1 Max Planck Institute for Medical Research, Jahnstr. 29, 69120 Heidelberg,
Germany
2 Johns Hopkins University, Department of Biology, 3400 North Charles Street,
21218 Baltimore, MD, USA
* Author for correspondence (e-mail: hutter{at}mpimf-heidelberg.mpg.de)
Accepted 1 May 2003
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SUMMARY |
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Key words: zag-1, C. elegans, Neuronal differentiation, Axon guidance
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INTRODUCTION |
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A number of transcription factors are implicated in various steps of
neuronal differentiation, starting with the selection of neuronal precursors
to the specification of neuronal subtypes
(Bertrand et al., 2002;
Brunet and Pattyn, 2002
;
Dubois and Vincent, 2001
;
Goulding, 1998
;
Lee and Pfaff, 2001
;
Marquardt and Pfaff, 2001
;
Shirasaki and Pfaff, 2002
). An
extensively studied family that affects aspects of neuronal differentiation
are the LIM-homeodomain transcription factors, which define
motorneuron-subtype identities in a combinatorial fashion (reviewed by
Jacob et al., 2001
;
Shirasaki and Pfaff, 2002
).
However, expression of LIM-homeodomain transcription factors is restricted to
only a few subsets of neurons, so that LIM proteins alone cannot explain how
hundreds of different neuronal subtypes are specified. A few other
transcription factors are known to be involved in neuronal subtype
specification (Brunet and Pattyn,
2002
; Dubois and Vincent,
2001
; Lee and Pfaff,
2001
; Marquardt and Pfaff,
2001
), but a direct link between these and genes involved in
axonal pathfinding has been made rarely
(Erkman et al., 2000
). This
leaves the problem of how a particular class of neurons express the
appropriate set of axon-guidance receptors and signal-transduction components
largely unresolved.
Neurons and their processes can be visualized in vivo in C.
elegans with GFP markers (Chalfie et
al., 1994). This allows new types of genetic screens, in which
mutants with axon guidance defects can be isolated directly by selecting for
animals with visible axon outgrowth defects in GFP-labelled neurons. We
isolated mutants with defects in the outgrowth of interneuron axons in C.
elegans (H.H., unpublished). Here we describe one of these mutants
defining the gene zag-1. Mutants in zag-1 show
characteristic defects in the navigation of interneuron axons, ranging from
fasciculation defects in the ventral cord to completely misrouted axons that
extend inappropriately along the side of the animal. Furthermore,
zag-1-mutant animals ectopically express the glr-1::GFP
marker in additional neurons, indicating that the specification of
neurotransmitter subtypes, in this case expression of the glutamate receptor
glr-1, is compromised. Various classes of motorneurons also show
characteristic axon outgrowth defects, such as ventral cord fasciculation
defects, premature and incomplete branching of commissures, and misexpression
or lack of expression of cell-type-specific markers. A deletion allele of
zag-1, which was isolated from our deletion library, has additional
defects in pharynx development leading to an inability to feed and,
consequently, larval lethality. zag-1 encodes a putative
transcription factor with N- and C-terminal Zn-finger clusters and a
homeodomain in between, hence the name zag-1 (Zn finger involved in
axon guidance). zag-1 is expressed transiently in a large number of
postmitotic neurons, indicating that this gene plays an important role in
controlling aspects of neuronal differentiation.
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MATERIALS AND METHODS |
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The following GFP-reporter strains were used for analysis of axonal defects and misexpression: evIn82A[unc-129::GFP], oxIs12[unc-47::GFPNTX, lin-15(+)], wdIs6[del-1::GFP, dpy-20(+)], evIs111[F25B3.3::GFP], gvEx173[opt-3::GFP, rol-6(su1006)], kyIs51[odr-2::GFP, lin-15(+)], ccIs4251[myo-3:DFP, myo-3:mitoGFP, dpy-20(+)] I; him-8(e1489), rhIs4[glr-1::GFP; dpy-20(+)], rhIs7[unc-4::GFP; rol-6(su1006)], rhIs11[unc-3::GFP; rol-6(su1006)], rhIs12[sra-6::GFP; dpy-20(+)], rhIs18[epi-1::GFP nuclear, dpy-20(+)], hdIs1[unc-53::GFP, rol-6(su1006)], hdIs10[unc-129::CFP, glr-1::YFP, unc-47::DsRed, hsp16::rol-6] V, hdIs14[odr-2::CFP, unc-129::YFP, glr-1::DsRed, hsp-16::rol-6], hdIs8[him-4:GFP, rol-6(su1006)].
