Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA
* Author for correspondence (e-mail: or38{at}columbia.edu)
Accepted 30 October 2002
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SUMMARY |
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Key words: C. elegans, zig, LIM homeobox, Heterochronic, Axon maintenance
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INTRODUCTION |
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Instances in which neuronal phenotypes are modified in a developmentally
programmed, stereotyped temporal manner that occurs significantly past the
axon outgrowth and target recognition stage include sexual differentiation in
the mammalian brain at defined postnatal stages
(MacLusky and Naftolin, 1981),
remodeling of the insect CNS during metamorphosis
(Levine et al., 1995
) and
synaptic rewiring of postmitotic motor neurons in the ventral nerve cord (VNC)
of C. elegans (White et al.,
1978
). In all three circumstances, temporally defined alterations
in gene expression are presumably mediated through hormones and their effector
transcription factors to eventually cause a structural remodeling of
individual groups of neurons (Levine et
al., 1995
; MacLusky and
Naftolin, 1981
; Zhou and
Walthall, 1998
).
We describe our analysis of another neural gene expression program that is
not only under tight spatial, but also under stereotyped temporal control.
Unlike the cases described above, these dynamics in gene expression do not
serve to induce remodeling of the neuron but provide the neuron with a novel
and specific capacity at a defined time in its life. The case studied is the
PVT interneuron located in the VNC of C. elegans
(Fig. 1). Using cell ablation
techniques, PVT was recently found to have two temporally separable roles in
axonal patterning in the VNC. In mid-embryonic stages, PVT is required for
axon attraction into the VNC (Ren et al.,
1999). Surprisingly, at the postembryonic larval L1 stage, PVT was
found to have an additional role in non-autonomously maintaining the intact
axon scaffolding in the VNC, such that loss of PVT causes axon `flip-overs'
across the ventral midline (Fig.
1) (Aurelio et al.,
2002
).
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These two temporally separated functions of PVT correlate with the
execution of a temporally controlled, biphasic gene expression program. In
mid-embryogenesis, during the development of the VNC, PVT transiently
expresses the UNC-6/Netrin cue to attract axons into the VNC
(Wadsworth et al., 1996).
Several hours later, in the larval L1 stage, PVT initiates the expression of
several members of the zig gene family
(Fig. 1)
(Aurelio et al., 2002
).
zig genes encode small transmembrane (zig-1) or secreted
(zig-2 to zig-8) proteins composed exclusively of two
immunoglobulin domains. Six zig genes are expressed in restricted
domains of the nervous system, while the two remaining family members are
expressed outside the nervous system. The expression of all six neuronally
expressed zig genes overlaps in the PVT interneuron. The onset of
expression of several of the zig genes in the PVT neuron in the L1
stage precisely correlates with the requirement for PVT in maintaining axonal
organization of the VNC (Aurelio et al.,
2002
). Consistent with the hypothesis that zig genes
mediate the maintenance function of PVT, we found that a deletion in the
zig-4 locus affects the maintenance of axonal position of a subset of
those neurons that are affected by PVT ablation
(Aurelio et al., 2002
),
suggesting that different zig family members are required to maintain
the positioning of distinct subsets of VNC axons.
What are the molecular mechanisms that underlie the biphasic gene expression program in PVT and, specifically, what are the factors that confer the precise spatial and temporal aspects of zig gene expression? We used postembryonically expressed zig gene reporter gene constructs as tools to investigate these mechanisms. We define three factors that are required for the execution of the temporal and spatial aspect of zig gene expression and show that their absence not only disrupts zig gene expression but also leads to defects in the maintenance of axon positioning in the VNC. Furthermore, we find that spatiotemporal misexpression of one of the zig genes, zig-4, causes developmental defects in the VNC, suggesting that the normal spatiotemporal control of zig gene expression serves to dedicate them to a role in axon maintenance and to prevent them from interfering with axon outgrowth.
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MATERIALS AND METHODS |
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Reporter strains
PVT cell fate markers used in this study are syIs7: Is[gpa-2::lacZ;
dpy-20(+)] (Zwaal et al.,
1997), otIs90: Is[pin-2::gfp; rol-6(d)]
(Hobert et al., 1999a
),
otIs85: Is[srq-1::gfp; rol-6(d)] and otIs39:
Is[unc47
::gfp; lin-15(+)]. The srq-1 reporter is a translational
fusion constructed from a 4.4 kb genomic fragment, encompassing 2.2 kb of
sequence upstream of the predicted ATG and 2.2 kb of exonic and intronic
sequences of the predicted serpentine receptor gene F59B2.13 gene.
