounds, but the expression levels for these individual isoforms are not resolvable at
the level of Northern analysis. The results suggest the relative daf-12 steady state mRNA
isoform levels are not altered by genes from the TGF-beta and insulin-like signaling
pathways. Perhaps the alteration of function occurs via protein interactions such as the
one described between Smad3 and the vitamin D receptor (Yanagisawa et al., 1999).
Interestingly, the daf-3 expression pattern overlaps with that of daf-12 and is thus a
candidate Smad for interaction with DAF-12 (Patterson et al., 1998).
DAF-12 has sequence similarities in the ligand binding domain to other nuclear
hormone receptors, but is less highly conserved in the DNA binding domain. All C.
elegans nuclear hormone receptors, including daf-12, are defined as orphans because a
ligand for these receptors has yet to be identified or does not exist. There are similarities
between the tj motif of the ligand binding domain of DAF-12 and a subfamily (group II)
of nuclear hormone receptors including the human vitamin D receptor (amino acids 245-
276). The t, motif is a putative transcriptional inactivation domain that is relieved by
ligand binding (Forman and Samuels, 1990). Dimerization domains are important in
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44
formation of interfaces that could interact with specific partners and bind as hetero- or
homodimers on hormone response element sequences in the DNA (Beato, 1989; Evans,
1988; Green and Chambon, 1988; Yamamoto, 1985). Overall, the putative DAF-12
dimerization domain is not well conserved, but some similarities are seen in key
hydrophobic residues in repeat #2 and #7 of the human VDR.
daf-12 is present throughout development and could act as a competence factor in
reproductive growth conditions and adult longevity. As stated, regulation of the dauer
traits is confined to the DNA binding region by mutant analysis. The separable
phenotypes of abnormal cell migration and abnormal cell lineage result from defects in
the ligand binding domain (Antebi et al., 2000) perhaps altering dimerization strength,
partner choice, or ligand binding. Dauer larvae formation is regulated in a temporal
fashion by the genes lin-4, lin-14, lin28 and lin-29 (Liu and Ambros, 1989). Epistasis
analysis suggests daf-12 is involved with the lin-14 and lin-28 genes (Antebi et al., 1998).
Therefore, certain daf-12 alleles that display the abnormal cell migration phenotype at the
L2/L3 division may be affecting some part of the Lin genes in this network. However,
the molecular details of this interaction remain to be determined. In addition, daf-3 r.gfp
expression is detected in the distal tip cells (Patterson, et al. 1997) suggesting multiple
genes in the dauer formation pathway may be regulating migration of the distal tip cells.
The DAF-12 isoforms may be distinct in their function: the Al and or A2 iso form could
be necessary for dauer formation in particular tissues or at specific times, while the B
isoform may be autoregulatory. The hypervariable domain differs between Al and A2,
but there is no obvious functional domain in the additional 59 amino acids of A l. One
possibility for a functional difference may be in the time and place that A l or A2 is
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45
expressed. The daf-12 B isoform could act with a dominant negative-like function in
dauer formation similar to the non-DNA binding domain form of the E75B hormone
receptor from Drosophila which has been demonstrated to act as a dominant negative in
ecdysone induction of early genes (White et al., 1997). SHP, a murine orphan nuclear
hormone receptor, lacks a DNA binding domain and was demonstrated to interact with
and inhibit transactivation of some nuclear hormone receptor family members (Seol et al.,
1996).
The DAF-12 protein sequence is most similar throughout the entire Al iso form to
a steroid/thyroid hormone receptor from Strongyloides stercoralis, a human parasite.
This gene is speculated to be involved in the accelerated development of this parasite
during its auto-infection cycle (Siddiqui et al., 2000). DAF-12 shows similarity in the
DNA binding domain to a C. elegans hormone receptor nhr-48, a gene with unknown
function (Sluder et al., 1999). DAF-12 has similarities in the DNA binding and ligand
binding domains to the Drosophila melanogaster gene DHR96 that was cloned by a
degenerate PCR approach to identify hormone receptor genes (Fisk and Thummel, 1995).
The DHR96 gene was found to be inducible by 20-hydroxyecdysone, the hormone
known to be responsible for molting through successive stages in insects (Fisk and
Thummel. 1995; Riddiford, 1993). DHR96 produces two transcripts, 2.8 and 0.6 kb
which are expressed equally and increase expression at 106 hours of development.
Homology is also seen with nhr-8, a gene identified as a nematode hormone receptor
required for gut function that may interact with genes in the dauer formation pathway to
direct the dauer decision (Lindbloom and Sluder, personal communication). DAF-12 is
more distantly related to quail, rat, mouse and human Vitamin D receptors in both the
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46
DNA binding domain and ligand binding domain. The vitamin D receptors from rats and
humans are known to be activated by 1,25 dihydroxyvitamin D3 and interact with
chromatin to increase transcription of downstream targets. Downstream effects include
regulation of calcium transport in intestine, mobilization of calcium from bone and
reabsorption of calcium in kidney (Burmester et al., 1988). Vitamin D receptor null
mutant mice display retarded growth, skin and bone abnormalities, and immature uteri
after they are weaned, and most homozygous animals die at 15 weeks (Yoshizawa et al.,
1997). Null thyroid hormone receptor mutant mice, lacking both T3 receptors, have
decreased body growth and bone length, poor female fertility, and a hyperactive
thyrotropes (Gothe et al., 1999). Thus, regulation of growth is a common feature of
members of the nuclear hormone receptor family related to DAF-12.
The DAF-12 Al protein sequence was used to search the PROSITE database to
identify putative functional domains, in addition to those of classic nuclear hormone
receptors described above. Two cAMP/cGMP dependent protein kinase phosphorylation
sites were identified. 32KRVT36 is present in only the Al isoform and is located in the
hypervariable domain, while 197RKNS200 is in the AI and A2 iso forms near the end of
the DNA binding domain. The daf-1 1 gene, which is suppressed by daf-12 and
genetically functions upstream of daf-12, is homologous to guanylate cyclase which is
known to convert GTP to cGMP (Riddle and Albert, 1997). DAF-12 A l, A2 and B have
a consensus tyrosine kinase phosphorylation site 654KKNELAVY661 located at the C
terminal region of the hinge region. A BLAST search reveals this phosphorylation
sequence is unique to DAF-12 and a hypothetical protein from Treponema pallaidum
(Altschul et al., 1997). These key sites located within the DAF-12 nuclear hormone
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Al
receptor may be important functional domains that could interact with other d a f gene
protein products to dictate certain aspects of dauer formation and adult longevity.
Presented here are the molecular details of the daf-12 gene and demonstration that
it has the hallmarks of a nuclear hormone receptor. The integration of multiple signals
and a continuous reassessment of environmental conditions are necessary for C. elegans
to grow to adulthood when conditions are favorable for progeny production. A steroid
hormone receptor that is produced throughout development may be a key regulatory
molecule to coordinate the multiple signals for reproductive growth, up-regulate genes
important for the dauer decision, and modulate the daf-2 pathway function to regulate
adult life span.
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Chapter 2
48
A Longitudinal Analysis of Adult
Neurons in Caenorhabditis elegans
Introduction
In Caenorhabditis elegans many behaviors are mediated by the nervous system
including foraging, response to mechanical and chemical stimuli, and egg laying (Chalfie
et al., 1994). In other neuronally regulated behaviors such as movement, pharyngeal
pumping and defecation, age-associated declines have been described (Bolanowski et al.,
1981; Croll et al., 1977; Gems etal., 1998; Kenyon et al., 1993). To determine if there is
a relationship between the decline in these behaviors and the status of neurons, it is
necessary to follow individual neurons in individual animals as they age. Previous
studies following neuron number in rodents and humans have used a cross sectional
design and the correlative conclusions drawn from these experiments are controversial
with regard to whether or not there is a decline in neuron number with advancing age
(Calhoun et al.. 1998; Ohm et al., 1997; Pakkenberg and Gundersen, 1997; Rapp and
Gallagher, 1996; Smith etal., 1999; West, 1993).
The methods used to count neurons in mammals have improved over time to
provide a more reliable estimate of neuron number (Long et al., 1999). However, using
mammals as model systems to address the question of neuron status with increasing age
is limited because the same individual cannot be sampled at multiple ages. It is therefore
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49
not possible to accurately determine if changes in neurons occur over the span oflife.
Thus, to follow neurons in a longitudinal manner in the same individual a different
system is needed. C. elegans has several distinct advantages as a model system to
determine neuron status with increasing age such as the ability to illuminate specific
neurons with the GFP in live animals.
