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First published online 3 August 2006
doi: 10.1242/dev.02494
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1 Howard Hughes Medical Institute, Department of Biochemistry and Molecular
Biophysics, Columbia University Medical Center, 701 W. 168th Street, New York,
NY 10032, USA.
2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
Ontario K1H 8M5, Canada.
3 Howard Hughes Medical Institute, Department of Biochemistry and Molecular
Biophysics, Center for Computational Biology and Bioinformatics, Columbia
University Medical Center, 1130 St Nicholas Avenue, Room 815, New York, NY
10032, USA.
4 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY
14853, USA.
* Author for correspondence (e-mail: or38{at}columbia.edu)
Accepted 13 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Caenorhabditis elegans, Left/right asymmetry, Neuronal cell fate, Zn-finger transcription factor, FH2 domain
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The mechanisms controlling the gene expression profile of a
single cell are sometimes surprisingly complex. Feedback, feed-forward and
multi-tier parallel pathways are required to attain, stabilize and maintain
the complete signature of a cell-specific program of gene expression. Though
these types of regulatory networks have been rigorously described in single
cell organisms (e.g. Lee et al.,
2002; Shen-Orr et al.,
2002), a thorough analysis of complex regulatory networks in
multicellular organisms has only been described in few isolated examples
(Davidson, 2001). In complex
metazoan organisms, the functional analysis of gene regulatory networks are
often complicated by various factors, including the pleiotropies caused by
mutations in regulatory genes and the unavailability of a sufficient number of
molecular markers that define cellular fates on a single cell level.
To address the mechanisms controlling single cell-specific gene expression
programs, we have characterized the neuronal subclass diversification process
executed by the ASE class of gustatory neurons in C. elegans. This
neuron class consists of a pair of two bilaterally symmetric neurons, ASE left
(ASEL) and ASE right (ASER) (Fig.
1A). ASEL is the primary sodium sensor, whereas ASER is the
primary Cl- and K+ sensor
(Pierce-Shimomura et al.,
2001). The left/right asymmetric (`lateral') separation of
chemosensory capacities endows the worms with the ability to discriminate
between distinct chemosensory cues and thereby widens its chemosensory
repertoire. Other than these functional differences, the ASE pair of neurons
are bilaterally symmetric by anatomical criteria such as cell body position,
axodendritic structure and synaptic connectivity
(White et al., 1986).
ASEL and ASER subclass diversification is defined by an array of left/right
asymmetrically expressed cell fate markers including specific subsets of
guanylyl cyclase receptors, encoded by GCY genes, and FMRFamide-type
neuropeptides, encoded by FLP genes, (Fig.
1A) (Johnston et al.,
2005; Ortiz et al.,
2006; Yu et al.,
1997). We have previously shown that a network of micro RNAs
(miRNAs) and transcription factors controls ASEL/R cell fate diversification
(Fig. 1A)
(Chang et al., 2004;
Chang et al., 2003;
Hobert et al., 1999;
Johnston and Hobert, 2003;
Johnston et al., 2005;
Johnston and Hobert, 2005). A
bi-stable feedback loop constituted by these regulatory factors is an
essential network motif required for the establishment and stabilization of
lateral ASE fate. In this loop, ASEL-specific inducer genes, such as the
lsy-6 miRNA and die-1 transcription factor, activate
expression of other ASEL-specific inducer and effector genes and repress
ASER-specific inducer and terminal genes. By contrast, ASER-specific inducer
genes such as the mir-273 miRNA and cog-1 transcription
factor control expression of ASER-specific genes and repression of
ASEL-specific genes. Genetic experiments indicate that lsy-6 and its
upstream activator, the Zn-finger transcription factor lsy-2,
provides the input into the loop, whereas the output of the loop is provided
by the Zn-finger transcription factor die-1
(Johnston et al., 2005;
Johnston and Hobert, 2005)
(Fig. 1A).