The construct was generated using a PCR fusion protocol
(Hobert, 2002
). A
corresponding F59B2.13::lacZ construct had been constructed and
analyzed by Ian Hope in the course of a genome-wide expression pattern
analysis (Lynch et al., 1995
).
The otIs39 integrant derives from juEx60
(Eastman et al., 1999
) and
contains a promoter fragment from the unc-47 gene. The pan-neuronal
marker evIs111 (integrated F25B3.3::gfp); the PVQ marker
oyIs14 (integrated sra-6::gfp); and the zig::gfp
reporter gene constructs otIs25 (zig-1::gfp), otIs7
(zig-2::gfp), otIs14 (zig-3::gfp), otIs20
(zig-4::gfp), otIs11 (zig-5::gfp) and
otIs96 (zig-8::gfp) have been described previously
(Altun-Gultekin et al., 2001
;
Aurelio et al., 2002
). The
lim-6r::gfp expression construct is a full length translational
fusion of the lim-6 locus to gfp and contains all the
regulatory regions required for rescuing lim-6 mutant phenotypes
(Hobert et al., 1999b
);
expression in PVT was scored in an extrachromosomal line, otEx406.
The ceh-14::gfp construct was engineered using a PCR fusion protocol
(Hobert, 2002
) and encompasses
3.75 kb of promoter and parts of the first exons. A similar promoter fragment
has previously been described to faithfully reflect the expression of the
endogenous gene (Cassata et al.,
2000
). Expression of this construct was monitored in the
chromosomally integrated line otIs126.
Scoring of neuroanatomical defects and reporter gene expression
Reporter gene expression levels and neuroanatomical defects were assayed
with the exception of the extrachromosomal lim-6r::gfp array
using chromosomally integrated gfp or lacZ
expression constructs (see above). The integrated arrays were crossed into the
respective mutant backgrounds, thus allowing a direct comparison between
otherwise isogenic backgrounds. If not indicated otherwise, reporter gene
expression was scored in well-fed animals. zig-4::gfp expression was
also scored in starved animals (see text). For these starvation experiments,
eggs were prepared from otIs20 (zig-4::gfp) animals using a
standard bleaching protocol, plated onto non-seeded plates and scored for
zig-4 expression in PVT several days later.
To score neuroanatomical defects at the early L1 stage (when PVT has yet no maintenance role), eggs were prepared by bleaching and freshly hatched L1s scored 2 hours after the preparation of the eggs. With regard to neuroanatomical defects, we focused on the description of defects in the VNC. We found that in various mutant backgrounds (e.g. lim-6 ceh-14) neuroanatomical defects can also be observed in other axon fascicles.
Laser ablation
The nucleus of PVT was identified based on its characteristic position and
morphology by Nomarski optics. Ablation was performed using a LSI VSL-337
laser as previously described (Aurelio et
al., 2002; Bargmann and Avery,
1995
).
Ectopic zig-4 expression
A genomic fragment of the zig-4 locus ranging from its start to
stop codon and including all introns was amplified with restriction sites on
either end of the amplicon. The amplicon was cloned behind a 1.3 kb promoter
fragment from the upstream regulatory region of the flp-1 gene
(Nelson et al., 1998) and a
2.4 kb fragment from the muscle-specific myo-3 promoter
(Okkema et al., 1993
). When
fused to gfp, the 1.3 kb flp-1 promoter (-1358 to -9
relative to ATG) is exclusively active in the AVKL and AVKR neurons. All
expression constructs were injected into oyIs14 animals at 50
ng/µl using ceh-22::gfp
(Okkema and Fire, 1994
) at 50
ng/µl as an injection marker.
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RESULTS |
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Each one of the PVT-expressed zig genes is also expressed in a
restricted subset of other cell types
(Aurelio et al., 2002), yet
lim-6 ceh-14 double null mutants only show an effect on zig
gene expression in PVT (Fig. 2B
and data not shown). As shown in Fig.