An initial framework within which to place longitudinal experimental results has
been created after analysis of morphometric measurements of aging C. elegans (M. Tom
and P.L. Larsen, unpublished). This analysis proposed the animals progress through four
phases of adulthood. Use of long-lived Age mutants suggests that underlying genes and
mechanisms exist that control the animal's progression through phenotypically distinct
categories as it ages. The four distinct categories are termed Ml through M4. In contrast
to development, which is chronologically tightly regulated, the amount of time an
individual spends in a particular adult phase varies from individual to individual. After
the animal molts from the L4 stage and becomes an adult, it enters the Maturation (Ml)
phase. In this phase the animal grows and produces offspring. After the animal ceases
production o f eggs and reaches its full size, it enters the Maintenance (M2) phase and is
an active adult. As the animal ages it will eventually display substantial decline in
function termed the Moribund (M3) phase. In this phase the animals become
increasingly frail, are eventually very decrepit, and are close to the ends o f their lives.
When the animal ceases pharyngeal pumping and movement it is considered dead, which
is called the Mortality (M4) phase.
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50
Genetic analysis has revealed mutations in several different genes can increase the
life span of C. elegans. The age-1 (ageing abnormal) gene was originally identified in a
screen for animals that displayed an extended life span phenotype (Friedman and
Johnson, 1988; Johnson, 1990; Klass, 1983). Subsequently, the age-2 gene was found to
slightly extend the life span of the animals when mutated (Yang and Wilson, 1999). Two
genes that were originally isolated based on their abnormal dauer formation phenotypes,
daf-2 and daf-23 (dauer/ormation abnormal) display an increased adult life span
phenotype when mutated (Kenyon et al., 1993; Larsen et al., 1995). The age-1 and daf-
23 genes were subsequently shown to be allelic and the gene is now known as age-1
(Malone et al.. 1996; Morris et al.. 1996). The life span extension phenotypes of daf-2
and age-I mutants require wild-type daf-16 and daf-18 genes (Kenyon et al., 1993;
Larsen et al., 1995). The clk-1, clk-2, clk-3 (clock abnormal) and gro-I (growth rate
abnormal) mutants have altered developmental timing and display an extended life span
(Lakowski and Hekimi, 1996; Wong et al., 1995). Certain mutant alleles o f spe-26
(spermatogenesis abnormal) accumulate defective spermatocytes and display an
increased life span (Van Voorhies, 1992). The identification of these genes has provided
some insight into the mechanisms of aging in C. elegans, but the details of how these
genes interact has yet to be elucidated.
Molecular characterization of the genes involved in life span has resulted in the
hypothesis that an insulin-like signaling pathway plays a role in life span determination.
The age-1 gene is homologous to a phophotidyIinositol-3-kinase (Morris et al., 1996), the
daf-2 gene resembles an insulin/insulin-like growth factor receptor (Kimura et al., 1997),
the daf-16 gene is similar to a forkhead transcription factor (Lin, et al, 1997; Ogg et al.,
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51
1997) and the daf-18 gene is similar to a human tumor suppressor homolog PTEN (Gil et
al., 1999; Ogg and Ruvkun, 1998). A model for signaling has been proposed based on
genetic evidence and the molecular identities of the genes in the pathway (see Chapter 1),
but biochemical activities have yet to be established.
Laser ablation of the C. elegans germ line precursor cells (Z2 and Z3) results in a
life span extension suggesting a signal related to the germ line shortens or limits life span
(Hsin and Kenyon, 1999). DAF-16 is required for this life span extension and the Age
phenotype of daf-2 is enhanced by laser ablation of the Z2 and Z3 cells (Hsin and
Kenyon, 1999). These results suggest the germ line signal may act through the insulin
like pathway to regulate life span. In addition, animals with mutations in genes which
encode components important for sensory cilia display an extended life span phenotype
suggesting they are also required for normal aging (Apfeld and Kenyon, 1999). Several
genes and cells have been identified to be important for regulating aging in C. elegans.
What remains to be determined are the mechanisms involved in the aging process.
The attributes of C. elegans make it an excellent model system to determine if
there is a relationship between the age-associated decline in behaviors, such as
movement, and the status of neurons. The experiments presented in this chapter are the
first to follow neurons in C. elegans individuals in a longitudinal manner. Following
specific classes of neurons, this work shows that for the majority of individuals there is
no change in the number of GABA (unc-25::gfp) or dopamine {cat-2::gfp) neurons
expressing GFP with age. The small number of animals that did display changes were in
specific neurons. Assuming that the endogenous gene and the transgene are similarly
regulated, absence of GFP expression may indicate functional loss because the unc-25
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52
and cat-2 genes encode enzymes necessary for neurotransmitter biosynthesis. No
animals were observed to lose GFP expression in GABA or dopamine neurons when the
daf-2 mutation was present. Using this longitudinal approach, the conclusion is that loss
of GFP expression is not widespread and the daf-2 mutations are neural protective.
Methods
Strains used
Strains used in this experiment include: N2 Bristol, ju ls 7 [«nc-25::GFP; lin-
15(+)l daf-2(m4l). daf-2(el370J, him-5(el490); nisi 18 [cat-2::G¥P; lin-15(+)]. The
juls7 strain (CZ520) is a gift of Dr. Yishi Jin, University of California, Santa Cruz. The
him-5; nisi 18 strain (EM641) is a gift of Dr. Robyn Lints of the Albert Einstein School
of Medicine.
Culturing and handling of animals
Standard procedures were used for cultivating the nematodes (Brenner, 1974).
Synchronous cultures were initiated with eggs obtained by using hypochlorite treatment
on gravid adults (Sulston and Hodgkin, 1988). Strains containing daf-2 were raised at
15”C until they reached the L4 stage. To maintain age synchrony, adults were moved to
fresh plates on a daily basis. These progeny were grown until the mid to late L4 stage
and picked to plates in groups of 20-30 animals. These groups of animals were aged at
20 “ C and moved on a daily basis to fresh plates until they reached day 6 of adulthood
(approximate post-reproductive age). Animals that were not actively moving, had
internally hatching animals (bags) or exploded vulvas were excluded from the study.
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53
Scoring GFP expression
Individual animals were raised in cohorts (approximately 20 individuals/cohort),
examined for GFP expression at day 6 of adulthood and again just before death.
Mounting pads were constructed of 5% noble agar on glass cover slips. Molten agar was
dripped from a Pasteur pipette. A second cover slip was immediately dropped on top of
the agar to create the thin pad. After removal of one cover slip, a small drop of M9 (1-2
microliters) was placed on the agar pad. An individual animal was placed in the M9,
covered with a second cover slip and was observed at 100X with differential image
contrast (DIC) optics. The animals were exposed to ultraviolet light for a period of no
more than 30 seconds to determine their GFP expression. The animals were examined in
three different fields-the head, vulval, and tail regions-and in all the focal planes. The
individual neurons were identified according to published literature (Mclntire et al.,
1993; Sulston et al., 1975; White et al., 1986) and by using landmarks for a particular
neuron such as the pharyngeal bulbs, vulva, and commisures. Immediately after the GFP
expression pattern was determined for each individual, the cover slip was moved back to
the light microscope. The slide was re-inverted, the top cover slip was removed and M9
was added to the individual animals. Using a mouth pipette, individual animals were
moved from the M9 on the cover slip to freshly seeded 60mm NGM agar plates and
examined a short time later to determine if the animal survived the procedure. Single
animals were moved to individual plates after their GFP expression was determined at
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54
day 6 of adulthood. Only animals that were scored as expressing GFP in all the 26
GABAergic neurons for the juls7 strains and GFP in all the 8 dopaminergic neurons for
the him-5; nisi 18 strains at day 6 of adulthood were included in the study.
Scoring for fitness
The fitness of the individuals was determined on a daily basis by scoring them for
movement at approximately 24 hour intervals. A qualitative scale was used when
following animals to score fitness. An animal was assigned to the category of stimulated
if it moved about in response to the plate being placed on the stage of the light
microscope. These animals move about the plate in response to this vibration stimulus.
An animal was assigned to the sedentary category if it did not respond to the vibration of
the plate when it was placed upon the microscope stage. Such an animal required
prodding with a platinum wire pick to be stimulated to move about the plate. An animal
was assigned to the slight category if it did not respond to the vibration of the plate or to
being prodded with the pick. Such an animal is stationary, but can wag its head and or its
tail in response to the prodding.
Second scoring of GFP in slight animals
When animals reach the slight category as described above, they are close to the
end of their lives and were re-examined for GFP expression. Animals were picked with a
platinum wire pick from their individual plate and placed in an M9 drop on an agar pad.
The GFP expression was visualized as described above. If an animal was not pumping its
pharynx it was considered dead and not included in the study. Those animals that were
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55
observed to not be expressing GFP in a particular neuron were rotated and observed a
second time to confirm the loss of expression in the particular neuron(s). After scoring
the expression pattern for each individual, animals were moved from the agar pad to their
individual plates. Animals were then scored for viability within 24 hours of this second
mounting procedure.