All strains were cultured at 20°C using standard methods.
GFP markers
To generate GFP-reporter constructs, promoter sequences were amplified by
PCR and cloned into GFP vectors: epi-1::GFP: 2.8 kb upstream of ATG cloned in
pPD96.62 (nuclear localized GFP, gift from A. Fire). All other markers (unc-3,
unc-4, unc-47, unc-53, unc-129, glr-1, him-4, odr-2, opt-3, F25B3.3, myo-3 are
sra-6) were derived from previously published constructs. For some, the GFP
coding region was replaced with a different GFP variant. Variants used are (A.
Fire vector kit): GFP, S65C mutation; CFP, Y66W, N146I, M153T, V163A; YFP,
S65G, V68A, S72A, T203Y; DsRed, DsRed from Clontech vector pDsRed.
Mutant isolation
The zag-1(rh315) allele was isolated after EMS mutagenesis of
rhIs4[glr-1::GFP] animals in a nonclonal screen for animals with axon
outgrowth defects in glr-1-expressing interneurons. The deletion
mutant hd16 was isolated from an EMS-mutagenised library using a
poison primer approach targeting the first exon
(Edgley et al., 2002).
Mapping and cosmid rescue
zag-1(rh315) was mapped to a region between unc-17(e113)
and dpy-13(e184) on LG IV by classical genetic methods. For further
mapping using single nucleotide polymorphisms zag-1(rh315)
dpy-13(e184) recombinants were crossed into the CB4856 wild type strain.
F2 animals having lost one of the markers were scored for the presence of a
SNP marker on cosmid W03D2. Transgenic animals injected with a cosmid pool
(K09B3, M02B7, F28F9, T08C8, F37C4, K02F11, F26F6, T17A2) at 10 ng/µl each
and pRF4 (rol-6(su1006)) at 25 ng/µl) or the individual cosmids
were analysed for rescue of movement and axonal defects. PCR fragments
containing the entire coding region of F28F9.1 or F28F9.4 plus 4.5 kb or 3.5
kb upstream were also tested for their rescuing abilities.
Sequencing
PCR fragments containing either exons 1-4 or exons 5-7 were generated for
sequencing to identify the mutation in zag-1(rh315). cDNA clones
yk168d11, yk281e5, yk312a9, yk556a12, yk621g7, and yk621g7 were excised
according to the manufacturer's instructions (Stratagene) and used for
restriction analysis and sequencing. The deletion in zag-1(hd16) was
defined by sequencing a 1.7 kb PCR fragment starting 0.75 kb upstream of
the first ATG. The deletion in zag-1(hd16) is 516 bp in size and
starting 50 bp downstream of the ATG.
Analysis of zag-1 expression
To generate reporter constructs we used the GatewayTM cloning
system according to the manufacturer's instructions (Life Technologies). A PCR
fragment containing 4.7 kb upstream region of zag-1 was cloned into
an entry vector and recombined with a destination vector containing YFP to
create Pzag-1::YFP. Similarly, the same upstream region plus the
entire coding region was fused to the N-terminus of YFP. Transgenic animals
were generated by injecting 20 ng µl-1 reporter plasmid plus 50
ng µl-1 pRF4. Arrays were integrated using UV-irradiation,
followed by two backcrosses with N2.
Analysis of neuronal defects
Animals were grown at 20°C and analysed as either newly hatched L1
larvae or adult animals from a growing population. For taking images, animals
were incubated with 100 mM NaN3 in M9 buffer for 1 hour and mounted
on agar pads. Stacks of confocal images with 0.3-0.4 µm vertical pitch were
recorded with a Leica TCS SP2 microscope. Maximum intensity projections of all
images from a given animal were generated using the Imaris 3.1 software
package.