3, the impact of lim-6 ceh-14 is strongest on those
zig genes that are expressed strictly postembryonically (zig-2,
zig-3, zig-4, zig-8), although it is weaker, but still significant, on
those zig genes that already show some expression in embryos
(zig-5::gfp, completely penetrant embryonic expression in wild type;
zig-1::gfp, incompletely penetrant embryonic expression in wild type)
(Aurelio et al., 2002
).
Given the significant loss of zig gene expression in PVT in
lim-6 ceh-14 double mutants, we next examined whether the generation
and overall cell fate of PVT is grossly affected in lim-6 ceh-14
mutants. To this end, we monitored the expression of several PVT cell fate
markers, namely the GTPase gpa-2
(Zwaal et al., 1997), the
LIM-only gene pin-2 (Hobert et
al., 1999a
), the serpentine receptor srq-1 (see Materials
and Methods) and the unc-47 GABA transporter gene
(Eastman et al., 1999
). All of
these markers are expressed normally in lim-6 ceh-14 double mutants
(Fig. 4). In addition, as
assessed with the unc-47
::gfp marker, PVT axon morphology
appears unaffected (data not shown). Last, the embryonic,
unc-6/Netrin-mediated role of PVT in attraction of axons from tail
ganglia into the VNC (Ren et al.,
1999
) is unaffected in lim-6 ceh-14 double mutants, as
visualization of several of these axons in lim-6 ceh-14 mutants
reveals no defect (data not shown). Taken together, these data suggest that in
the absence of the two LIM homeobox genes, lim-6 and ceh-14,
PVT adopts its correct cell fate, i.e. displays all known features of its
terminal differentiation program, but is incapable of initiating expression of
zig genes in the L1 stage.
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Although both lim-6 and ceh-14 are jointly required for
initiation of zig gene expression, they are not jointly sufficient to
induce zig gene expression for the following two reasons. First, both
lim-6 and ceh-14 are already expressed embryonically
(Fig. 2A) and hence
significantly precede the onset of zig gene expression; second,
pan-neuronal misexpression of lim-6 under control of the
unc-119 promoter yields co-expression of lim-6 and
ceh-14 in several neurons (ceh-14 is expressed in a total of
18 neurons) (Cassata et al.,
2000), yet does not cause ectopic zig-4 expression in any
of these cells (data not shown).
We also tested whether other transcription factors previously shown to be
expressed in PVT, namely the nuclear hormone receptor fax-1
(Much et al., 2000) and the
paired-type homeobox gene unc-42
(Baran et al., 1999
), affect
zig gene expression. By crossing various zig::gfp reporter
gene integrants with fax-1 and unc-42 single mutants and
with lim-6; unc-42 double mutants, we found this not to be the case
(data not shown).
Ventral nerve cord defects in lim-6 ceh-14 double
mutants
A loss-of-function mutation in zig-4 or postembryonic laser
removal of PVT, the cellular source of zig-4, causes a
disorganization of the left and right axonal tracts in the VNC, characterized
by a `flip-over' of embryonically established axonal tracts into the opposite
fascicle (Fig. 1)
(Aurelio et al., 2002). The
disruption of zig gene expression in PVT in lim-6 ceh-14
double mutant animals would thus be expected to cause similar disruptions of
VNC architecture. To investigate this point, we crossed lim-6 ceh-14
double null mutants with transgenic animals in which either the whole VNC or
individual neurons within the VNC are labeled with gfp. Using a
pan-neuronal marker, we indeed observed left/right axon flip-overs from one
fascicle into the opposite fascicle in the VNC of lim-6 ceh-14 double
mutant animals (Fig. 5, Table 1).
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The VNC axon flip-over defect in lim-6 ceh-14 animals manifests itself only at stages past the late L1 stage, while freshly hatched animals show no mutant phenotype (Table 1). Hence, lim-6 ceh-14 mutants show axonal maintenance defects in the VNC that resemble those in PVT ablated and zig-4 mutant animals not only in overall appearance but also in their temporal profile.
