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RESEARCH PAPER
Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA
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Abstract |
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[Keywords: C. elegans; left/right asymmetry; transcription factor; homeobox]
Received May 30, 2003; revised version accepted July 9, 2003.
).
However, specific deviations from this bilateral symmetry are apparent at two
different levels. First, individual organs such as the heart, stomach, and
spleen are placed in a left/right (L/R) asymmetric manner in many organisms
(Ludwig 1932). Second, organs
or morphological features that display an inherent pattern of bilateral
symmetry can show defined deviations from symmetry. Such symmetry breakages
are evident in the structure of seemingly bilaterally symmetric nervous
systems of several species, from invertebrates to humans. In vertebrates,
neuronal L/R asymmetry is evident in the different sizes of bilaterally
positioned structures, such as the left and right temporal lobes of the human
brain (Galaburda 1991) or the
diencephalic habenular nuclei in several amphibian brains
(Ludwig 1932). More recently,
L/R-specific gene expression patterns have been observed in the brains of
several vertebrate species (Ramsdell and
Yost 1998; Mercola and Levin
2001) and, in zebrafish, have been shown to correlate with the
determination of L/R-specific features of diencephalic structures
(Concha et al. 2000;
Essner et al. 2000;
Liang et al. 2000). Smaller
sized invertebrate brains, in which individual cell types can be more easily
identified and characterized, also reveal L/R asymmetries in otherwise largely
bilaterally symmetric ganglia. These asymmetries include L/R-specific
positioning of individual cells, L/R-specific cell death, and L/R-specific
gene expression profiles (for review, see
Hobert et al. 2002). Of the
two most widely used invertebrate model systems that are amenable to genetic
studies, Drosophila melanogaster and Caenorhabditis elegans,
neuronal L/R asymmetries have only been observed in C. elegans, thus
making it the prime choice to genetically dissect the development of neuronal
L/R asymmetry.
Evidence in vertebrates and the nematode C. elegans suggests that
bilateral neuronal structures may diversify with a L/R-specific bias as a
means to increase the functional capacities of the nervous system. In humans,
certain brain functions, such as language and attention, are highly
lateralized (Davidson and Hugdahl
1994). Nervous system functions in other vertebrate species,
including rodents (Glick and Ross
1981) and fish (Miklosi et al.
1997), also display a lateral bias. In C. elegans,
laterality has been observed in chemosensory capacities, specifically in the
AWC and ASE neuron classes
(Pierce-Shimomura et al. 2001;
Wes and Bargmann 2001). The
AWC odorsensory neuron class consists of two neurons, AWC left and AWC right
(AWCL and AWCR, respectively; Fig.
1), whose symmetries extend to many differentiated features of the
neurons including cell position, axonal and dendritic morphology, outgrowth
and placement, and synaptic connectivity
(Fig. 1;
White et al. 1986). In
addition, both cells express similar sets of genes and are both required for
chemotaxis to specific odorants (Bargmann
et al. 1993). However, a putative G-protein coupled olfactory
receptor, encoded by the str-2 gene, was found to be expressed
asymmetrically in these neurons (Fig.
1; Troemel et al.
1999). Induction of str-2 occurs stochastically through a
calcium-signaling pathway in either the left or the right cell, but never in
both (Troemel et al. 1999;
Sagasti et al. 2001). The
functional significance of this stochastic distribution is demonstrated by the
fact that mutants defective for str-2 asymmetry show
odor-discrimination defects (Wes and
Bargmann 2001).
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). The phenomenon
of antisymmetry is contrasted with "directional asymmetry",
characterized by the sidedness of morphological features not being randomly
distributed within a population, but strongly biased to one side of each
individual (Palmer 1996). A
clear case of directional asymmetry is displayed by the main class of C.
elegans gustatory neurons called ASE. Like the AWC odorsensory class, the
ASE neuron class consists of two bilaterally symmetric cells, called ASEL
(ASE left) and ASER (ASE right),
that are symmetric with regard to cell position, axonal and dendritic
morphology, and patterns of synaptic connectivity
(Fig. 1;
White et al. 1986). However,
three putative sensory receptors of the guanyl cyclase receptor family are
asymmetrically expressed in ASEL (gcy-6 and gcy-7) or ASER
(gcy-5; Fig. 1;
Yu et al. 1997). Laser
ablation of ASEL or ASER revealed that these asymmetric gene expression
profiles correlate with functional asymmetry of the two neurons; each neuron
is responsible for detecting a distinct class of water-soluble chemicals
(Pierce-Shimomura et al.
2001). The functional significance of this L/R separation of
chemosensory capacities was revealed through the analysis of lim-6
mutant animals in which the ASEL neuron, which normally senses sodium, but not
chloride, now adopts the chloride-sensing feature of the ASER neuron
(Hobert et al. 1999;
Pierce-Shimomura et al. 2001).
Hence, in lim-6 mutant animals, ASEL has the capacity to sense both
sodium and chloride. These animals fail to effectively discriminate between
the two chemicals; that is, they fail to sense one ion in the presence of the
other (Pierce-Shimomura et al.
2001). The L/R separation of the chemosensory capacities of the
ASE neurons in wild-type animals therefore increases the chemosensory
capacities of the animals.
What are the cellular and molecular mechanisms that lead to functional
diversification of ASEL and ASER? We have previously shown that the
lim-6 LIM homeobox gene is required to repress expression of the
ASER-specific guanyl-cyclase gene gcy-5 in ASEL. However,
lim-6 is only one of presumably many factors that establishes
asymmetry because first, lim-6 itself is already asymmetrically
expressed in ASEL but not in ASER (Hobert
et al. 1999) and second, ASEL-specific expression of the guanyl
cyclase genes gcy-6 and gcy-7 is unaffected in
lim-6 null mutants (Hobert et al.