Genetics
To introduce the daf-2 mutation into the transgenic lines juls7 and him-5; nisi 18,
the following methods were used. Two to five ju Is7 hermaphrodites were placed on a
plate with 8-10 N2 males and allowed to mate. The heterozygous juls7 males or him-5;
nisi 18 males were grown to the L4-young adult stage. Eight to 10 of these males were
placed on a plate with 2 to 5 daf-2(m4l) or daf-2(el370) hermaphrodites at 25.5*C.
Individual L4 FI hermaphrodites were grown at 25.5°C. The F2 animals were grown for
2 days at 25.5"C. To select for homozygous daf-2 progeny, animals were removed from
the plates in liquid by adding approximately 5ml of M9 to the plate and washing the
animals into plastic centrifuge tubes. Animals were spun down and the majority of the
liquid was removed and 0.1 % SDS in M9 was added to each tube. The centrifuge tubes
were then spun down and each population was then washed three times in M9. Animals
were then pipetted onto plates in a small volume of liquid and incubated at 15* C to allow
the surviving dauer larvae to resume development. Animals that had recovered from the
dauer stage were then examined for GFP expression. Individuals expressing GFP were
cloned to establish lines. The Daf-c, Age and GFP expression phenotypes were
confirmed for the double mutant lines.
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Life spans
Life spans of animals that were not examined for GFP expression were
determined by picking groups of approximately 20 mid-L4 stage animals to 60mm NGM
plates. These animals were moved daily to fresh plates throughout the reproductive
phase in order to separate them from the offspring. When the animals ceased laying
eggs, they were moved to fresh plates every 2-3 days. Animals were scored daily to
determine if they were alive. An animal was considered dead when it did not move in
response to being prodded with a platinum wire pick and the pharyngeal pumping had
ceased.
Heat shock to kill animals
Gravid adult animals were picked to plates that were then wrapped with parafilm
and floated in a 42“ C water bath for 25 minutes. Animals were examined on the
dissecting microscope to ascertain that they were dead. Animals were then mounted on
an agar pad and GFP expression was visualized at various time points after the heat
shock.
Laser ablation
Adult animals were mounted on agar pads that contained 1-2 pM sodium azide.
Individual GFP neurons were identified using UV light and differential image contrast
(DIC) optics. Neurons were ablated with a nitrogen laser microbeam (coumarin 440 nm
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57
dye) and recovered to NGM plates and incubated at 20‘C. Animals were re-mounted and
examined for GFP expression in the particular ablated neuron at various time points after
the surgery.
Statistical Analysis
All life span statistics were calculated with SAS 7.0 or Statview 5.0. The non-
parametric analyses of survival curves, mean life spans, chi-square and P values were
used.
Results
Neuronal GFP expression with age
GABA neurons are involved in the control of muscles; some function to promote
movement in a smooth sinusoidal wave-like motion and others are involved in the
defecation cycle (Mclntire et al.. 1993). The tinc-25 gene was shown to encode a
homolog of the biosynthetic enzyme glutamic acid decarboxylase (GAD) (Jin et al.,
1999). The amino acid glutamate is converted to the neurotransmitter gamma-amino
butyric acid (GABA) by the GAD enzyme (Martin and Rimvall, 1993). The strain
CZ520 contains an integrated chromosomal array of the unc-25::gfp gene fusion. The
unc-25::gfp plasmid was constructed by fusing the gfp in frame with the N terminus of
the unc-25 gene (Hallam and Jin, 1998). Adult ju Is7 hermaphrodites express the GFP in
all of the 26 GABA neurons that were previously identified by antibody staining with
antisera to GABA (Mclntire et al., 1993).
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58
juls7 animals were grown until they reached day six of adulthood. These animals
were examined for GFP expression in the 26 GABA neurons, and only those animals in
which all 26 neurons could clearly be distinguished were included in the experiment.
The animals in which all 26 neurons were not clearly distinguishable were not necessarily
expressing GFP in less than 26 neurons but the way in which these animals were
mounted could have prevented visualization of all of the neurons.
After visualization of GFP at day 6 of adulthood, individuals were then scored
daily for fitness roughly every 24 hours until they reached the slight category. Animals in
this fitness category do not advance in response to being prodded with a platinum wire
pick, but are able to move either their head or tails and display pharyngeal pumping.
Animals were aged in 19 cohorts and 58/160 of the juls7 animals had progressed through
each fitness category and died before they could be examined for GFP expression. The
102 juls7 animals examined for GFP expression were scored slight at an age o f 10 to 22
days of adulthood. 86 of the 102 (84%) individuals that were examined were still
expressing GFP in all of the 26 GABA neurons (Figure 10). Most of the aged animals
had an approximate equal intensity of expression compared to the day 6 individuals as
depicted in Figure 10. Approximately 10% of the aged animals showed a fainter
intensity of expression, yet it was still detectable in the 26 GABA neurons.
16 o f the 102 (16%) individual animals were expressing GFP in a reduced number
o f neurons (Figure 11). O f the 16 animals that had an altered GFP expression pattern, 15
animals were no longer expressing GFP in the RIS neuron. The RIS neuron is an
intemeuron located in the head region (White et al., 1986). RIS makes a gap junction
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59
Figure 10: Photomicrographs of GFP neurons in adult juls7 individuals. A: young adult
juls7, ventral side left, expressing GFP in RME neurons (RMEL, RMER, RMED, and
RMEV). AVL and RIS neurons. B: senescent juls7 adult, ventral side down, expressing
GFP in the same head neurons. C: young adult juls7, ventral side right, expressing GFP
in RME's, AVL and RIS neurons. D: senescent juls? adult, ventral side right, faintly
expressing GFP in RME's and AVL; no GFP is detected in RIS neuron.
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60
A RME's
AVL
i t
RIS
B
RME's
y
AVL
C
RME's
AVL
* RIS
D
RME's
AVL
RIS
/ i ' : - (omtotectab**]
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61
26 GFP cells
less GFP cells
juls7 juls7;daf-2(m41)
Genotype
juls 7;daf-2(e1370)
Figure 11: GFP expression in GABA neurons in moribund individuals. Animals were
examined for expression when they entered the slight phase. Individuals expressing GFP
in all 26 GABA neurons are scored "26 GFP cells," while those expressing GFP in less
than 26 cells are scored as "less GFP cells."
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62
connection to AVJ and has synaptic outputs to the AVE, RIM, RMD, RIB, CEP, and
AVK neurons (White et al., 1986). AVK, AVE, and RIB are intemeurons, RIM and
RMD are motor neurons, and CEP neurons have ciliated endings in the cephalic sensilla
(White etal., 1986).
Five of the 16 animals lost expression in the AVL neuron that is located anterior
to the RIS neuron near the terminal bulb of the pharynx. One of the 16 animals lost
expression in the pair of RME neurons, RMEL and RMER that are located in the head.
One animal lost expression in the VD1, VD2 and DDl neurons, a trio located along the
ventral nerve cord. Unexpectedly, animals that displayed a change in GFP expression in
the slight category were not only those of advanced ages but rather ranged in ages from
11 to 23 days of adulthood (Figure 12). After the expression pattern was determined
when in the slight category, some of the animals (40/102) were then returned to NGM
plates and scored for survival after an approximate 24 hour time period. All of these
animals were dead within 24 hours.
To obtain longer lived animals, double mutant strains of juls? and different alleles
of daf-2 were constructed. The Age phenotype o f the daf-2 alleles approximately doubles
the adult life span. The ju lsl; daf-2(m4l) and juls7; daf-2(eI370) strains were verified
for their dauer constitutive (Daf-c), Age and GFP expression phenotypes at 25.5°C. The
daf-2(m4l) mutation did not affect the GFP expression at day 6. One hundred ninety
three juls7; daf-2(m4l) animals were aged in 20 cohorts to day 6 of adulthood and their
expression pattern examined. Animals were scored for movement on a daily basis as an
indicator of fitness and the slight category was reached at 11 to 38 days o f adulthood.
One hundred eleven o f the 193 animals were examined for GFP expression when they
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63
£
3
■ o
»
B
e
a >
a
E
16
14
12
10
26 GFP cells
less GFP cells
I I I H 11
III
rl 1
■ II *
til .1
t j j | j M
10 11 12 13 14 15 16 17 18 19 20 21 22
Age when slight (days)
Figure 12: Individual expression profile for juls7. One hundred two individuals were
examined for GFP expression in GABA neurons when they were in the slight fitness
category.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
reached the slight fitness category. None of the 111 juls7; daf-2(m4l) animals displayed
a change in the number of neurons expressing GFP compared to day 6 o f adulthood
(Figures 11 & 13). In contrast to some of the juls7 animals, most juls7; daf-2(m4l)
animals with advanced ages appeared to have an approximate equal intensity of GFP
expression compared to day 6. 141 juls7; daf-2(el370) individuals were aged in 17
cohorts to day 6 of adulthood and 102 were scored when they reached the slight category.