In summary, VNC axon flip-over defects are present in the lim-6 ceh-14 double mutant, but not in the single mutants; the expression of lim-6 and ceh-14 exclusively overlaps in PVT; PVT loses the postembryonic zig gene expression in lim-6 ceh-14 double mutants; and the VNC defects show a zig-4() and PVT()-like characteristic postembryonic profile. These points strongly suggest that lim-6 and ceh-14 act in PVT to affect VNC maintenance by regulating zig gene expression.
ceh-14 acts outside PVT to suppress the
zig-4-induced axon flip-over phenotype
Examination of individual axons in the VNC in lim-6 ceh-14 double
mutant animals lead to a surprising observation. Although the axons of the
PVQL/R neurons flip into the opposite site of the VNC in zig-4 mutant
animals, no such defect above background level can be observed in lim-6
ceh-14 double mutant animals, in which zig-4 expression is
downregulated (Fig. 5B,
Fig. 6). This observation could
be explained by zig-4 expression levels and hence zig-4
activity not being as significantly affected in lim-6 ceh-14 double
mutants as in the zig-4 null mutant. Alternatively, lim-6
and/or ceh-14 gene activity could be required for the PVQL/R
flip-over event to occur in the absence of zig-4. To address this
issue, we conducted a genetic epistasis test in which we analyzed PVQL/R
neuroanatomy in lim-6(nr2073) ceh-14(ch3) zig-4(gk34) triple null
mutant animals. We found that the zig-4 mutant phenotype is
suppressed in these triple mutant animals
(Fig. 6).
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We next asked whether lim-6 ceh-14 act in PVT to suppress the zig-4 mutant phenotype. It could, for example, be envisioned that expression of a factor X is repressed by the LIM-6 and/or CEH-14 proteins in PVT and that its derepression in lim-6 ceh-14 mutants prevents an axon flip-over caused by the absence of zig-4. Alternatively, lim-6 and/or ceh-14 could act outside of PVT to suppress the zig-4 mutant phenotype. To distinguish between these possibilities we laser ablated PVT in lim-6 ceh-14 double mutant animals. While PVT ablation in wild-type animals causes PVQL/R axon flip-overs, the same ablation in lim-6 ceh-14 animals causes no PVQL/R flip-overs above background levels (Fig. 6). Hence, lim-6 and ceh-14 must act outside of PVT to suppress the PVT() or zig-4()-induced axon flip-over.
Although the above mentioned experiments were all made with a lim-6
ceh-14 double null mutant, it could be envisioned that one of the two LIM
homeobox genes alone is sufficient to suppress the zig-4 induced
axonal flip-over of PVQL/R. In contrast to lim-6, which in the L1
stage is not expressed elsewhere in the VNC than in PVT, ceh-14 is a
good candidate as it is normally expressed in PVQL/R
(Fig. 1)
(Cassata et al., 2000). It was
thus conceivable that ceh-14 is required for PVQL/R to flip into the
opposite cord, e.g. through regulating the expression of homophilic adhesion
molecules, which we have previously implicated in playing a critical role in
the axon flip-over process (Aurelio et al.,
2002
). We indeed found that in ceh-14 zig-4 double null
mutant animals, but not in lim-6 zig-4 double null mutant animals,
the zig-4-mediated PVQL/R axon flip-over phenotype is suppressed
(Fig. 6).
Taken together, our data suggests that lim-6 and ceh-14 probably act in PVT to affect zig gene expression and that ceh-14 has an additional role outside of PVT, possibly within PVQL/R, to enable axon flip-over to occur in the absence of a functional axon maintenance mechanism.
zig gene expression also depends on a lim-6
ceh-14-independent, intrinsic timing mechanism
Previous studies focused on postembryonic aspects of lim-6 and
ceh-14 expression (Cassata et
al., 2000; Hobert et al.,
1999b
). We examined the temporal dynamic of lim-6 and
ceh-14 reporter gene expression in PVT in more detail, and found both
genes to be expressed already in mid-embryonic stages
(Fig. 2A) and maintained
throughout the life of the animal (data not shown). Considering the
postembryonic expression of most zig genes (zig-1, zig-2, zig-3,
zig-4, zig-8) in PVT, the embryonic expression of lim-6 and
ceh-14 indicates that these genes are insufficient to provide the
temporal trigger for onset of zig gene expression at the L1 stage.