Feeding experiments
1 ml of a 0.02% suspension of fluorescent latex beads (100 nm diameter,
Molecular Probes) in OP50-containing medium was added to a mixed population of
almost starving animals (progeny of a zag-1(hd16)/hdIs14 IV parent)
on a small Petri dish. After 1 hour incubation with occasional agitation,
animals were transferred to agar pads on microscope slides and observed with
DIC and epifluorescence microscopy. Images were recorded with a Princeton
Instruments MicroMAX cooled CCD camera using the Metamorph 4.1 Imaging
software package. To investigate uptake of nonparticulate markers, 10 µM
FM1-43 (Molecular Probes) in M9 buffer was applied as above and analyzed after
either 1 or 4 hours.
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RESULTS |
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Orthologs of zag-1 are found in Drosophila and
vertebrates. The Drosophila protein is called ZFH-1
(Fortini et al., 1991). The
vertebrate proteins are
EF1 in chick
(Funahashi et al., 1993
),
MEB1/
EF1 in mouse (Genetta and
Kadesch, 1996
; Sekido et al.,
1994
), AREB6/ZEB-1/nil-2-a in humans
(Genetta et al., 1994
;
Watanabe et al., 1993
;
Williams et al., 1991
) and
Zfhx1 in zebrafish (Muraoka et al.,
2000
). Each of these proteins has a cluster of Zn fingers at both
ends and a single homeodomain in the center of the protein
(Fig. 1D). The C-terminal
Zn-finger cluster consists of three fingers in all proteins, whereas the
number of fingers in the N-terminal cluster varies from two (ZAG-1) to five
(ZFH-1, long isoform).
EF1 has been shown to act as transciptional
repressor and interact with a corepressor called CtBP1. The CtBP1-binding site
downstream of the homeodomain (consensus sequence PLDLS/T) is also conserved
in ZAG-1, and a CtBP1 homolog is found in the C. elegans genome
(F49E10.5 Wormpep).
ZAG-1 and its homologs have a high degree of sequence identity (80% identity) in the Zn fingers (Fig. 1C). The homeodomain is also fairly well conserved (61% similarity), but sequences between these structural motifs have little similarity. A paralog, SIP1/ZEB2, has been identified in vertebrates, indicating that there was a gene duplication in the vertebrate lineage.
Expression of zag-1
We generated several YFP-reporter constructs to study the expression
pattern of zag-1. First we cloned the upstream regulatory region,
which is sufficient to rescue the defects in zag-1-mutant animals,
into a YFP-vector (Pzag-1::YFP). Second, we cloned the upstream and
entire coding region into a vector fusing YFP to the C-terminus of ZAG-1
(zag-1::YFP). With both constructs we generated transgenic lines that
were stably integrated into the genome and used for expression analysis.
We found the Pzag-1::YFP construct was expressed predominantly in neurons in head and tail ganglia, starting approximately midway through embryogenesis (Fig. 2A). In some of these neurons expression was maintained throughout development (Fig. 2I). Additional expression was found consistently in the intestinal and anal depressor muscles during all life stages (Fig. 2L) as well as occasionally in body-wall muscles during embryogenesis.
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ZAG-1 regulates its own expression
The Pzag-1::YFP construct was crossed into the
zag-1(rh315) mutant to analyze the potential effects of
zag-1 on its own expression. We found that expression of the
Pzag-1::YFP construct in several classes of neurons, most notably
motorneurons in the ventral cord, was not downregulated in
zag-1(rh315)-mutant animals (Fig.