These individuals were from 15 to 45 days old. O f the 102 slight individuals that were
examined none had a change in GFP expression in the 26 GABA neurons (Figures 11 &
14). Similar to juls7; daf-2(m4l) animals, most ju Is7; daf-2(eI370) animals with
advanced ages appeared to express GFP at an approximately equal intensity, compared to
day 6 of adulthood. Some of the juls7; daf-2(m4l) (20/111) and juls7; daf-2(eI370)
(25/102) animals were placed back onto NGM plates after their GFP expression was
determined at the slight category. Similar to juls7, none of these individuals were alive
twenty four hours later. It is apparent that juls7 animals with the daf-2(el370) mutation
live longer than those with the daf-2(m4l) mutation (Figure 15). This graph also shows
that animals display the slight phenotype across a wide range of ages.
To determine what happens to expression in a different class of neurons,
dopaminergic neurons were followed with age. By laser ablation experiments,
dopaminergic neurons were demonstrated to be involved in locomotion (Sawin, 1996).
The cat-2 gene encodes a homolog of the gene tyrosine hydroxylase, a rate-limiting
enzyme necessary for dopamine biosynthesis from the amino acid tyrosine (Lints and
Emmons, 1999). The strain EM641 contains an integrated chromosomal array o f the
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65
■ 26 GFP cells
■ less GFP cells
- - - -
L
L
t
- - - -
1 1
I 1
t
H i Hi i i i i l i I I Q ■■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ! Mi I i i I Mi ■
11 14 17 20 23 26 29 32 35 38
Age when slight (days)
Figure 13: Individual expression profile for ju lsl; daf-2(m41). One hundred eleven
individuals were examined for GFP expression in GABA neurons when they entered the
slight fitness category.
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66
8
26 GFP cells
less GFP cells
6
13
3
1 4
c
0 )
A
E
i J_L
-L..1 I
13 16 19 22 25 28 31 34 37 40 43 46 49 52
Age when slight (days)
Figure 14: Individual expression profile for juls7; daf-2(el370). One hundred two
individuals were examined for GFP expression in GABA neurons when they entered the
slight fitness category.
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67
120
100
J2 80
a a a a a a A
• • /
A 4 4
• • ■ ■
A A *
A
■
O 60
E
C 0
40
20
• ■
• ■
• juls7
m juls7; daf-2(m41)
a juls7; daf-2(e1370)
4 a a a
AA
0 *—A*1
J ------------ 1 -------------1 -------------1 ________ I ________ I ________ L
10 15 20 25 30 35 40 45 50 55 60
Age when slight (days)
Figure 15: Cumulative number of animals dying over time for juls7 and juls7; daf-2
strains. juls7 n=l02,juls7; daf-2(m4l) n=l 11, andjuls7; daf-2(el370) n=102.
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68
cat-2::gfp gene fusion. The cat-2::gfp plasmid contains 1.5 kb o f upstream sequence and
the first 3 exons of the cat-2 gene fused to the gfp gene. Hillel Schwartz, from the
laboratory o f Dr. Robert Horvitz at MIT, generated animals carrying the integrated
chromosomal array nisi 18. The him-5(el490) mutation, which results in a higher
incidence of males (33% at 20°C) (Hodgkin et al., 1979) was crossed into the nisi 18
strain, him-5; nisi 18 hermaphrodites express GFP in 8 dopaminergic neurons, 3 pairs
located in the head and I pair posterior to the vulva, him-5; nisi 18 males express GFP in
these same 8 neurons as well as an additional 6 dopaminergic neurons located in the tail
region.
One hundred ninety four him-5; nisi 18 hermaphrodites were aged in 22 cohorts
until they reached day 6 o f adulthood and GFP was visualized in the 8 neurons. Only
those animals in which all 8 dopaminergic neurons could be clearly distinguished were
included in the experiment. There were 95 animals examined for GFP expression when
they were in the fitness cateogry. These 95 animals reached this category at 11 to 29
days o f adulthood. Ninety of the 95 (95%) him-5; nisi 18 hermaphrodites displayed no
change in the number of neurons expressing GFP. Five of 95 (5%) animals were found
in which GFP was expressed in less than 8 dopamine neurons (Figure 16). Of the 5
animals that had a reduction in the number of GFP expressing neurons, 2 animals were 12
days old, 2 animals were 13 days old, and 1 animal was 14 days old (Figure 17). This is
a different pattern than for the juls? strain in which there were more animals that
displayed a loss of GFP at a wider range o f adult ages. Some (18/92) o f the him-5;
nisi 18 animals were then placed back onto NGM agar plates. None of these animals
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69
V)
(8
3
TJ
>
e
o
©
.Q
E
3
z
100
80
60
8 GFP cells
less GFP cells
« 40
20
him-S; nls118
daf-2(m41);
nls118
Genotype
daf-2(e1370);
nls118
Figure 16: GFP expression with age in dopamine neurons. Animals were re-examined
for expression when they were in the slight category. Individuals expressing GFP in all 8
dopamine neurons are scored "8 GFP cells," while those expressing GFP in less than 8
cells are scored as "less GFP cells."
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70
were alive when they were examined after 24 hours. Four of the 5 him-5; nisi 18 animals
lost GFP expression in the CEPDR neuron. In addition to displaying a loss of expression
in the CEPDR neuron, four animals lost expression in the CEPDL neuron (see Table 1).
Three of the five animals lost expression in the CEPVL/CEPVR neuron pair and the
remaining two animals lost one pair. One individual showed a loss o f expression in the
ADEL and ADER neuron pair located posterior of the vulva. This individual also had
losses in expression in the CEPDR and CEPVR neurons, but not in their respective left
side partners (CEPDL and CEPVL).
To address the question of what happens to GFP expression in the hermaphrodite
dopaminergic neurons in individuals with more advanced ages, daf-2 mutations were
introduced into the him-5; nisi 18 animals. These long-lived strains were constructed as
described in the methods section. The daf-2(m41); nisi 18 and daf-2(el370); nisi 18
strains were verified for the Daf-c, Age and GFP expression phenotypes at 25.5’C.
Individuals were aged to day 6 o f adulthood and the daf-2(m4l) and daf-2(eI370)
mutations were observed to not affect the nisi 18 GFP expression pattern o f the young
adult animals. One hundred ninety three daf-2(m4I); nisi 18 individuals were aged in 21
cohorts and examined at day 6 o f adulthood for GFP expression in the dopaminergic
neurons. Ninety two of these 193 individuals were examined for GFP expression when
in the slight category at ages of 13 to 38 days. None of the daf-2(m4l); nisi 18 displayed
a change in the number of neurons expressing GFP (Figures 16 & 18). The GFP
expression of the him-5; nisi 18 in the slight category was more faint than compared to
day 6 o f adulthood and in some older individuals was very faint but still detectable.
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71
«
75
s
•o
‘>
c
< k
o
k
o
18
16
■ 8 GFP cells
14
less GFP cells
12
10
8
6
4
2
0
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Age when slight (days)
Figure 17: Individual expression profile for him-5; nisi 18. 95 individuals were
examined for GFP expression in dopamine neurons when they entered the slight fitness
category.
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72
NEURONS EXPRESSING GFP
CEPDR CEPDL CEPVR CEPVL ADEL ADER
INDIVIDUAL
C.2
- + - + - -
G.4
- - - - + +
H.4
- - - - + +
T.I + - + - + +
U.6 - - - - + +
Table 1: The 5 him-5; nisi 18 individuals that had a change in the number of neurons
expressing GFP. The detection of GFP expression is indicated by a positive (+).
Undetectable GFP is indicated by a negative (-). GFP was detected in the fourth pair of
dopaminergic neurons, PDEL 8c PDER, for all of these individuals.
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73
The majority of the juls7; daf-2(m41) individuals displayed bright GFP expression in
GABA neurons in the slight category which differed from the faint GFP fluorescence in
the old him-5; nisi 18. One hundred sixty seven daf-2(eI370); nisi 18 were aged in 18
cohorts and examined at day 6 for GFP expression and 92 of these 167 animals had
reached the slight phase at a range of 15 to 51 days of adulthood. None of the 92 daf-
2(el370); nisi 18 animals showed a change in the number of neurons expressing GFP
(Figures 16 & 19). Similar to daf-2(m41)\ nisi 18 animals, those with the daf-2(el370);
nisi 18 mutation displayed a more faint GFP expression pattern in slight animals,
compared to day 6 of adulthood. Some (18/92) of the daf-2(m41); nisi 18 and (17/92)
daf-2(eI370); nisi 18 animals, after being examined while in the slight category, were
placed back onto NGM agar plates and all were dead within 24 hours. The animals with
the daf-2(el370) mutation lived longer than those with the daf-2(m41) mutation at 20' C
as expected (Figure 20).
Movement as an indicator of fitness
After animals were examined for GFP expression at day 6 of adulthood,
individuals were moved to NGM plates with OP50 and incubated at 20'C.