In this paper, we describe the cloning and characterization of fozi-1, a novel protein containing two Zn fingers and a single FH2 domain. This gene plays a role downstream of the bi-stable feedback loop to repress expression of ASEL-specific effector genes in ASER. We anticipate that the complex regulatory network described here will provide a paradigm for subtype fate specification in other cellular contexts.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Chang et al.,
2003; Hobert et al.,
1999; Hodgkin and Doniach,
1997; Johnston and Hobert,
2003; Johnston et al.,
2005; Simmer et al.,
2002).
List of new transgenes
All contain the elt-2::gfp
(Fukushige et al., 1998) as
injection marker and are named as follows: otEx2179-2182, Ex[fozi-1
genomic; elt-2::gfp]; otEx2190-2193, Ex[fozi-1::gfp;
elt-2::gfp]; otEx2512-2515, Ex[gcy-5prom::fozi-1cDNA::gfp;
elt-2::gfp]; otEx2523-2526,
Ex[gcy-5prom::fozi-1cDNA
Zn-finger::gfp;
elt-2::gfp]; otEx2516, 2518, 2520,
Ex[gcy-5prom::fozi-1cDNAFH2::gfp; elt-2::gfp];
and otEx2534, Ex[ceh-36prom::fozi-1cDNA::gfp;
elt-2::gfp].
DNA for transgenic lines
The genomic fozi-1 locus was amplified from N2 genomic DNA using
the primers 5'-CACCCCAAGATGGTAGTAATCC and
5'-GAAGAACTGGACAATTCGG.
fozi-1::gfp
fozi-1::gfp was generated by PCR fusion
(Hobert, 2002). The first PCRs
were carried out on genomic DNA with 5'-GGAGTGGACGATGACATTGTG and
5'-AGTCGACCTGCAGGCATGCAAGCTAGGAGACGAGACATTGATGTG and on the gfp
sequence in pP95.75 with
5'-CACATCAATGTCTCGTCTCCTAGCTTGCATGCCTGCAGGTCGACT and primer D
(Hobert, 2002). The fusion PCR
was carried out with the non-nested primer 5'-GGAGTGGACGATGACATTGTG and
primer D* (Hobert,
2002).
gcy-5prom::fozi-1cDNA::gfp and ceh-36prom::fozi-1cDNA::gfp
The fozi-1-coding sequence from start to stop codon was PCR
amplified from the EST clone yk288g3 with the primers
5'-TTGGATCCATGATGCTTGCATCATCAGCG and
5'-AATGGCCAAGGAGACGAGACATTGATGTG. The amplicon was cloned into
gcy-5prom::gfp and ceh-36prom::gfp
vector constructs that will be described elsewhere in detail.
gcy-5prom::fozi-1cDNAZn-finger::gfp
The construct gcy-5prom::fozi-1cDNA::gfp was mutated by
standard mutagenesis using the primers
5'-CTATCCCTGTACATTTCAATATgGATTAGCGGGTGACCCGATCAG and
5'-CTGATCGGGTCACCCGCTAATCcATATTGAAATGTACAGGGATAG.
gcy-5prom::fozi-1cDNAFH2::gfp
Truncated fozi-1 was amplified from the fozi-1 EST clone
yk288g3 using the primers 5'-ttggatccATGATGCTTGCATCATCAGCG and
5'-aatggccaAATTGGCTGAATCGGAATTATAGATGATGACAAGG. The resulting amplicon
was subcloned into the gcy-5prom::gfp vector
construct.
Genetics
The genetic screen from which ot61 was isolated has been described
before (Johnston et al.,
2005). Briefly, animals carrying the otIs3 transgene,
which express gcy-7prom::gfp exclusively in ASEL, were
mutagenized with EMS. F1 progeny were singled and F2 progeny were scored under
a Zeiss SV6 fluorescent dissecting microscope. Mutant mapping was carried out
using a combination of single nucleotide polymorphisms from the Hawaiian
wild-type isolate CB4856 (Hodgkin and
Doniach, 1997) and three-factor mapping with the visible markers
dpy-17 and unc-49.