2J,K). These neurons expressed zag-1 throughout the
entire life cycle, indicating that downregulation of zag-1 depends on
the presence of intact ZAG-1 protein. The vertebrate homolog of ZAG-1,
EF1, has been shown to bind to E-box-sequence motifs (CACCTG) with each
of its Zn fingers. Pairs of such motifs are found in the upstream regulatory
region and the first intron of the zag-1 gene (data not shown),
indicating that this repression could be mediated directly, by ZAG-1 binding
to its own promoter.
zag-1 mutants show defects in axonal outgrowth, branching
and fasciculation
The ventral cord contains essential components of the motor circuit and
consists of two axon bundles flanking the ventral midline. Interneuron axons
enter the ventral cord from the anterior after exiting the nerve ring on the
ventral side. Almost all axons on the left side cross over to run in the right
axon tract, which leads to a highly asymmetrical ventral cord with many more
axons running in the right than the left fascicle.
zag-1(rh315)-mutant animals showed penetrant axon outgrowth defects
in glr-1::GFP-expressing interneurons
(Table 1). First, not all axons
followed their normal trajectory from cell bodies in the head ganglia towards
the nerve ring and further on into the ventral cord. Instead, axons extended
abnormally in lateral positions, often wandering between lateral axon tracts
(Fig. 3B). Occasionally,
additional processes were sent out from the cell body, which sometimes formed
ectopic branches. Axons reaching the ventral cord frequently had fasciculation
problems in the ventral cord and crossed back and forth between the right and
left axon tracts (Fig. 3D).
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AVE-interneuron axons labeled with opt-3::GFP normally cross the midline to the contralateral side in the nerve ring, turn posterior and enter the ventral cord. In 16% of zag-1-mutant animals they never entered the ventral cord and, instead, ran in a lateral position. These axonal defects in various classes of neurons that are part of the motor circuit led to variable movement defects in zag-1(rh315) mutants. Animals were able to move forward and backward, but were significantly uncoordinated in their movement. In some animals, parts of the body appeared stiff and other animals tended to coil, whereas a few moved without apparent problem. This variability in movement defects reflects the variability in the axonal-outgrowth defects seen in zag-1(rh315).
zag-1 regulates neuronal differentiation
When analysing axonal defects in zag-1(rh315) mutants we noticed
that additional cells expressed the glr-1::GFP marker. In head, as
well as tail, ganglia a number of extra cells expressed glr-1::GFP
(Fig. 3B,F). Among these were,
occasionally, motorneurons and one or two cells in the PDE cluster, most
likely PVD and occasionally PVM (Table
2). Some cells, such as RID and PDB, could also be identified by
the position of their cell body and axon trajectory
(Fig. 3F), indicating that
these neurons retained major aspects of their original identity. The same was
true for motorneurons that misexpress glr-1::GFP. Typically, these
sent out commissures towards the dorsal cord, just as normal motorneurons do
(Fig. 3F).
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Various motorneuron markers were also misexpressed in zag-1(rh315) mutant animals (Table 2). We found unc-129::GFP expressed ectopically in head neurons, a defect also seen in unc-130 mutants. Some of the DA-type motorneurons failed to express the unc-3::GFP and unc-4::GFP markers (Fig. 3L). Conversely, unc-129::GFP was expressed consistently at high levels in DA8 and DA9, cells that do not express this marker in adult, wild-type animals. Other motorneuron markers, such as unc-47::GFP, were expressed normally in GABAergic motorneurons although, occasionally, some cells had lower levels of expression. Similarly, expression of del-1::GFP in VA and VB motorneurons was only changed to a minor degree. A pan-neuronal marker (F25B3.3::GFP) is expressed in the normal number of motorneurons in L1 larvae, again indicating that the failure in expression of some markers is not caused by the absence of the cells (Table 2). Finally, several other neuronal markers expressed in subsets of sensory and interneurons, including odr-2::GFP and opt-3::GFP, were also expressed normally, indicating that not all classes of neurons are affected in zag-1(rh315).