Approximately every 24 hours the animals were scored for fitness based on their
movement and given a score as described in methods. Fitness is described here as
stimulated, sedentary, or slight. Animals were moved in groups of 20 to 30 from the L4
stage until day 6 of adulthood. Between the L4 stage and day 6 o f adulthood, these
animals were not scored for fitness on an individual basis.
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74
J2
«
3
■ o
>
0 )
.a
E
10
■ 8 GFP cells
B less GFP cells
8
6
4
2
0
Age when slight (days)
Figure 18: Daily expression profile for daf-2(m4l); nisi 18. Ninety two individuals were
examined for GFP expression in dopamine neurons when they entered the slight fitness
category.
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75
12
■ 8 GFP cells
B less GFP celis
10
2
I D 1 1 ' ' ' 11 ' i i i 1
15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
Age when slight (days)
Figure 19: Daily expression profile of daf-2(eI370); nisi 18. Ninety two individuals
were examined for GFP expression in dopamine neurons when they entered the slight
Fitness category.
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76
100
80
M
E
60
a
co
20
:X ♦ ♦ ♦ ♦ * * * * * * * * *
x ♦♦
x w *
X ♦ **
♦ * *
▼
x him-5; n isi 18
♦ daf-2(m41); nls118
* daf-2(e1370); nls118
-I______l ______ I ______ l ______ L
10 15 20 25 30 35 40
Age w hen slight (days)
45 50 55
Figure 20: Cumulative number of animals dying over time for him-5; nisi 18 and daf-2;
nisi 18 strains, him-5; nisi 18 n=95, daf-2(m4I); nisi 18 n=92 and daf-2(eI370); nisi 18
n=92.
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77
phenotype on day 6, so it is assumed that they were also in this category in the days prior
to this. As shown in Figure 21 ,juls7 animals live the majority of their lives in the
stimulated category as active adults, an average of 12.9 ± 2.9 days. These animals were
in the sedentary category for an average of 1.6 ± 1.0 days before they become slight and
die. For some of the animals, the graph depicts that no time is spent in the sedentary
phase. These animals probably were in the sedentary phase for a period of less than 24
hours, and therefore animals were not observed as such, due to the intervals of the
scoring times. All o f the animals that were scored died within 24 hours of being
examined for GFP expression in the slight category, so the graph depicts that all of the
individuals spend 1 day in the slight category. juls7; daf-2(m41) animals display similar
fitness patterns to that of juls? animals in that they live the majority of their lives as
active adults (Figure 22). Animals with the dcif-2(m4l) mutation spend an average of
16.6 ± 4.7 days in the stimulated phase. The juls 7; daf-2(m41) animals then enter the
sedentary category for an average o f 1.6 ± 1.3 days then become slight and die. Similar
to juls7 animals, most of these individuals were not observed in the sedentary phase, but
the time the animal spends in this phase is most likely shorter than the interval for scoring
these animals, ju ls 7; daf-2(el370) individuals live an average of 20.5 ± 4.2 days in the
stimulated phase. Many have an extended sedentary phase with the average being 11.3 ±
7.2 days (Figure 23). The few that have no observed sedentary phase were relatively
short lived. These animals live a higher percentage of their lives not moving about the
plate (in the absence of physical stimulus). The ju ls 7; daf-2(e!370) animals lie on the
plate in a curled up manner with their heads close to their tails. After they are prodded
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22
78
■ stimulated
□ sedentary
■ slight
Percent of life in category
Figure 21: Movement as an indicator of fitness for juls 7. Each horizontal bar represents
one o f the 102 individuals scored in the experiment. Bars are stacked on the Y-axis
according to advancing age at death. The X-axis represents percent o f an individual's life
spent in a fitness category. Individuals with white dotted lines had less than 26 GFP
cells.
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■ stimulated
□ sedentary
■ slight___
0 25 50 75 100
Percent of life in category
Figure 22: Movement as an indicator of fitness for juls7; daf-2(m4l). Each horizontal
bar represents one o f the 111 individuals scored in the experiment. Bars are stacked on
the Y-axis according to advancing age at death. The X-axis represents percent of an
individual's life spent in a fitness category.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
■ stimulated
□ sedentary
■ slight I
0 25 50 75 100
Percent of life in category
Figure 23: Movement as an indicator of fitness for ju lsl; daf-2(eI370). Each horizontal
bar represents one of the 102 individuals scored in the experiment. Bars are stacked on
the Y-axis according to advancing age at death. The X-axis represents percent o f an
individual's life spent in a fitness category.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
81
with a platinum wire pick, they move similar to the way in which active adults in the
stimulated phase would. However, when they are examined after the 24 hour interval,
these individuals are again curled up and are not actively moving about the plate. These
juls7; daf-2(eI370) animals display a slight category pattern similar to the juls7 and
ju ls 7; daf-2(m4l) individuals.
him5; nisi 18 animals spend the majority o f their lives, an average of 15.3 ± 4.4
days, in the stimulated category (Figure 24). The him-5; nisi 18 animals entered the
sedentary category for 2.4 ± 3.3 days. dcif-2(m41); nisi 18 animals lived an average of
19.0 ± 5.5 days in the stimulated phase and 2.6 ± 1.8 days in the sedentary phase (Figure
25). The fitness graph of these animals looks similar to juls7; daf-2(m4I) animals.
These animals are lived to more advanced ages but overall their fitness graphs look very
similar to strains without a daf-2 mutation. The daf-2(eI370); nisi 18 animals lived an
average of 18.8 ± 4.5 days in the stimulated fitness category while an average o f 9.5 ± 5.8
days was spent in the sedentary category (Figure 26). These animals appeared similar to
the ju ls 7; daf-2(eI370) strain in that they display a curled up, U-shaped Unc phenotype.
Upon stimulation with the platinum wire pick, the daf-2(eI370); nisi 18 animals move
fairly normally. When the animals were scored after 24 hours, they had returned to the
still, curled up, U-shaped position and responded again when prodded. The assumption is
that approximately 1/3 of these animals spent less than 24 hours in the sedentary
category, though it was not observed due to the scoring intervals.
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82
■ stim ulated
□ sedentary
■ slight
Percent of life in category
Figure 24: Movement as an indicator o f fitness for him-5; nisi 18. Each horizontal bar
represents one of the 95 individuals scored in the experiment. Bars are stacked on the Y-
axis according to advancing age at death. The X-axis represents percent o f an
individual's life spent in a fitness category. Those individuals with a white dotted line in
the slight category displayed a change in the number of dopamine neurons expressing
GFP.
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83
41
0)
>*
< 0
■o
(0
0)
■o
< 0
4)
O)
<
20
stim ulated
□ sedentary
13
Percent of life in category
Figure 25: Movement as an indicator of fitness for daf-2(m41); nisi 18. Each horizontal
bar represents one of the 92 individuals scored in the experiment. Bars are stacked on the
Y-axis according to advancing age at death. The X-axis represents percent of an
individual's life spent in a fitness category.
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84
■ stimulated
□ sedentary
■ slight
0 25 50 75 100
Percent of life in category
Figure 26: Movement as an indicator of fitness for daf-2(el370); nisi 18. Each
horizontal bar represents one of the 92 individuals scored in the experiment. Bars are
stacked on the Y-axis according to advancing age at death. The X-axis represents percent
of an individual's life spent in a fitness category.
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85
Determination o f adult life span
In order to perform these experiments, exposure to ultraviolet light for a short
period of time to visualize GFP expression was required. Experiments were conducted to
determine whether this manipulation and exposure to UV light affected the life span of
the animals. The life spans were determined following standard procedures, as described
in the methods section. Non-manipulated animals were neither placed on agar pad cover
slips nor exposed to UV light. The manipulated animals are those from the experiments
presented in the previous section. The age of death for the manipulated animals is the
sum o f days lived until the slight category plus 1 day.
For the ju ls? strain. 160 manipulated animals were observed. The mean life span
o f the animals that were examined for GFP expression is 13.9 ± 0.3 days. The earliest
day an animal died was day 7 and the maximum life span for this set of animals was 23
days. Of the 78 non-manipulated juls? animals that were observed in this experiment, 16
(20%) were censored due to internal hatching of larvae, an exploded vulva, or if they
desiccated on the sides of the plate. The censored mean life span o f the non-manipulated
animals was 14.2 ± 0.3 days. The minimum life span is 8 days and maximum 19 days
(Figure 27A). A Mantel-Cox Iogrank test comparing the manipulated juls? animals to
the non-manipulated juls7 animals results in a chi-square value o f 0.34 and a P-value of
0.56 (Table 2).