1999). In order to elucidate the molecular mechanisms required to
restrict expression of lim-6 and the guanyl cyclase genes in an
asymmetric manner to just one of the two ASE neurons, we have undertaken a
genetic screen to uncover mutants that show symmetrization of normally
asymmetric ASE(L/R) features and report here the molecular identity of a
subset of these mutants. Our study thus provides novel insights into the as
yet poorly understood mechanisms of L/R diversification in the nervous
system.
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Results |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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We first tested whether the determination of directional asymmetry in the ASE(L/R) gustatory neurons shares mechanistic similarities with the antisymmetric gene expression pattern of str-2 in the AWC(L/R) odorsensory neurons. Mutants that were previously shown to affect antisymmetry of str-2 gene expression in AWC(L/R) fall into three broad categories.
First, mutations that disrupt axon guidance cause str-2 asymmetry
defects (Troemel et al. 1999).
This defect has been attributed to a failure of the establishment of a direct
cell–cell contact between the axons of AWCL and AWCR, which normally
meet at the dorsal midline, run in close proximity to one another, and make
reciprocal synaptic contacts with one another
(White et al. 1986). Although
making no reciprocal synaptic contact
(White et al. 1986), the axons
of ASEL and ASER also run in close proximity to one another after meeting at
the dorsal midline (D. Hall and O. Hobert, unpubl.). Disruption of these
contacts through the use of the same set of axon guidance mutants that disrupt
asymmetric str-2 expression in AWCL/R has, however, no effect on
asymmetric reporter gene expression in ASE(L/R)
(Fig. 1; Supplementary Table
1).
Second, mutations that disrupt calcium signaling and mitogen-activated
protein kinase (MAPK) signaling within AWC(L/R) cause str-2 asymmetry
defects (Troemel et al. 1999;
Sagasti et al. 2001). The same
set of signaling mutants has no effect on asymmetric reporter gene expression
in ASE(L/R) (Fig. 1;
Supplementary Table 1).
Third, antisymmetric str-2 expression in AWC(L/R) was found to be
affected in mutants that disrupt olfactory sensory processing
(Troemel et al. 1999). We
tested whether more than a dozen mutants that disrupt taste perception affect
ASE(L/R) asymmetry. With the exception of che-1 (described
following), we found this not to be the case
(Fig. 1; Supplementary Table
1).
Last, as we will describe following, we have identified mutant alleles in which ASE(L/R) asymmetry is affected, but AWC(L/R) is not (Fig. 1). Consistent with their distinct appearance (antisymmetry vs. directional asymmetry), we conclude that the mechanisms of establishment of AWC(L/R) and ASE(L/R) asymmetry are genetically separable.
Identification of genes that affect ASE(L/R) asymmetry
Besides the mutant backgrounds mentioned earlier, we tested a variety of
candidate genes for an effect on asymmetric expression of ASEL markers,
including lin-12/Notch, receptor tyrosine kinase (RTK)-,
transforming growth factor (TGF
)-, and Wnt-signaling
mutants and various transcription factors and other known patterning mutants
and did not observe any defects (Supplementary Table 1). Given the previously
reported impact of TGF-like signaling on the determination of L/R
asymmetry in vertebrate organ and brain development
(Ramsdell and Yost 1998;
Mercola and Levin 2001), the
absence of a defect in null mutants of the daf-4 gene, which codes
for the sole type II TGF-receptor protein in the C. elegans
genome (Estevez et al. 1993;
Ruvkun and Hobert 1998), is of
interest because it points to a different mechanism of regulation of L/R
asymmetry.
Using transgenic reporter strains that express green fluorescent protein (gfp) exclusively in ASEL (lim-6::gfp and gcy-7::gfp), we then conducted unbiased genetic screens for mutants that display defects in asymmetric ASE marker gene expression (see Materials and Methods). Mutants derived from these screens were examined for defects in asymmetric expression of three ASE asymmetry markers, lim-6::gfp and gcy-7::gfp for ASEL and gcy-5::gfp for ASER. Because two ASEL markers, lim-6::gfp and gcy-7::gfp, show identical behaviors in all mutant backgrounds tested, we have not included a third ASEL marker, gcy-6::gfp, in our analysis but assume that it behaves similarly to lim-6::gfp and gcy-7::gfp. Consistent with this notion, lim-6, gcy-7 and gcy-6 contain significant patches of sequence similarity in their cis-regulatory regions (data not shown).
We retrieved three classes of mutants from our screen. In class I and class II mutants, the overall identity of the ASE neurons, as assessed by cell position, axon morphology, and bilaterally symmetric gene expression profiles is unaffected. In class I mutants, however, the ASEL markers lim-6::gfp and gcy-7::gfp are expressed in both ASEL and ASER, and gcy-5::gfp expression is concomitantly lost in ASER ("two ASEL"-phenotype; see Fig. 2 for examples). In class II mutants, lim-6::gfp and gcy-7::gfp fail to be expressed in ASEL, and there is concomitant ectopic expression of gcy-5::gfp in ASEL ("two ASER"-phenotype; see Fig. 5, below, for examples). Last, in class III mutants, the ASE neurons are generated but lackthe expression of several identity-determining markers, including all three ASEL- and ASER-specific asymmetry markers as well as bilaterally symmetric markers (see Fig. 8, below, for examples; Fig. 9A, below, shows a schematic summary of all mutant phenotypes in single and double mutants). We termed mutants from the first two classes "lsy" mutants (pronounced "lousy"), for lim-6 symmetry mutant. We also noted that lsy mutants do not disrupt the L/R asymmetric placement of a specific unilateral neuron (RIS) or the L/R asymmetric migration of the Q neuroblasts (data not shown).