The putative null allele, zag-1(hd16), has axon guidance defects comparable to zag-1(rh315) mutants when assayed with a glr-1::GFP reporter (Fig. 4C, Table 1). Ectopic expression of glr-1::GFP in motorneurons was seen in the majority of animals (64% in 0-2 cells, 36% in ≥3 cells, n=67). With a motorneuron marker (unc-129::GFP) that was expressed in the normal number of cells in zag-1(rh315), we saw partial loss of expression in zag-1(hd16)-mutant animals (43% in 7-10 cells, 57% in ≤6 cells, n=67). With the D-type motorneuron marker unc-47::GFP, we saw no changes in expression but very pronounced axon outgrowth defects (Fig.4D, Table 1), including premature termination of outgrowth that led to gaps in the ventral cord. In addition, a few motorneuron cell bodies are mispositioned in the ventral cord, most prominently DD2 lying close to DD1 in 95% of animals (n=77). This indicates that some residual function for neuronal development is still present in zag-1(rh315).
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Because the zag-1(rh315) mutation leaves a large part of the protein intact, it is not clear whether zag-1(rh315) represents a complete loss-of-function allele. We therefore isolated a deletion allele from our deletion library using primers targeting the first exon. This 517 bp deletion removes part of the first exon (starting at codon 18) and most of the first intron, introducing a frame shift and a stop codon shortly thereafter (Fig. 1A). We expect this mutant to produce no functional product from the zag-1 locus. We were unable to obtain homozygous mutant animals, even after two rounds of outcrossing, indicating that the zag-1(hd16) deletion might be lethal. Balanced unc-17 dpy-13/zag-1(hd16) IV and hdIs14[unc-129::YFP]/zag-1(hd16) IV strains indeed segregated early-larval-lethal animals. Overall, these animals have normal body morphology with all organs developed. They are severely uncoordinated and tend to coil. Even on plates with food they adopt a starved appearance and die after a few days, indicating that the animals are unable to either take up or digest food. Feeding zag-1(hd16) mutants with fluorescent beads (0.1 µm), we observed that the beads inevitably stuck in the anterior part of the pharynx (procorpus), suggesting that the animals are unable to swallow food (Fig. 4B). Similar observations were made when fluid-phase markers were used for feeding. No obvious morphological defects in the pharynx were observed by light microscopy. Pharynx-muscle-specific markers like myo-2::GFP are expressed normally (data not shown). Pharyngeal muscles are able to contract in zag-1(hd16), however the contractions were weaker and did not lead to the characteristic opening and closing of the lumen of the isthmus. The lethality of zag-1(hd16) can be rescued by the zag-1::YFP transgene, which also rescued the neuronal defects in zag-1(rh315), indicating that the lethality is, indeed, caused by deletion in the zag-1 gene (data not shown). Transheterozygous zag-1(rh315)/zag-1(hd16) animals can survive (20% survivors), but grow slowly and are severely uncoordinated, indicating that they still have some feeding problems and, probably, have even stronger neuronal defects than zag-1(rh315) alone.
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DISCUSSION |
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EF1 binds to CACCT sequences and is thought to compete with basic
helix-loop-helix (bHLH) transcription factors for binding to E2-boxes
(sequence, CACCTG), thereby preventing gene transcription
(Sekido et al., 1994
). Two,
independent, repression domains of
EF1/ZEB outside the Zn fingers have
been shown to be sufficient to repress genes regulating either hematopoetic or
muscle-specific differentiation pathways
(Postigo and Dean, 1999a
). One
of these domains contains a PLDLS motif, which was shown to recruit the
CtBP1/2 corepressor (Furusawa et al.,
1999
; Postigo and Dean,
1999b
). This indicates that
EF1/ZEB can act in more than
one way as repressor (Ikeda and Kawakami,
1995
; Postigo and Dean,
1999a
; Remacle et al.,
1999
; Sekido et al.,
1997
). The key sequence elements essential for
EF1/ZEB
repressor function, the Zn-finger cluster and the CtBP-corepressor binding
site, are highly conserved in ZAG-1, with a high degree of sequence identity.