For the juls?; daf-2(m4l) strain 193 manipulated animals were scored. The mean
life span of this strain when manipulated is 15.7 ± 0.4 days. The minimum life span is 7
days and the maximum 39 days. 80 non-manipulated animals were observed and 28
(35%) were censored. The mean life span of the non-manipulated juls?; daf-2(m4l)
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86
i onorrrary^
0.8
juls7
0.6
0.4
0.2
O V
10 19 20 25 30 39 40 45
A d u lt M t
50 55 60
B
0.6
0.6
0.4 f-
0.2
juts7; daf-2(m41)
a
a m
□
CD
“b
0 i ---- 1 --- I --L I ____________ I » » ■
0 5 10 15 20 25 30 35 40 45 50 55 60
A d u lt a g a
m
e
o
s
0.6
0.6
0.4 -
0.2
juls7; d*f-2(e1370)
i— i- i i » i i i
0 5 10 15 20 25 30 35 40 45 50 55 60
Adult agd
Figure 27: Survival curves comparing manipulated animals to non-manipulated animals.
Adult age is in days. A: juls7, B: juls7; daf-2(m41) m dC :juIs7; daf-2(el370). Filled
symbols represent manipulated animals, open symbols represent non-manipulated
animals.
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87
STRAIN MEAN
75,h
PERCENTILE N Censored
juls 7 man. 13.9 ± 0.3 16 ±0.5 160 0*
juls 7 non-man. 14.2 ± 0.3 16 ±0.4 78 16
julsl;daf-2(m41) man. 15.7 ± 0.4 18 ±0.8 193 0*
ju h 7 ;daf-2(m4l) non-man. 21.5 ± 1.0 28 ± 1.4 80 28
juls7;daf-2(el370) man. 26.6 ± 1.1 38 ± 1.1 141 0*
juls7;daj-2(el370) non-man. 30.3 ± 1.1 38 ± 0.4 99 26
N2 non-man. 19.2 ±0.7 22 ± 3.0 60 8
daf-2(m4I) non-man. 21.5 ± 0.9 26 ± 1.6 120 41
daf-2(el370) non-man. 37.3 ± 2.1 47 ± 1.9 80 36
Manipulated vs. Non-manipu ated comparisons
^ STRAIN CHI-SQUARE P-VALUE
juls 7 0.34 0.56
juls 7;daf-2(m41) 26.7 <0.001
juls 7;daf-2(e 13 70) 0.01 0.92
Non-Manipulated comparisons
STRAIN CHI-SQUARE P-VALUE
N2 vs. 43.9 <0.001
juls7
daf-2 (m4l) vs.
juls7;daf-2(m4l)
0.18 .6713
daf-2(eI370) vs,
juls 7;daf-2(el3 70)
24.5 <0.001
Table 2: Adult life span statistics for juls7 and juls 7; daf-2 strains. Man.= manipulated;
non-man. = non-manipulated. Mean life spans are in days. 75th percentile is the age
when the fraction of adults alive reaches 0.25. Chi-square and P values calculated using
Mantel-Cox logrank test. * Most censoring occurs between days 1 to 6. This set was not
scored until after day 6.
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88
animals is 21.5 ± 1.0 days. The minimum life span of the non-manipulated animals is 9
days and the maximum is 36 days (Figure 27B). A Mantel-Cox logrank test of these two
groups of animals results in a chi-square value of 26.7 and a P-value of <0.001 (Table 2),
suggesting that the longer non-manipulated mean life span is significant.
For juls7; daf-2(eI370), 141 manipulated animals were observed and their mean
life span was 26.6 ±1.1 days. The minimum life span was 7 days and maximum was 55
days. For 99 non-manipulated juls7; daf-2(el37Q) animals, 26 (26%) were censored.
The mean life span of the non-manipulated animals is 30.3 ±1.1 days. The minimum life
span was 8 days and the maximum 42 days (Figure 27C). A Mantel-Cox logrank test
results in a chi-square value o f .01 and a P-value of 0.91 (Table 2).
For the him-5; nisi 18 manipulated animals, 194 individuals were scored resulting
in a mean life span of 13.5 ± 0.4 days. The minimum life span was 7 days and maximum
was 30 days. 100 non-manipulated animals were scored and 28 (28%) were censored.
The mean life span of the him-5; nisi 18 non-manipulated animals is 15.2 ± 0.5 days.
The range of life spans was a minimum of 9 days and a maximum of 26 days (Figure
28A). A Mantel-Cox logrank test comparing manipulated and non-manipulated results in
a chi-square value of 2.5 and a P-value of 0.11 (Table 3).
The mean life span for 193 daf-2(m4I); nisi 18 manipulated animals was 16.0 ±
0.5 days. The range o f life spans was from 7 days to 41 days. O f 100 non-manipulated
animals, 27 (27%) are censored. The mean life span of the non-manipulated daf-2(m4I);
him-5; nisi 18 was 19.2 ± 1.2 days. The non-manipulated animals lived a range o f 8 to
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89
0 .1
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Figure 28: Survival curves comparing manipulated animals to non-manipulated animals.
Adult age is in days. A: him-5; n isi 18, B: daf-2(m41); nisi 18 and C: daf-2(el370);
n isi 18. Filled symbols represent manipulated animals, open symbols represent non
manipulated animals.
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90
STRAIN MEAN
7 5 * '*
PERCENTILE N Censored
him-5; nisi 18 man. 13.5 ±0.4 16 ± 1.0 194 0*
him-5; nisi 18 non-man. 16.2 ±0.5 17 ±0.8 100 28
daf-2(m4l); nisi 18 man. 16.0 ±0.5 20 ± 0.6 193 0*
daf-2(m41); nisi 18 non-man. 19.2 ± 1.2 23 ± 1.0 100 27
daf-2(e!370); nisi 18 man. 21.2 ±0.7 27 ± 1.2 167 0*
daf-2(e!370); nisi 18 non-man. 20.5 ± 2.4 22 ± 1.9 60 32
N2 non-man. 19.2 ±0.7 22 ± 3.0 60 8
daf-2(m41) non-man. 21.5 ±0.9 26 ± 1.6 120 41
daf-2(el370) non-man. 37.3 ±2.1 47 ± 1.9 80 36
Manipulated vs. Non-manipu ated comparisons
STRAIN CHI-SQUARE P-VALUE
him-5; nisi 18 2.54 0.11
daf-2(m41); nisi 18 8.56 0.003
daf-2(el370); nisi 18 0.29 0.59
Non-Manipulated comparisons
STRAIN CHI-SQUARE P-VALUE
N2 vs.
him-5;nlsl 18
18.35 <0.001
daf-2 (m41) vs.
daf-2(m41); nisi 18
0.600 0.46
daf-2(e!370) vs,
daf-2(e!370); nisi 18
20.93 <0.001
Table 3: Adult life span statistics for him-5; nisi 18 and daf-2; nisi 18 strains. Man.=
manipulated; non-man. = non-manipulated. Mean life spans are in days. 75th percentile
is the age when the fraction of adults alive reaches 0.25. Chi-square and P values
calculated using Mantel-Cox logrank test. * Most censoring occurs between days 1 to 6.
This set was not scored until after day 6.
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91
46 days (Figure 28B). The Mantel-Cox logrank test results in a chi-square value of 8.6
and a P-value o f .003 (Table 3), suggesting that the non-manipulated mean life span is
significantly longer.
One hundred sixty seven daf-2(eI370); nisi 18 manipulated animals were scored
and the mean life span was 21.2 ± 0.7 days. These manipulated animals live a range of 7
to 51 days. O f the 60 non-manipulated daf-2(e!370); nisi 18 animals, 32 (53%) were
censored resulting a mean life span of 20.5 ± 2.4 days. The non-manipulated animals
lived a minimum of 8 days and a maximum of 54 days (Figure 28C). A Mantel-Cox
logrank test results in a chi-square value of 0.30 and a P-value of .59 (Table 3).
The life spans of 60 N2 (wild-type) non-manipulated animals were determined.
The mean was 19.2 ± 0.7 days. The animals live a range of 4 to 31 days and 8 of the 60
individuals (13%) were censored. A Mantel-Cox logrank test was used to compare this
wild type strain to that of juls7 and results in a chi-square value of 43.9 and a P-value of
<0.001 (Table 2). Comparing the wild type strain to the him-5; nisi 18 strain shows a
chi-square value of 2.5 and a P-value of 0.11 (Table 3). Survival curves comparing N2 to
juIsT and N2 to him-5; nisi 18 are shown in Figure 29.
Life spans for non-manipulated daf-2(m41) and daf-2(el370) were determined.
One hundred twenty daf-2(m41) individuals were followed and 41 of them were censored
(34%). The mean life span of the daf-2(m4l) non-manipulated animals is 21.5 ± 0.9
days. The minimum life span was 3 days and the maximum is 39 days. Comparison of
this daf-2(m41) strain to the juls7; daf-2(m4l) non-manipulated strain results in a chi-
square value o f 0.18 and a P-value of 0.67 (Table 2). Comparison of the non-
manipulated daf-2(m4I) to the non-manipulated daf-2(m41); nisi 18 shows a chi-square
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92
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5 10 15 20 25 30
Adult age
35
Figure 29: Survival curves comparing different genotypes o f non-manipulated animals.