Although we can not exclude the possibility that the lim-6 and
ceh-14 reporter gene constructs do not accurately reflect endogenous
gene expression, the reporter gene results provided us with sufficient
motivation to search for other, temporal parameters possibly involved in
timing zig gene expression.
An important temporal trigger for initiation and progression of larval development is the feeding state of the animal. In the absence of an external food source, animals arrest after hatching at the L1 stage. Hence, we considered the possibility that a food-dependent signal, possibly a hormonal signal, is involved in determining the timing of zig gene expression. By cultivating transgenic zig-4::gfp animals (otIs20) on plates that contain no food, we found that 20/20 L1 animals show zig-4::gfp in PVT under starvation conditions (see Materials and Methods). This results suggests that zig-4::gfp expression is not triggered through an extrinsic, food-dependent signal, but rather depends on an intrinsic timer.
The heterochronic timer lin-14 regulates zig-4 gene
expression
In C. elegans and possibly other animals, timing of developmental
events depends on well defined heterochronic genes
(Ambros, 2000;
Rougvie, 2001
;
Slack and Ruvkun, 1997
).
Heterochronic genes act at defined larval stages and their aberrant activity
(either through loss or ectopic expression) causes either precocious or
delayed execution of developmental events. The lin-14 gene codes for
a ubiquitously expressed nuclear protein, which is so far the only
heterochronic factor known to be required within the L1 stage to ensure the
correct execution of L1-specific blast cell divisions
(Ambros and Horvitz, 1984
;
Ambros and Horvitz, 1987
;
Ruvkun and Giusto, 1989
) and
neuronal rewiring events (Hallam and Jin,
1998
). To investigate whether the role of this heterochronic gene
also extends to the timing of a postmitotic gene expression program, we
crossed a representative zig gene reporter, chromosomally integrated
zig-4::gfp, with three different loss-of-function alleles of
lin-14. We focused on the zig-4 gene as it is the only
zig gene for which a mutant phenotype is available so far and which
would therefore allow us to compare its mutant phenotype with potential
lin-14 mutant phenotypes (see below). We found that the L1-specific
onset of zig-4::gfp expression is abolished in all lin-14
loss-of-function alleles tested (Fig.
7A, Table 2).
Notably, onset of expression is not merely precociously executed or later
initiated in the L2 stage, but absent throughout all stages. lin-14
does not affect the generation or overall fate of PVT as the cell fate markers
unc-47
::gfp and pin-2::gfp show normal expression in
PVT in lin-14 mutant animals; moreover, the axon anatomy of PVT is
unaffected in lin-14 mutants (Fig.
7B).
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A lin-14 gain-of-function allele that causes LIN-14 protein to be
present at all larval stages (Ambros and
Horvitz, 1984; Arasu et al.,
1991
) has no effect on zig-4 expression
(Table 1). A heterochronic gene
that is required for L2 specific developmental events, lin-28
(Ambros and Horvitz, 1984
),
also has no effect on zig-4 expression
(Table 2). The period
homolog lin-42, which affects timing of L4 stages (Z. Liu, PhD
thesis, Massachusetts Institute of Technology, 1990), but whose expression has
been shown to peak in each individual larval stage
(Jeon et al., 1999
) leaves
zig-4 expression unaffected as well
(Table 2). Taken together,
while zig-4 is expressed throughout all larval stages well into
adulthood, it is specifically the passing through the L1 stage but not any
other larval stage that is required for initiation of zig gene
expression.
Expression of zig-4::gfp is not affected by lin-14 in cells other than PVT. Consistent with this observation, none of the other zig-4::gfp expressing cells (ASK, ASI, BAG, M2) show stage-specific regulation of zig-4 expression in wild-type animals.
lin-14 affects ventral nerve cord architecture
Loss of zig-4 expression in lin-14 mutant animals would
be expected to cause axon maintenance defects in the VNC of lin-14
mutants. We analyzed VNC architecture by crossing three different
loss-of-function alleles of lin-14 with transgenic animals that
express a gfp reporter either in all VNC neurons or in a selected
subset. As expected from the loss of zig-4 gene expression, we found
that lin-14 animals display axon flipovers in the VNC
(Fig. 8;
Table 3). The defects can be
observed with a pan-neuronal gfp marker as well as with the PVQL/R
specific marker and are thus very similar to defects observed in
zig-4 mutant animals. Heterochronic genes that affect later stages of
larval development do not affect VNC architecture (data not shown).