This makes it likely that ZAG-1 binds to the same consensus sequence as
EF1 and also acts as transcriptional repressor.
zag-1 expression and function in the mesoderm
The zag-1 gene is expressed in mesodermal tissues including the
pharynx, and the intestinal and anal depressor muscles
(Fig. 2F,I,J). Expression in
the pharynx is transient during embryogenesis, whereas expression in the
intestinal and anal depressor muscles was apparent throughout development.
zag-1 orthologs in Drosophila and vertebrates are also
expressed in the developing mesoderm, most notably in muscle cells
(Funahashi et al., 1993;
Lai et al., 1991
;
Takagi et al., 1998
), again
emphasizing the strong conservation in the expression of these proteins.
zfh-1 mutants in Drosophila have various defects in
mesodermal tissues, including body-wall muscle, heart and gonadal mesoderm
(Broihier et al., 1998
;
Lai et al., 1993
). In
zfh-1 mutants some muscle cells are missing whereas others are either
misplaced or disorganized.
EF1/ZEB in vertebrates has been shown to
interfere with muscle differentiation in transfected cells in culture by
counteracting the effect of bHLH proteins such as MyoD
(Postigo and Dean, 1997
;
Sekido et al., 1994
). The
mouse
EF1 (Zfhxla Mouse Genome Informatics)
gene is expressed in muscle cells during embryonic development, but no obvious
defect in muscle-cell differentiation was detected in
EF1-mutant mice
(Takagi et al., 1998
). In
C. elegans, zag-1 is the only homolog of
EF1/zfh-1. With the zag-1-promoter-GFP
construct we occasionally saw transient expression in body-wall-muscle cells
during embryogenesis. However, using several muscle markers we were unable to
detect any obvious defects in the differentiation of body-wall-muscle cells in
zag-1(rh315) mutants, indicating that ZAG-1/
EF1 might not be
essential for muscle differentiation in this animal. We did, however, find
subtle changes in the level of expression of muscle-specific markers in anal
depressor and sphincter muscles. zag-1 might have a role in
modulating expression of muscle genes, rather than acting as an all-or-none
switch for the expression of particular target genes in muscle cells.
The zag-1(rh315) mutation leads to a truncated protein with the N-terminal Zn fingers and the homeodomain intact, indicating that this might be a partial loss-of-function allele. This is confirmed by the observations that neuronal defects in zag-1(rh315) were recessive and that transheterozygous zag-1(rh315)/zag-1(hd16) animals had a phenotype stronger than zag-1(rh315) but weaker than zag-1(hd16), the putative null allele.
zag-1(hd16) mutants died with a starved appearance. We found that
the animals were, apparently, unable to swallow (food) particles efficiently.
Fluorescent beads fed to the animals stuck in the anterior part of the
pharynx, the procorpus, indicating that the pharynx did not function properly.
This defect could either be caused by defects in pharyngeal-muscle development
or by a failure in development or function of the pharyngeal M4 neuron that
has been shown to be essential for contractions of the isthmus and,
consequently, for passage of food into the terminal bulb
(Avery and Horvitz, 1987). One
of the pharyngeal cells expressing zag-1 is in a position consistent
with it being the M4 neuron. Other pharyngeal cells expressing zag-1
are most likely the m4 and m5 muscle cells, which leaves both possibilities
(either neuronal or muscle defect) open at the moment.
In the mouse, two mutants in the EF1 gene have been
generated. The
EF1
C727 mutation truncates
the protein and eliminates the C-terminal Zn-finger cluster. Mutant mice have
defects in thymus development and a greatly reduced number of T cells
(Higashi et al., 1997
).
EF1null(lacZ) mutants, where the entire protein is
eliminated, have additional defects in neural-crest-derived skeletal elements,
limb-bone and sternum development (Takagi
et al., 1998
), also suggesting that the truncated protein has some
residual activity.