Adult age is in days. A: N2 (wild-type) compared to juls7, B: N2 compared to him-5;
nisi 18.
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93
value of 0.60 and a P-value of 0.46 (Table 3). This suggests no statistically significant
difference between the daf-2(m41) strain and the GFP expressing strains carrying the daf-
2(m4l) mutation (Figure 30). Life spans for 80 non-manipulated daf-2(el370)
individuals were determined and 36 (45%) of them were censored. The mean life span of
the daf-2(e!370) non-manipulated animals was 37.3 ± 2.1 days. The minimum life span
was 11 days and the maximum 60 days. Comparison of the daf-2(el370) strain to the
juls7; daf-2(el37Q) strain results in a chi-square of 24.50 and a P-value of <0.001 (Table
2). Comparison of the daf-2(el370) strain to the daf-2(el370); nisi 18 strain results in a
chi-square value of 20.9 and a P-value of <0.001 (Table 2). Survival curves comparing
daf-2(eI370) to juls7; daf-2(el370) and daf-2(e!370) to daf-2(eI370); nisi 18 is shown
in Figure 31.
GFP florescence and death
Two artificial methods were used to cause death of either the animal or a neuron
to observe GFP expression over time proximal to the time of death. Two to four day old
adult ju Is7 hermaphrodites expressing GFP in GABA neurons were exposed to heat
shock to cause death. After incubation at 42“C for 25 minutes, none of the animals
exhibited movement or pharyngeal pumping. The animals were examined several times
for GFP fluorescence in the first hour after the heat shock and approximately every 4
hours after the initial examination. The majority of the animals (27/30) continued to
show GFP fluorescence in the expected cells for approximately 24 to 48 hours, although
the intensity becomes much weaker and less distinct after 6 hours compared to old
animals that are still alive. The young animals that underwent heat shock do not display
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94
9
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Figure 30: Survival curves comparing different genotypes o f non-manipulated animals.
Adult age is in days. A: daf-2 (m41) compared to juls7; daf-2(m4l) and B: daf-2(m4l)
compared to daf-2(m41); n isi 18.
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95
4
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Figure 31: Survival curves comparing different genotypes of non-manipulated animals.
Adult age is in days. A: daf-2 (eI370) compared to juls7; daf-2(el370) and B: daf-
2(el370) compared to daf-2(eI370); nisi 18.
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96
the more intense auto florescence associated throughout the body o f aged animals. Three
of the animals lost GFP expression in a few neurons (1 in RIS, 2 in DVB) but overall
GFP continues to fluoresce, although the localization is less distinct and appears to leak
out of the cell. The heat-shocked individuals display a different phenotype compared to
animals aged in a normal manner. The heat-shocked animals that die outside of the
bacterial lawn appear to be fairly intact with regard to the internal structures for up to 60
hours after the heat shock treatment. Those animals that die on the bacterial lawn begin
to break down at a more rapid rate (after approximately 24 hours). This phenotype
differs from that of aged animals in the slight category which appear to have more
internal structure disorganization and cuticle morphology changes.
Individual neuron cell bodies were ablated in juls7 adult animals to determine
when GFP fluorescence was reduced and eliminated. The RIS, DD4 and several tail
neurons were identified by GFP florescence and DIC optics and the individual neuron
was ablated with the laser. The RIS neuron was ablated in 8 individuals, the DD4 neuron
was ablated in 7 individuals, the VD12 in 2 animals, DD6 in 1 animal, VD13 in 2
individuals and DVB in 2 individuals. The majority of the operated animals moved
normally about the plate after recovery from the surgery. Some animals in which the
DD4 neuron was ablated had impaired movement. After the laser ablation, animals were
scored for GFP fluorescence in the particular neuron at three time points: immediately
after ablation, 1.5 hours after ablation and 4 hours after ablation. All of the individuals
lost GFP expression in the ablated neuron shortly after the laser treatment, while adjacent
neurons were still expressing GFP at the three different time points.
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97
Discussion
In these experiments, individual live animals were examined for GFP expression
in GABAergic and dopaminergic neurons to determine if the pattern changes as age
increases. Two transgenic lines were used that had either an integrated chromosomal
array of a gene encoding glutamic acid decarboxylase or tyrosine hydroxylase fused to
the green fluorescent protein gene. Therefore, the focus is on expression of biosynthetic
enzymes for specific neurotransmitters. Animals carrying these arrays express GFP in
either the 26 GABA neurons or in 8 dopaminergic neurons, respectively. In both strains,
comparison of GFP expression in individuals as a young adult and again just before death
from old age showed no change in the GFP expressing neurons number for the majority
o f the animals examined. Two mutant alleles of the daf-2 gene were introduced in the
strains expressing GFP in the GABA and dopaminergic neurons. Animals with a daf-2
mutation showed the expected GFP expression pattern and increased life span. None of
the animals examined with daf-2 mutation displayed a change in the number of neurons
expressing GFP in the old animals. The life spans of the animals that were manipulated
and exposed to UV light to determine their GFP expression pattern were compared to the
life spans of animals that were not exposed to this treatment. The mean life spans for
these two different groups are statistically similar for animals with a wild-type
background and those with the daf-2 (el370) mutation, but animals with the daf-2(m41)
mutation have statistically different mean life spans. Individual animals were also scored
for fitness on a daily basis based on movement criteria. For both strains that express GFP
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98
in GABA and dopaminergic neurons, animals with a wild type or daf-2(m4l) background
were found to spend the majority of their lives as active adults before they showed a
decline in fitness. Most animals with the daf-2(el370) mutation were found to spend a
large portion of their lives sedentary and the longer lived individuals spend a greater
percentage o f their lives sedentary rather than active. The life spans of the GABA and
dopamine GFP expressing strains were statistically shorter compared to wild type, non-
transgenic animals. When these GFP strains carry the daf-2(m4l) mutation there is no
significant difference in life span when compared to the daf-2(m41) single mutant.
However, when the integrated transgenic lines carry the daf-2(e 1370) mutation, the life
span is statistically shorter compared to the daf-2(el370) single mutant.
Those animals with fewer GFP expressing neurons displayed loss in a limited
number of specific neurons. The experimental design determined if there was a reduction
in the number of neurons expressing GFP with age, and it not assumed that loss of GFP
expression represents death of neurons, although death may occur. In the jitls7 strain, the
gene being monitored encodes a biosynthetic enzyme so loss of expression could
represent failure of this enzyme to be produced in the particular neuron. This could
represent loss of the full neural differentiated state or general loss of cellular biosynthetic
capabilities. If function is sufficiently compromised this could lead to neuronal death.
Almost all (15/16) of the individuals lost GFP expression in the RIS neuron. RIS is an
intemeuron located in the head and laser ablation of this neuron results in no visible
phenotype suggesting this neuron alone does not control behavior traits nor is it crucial
for survival (Mclntire et al., 1993). This intemeuron has synaptic output to several
classes of motor neurons (RIM & RMD). Loss of expression of the GABA synthetic
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99
enzyme in this neuron could be a marker for decline in function o f other (non-GABA)
neurons. The juls7 animals that show a change in GFP expression died at adult ages
from day 12 to day 21. Animals lose expression in this neuron sometime after day 6 but
the actual time point is unknown. Experiments were performed with more frequent
scoring of GFP and this was found to dramatically shorten life span (D. Bota, M. Tom,
and P.L. Larsen, unpublished observations). The loss of GFP expression could be
particular for unc-25::gfp expression or it could be particular for the RIS neuron. This
could be tested with another transgene which illuminates the RIS neuron. A limited
number of animals show loss of expression in the AVL neuron and laser ablation of this
neuron results in a decrease in the frequency of enteric muscle contractions (Mclntire et
al., 1993). Decline in the function of this neuron could be related to the age associated
decrease in the frequency of defecation (Bolanowski et al., 1981; Croll et al., 1977).
Some animals show a loss of GFP expression in the RMEL and RMER neurons. This
pair of neurons, located in the head region, were demonstrated to be involved in foraging
by laser ablation experiments (Mclntire et al., 1993). Loss of GFP expression in the
VD1, VD2, and DD1 neurons was visualized in one animal. These neurons, by laser
ablation experiments, were determined to be involved in movement of the animal
(Mclntire et al., 1993). Several groups have shown a decrease in the frequency of
movement with increasing age (Bolanowski et al., 1981; Croll et al., 1977) and decline in
neurons involved in movement could be a mechanism to explain this behavioral
deterioration. The small number of animals with loss of expression in these neurons
could be attributable to the phenotypes visualized in the slight animals. However, even
animals that displayed no change in the number of neurons expressing GFP displayed a
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100
similar phenotype as they reached the ends of their lives. It may be that decline with age
is due to neuron signal propagation steps subsequent to GABA synthesis. The slight
phenotype restricted the examination of phenotypes as described after laser ablation in
movement, foraging, or defecation because the animals are extremely frail.
him-5; nisi 18 animals express GFP in 8 dopaminergic neurons in hermaphrodites.