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Class I mutants (`two ASEL') affect the ASE(L/R)-expressed COG-1 and
UNC-37 transcription factors
The four class I lsy mutants ot28, ot38, ot62, and
ot59 define two complementation groups
(Fig. 2). Through
single-nucleotide polymorphism (SNP) mapping, complementation testing,
transformation rescue, and allele sequencing, we demonstrated that ot28,
ot38, and ot62 are allelic to cog-1 (Figs.
3A,
4). cog-1 was recently
shown to code for a homeobox gene orthologous to the vertebrate Nkx6.1 and
Nkx6.2 genes and to be involved in vulval patterning
(Palmer et al. 2002). The
previously described cog-1 alleles, sy607 and
sy275, also display a lsy phenotype
(Fig. 2B).
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In vitro binding assays revealed that the vertebrate orthologs of COG-1
interact through the conserved engrailed homolog (eh1) domain with the
transcriptional corepressor Groucho (Muhr
et al. 2001). The C. elegans ortholog of Groucho,
unc-37, maps to a chromosomal region on linkage group I
(Pflugrad et al. 1997), to
which we mapped another asymmetry mutant, ot59, with a lsy
phenotype similar to our cog-1 alleles
(Fig. 2). ot59 animals
also displayed a characteristic unc-37-like locomotory defect. We
sequenced the unc-37 gene in ot59 mutant animals and found
an early splice site mutation (Fig.
3A). The canonical allele of unc-37, e262, also shows a
class I lsy phenotype, which can be rescued through the introduction
of the wild-type unc-37 locus
(Fig. 4A).
cog-1 and unc-37 interact genetically
UNC-37 has recently been shown to interact with the engrailed homology
(eh1) domain of the UNC-4 homeodomain protein
(Winnier et al. 1999). The eh1
domain is also conserved in COG-1 (Fig.
2A). Moreover, the vertebrate orthologs of cog-1 and
unc-37, Nkx6.1 and Grg4/Groucho, directly interact in vitro through
the eh1 domain (Fig. 2A;
Muhr et al. 2001), suggesting
that COG-1 and UNC-37 may also directly interact to affect ASE asymmetry. To
corroborate this notion, we examined a potential genetic interaction between
cog-1 and unc-37. Lowering the dose of either cog-1
or unc-37 through placing a wild-type copy of the respective gene
over a hypomorphic allele has no effect on asymmetry (0 out of 115
cog-1(ot28)/+ animals show ectopic gcy-7::gfp
expression in ASER; 0/84 unc-37(e262)/+ animals show defects). If,
however, the dosage of both genes is simultaneously reduced in a
transheterozygous state, a significant asymmetry defect becomes obvious [26/95
unc-37(e262)/+; +/cog-1(ot28) animals show ectopic
gcy-7::gfp expression in ASER]. Later we report that
cog-1 and unc-37 act in a similar cell to affect asymmetry,
thus leading us to conclude that, like their vertebrate orthologs, COG-1 and
UNC-37 are likely to physically associate to regulate asymmetric patterns of
gene expression.
unc-37 and cog-1 expression in the ASE neurons
Consistent with the ubiquitous expression of its vertebrate and fly
orthologs, unc-37/Groucho was previously reported to be broadly
expressed (Pflugrad et al.
1997); however, its expression was not specifically examined in
individual head neurons. We therefore examined the expression of
unc-37 in ASE(L/R) through the use of an UNC-37::GFP translational
reporter (Kelly et al. 1997),
which we found to rescue the asymmetry defects of unc-37 mutants
(Figs. 3B,
4A). A red fluorescent protein
rfp-expressing transgene, otIs131, which is exclusively
expressed in larval ASE(L/R) neurons, shows cofluorescence with the
unc-37::gfp reporter signal
(Fig. 3B), thus demonstrating
unc-37 expression in ASEL and ASER.
A cog-1::gfp reporter gene fusion was previously reported
to be expressed in three classes of head sensory neurons, including ASEL and
ASER (Palmer et al. 2002).
Using previously described reporter lines as well as newly constructed
reporter lines, we made the intriguing observation that all transgenic lines
tested show significantly higher levels of cog-1 expression in ASER
versus ASEL (Fig. 3B,C). Higher
levels of expression of cog-1 can be observed with a rescuing
cog-1::gfp reporter gene fusion, which contains the complete
coding region of cog-1, as well as with reporter gene fusions in
which only the promoter of cog-1 is fused to gfp
(Fig. 3C). This observation
indicates that differential expression of cog-1 in ASER versus ASEL
is mediated via the transcriptional level.
cog-1 and unc-37 act in ASER and through lim-6
to affect gcy-5 expression
We first focused on the effects of cog-1 and unc-37 mutations in ASER, which loses gcy-5 expression (ASER marker) and gains lim-6 expression (ASEL marker) in the respective mutants. First, we tested whether cog-1 and unc-37 function is cell autonomous. We found that cDNAs of unc-37 and cog-1 driven by a postmitotic, ASER-specific transcriptional regulatory element derived from the gcy-5 locus rescue the respective mutant phenotypes (Fig. 4A), indicating that these two genes may act postmitotically in ASER to repress ASEL-specific features.