For EF1 it has been shown that binding of both Zn-finger clusters is
necessary for efficient transcriptional repression at some promoters
(Remacle et al., 1999
;
Sekido et al., 1994
). By
contrast, the N-terminal and C-terminal fingers alone can bind to their
respective target sequences, so that a truncated form of
EF1/ZAG-1 with
just one cluster of Zn fingers intact might still successfully repress
transcription at other target sites. Binding studies with
EF1/AREB6 and
SIP1 suggest models in which binding to target sites with either one or two Zn
fingers leads to different effects on the transcription of target genes
(Ikeda and Kawakami, 1995
;
Remacle et al., 1999
). This
might provide an explanation for the additional phenotypes observed with null
mutants in mice and C. elegans.
zag-1 controls aspects of neuronal differentiation
We found zag-1 expressed predominantly in the developing nervous
system. Expression started soon after neurons became postmitotic, peaked
during the period when neurons differentiated and faded away in most neurons
when embryogenesis was complete. Expression of zag-1 is highly
dynamic and occurs in many different classes of neurons, arguing against a
role of zag-1 in the specification of particular neuron types. This
is in contrast to the function of other transcription factors like
LIM-homeodomain proteins, which are expressed in a more restrictive way and
are thought to act in combination to specify subtype identities of
motorneurons in vertebrates (Goulding,
1998; Jacob et al.,
2001
). zag-1 orthologs in Drosophila and
vertebrates are also expressed prominently in the developing nervous system
(Lai et al., 1991
;
Takagi et al., 1998
). However,
no neuronal defects have been described in the corresponding mouse or
Drosophila mutants (Broihier et
al., 1998
; Higashi et al.,
1997
; Lai et al.,
1993
; Su et al.,
1999
; Takagi et al.,
1998
). Our data show that zag-1 plays an important role
in neuronal differentiation in C. elegans. Because of the strong
conservation in sequence and expression of the zag-1 homologs, we
strongly suspect that
EF1/ZEB/AREB6 might have a similar, but so far
undetected, role in neuronal development in vertebrates.
We found that zag-1 expression itself was affected by the absence
of functional ZAG-1 protein. The zag-1-promoter-GFP construct, which
closely reflects the dynamic expression of zag-1 in wild type, is not
downregulated in zag-1(rh315) mutants during postembryonic
development, indicating that zag-1 negatively regulates its own
expression and seems to shut down its own expression when it is no longer
required. We found closely spaced pairs of putative ZAG-1/EF1 binding
sites in the promoter region of the zag-1 gene, indicating that this
effect could be due to ZAG-1 binding to its own promoter.
zag-1-mutant animals had several defects indicative of incomplete neuronal differentiation. First, cell-type-specific markers were not expressed properly in zag-1 mutant animals. Affected are genes determining neurotransmitter properties, including glr-1, a glutamate receptor gene, and chemosensory receptor genes such as sra-6. These genes encode terminal differentiation products that are characteristic of some subtypes of neurons. In zag-1 mutants, either too few cells express these markers (sra-6) or additional cells express the marker (glr-1). Typically, cells that fail to express a particular marker still express other neuronal markers and also have the characteristic appearance of neurons, indicating that neuronal cell lineages are normal and that zag-1 does not affect the generation of neurons per se. Furthermore these changes in gene expression do not seem to reflect a switch in neuronal identity because extra neurons expressing the glr-1 marker like the PDB and RID neurons send out their axons along their normal (often unique) paths. Genes controlling axonal outgrowth are still, apparently, expressed correctly in these cells, suggesting that neuronal identity has not been lost completely. Conversely, in other neurons, such as the D-type motorneurons, the expression of particular cell-type-specific markers is normal, but axon outgrowth is affected, again indicating that only some aspects of neuronal identity are disturbed.
The most prominent defects in zag-1 mutant animals are characteristic axon outgrowth defects. Interneurons that express glr-1 have defects in the navigation through the nerve ring towards the ventral cord and fasciculation defects in the ventral cord. Motorneuron axons also exhibit fasciculation defects in the ventral cord, as well as commissure outgrowth defects; commissures grow out on the wrong side, sometimes fail to reach the dorsal cord, and, occasionally, either branch prematurely before reaching the cord or fail to branch after reaching the dorsal cord. These defects could be caused by either a failure in the proper expression of extracellular guidance cues or be intrinsic problems of particular neurons in responding to guidance cues. The expression of zag-1 in neurons shortly before the time of axon outgrowth suggests that zag-1 directly or indirectly affects the transcription of genes that are important for responding to axon guidance signals. These genes could be either receptors for particular guidance cues or components of the signal transduction machinery that integrates guidance signals.