The dopaminergic neurons were demonstrated to be involved in foraging by individual
cell laser ablation (Sawin, 1996). O f the 95 animals examined, 5 individuals displayed
changes in the number neurons expressing GFP. For three of the individuals, reduction
of GFP expression was restricted to the CEP neurons (CEPDL, CEPDR, CEPVL and
CEPVR). Interestingly, these neurons have synaptic input from the GABAergic RIS
neuron (White et al., 1986), the neuron in which GFP expression was lost in 15/16 of
aged juls7 individuals. One individual had lost expression only in the dorsal pair of CEP
neurons, but not in the ventral CEP neurons. Another individual lost expression the
neurons posterior to the vulva, ADEL & ADER along with CEPDR and CEPVR but not
their respective partners. Although there were only 5 him5; nisi 18 individuals identified
with expression in less GFP cells with age, almost all of these individuals did lose
expression specifically in CEPD neurons. The animals with changes in the number of
GFP expressing cells all died at fairly young ages from day 13 and day 15 of adulthood.
This is different from the juls7 animals that displayed changes in the number of GFP
expressing cells which died within a larger range of ages. This may indicate these
dopaminergic neurons, particularly the CEP's, could be involved in some aspect critical
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101
for survival at a particular time point o f adulthood. The decline in foraging neurons
could result in a change in feeding behavior such as a decrease in pharangyeal pumping
as is seen with increasing age (Gems et al., 1998; Kenyon et al., 1993).
For juls? and him-5; nisi 18 animals, most of the longest lived individuals showed
no change in the number of GFP-expressing neurons. To determine if extending the life
span of the animals had an affect on GFP expression with increasing age, two mutant
alleles of the daf-2 gene that double adult life span were studied in these GFP lines. In all
of the aged ju ls 7 and him-5; nisi 18 animals examined with either daf-2(m4l) or daf-
2(el370), no change in the number of neurons expressing GFP was detected. The daf-2
gene has been cloned and encodes a homolog of an insulin/insulin-like growth factor
receptor (Kimura et al., 1997). The mutations in the daf-2 gene, which increase the life
span of the animal, appear to afford neural protection as measured by prevention of loss
of the number of GFP expresing cells. It would be interesting to construct strains that
have an enhanced life span that are not involved in the insulin-like signaling pathway to
determine whether or not these Age animals show decreases in the number o f GFP
expressing neurons. Alternatively, the daf-2 mutation could be affecting some aspect of
GFP expression independent of life span. This could be an interaction between mutant
DAF-2 function and the GFP transcript or protein in vivo that is promoting stable
expression of GFP in older animals. Another possibility is that some animals with a daf-
2 mutation actually do show a change in the number of GFP expressing cells with age.
but the particular samples were not large enough in number to detect the rare events. In
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102
addition, the daf-2(m4I) mutation results in temperature sensitivity making it borderline
for life span extension at 20° C, and therefore could contribute to the increased variablity
that was observed between trials (Gems, et al., 1998)
Handling of the animals could have had a negative impact on their life spans.
Animals that were examined for GFP expression were moved from their plates to agar
pads containing a small drop of M9. This glass cover slip was then placed on the
microscope stage to examine the animal. After the proper landmarks in the animal were
found using differential image contrast optics, the animals were exposed to ultra violet
light to determine their GFP expression pattern. Animals were then transferred back to
their plates in M9 liquid. The life spans of the manipulated animals were analyzed and
compared to the life span of non-manipulated populations. The life spans o f each of the
strains used were determined using standard methods for the non-manipulated groups.
The survival statistics including mean life spans, chi square and P-values for the
manipulated and non-manipulated groups of each strain were calculated. For the juls7,
juls7; daf-2(el370), him-5; nisi 18, daf-2(el370); nisi 18 strains the chi square and P-
values indicate acceptance of the null hypothesis that there is no difference in the life
span of these two groups of animals. This would indicate that the manipulation and
exposure to UV light in the manipulated animals did not significantly affect the life spans
in these particular strains. For the ju ls 7; daf-2(m4l) and daf-2(m41); nisi 18 animals, the
chi square and P-values indicate rejection of the null hypothesis to suggest the
manipulated animals have a significantly shorter mean life span than the non-manipulated
animals. The manipulated animals with the daf-2(m4I) mutation may be more
susceptible to the manipulation and UV light exposure which results in a shorter life
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103
span. The manipulated animals must recover from being on an agar pad for a short time
and could be more prone to death after this manipulation. When non-manipulated
animals are near the ends of their lives, they are only prodded with a platinum wire pick
to determine if they are viable. The difference in treatment between the two groups could
be attributed to the difference in the life spans.
Comparing the life spans of the non-manipulated wild type animals to that o f the
strains expressing GFP in neurons, the P value indicates a statistically shorter life span
for both juls? and him-5; nisi 18. These strains carry integrated chromosomal arrays and
the insertion sites of the gfp fusion genes may shorten the life span. In addition, to
generate the ju ls 7 and nisi 18 strains, transgenic animals carrying extrachromosomal
arrays are exposed to mutagenic agents to promote the chromosomal integration event.
Even though the animals are back-crossed, they could contain a mutation which results in
a shorter life span. Comparison of the life spans of the double mutant strains juls7; daf-
2(m4I) and daf-2(m4l); nisi 18 to the daf-2(m41) mutants shows no significant
difference. However, the ju ls/; daf-2(el370) and dcif-2(el370); nisi 18 strains have a
significantly shorter life span compared to daf-2(el370). This result suggests the juls7
and nisi 18 mutations partially suppress the Age phenotype of the daf-2 (el3 70) animals.
Previous aging studies in C. elegans have focused on populations to determine
life span and movement. In these studies, a relationship between increasing age and the
declining frequency o f movement was found (Bolanowski et al., 1981; Croll et al., 1977;
Russell and Russell, 1975). The experiments presented here are the first in which
collection of life span and fitness data is at the level of the individual. After animals were
mounted and their GFP expression pattern was determined at day 6 of adulthood, animals
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104
were placed on individual plates and scored at approximately 24 hours for movement.
The animals were given a score of fitness based on the way in which they responded to
stimuli as previously described in the methods section. This fitness data was collected on
a daily basis for all the animals that were scored for GFP expression. The juls7 and
juls7; daf-2(m41) animals have similar profiles in that they live the majority of their lives
as active adults (stimulated category). However, the juls7; daf-2(e!370) animals are
different and some animals live more than half of their lives as non-active adults
(sedentary category). These juls 7; daf-2(eI370) animals display a slightly uncoordinated
(Unc) phenotype in that the animals do not move in response to the vibration of the plate
and lie in a slight U or semi-circle shape. When prodded, they respond by crawling about
the plate like an animal in the stimulated phase. This phenotype has been previously
described for daf-2(eI370), in which groups of animals exhibited reduced movement
after approximately 19 days at 22.5"C and 4 days at 25.5°C (Gems et al., 1998).
However, data to determine the actual amount of time an aging individual spends in this
particular phase has not been previously collected. The amount of time an animal with
the daf-2 (el370) mutation is sedentary is much longer than that of wild type or daf-
2(m4I). The movement pattern is due to the daf-2 allele present since the him-5; nisi 18
or daf-2(m4l); nisi 18; individuals lived the majority of their lives as active adults
whereas the daf-2(e!370); nisi 18 animals displayed an extended sedentary category in
which they displayed the U-shaped, Unc phenotype. The daf-2(m4l) molecular lesion
was identified as a point mutation in the ligand binding domain (H. Yu and P. L. Larsen,
unpublished) while the daf-2(el370) point mutation is located in the tyrosine kinase
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105
domain (Kimura et al., 1997). The nature of the molecular lesion may affect some aspect
of insulin-like signaling critical for the phenotypes seen in these individuals.
The experiments presented here demonstrate that in two different classes of
neurons, the majority of C. elegans do not have fewer GFP expressing cells when very
old. These experiments also suggest a role for the daf-2 mutation in healthy neuronal
aging. These experiments demonstrate that C. elegans can be followed in a longitudinal
manner with a marker for status of neurons in individuals as they age. This design can be
generalized using the gfp gene fused to another gene of interest as a means to illuminate
specific types of neurons or other cell types in the animals and look for changes in the
marker tissue as a function of age. This design allows for visualization of cells in live
animals and also allows for genetic manipulation to investigate the role of particular
genes in aging.
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Creator Snow, Mark Irvin (author)

Core Title Development and aging in Caenorhabditis elegans: Gene structure and expression of daf-12 and a longitudinal study of neurons in long-lived individuals

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Advisor Arnheim, Norman (committee chair), Finkel, Steven E. (committee member), Moses, Kevin (committee member), Trump, Gary (committee member), Warrior, Rahul (committee member)

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