Second, we examined whether the effect of cog-1 and
unc-37 on asymmetric gcy-5 expression is mediated through
the regulation of the homeobox gene lim-6. lim-6, which is normally
expressed exclusively in ASEL, is required to repress gcy-5
expression in ASEL (Hobert et al.
1999); in cog-1 and unc-37 mutants,
lim-6 is ectopically expressed in ASER, and gcy-5 expression
is concomitantly lost in ASER. To ask whether this loss of gcy-5
expression is due to ectopic lim-6 expression, we eliminated
lim-6 in an unc-37 mutant and in a cog-1 mutant
background. We find that in both cog-1; lim-6 and unc-37;
lim-6 double mutants, gcy-5 expression in ASER reappears
(Fig. 4B). Hence, in wild-type
animals, cog-1 and unc-37 allow gcy-5 to be
expressed through inhibiting the expression of the gcy-5 repressor
lim-6. The lack of a perfect correlation between gain of
lim-6 expression (e.g., 53% of unc-37 mutant animals gain
lim-6 in ASER, but only 32% concomitantly lose gcy-5
expression; Fig. 2B) may be a
reflection of a need for a threshold level of ectopic lim-6
expression to achieve gcy-5 repression; this level may not be reached
in all animals.
After having shown that lim-6 is required to repress gcy-5 expression, we next tested whether lim-6 alone is sufficient to repress gcy-5 expression. To this end, we expressed lim-6 in both ASEL and ASER (and in all other neurons of the nervous system, using the unc-119 promoter; see Materials and Methods) in a lim-6 null mutant background. We found that, in these transgenic animals, the loss of gcy-5 repression in ASEL is rescued, confirming that this promoter produces sufficient levels of a functional LIM-6 protein (Fig. 4C). However, in none of the animals in which the repression of gcy-5 in ASEL is reestablished can we observe a concomitant repression of gcy-5 expression in ASER. The requirement but lack of sufficiency for lim-6 function suggests that the ectopic lim-6 expression observed in cog-1 and unc-37 mutants is necessary, but not sufficient, to repress gcy-5 expression. Other factors must similarly be derepressed in ASER in cog-1 and unc-37 mutants to allow lim-6 to repress gcy-5 expression.
Overexpression of cog-1 can convert ASEL to
ASER
We next asked whether raising the levels of cog-1 in ASEL may be sufficient to repress ASEL features (lim-6) and allow ASER features (gcy-5) to appear. We used two approaches: First, we generated multicopy arrays of the cog-1 locus and second, we expressed a cog-1 cDNA under control of the gcy-7 promoter, which is active in ASEL and ASER embryonically and becomes restricted to ASEL postembryonically. We found that transgenic wild-type animals expressing either of these two constructs show repression of lim-6 expression in ASEL and a concomitant gain of the normally ASER-specific marker gcy-5 expression in ASEL (Fig. 4D).
In conclusion, cog-1 activity appears to be tightly regulated in ASER versus ASEL. In wild-type animals, cog-1 is present in ASEL and ASER, but effective as a repressor of lim-6 and gcy-7 only in ASER, possibly because of higher levels of expression. Raising the activity of cog-1 in ASEL through overexpression reveals that cog-1 can act as a repressor in ASEL as well. Following we describe a set of transcription factors that are intimately tied to the differential activity of COG-1/UNC-37.
Class II mutants (`two ASER') affect the LIN-49 and CEH-36
transcription factors
In order to better understand how cog-1 and unc-37 affect asymmetric lim-6 expression, we molecularly characterized class II asymmetry mutants derived from our screen. Those mutants display an opposite phenotype to cog-1 and unc-37; that is, they display a "two ASER" phenotype (Fig. 5A,B). Bilaterally symmetric features of ASE(L/R), such as the expression of the flp-6 gene, are unaffected in class II mutants (data not shown), suggesting that these mutants specifically disrupt the asymmetric gene expression programs. We will first describe two of these class II genes and in the next section we will describe their genetic interactions with cog-1 and unc-37.
lin-49: Through SNP mapping, complementation testing, allele
sequencing, and transformation rescue, we found that the three recessive
ot69, ot74, and ot78 alleles are mutations in the
lin-49 gene, which codes for a predicted nuclear protein with two
plant homeodomain (PHD)-finger domains and a bromodomain
(Chamberlin and Thomas 2000).
The PHD-finger domains, found in many chromatin-associated proteins
(Aasland et al. 1995), and the
bromodomain, an acetyllysine-binding domain
(Dyson et al. 2001), indicate
that the LIN-49 protein may be a general transcriptional cofactor involved in
chromatin remodeling. Consistent with this notion, lin-49 was
reported to be broadly expressed and shown to affect the development of
several tissue types including the gut, the egg-laying system, and male mating
structures (Chamberlin and Thomas
2000). We corroborated the broad and possibly ubiquitous
expression of lin-49 using a gfp fusion construct that
rescues the mutant phenotype (Fig.
6C).
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ceh-36: Through SNP mapping, allele sequencing, and transformation
rescue, we found that ot79 is an allele of the previously
uncharacterized ceh-36 gene, one of three orthodenticle-type homeobox
genes predicted in the C. elegans genome
(Fig. 6A,B;
Ruvkun and Hobert 1998). The
ot79 allele is completely recessive but unlikely to be a null allele
because the truncation introduced by the premature stop codon still leaves the
homeodomain intact (Fig. 6A). A
ceh-36::rfp fusion construct that is capable of rescuing the
mutant phenotype (Fig. 6C), as
well as a fusion of the ceh-36 upstream regulatory region to
gfp, showed exclusive expression in two pairs of head sensory neurons
in postembryonic animals, one being ASE(L/R)
(Fig. 6D; A. Lanjuin and P.