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The VNC defects in lin-14 mutant animals display temporally
dynamic axonal defects. Freshly hatched lin-14 animals show no
defects above background level, while animals at later larval and adult stages
display significant defects (Table
3). These temporal dynamics precisely reflect the temporal
dynamics observed in zig-4 mutants animals and are also consistent
with the observation that PVT is specifically required in the L1 stage to
affect VNC architecture (Aurelio et al.,
2002).
lin-14 is ubiquitously expressed in neuronal and non-neuronal
cells (Ruvkun and Giusto,
1989). Its expression is initiated in embryogenesis, peaks right
after hatching, then rapidly fades and is absent by the L2 stage
(Ruvkun and Giusto, 1989
). To
affect VNC architecture, lin-14 could conceivably act in neurons or,
alternatively, in the underlying hypodermis. The later possibility seems
unlikely, because a null mutation in the lin-28 gene, which
diminishes LIN-14 protein levels in the hypodermis, but not in neurons
(Arasu et al., 1991
), has no
effect on VNC architecture (Table
2). As we have so far been unable to express lin-14 under
control of a PVT-specific promoter, we sought to further narrow the focus of
action of lin-14 action by conducting a laser ablation experiment. We
reasoned that if lin-14 acts in a PVT-dependent manner, laser
ablation of PVT in a lin-14 mutant background would not enhance the
lin-14 mutant phenotype. By contrast, if lin-14 were to act
independently of PVT in some other cell, laser ablation of PVT would enhance
the lin-14 mutant phenotype. Laser ablation of PVT causes a 36.4%
penetrant PVQL/R phenotype (Fig.
6). We found that 38.1% of lin-14(n179) animals in which
PVT was ablated showed defects in PVQL/R axon positioning
(Table 3). This number is
virtually the same as the 40.8% defects observed in unoperated
lin-14(n179) animals (Table
3). Although this result does not conclusively prove that
lin-14 acts within PVT, it provides strong suggestive evidence that
lin-14 activity is mediated through PVT to affect VNC structure.
Together with the effect of lin-14 on zig-4 expression, we
conclude that the lin-14 defects are probably due to absent
zig gene expression in PVT.
Ectopic expression of zig-4 causes VNC defects
We have defined three factors, lim-6, ceh-14 and lin-14
that are required for the correct spatiotemporal expression of zig-4.
We investigated the consequences of overriding the spatiotemporal control of
zig-4 mediated through these factors by ectopically expressing
zig-4 under the control of heterologous promoters at earlier time
points and at different locations in the VNC. To this end, we made use of the
promoter fragments from the flp-1 and myo-3 genes
(Nelson et al., 1998;
Okkema et al., 1993
). The
flp-1 promoter fragment is exclusively active in the AVKL and AVKR
neurons (see Materials and Methods), which send axons along both sides of the
VNC, while the myo-3 promoter is active in body wall muscle cells
which abut the left and right VNC and send muscle arms into the VNC. As
assessed through analysis of the expression of corresponding gfp
fusion constructs, both promoters are already embryonically active (data not
shown). We find that adult animals that misexpress zig-4 under
control of either of the two promoters show mispositioned PVQL/R axons in the
VNC (Fig. 9). The observed
defects could be caused through two distinct mechanisms. In one scenario,
precise levels and/or a defined localization of the endogenous ZIG-4 protein
is required in the L1 stage to ensure axon maintenance and ectopic ZIG-4
expression obscures this finely tuned distribution. In an alternative
scenario, ectopically expressed ZIG-4 may already act in embryonic stages to
affect PVQL/R axon outgrowth. In an attempt to distinguish between these two
possibilities, we assessed PVQL/R axon anatomy right after hatching, that is,
before PVT and zig-4 are required to maintain axon anatomy in the
VNC. We find that animals that ectopically express zig-4 already
display defects at this early stage (Fig.
9). We thus conclude that ectopically expressed zig-4
affects development of the VNC, probably during the axon outgrowth stages.