zag-1 transcriptional repressor acts in a regulatory network
for neuronal differentiation
An increasing number of transcription factors are known to affect different
steps of neuronal differentiation in C. elegans. Several genes have
been identified that affect the division pattern of neuroblasts and, hence,
the generation of neurons. This frequently leads to loss of particular classes
of neurons, due either to premature withdrawal of neuroblasts from the cell
cycle, as in cnd-1 mutants
(Hallam et al., 2000), failure
to generate neuronal lineages, as in lin-32
(Chalfie and Au, 1989
), and to
changes in the lineage program itself, as in unc-86
(Chalfie et al., 1981
) and
pag-3 mutants (Cameron et al.,
2002
). zag-1 seems to act downstream of these early
events in neuronal specification, because neurons are generated in
zag-1 mutants and seem to differentiate with neuronal properties.
Next in the hierarchy of neuronal differentiation are transcription factors
that affect the differentiation of particular classes of neurons, like touch
cells in mec-3 mutants (Mitani et
al., 1993) and D-type motorneurons in unc-30 mutants
(Jin et al., 1994
;
McIntire et al., 1993
). MEC-3
has been shown to directly regulate the expression of the mechanosensory
specific genes mec-4 and mec-7
(Duggan et al., 1998
),
providing a direct link between a transcription factor and terminal
differentiation products of certain subtypes of neurons. There are also a few
examples where neurons switch identities. The AWB sensory neuron adopts an AWC
fate in lim-4 mutants (Sagasti et
al., 1999
), and several different neurons adopt a CEM fate in
cfi-1 mutants (Shaham and
Bargmann, 2002
). All known aspects of the differention of
particular classes of neurons are affected in mutants in the above-mentioned
genes, which, again, is different from the situation in zag-1
mutants, where neuronal identities are not completely lost or changed.
The partial loss of neuronal identity in zag-1mutants is more
reminiscent of the situation found in unc-42, which affects
expression of glutamate-receptor subunits in a subset of interneurons of the
motor circuit (Baran et al.,
1999). zag-1 phenotypes are also reminiscent of defects
in the nuclear hormone receptor fax-1, which is required for correct
pathfinding of axons extending in the left axon tract of the ventral cord, and
also correct expression of a peptide neurotransmitter precursor
(Much et al., 2000
). Distinct
aspects of neuronal identity are also affected in unc-4 mutants,
where VA motorneurons receive input from interneurons that would normally
connect to VB motorneurons but retain VA-specific output and axon trajectories
(Miller and Niemeyer, 1995
;
Miller et al., 1992
;
White et al., 1992
). UNC-4, a
homeodomain transcription factor, is thought to be involved specifically in
defining synaptic input for one class of motorneuron axons, again illustrating
that different aspects of neuronal differentiation can be under independent
transcriptional control.
zag-1 probably acts as a coregulator, most likely in combination with various other transcription factors in different neurons because many features of neuronal identities are still normal in zag-1 mutants. Dissecting the components of this transcriptional network further will lead to a better understanding of how distinct features of neurons appear during their differentiation. In vertebrates zag-1 homologs have been shown to block the function of bHLH proteins in myogenic development. Therefore it will be especially interesting to study the interactions between zag-1 and neuronally expressed bHLH proteins, which constitute a fairly large family of transcription factors in C. elegans, with more than 20 members.
It is possible that zag-1 acts at different levels in the differentiation pathway of neurons because it is found in many neurons only at late stages of differentiation. We found several terminal differentiation markers were expressed ectopically in zag-1 mutants, and it is possible that zag-1 acts directly to repress expression of some of these genes in inappropriate places. The most prominent defects in zag-1-mutant animals are axon guidance defects. Many of these defects affect particular guidance decisions, implying that zag-1 might regulate genes that are essential for the response to particular guidance cues. The identification of target genes whose expression is controlled by zag-1 might lead to the identification of novel, key regulators of axon guidance.
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ACKNOWLEDGMENTS |
---|
The GenBank Accession Number for the zag-1 cDNA is AY289599.
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