Sengupta, pers. comm.).
Because both lin-49 and ceh-36 mutants have similar effects on lim-6 and gcy gene expression, we considered whether they may do so in a sequential manner through activating each other's expression. Since we consider it highly unlikely that a broadly expressed transcriptional cofactor is under control of the 2-neuron-specific transcription factor ceh-36, we only tested the possibility that ceh-36 expression may be regulated by lin-49. We found that a ceh-36::gfp reporter construct is normally expressed in lin-49 null mutants; it is also unaffected in cog-1 and unc-37 mutants (data not shown). Following we present genetic epistasis data that indicate that ceh-36 and lin-49 show very similar patterns of interaction with other transcription factors, suggesting that CEH-36 and LIN-49 may act together, possibly in a complex analogous to COG-1 and UNC-37.
The cog-1/unc-37 repressor complex antagonizes the
ceh-36/lin-49-mediated activation of lim-6 expression
We have shown that lin-49 and ceh-36 are required for lim-6 and gcy-7 expression in ASEL. Yet ceh-36 (and the ubiquitously expressed lin-49 gene) is expressed in both ASEL and ASER. What prevents ceh-36 and lin-49 from activating lim-6 expression in ASER? One possibility is that their activity is antagonized in ASER by the transcriptional repressors cog-1 and unc-37. We tested this hypothesis by asking whether lowering the activity of cog-1 and unc-37 now allows lin-49 and ceh-36 to activate lim-6 expression in ASER. To this end, we constructed a variety of double mutant animals. We find that ectopic expression of lim-6 in ASER in cog-1 and unc-37 mutants requires the activity of both lin-49 and ceh-36 because, in either double mutant combination, ectopic lim-6 expression in ASER is diminished (Fig. 7A). The model of an antagonism between cog-1/unc-37 and ceh-36/lin-49 is further corroborated by two experiments. First, as described earlier, raising the levels of cog-1 in ASEL (which normally expresses lower levels of cog-1 than does ASER) counteracts the normal activation of lim-6 by ceh-36 and lin-49 in ASEL and hence leads to a loss of lim-6 expression (Fig. 4D; see also model in Fig. 7C). Second, we find that reduction of ceh-36 or lin-49 activity does not merely lead to a loss of transcriptional activation of lim-6 in ASEL but, to the contrary, leads to a cog-1-dependent repression of lim-6. This is because, if we lower cog-1 activity in a ceh-36 or lin-49 hypomorphic background, lim-6 expression in ASEL is at least partially, if not completely, restored (Fig. 7B,C; a concomitant repression of gcy-5 in ASER that correlates with restored lim-6 expression can also be observed; data not shown). This experiment corroborates our observation of low levels of cog-1 expression in ASEL, whose repressive activity in wild-type animals seems to be antagonized by ceh-36 and lin-49 activity; lowering of ceh-36 and lin-49 activity hence allows cog-1 to repress lim-6.
Also consistent with the notion of cog-1/unc-37 and ceh-36/lin-49 acting in parallel rather than sequentially, we find that L/R asymmetric expression of a cog-1 reporter gene construct is unaffected in ceh-36 and lin-49 mutants and that ceh-36 reporter gene expression in a cog-1 and unc-37 mutant background is unaffected (data not shown).
One way to fit this genetic data into a molecular model is to suppose that a COG-1/UNC-37 repressor complex competes with CEH-36 and LIN-49 for control over the activity of the lim-6 and gcy-7 promoters (or the promoter of an intermediary factor that positively regulates these factors). In ASER, the COG-1/UNC-37 complex prevails over CEH-36 and LIN-49; loss of the COG-1/UNC-37 complex in ASER hence reveals the activity of CEH-36 and LIN-49. In ASEL, however, CEH-36 and LIN-49 prevail over the COG-1/UNC-37 complex, likely because of the lower expression of COG-1 in ASEL; the loss of CEH-36 and LIN-49 hence reveals the activity of COG-1/UNC-37 (Figs. 7C, 9).
Although ceh-36 and lin-49 show no mutant phenotype in the transheterozygous state (data not shown), we hypothesize that both proteins act in a common transcriptional activation complex because both genes behave indistinguishably in genetic interaction tests, loss of either causes the same effects on the expression of downstream genes, they are both required for ASEL-marker expression in ASER in the absence of cog-1 or unc-37, and their reduction of activity in ASER is compensated by removal of cog-1. The domain structures of the two proteins are also supportive of a role for these proteins in a single transcriptional activation complex in which CEH-36 may provide the DNA binding specificity and LIN-49 the connection to the chromatin remodeling activities required for gene activation.
che-1 is a positive regulator of several ASEL and ASER
features
We have described earlier a set of genes that act as positive regulators of
gcy-7 and lim-6 in ASEL, with the latter factor being a
repressor of the ASER marker gcy-5. But what factor is required for
the activation of gcy-5 expression in ASER? In cog-1 and
unc-37/groucho mutants, gcy-5 expression in ASER is lost.
Groucho has previously been shown to be able to convert transcriptional
activators, such as Runt or Dorsal, into repressors
(Fisher and Caudy 1998). It
could have thus been envisioned that cog-1 is an
unc-37/groucho-independent activator of gcy-5 expression and
an unc-37/groucho-dependent repressor of lim-6. However, our
genetic interaction data show that this is not the case, because we can
restore gcy-5 expression in cog-1 mutants if we remove the
lim-6 gene. cog-1 thus "activates"
gcy-5 expression through repressing the gcy-5 repressor
lim-6.