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Several of the transgenic lines revealed another intriguing aspect of zig-4 function. A subset of the transgenic lines (flp-1 promoter: line #2 and #3, myo-3 promoter: line #2) show no developmental defects, yet showed maintenance defects in the adult (Fig. 9). As levels of expression from independent extrachromosomal arrays can be highly variable, we consider it possible that these lines do not express enough zig-4 to cause embryonic defects, but enough zig-4 to interfere with the maintenance role of endogenous zig-4 at the L1 stage. In other words, ectopic zig-4 has two separable effects: it can interfere with axon outgrowth but it can also disrupt the normal function of zig-4 at postembryonic stages to affect VNC axon positioning. Taken together, the precise spatiotemporal control of zig-4 gene expression is necessary to prevent it from inappropriately interfering with VNC patterning at distinct stages.
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DISCUSSION |
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The lim-6 and ceh-14 LIM homeobox genes define the
spatial domain of zig gene expression. The well defined cooperative
action of several LIM homeobox genes in cell specification in the vertebrate
spinal cord (Sharma et al.,
1998; Tsuchida et al.,
1994
; Jessell,
2000
; Lee and Pfaff,
2001
) and in Drosophila
(Thor et al., 1999
),
collectively referred to as a LIM-code, and the biochemical evidence for
hetero- and homodimerization of individual LIM homeodomain proteins, including
the vertebrate LIM-6 ortholog LMX-1 and the vertebrate CEH-14 ortholog LHX3
(Jurata et al., 1998
;
Thaler et al., 2002
), suggest
a model in which LIM-6 and CEH-14 bind as heterodimers either directly to
zig gene promoters or regulate the expression of intermediary factors
that directly control zig gene expression. In the absence of either
LIM homeodomain protein alone, the other LIM homeodomain protein may still be
capable of activating its target gene(s) as a homodimer, thus explaining why
each single mutant has little observable effect on zig gene
expression.
Although lim-6 and ceh-14 are both jointly required for
zig gene expression, they are not sufficient to induce zig
gene expression, as inferred from the observation that the embryonic
expression of both genes is insufficient to yield embryonic zig gene
expression and from the observation that supplying lim-6 in other
ceh-14-expressing cells does not cause ectopic zig gene
expression. The effects of lim-6 and ceh-14 are thus
strictly dependent on the cellular context and strongly suggests the existence
either of other activators required in PVT to induce zig gene
expression or of repressors that prevent lim-6 and ceh-14
from activating zig genes in embryonic stages and/or other cell
types. The strict cellular context-dependence of target gene regulation by
lim-6 and ceh-14 is reminiscent of several other C.
elegans LIM homeobox genes, such as mec-3
(Duggan et al., 1998) or
ttx-3 (Altun-Gultekin et al.,
2001
). Each of these genes is required for expression of all
subtype characteristics of specific neuron classes, but incapable of inducing
these characteristics in many other cell types when ubiquitously expressed. A
similar context-dependent activity of the ceh-14 ortholog
Lhx3 has also recently been described in vertebrates
(Thaler et al., 2002
).
Although the expression of all neuronally expressed zig genes
overlaps uniquely in PVT, individual zig genes are expressed in cells
other than PVT. In those cells, zig gene expression does not appear
to be temporally regulated in a similar manner as it is in PVT. Moreover,
there is hardly any overlap of expression of zig genes,
lim-6 and ceh-14 in cells other than PVT. This is again
consistent with the previously discussed case of the ttx-3 homeobox
gene. All genes that are under control of ttx-3 in the AIY
interneuron class, are also expressed in cells other than AIY and are under
control of different transcription factors in those other cell types
(Altun-Gultekin et al., 2001).
This point again illustrates that zig gene regulation by LIM homeobox
genes in PVT falls into the general paradigm of context dependent regulation
of gene expression.
An interesting feature of lim-6 and ceh-14 activity is the specificity of their impact on mostly those genes that show a temporally regulated profile of expression: lim-6 and ceh-14 affect all postembryonically expressed zig genes, but exert a lesser effect on zig genes that show a certain component of embryonic expression and no effect on any other of the embryonically expressed PVT cell fate markers available. In classical developmental terms, lim-6 and ceh-14 thus appear to define the competence of the PVT cell to respond to a temporal cue.
One co-factor that either directly or indirectly contributes to
zig gene expression is the heterochronic factor LIN-14. This protein
defines the temporal domain of zig gene expression. Recent
experiments show that LIN-14 acts as a DNA-binding transcription factor (V.