Activation of gene expression through counteracting repression
automatically invokes the existence of a basal transcriptional activation
mechanism. We considered it possible that class III mutants that we have
retrieved from our screen may shed light on the issue of transcriptional
activation of ASER features. In all class III mutants gcy-5
expression is lost (Fig. 8B).
Because lim-6 expression is also lost in these mutants
(Fig. 8B), the loss of
gcy-5 expression cannot be due to ectopic expression of
lim-6 in ASER. We hence decided to undertake a molecular
characterization of class III mutants, which may define direct or indirect
activators of gcy-5 expression. We found that all class III mutants
fall into a single complementation group that is allelic to the che-1
locus (Fig. 8A). che-1
was recently shown to be a Zn-fingercontaining transcription factor related to
the Drosophila Glass transcriptional activator
(Uchida et al. 2003). We found
that in che-1 mutants not only are ASER markers lost, but also the
ASEL markers lim-6 and gcy-7
(Fig. 8B). We furthermore
examined expression of the ASE(L/R)-expressed cog-1 and
ceh-36 genes in che-1 mutants and found a complete loss of
their expression as well (data not shown). Another symmetric ASE(L/R) cell
fate marker, flp-6, also fails to be expressed in che-1
mutants (Fig. 8A,B). The loss
of expression of all of these genes is not a reflection of a failure of the
neuron to be generated or to adopt a neuronal fate, because a pan-neuronal
gfp marker is still expressed in ASE(L/R) and because the anatomy of
ASE is only partially affected in che-1 mutants
(Lewis and Hodgkin 1977;
Uchida et al. 2003). We
propose that che-1 is a permissive transcriptional regulator, located
at the top and at the lower levels of a transcriptional regulatory cascade
required to initiate asymmetric profiles of gene expression in ASEL and
ASER.
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). The genetic amenability of C. elegans enabled us
to identify a set of proteins involved in determining neuronal L/R patterning,
most of which are highly conserved across phylogeny. In C. elegans,
these factors serve to eventually segregate the expression of putative sensory
receptors encoded by the gcy genes into otherwise largely bilaterally
symmetric types of cells. The separation of distinct sensory capacities into
two separate, yet bilaterally symmetric cells, each of which has a similar set
of downstream synaptic partners (White et
al. 1986), is a prerequisite for the capacity of worms to
appropriately discriminate between distinct sensory inputs and hence to
navigate in a complex sensory environment
(Pierce-Shimomura et al.
2001). The increase in the discriminatory sensory potential
through asymmetric chemoreceptor gene expression is not only a feature of the
gustatory ASEL and ASER neurons, but also of the olfactory AWCL and AWCR
neurons (Wes and Bargmann
2001). Despite their similar purpose, our genetic analysis points
to the distinctiveness of the molecular mechanisms of establishment of AWC and
ASE asymmetries. Although in the former case, cell–cell contact,
calcium, and MAPK signaling are required to establish antisymmetric gene
expression pattern (Troemel et al.
1999; Sagasti et al.
2001), none of these events is required to establish directional
ASE asymmetry.
A molecular model that sheds some light onto the L/R asymmetric segregation
of chemosensory receptor expression has emerged from our genetic studies and
can be summarized as follows (Fig.
9). The Zn finger transcription factor CHE-1 acts at the top of
the regulatory hierarchy to determine subtype-specific and bilateral-symmetric
features of ASE, including the expression of terminal differentiation markers
such as the neuropeptide gene flp-6, cyclic nucleotide ion channels,
and orphan serpentine receptors (Uchida et
al. 2003; this paper). CHE-1 also triggers either directly or
indirectly the expression of factors that are required to determine the L/R
asymmetric state of ASEL and ASER, including cog-1, ceh-36, and
lim-6. These factors serve to modulate what we consider an
"ASER default state," which is defined by the expression of
gcy-5. This default state is also revealed through laser ablation
studies, demonstrating that the removal of putative signaling cells causes
ASEL to convert to the ASER state (R.J. Johnson Jr. and O. Hobert, unpubl.);
features of this default state, such as gcy-5 expression, may be
directly induced by CHE-1. After CHE-1 has induced CEH-36 and COG-1
expression, a putative CEH-36/LIN-49 complex activates expression of at least
two factors, a gcy gene (gcy-7) that defines ASEL properties
and a transcription factor (lim-6) that prevents the ASER-specific
gcy-5 gene from being expressed
(Fig. 9). Although the
bilaterally symmetrically expressed CEH-36/LIN-49 proteins (which may in
analogy to COG-1 and UNC-37 be acting in a complex) are capable of inducing
lim-6 and gcy-7 expression in both ASEL and ASER, they are
prevented from doing so in ASER through the activity of the COG-1/UNC-37
repressor complex, which thus helps preserve the ASER default state
(Fig. 9). The activity of
COG-1/UNC-37 antagonizes CEH-36/LIN-49 activity only in ASER because of higher
levels of COG-1 protein in ASER. Consequently, raising the levels of COG-1 in
ASEL can overcome the CEH-36/LIN-49-mediated induction of ASEL cell fate,
causing the adoption of ASER fate.
Our studies have revealed two basic transcriptional regulatory principles
in the novel context of L/R asymmetry determination. First, we have shown that
the expression of ASER fate (gcy-5 expression) is mediated through a
series of repressive transcriptional interactions; that is, COG-1 and UNC-37
repress lim-6 expression, which represses gcy-5 expression.
This transcriptional regulatory principle is reminiscent of the sequential
repressor scheme in the creation of cellular diversity in the vertebrate
spinal cord, which, intriguingly, involves the human orthologs of COG-1, the
Nkx6 proteins, and of unc-37, the Grg proteins
(Muhr et al. 2001). We have
provided further insight into the sequential repressor model by demonstrating
that a second transcriptional regulatory mechanism is at workin ASEL and ASER,
namely, a tightly balanced antagonistic relationship between a repressor
(COG-1/UNC-37) and a putative activator (CEH-36/LIN-49) complex. We do not
know at this point whether these two protein complexes functionally interact
on the lim-6 and gcy-7 promoter level, or whether they act
via intermediary factors. Under the assumption that they workon a common
target promoter, we can envision two molecular models for the mechanistic
basis of the antagonism. COG-1/UNC-37 and CEH-36/LIN-49 may directly compete
for a single binding site on their target gene promoter, or, alternatively,
they may occupy distinct sites on the promoter; different levels of COG-1 may
be read out as a differential binding site occupancy. We favor the latter
model because a preliminary mutational analysis of the gcy-7 promoter
identified two sites required for transcriptional activation in ASEL and a
separate site required for repression in ASER (S. Chang and O. Hobert,
unpubl.).
Our studies suggest that it is the differential levels of cog-1
activity in ASER versus ASEL in wild-type animals that are responsible for the
cellular diversification of ASEL and ASER fate. Moreover, our reporter gene
studies indicate that L/R differential activity of cog-1 is conferred
by differential transcription of cog-1 in ASER versus ASEL. Either a
repressor acts in ASEL to lower the levels of cog-1 transcription in
ASEL (e.g., through antagonizing CHE-1 mediated activation of cog-1
transcription) or, alternatively, an activator acts in ASER to increase
cog-1 levels (e.g., through assisting CHE-1-depending activation of
cog-1). The identification of the upstream regulator of
cog-1 expression will represent a further step toward understanding
the mechanism that is responsible for setting up asymmetric patterns of gene
expression. This mechanism may rely on intrinsically programmed lineage cues
and hence be a consequence of the initially highly asymmetric embryo
(Schnabel and Priess 1997).
Alternatively, asymmetry may be nonautonomously determined through an
"asymmetry-inducing" signal that acts past the stage of early
embryonic inductions, a notion that we find supported by preliminary cell
ablation data (R.J. Johnston Jr. and O. Hobert, unpubl.). Our candidate gene
approach has ruled out several canonical signaling molecules as determinants
in this event, including TGF-type signaling, found to be involved in
vertebrate L/R signaling (Mercola and
Levin 2001). We expect that the further molecular characterization
of as yet uncloned lsy mutants will provide us with a better
understanding of the development of L/R asymmetry in the nervous system.
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All strains were grown at 20°C and scored at room temperature as gravid adults if not otherwise indicated.
otIs6 and otIs114: Is[lim-6prom::gfp;
rol-6(d)]. Both integrants derive from a previously described
extrachromosomal line (Hobert et al.
1999). In contrast to previous nomenclature
(Hobert et al. 1999), we will
for the sake of simplicity from here on refer to
"lim-6prom::gfp" as
"lim-6::gfp."
otIs3: Is[gcy-7::gfp; lin-15(+)]. In
mid-embryogenesis, otIs3 is initially expressed in both ASEL and
ASER; expression becomes restricted to ASEL in late embryogenesis. This
integrant derives from an extrachromosomal line described in Yu et al.
(1997).
otIs125: Is[flp-6::gfp]. The extrachromosomal line from which this integrant was derived was a gift from C. Li.
otIs131: Is[gcy-7::rfp; rol-6(d)], derived from otEx445 (see following).
ntIs1: Is[gcy-5::gfp; lin-15(+)] [a gift from S.
Lockery; derived from an extrachromosomal line described in Yu et al.
(1997)]. Expression of
gfp is turned on exclusively in ASER in embryogenesis after the
generation of ASER.
kyIs140: Is[str-2::gfp; lin-15(+)]
(Troemel et al. 1999), a
5' fusion (i.e., containing only regulatory sequences upstream of the
start codon).
otEx445: Ex[gcy-7::rfp; rol-6(d)], a 5' fusion. Because of the delayed maturation of the RFP protein (compared with the GFP protein expressed from the otIs3 transgene mentioned earlier), GCY-7::RFP expression from this array can be observed in both ASEL and ASER until midlarval stages and becomes restricted to ASEL thereafter.
otEx862: Ex[ceh-36prom::gfp; rol-6(d)], a 5' fusion.
otEx1030, otEx1031: Ex[ceh-36::rfp; rol-6(d)], a translational fusion (i.e., containing upstream regulatory sequences as well as all exons and introns of the locus).
otEx863: Ex[lin-49prom::gfp; rol-6(d)], a 5' fusion.
otEx1025–1029: Ex[lin-49::gfp; rol-6(d)], a translational fusion.
syIs73: Is[pBP164 {cog-1prom::gfp; dpy-20(+)}],
a 5' fusion (Palmer et al.
2002).
syIs63: Is[cog-1::gfp; dpy-20(+)]
(Palmer et al. 2002), a
translational fusion.
otEx990: Ex[cog-1prom::gfp; rol-6(d)], a 5' fusion.
otEx1069–1071: Ex[cog-1prom::gfp], a 5' fusion.
otEx1006–1007: Ex[pBP159 (cog-1::gfp); rol-6(d)], a translational fusion.
otEx1066–1068: Ex[pBP159 (cog-1::gfp); unc-122::gfp], a translational fusion.