Ambros, personal communication), which prompts the speculation that LIN-14
directly binds to zig gene promoters, possibly in conjunction with
LIM homeodomain proteins. Initially, C. elegans heterochronic genes
were defined through their effect on developmental stage-specific cell
division events (Ambros and Horvitz,
1984). The first evidence that they may also act in postmitotic
processes in the nervous system was provided by the observation that loss of
lin-14 leads to a precocious re-wiring of D-type motor neurons, an
event that is normally only observed in late L1-stage animals
(Hallam and Jin, 1998
). Our
observation of a function of lin-14 in temporally controlled
induction of zig gene expression is qualitatively different from the
D-type motoneuron case. D-type motoneuron rewiring is executed in
lin-14 mutants, though at an inappropriate time; hence, LIN-14 acts
as a repressor of the rewiring process. By contrast, zig gene
expression in lin-14 mutants is not merely observed at an
inappropriate time, but largely absent; hence, LIN-14 acts as an activator in
PVT. In addition, while the molecular targets of LIN-14 that cause the D-type
rewiring defect are as yet elusive, the VNC axonal patterning that we observe
in lin-14 mutants is consistent with the notion that the zig
genes are (direct or indirect) effectors of lin-14 in axonal
patterning.
If LIN-14 is the temporal trigger for activation for zig gene
expression, what is the temporal mechanism that restricts LIN-14 activity to
the L1 stage? This question is particularly relevant as LIN-14 antibody
staining can already be observed during late embryogenesis
(Ruvkun and Giusto, 1989). Two
models could be envisioned: LIN-14 protein levels may have to accumulate to a
critical threshold level that does not occur until the L1 stage. Observable
embryonic expression may still be at subthreshold levels. Alternatively,
restriction of lin-14 activity to the L1 stage may be dictated
through the presence of a co-factor that is temporally regulated, such as a
nuclear hormone receptor; the secretion of its ligand may be coupled to
hatching of the embryo. The feeding state of the animal cannot be a
determinant as we found zig-4 expression to be normal in starved
animals.
We have previously suggested that zig genes may be required
specifically during the L1 stage as a stabilizing factor to ensure that
already established axonal tracts can cope with mechanical stress and changes
in the local molecular environment occurring in the VNC in the L1 stage
(Aurelio et al., 2002). But why
is it that zig genes are under control of an L1-specific timer and
not just simply expressed throughout embryonic, larval and adult stages, like
other PVT cell fate markers? We show that inappropriate expression of the
zig maintenance factors during embryonic development of the VNC
interferes with axonal patterning possibly through interfering with the axonal
outgrowth machinery. For example, during embryonic axon outgrowth, the
SAX-3/Robo protein is required to prevent PVQL/R axons from crossing the
midline inappropriately (Aurelio et al.,
2002
; Zallen et al.,
1998
). It is conceivable that precocious expression of the
Ig-domain containing ZIG-4 protein may interfere with the activity of SAX-3,
an Ig domain-containing transmembrane protein, thus causing the midline
crossover defects that we observe. The tight postembryonic temporal control of
zig gene expression in wild-type animals thus may serve to prevent
zig-4 from inappropriately acting to affect VNC development.
The observation that loss of a developmental timer, i.e. the LIN-14
protein, causes severe disruption of axonal organization in the VNC, presents
a striking example for the importance of precise temporal orchestration of
gene expression events in the nervous system. Does the concept of temporal
control of neural gene expression programs by heterochronic genes, as
described by Hallam and Jin for motoneuron rewiring and as described here in
this paper for zig genes, apply to species other than C.
elegans? The recent identification of temporally regulated microRNAs in
vertebrates (Banerjee and Slack,
2002; Pasquinelli et al.,
2000
), some of which are orthologous to C. elegans
heterochronic microRNAs (Lagos-Quintana et
al., 2002
; Pasquinelli et al.,
2000
) and the sequence similarity between heterochronic genes and
circadian clock genes (Jeon et al.,
1999
) (F. Slack, personal communication), suggests not just a
conservation of general concepts of heterochronic regulation of gene
expression but also a conservation on the mechanistic levels.
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ACKNOWLEDGMENTS |
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