PD8100: Ex[unc-37::gfp; rol-6]
(Kelly et al. 1997).
Mutant screen for lsy genes
otIs6 or otIs3 animals were mutagenized with ethyl
methanesulfonate (EMS) using standard procedures
(Brenner 1974). The progeny of
individually picked F1 animals derived from the mutagenized P0 population were
analyzed under a dissecting scope equipped with a fluorescent light source.
Using otIs6 as a marker, we screened through 3200 haploid genomes,
and using otIs3, we screened through 12,200 haploid genomes. A total
of 28 mutants were retrieved. Through chromosomal linkage and complementation
testing, we found these mutants to define 11 complementation groups; one
mutant has not yet been assigned to a group. Representatives from each
complementation group were backcrossed and transferred into several different
gfp transgenic backgrounds that assess gcy-5, gcy-7, and
lim-6 expression (otIs6, otIs3, otIs114, ntIs1).
SNP-based mapping
We made use of SNPs present in the Hawaiian C. elegans isolate
CB4856 identified by the Washington University Genome Sequencing Center and by
Ronald Plasterkand colleagues (Wicks et
al. 2001). The respective mutant strain was crossed with CB4856
and resultant mutant F2 progeny were singled out. Their progeny were lysed
using standard procedures, genomic fragments that included the SNP were
amplified by PCR, and the SNP was analyzed by RFLP. In order to facilitate the
identification of recombinants, mutant animals were in some cases marked with
a visible marker and mutant recombinants that lost the visible marker were
chosen for SNP analysis. che-1, cog-1, and lin-49 were
identified as lsy genes after initial SNP mapping of the lsy
alleles to chromosomal intervals that contained these genes (cog-1,
LGII, between K10H10 and Y53F4B; lin-49, LGIV, between cosmid D2096
and ZK596) and subsequent complementation testing and allele sequencing.
Positional cloning of ceh-36(ot79)
Of all transcription factors described in this paper, ceh-36 is the only one for which no mutant allele was previously reported. On mapping of the lsy mutants retrieved from our mutant screen, we noted that ot79 was linked tightly to a canonical linkage SNP on the right arm of chromosome X at 16.01 m.u. (R03E1; 0/92 recombinants from a cross with CB4856). Left- and right-hand boundaries were established at 15.42 m.u. (C33G3; 1/92 recombinants) and 17.28 m.u. (F23D12; 1/92 recombinants), respectively. This region includes 60 predicted protein-coding genes. Because at that point we had shown that numerous transcription factors play a role in asymmetry, we sequenced all four predicted transcription factors in this region (F28H6.2, C37E2.4, C37E2.5, K04C1.3) and found a mutation in the coding region of one of them, ceh-36/C37E2.4.
DNA constructs
Reporter gene constructs: gcy-7::rfp was constructed by
replacing gfp in the gcy-7::gfp construct with
dsRed2 (Clontech). gcy-7::gfp contained 1.3 kb of the
5' upstream regulatory region of the gcy-7 gene
(Yu et al. 1997). A
cog-1 transcriptional reporter was constructed by PCR fusion
(Hobert 2002) using 4.6 kb of
genomic region upstream of the ATG. A cog-1-rescuing gfp
plasmid (pBP159) was kindly provided by Takao Inoue and Paul Sternberg
[described by Palmer et al.
(2002)]. A lin-49
translational gfp reporter was constructed by PCR fusion
(Hobert 2002), including 2 kb
of 5' region (up to the preceding gene) and all exons and introns of
lin-49. A ceh-36 translational rfp reporter was
constructed by including 5.1 kb of 5' region and all exons and introns
of ceh-36; the rfp coding region derived from the dsRed2
(Clontech, Inc.) vector. The marker for all injections was
pRF4/rol-6(d) at either 50 or 100 ng/µL. The unc-37
translational gfp reporter was reported by Kelly et al.
(1997).
Heterologous expression constructs: Three promoters were used for
heterologous expression, unc-119
(Maduro and Pilgrim 1995),
gcy-5, and gcy-7 (Yu et
al. 1997). The latter two promoters had to be recloned; for
gcy-5, 3.1 kb of the 5' upstream regulatory region of the
gcy-5 gene were amplified from N2 genomic DNA using primers
containing BamHI sites at either end and subcloned into the
gfp vector pPD95.75 (a gift from A. Fire) to create
gcy-5::gfp; for gcy-7, 1.3 kb of the 5'
upstream regulatory region of the gcy-7 gene were amplified from N2
genomic DNA using primers with PstI/BamHI sites at either
end. The amplicon was subcloned into the gfp vector pPD95.75 to yield
gcy-7::gfp. A shorter deletion derivative,
gcy-7Hind::gfp, was constructed by releasing a
HindIII fragment from gcy-7::gfp, which left 188 bp
of the gcy-7 promoter, which still yielded completely penetrant ASEL
expression in adults. The gcy-5::cog-1 and
gcy-5::unc-37 rescuing plasmids were constructed by
releasing gfp from gcy-5::gfp and replacing it with
the cog-1a and unc-37 cDNAs. The unc-37 cDNA was
provided by David Miller (Pflugrad et al.
1997); the cog-1a cDNA was obtained by PCR from an
incomplete EST clone, providing the missing 5' sequence in the primer
sequence. The gcy-7::cog-1 expression plasmid was
constructed by replacing gfp from gcy-7Hind::gfp
with a cog-1a cDNA. unc-119::lim-6 was constructed
by amplifying the genomic lim-6 region from the start to the stop
codon and subcloning it into the pBY103 vector
(Maduro and Pilgrim 1995).
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