Biochemical analysis of the FOZI-1 FH2 domain
The cDNA encoding the FOZI-1 FH2 domain (residues 367-733) was subcloned by
standard techniques into pGEX-6P2 and expressed as a GST fusion protein in
E. coli strain BL21. FH2FOZI-1 protein was purified by
cleavage from the GST moiety (Copeland et
al., 2004). Protein purity was assessed by Coomassie Blue staining
of samples subjected to SDS-PAGE, concentration was determined by Bradford and
by OD280. The isolated FH2 domain (0.4 µM) was added to G-actin (4 µM,
5% pyrene-actin) for in vitro actin polymerization assays (Cytoskeleton)
according to the supplied protocol. For cross-linking, the purified
FH2FOZI-1 domain (20 nM) was incubated at room temperature for 60
minutes in HEK buffer with 30 µM of the crosslinker
Bis-maleimidohexane (Pierce) dissolved in DMSO or DMSO alone. A
second control sample was boiled for 10 minutes in 2% SDS prior to
crosslinking. The crosslinking reactions were quenched with 45 mM DTT and then
subjected to SDS-PAGE. The proteins were transferred to PVDF membranes and
visualized by immunoblotting with an anti-6xHis monoclonal antibody
(Clontech).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)]. The lsy mutants retrieved from past
genetic screens fall into three phenotypic categories.
). ;
Chang et al., 2003;
Johnston and Hobert, 2003;
Johnston and Hobert,
2005). ).
One recessive mutant allele identified in the screen, ot61,
exhibits a unique phenotype not described previously. All ot61 mutant
animals display de-repression of gcy-7prom::gfp in ASER
(Fig. 1B). However, although
the phenotype is completely penetrant (all animals show the defect), the
levels of aberrant gcy-7prom::gfp expression in ASER are
variable. Sometimes, the level of aberrant gcy-7prom::gfp
expression in ASER is equivalent to the normal and unaffected
gcy-7prom::gfp expression in ASEL, but more often, the
aberrant gcy-7prom::gfp expression level in ASER is not as
high as the gcy-7prom::gfp expression level in ASEL
(Fig. 1B,
Table 1). The fozi-1
mutant phenotype is therefore `variably expressive'. This variable
expressivity contrasts the previously described phenotype in the ASER neuron
of class I (`two ASEL') mutant animals in which the level of aberrant
gcy-7prom::gfp expression in ASER always reaches levels
indistinguishable from the level in ASEL (i.e. complete expressivity)
(Chang et al., 2003). To
determine whether this complete penetrance and partial expressivity applies to
other ASEL-specific fate markers, we crossed ot61 with animals
containing the ASEL-specific lim-6prom::gfp reporter
transgene and again observed de-repression of the reporter gene with complete
penetrance and variable expressivity (Fig.
1B).
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The predicted K01B6.1 gene structure was confirmed by analyzing an
expressed sequence tag provided by Y. Kohara. The protein encoded by
K01B6.1 contains three readily recognizable motifs
(Fig. 2B): (1) a Q-rich motif
often found in transcription factors (Titz
et al., 2006); (2) two C2H2 Zn fingers with a standard
CX2CX11-13HX4H spacing normally found in
DNA-binding transcription factors (Iuchi,
2001) (see Fig. S1 in the supplementary material); (3) a formin
homology 2 (FH2) domain, which is surprising as this domain is normally found
only in cytosolic actinpolymerizing proteins in which the domain catalyses
actin polymerization (Zigmond,
2004). Owing to the unusual combination of the formin homology 2
and zinc-finger domains, we have named the gene fozi-1 (pronounced
`fozzy-1').
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The ASER neuron adopts a `mixed fate' in fozi-1 mutants
After cloning fozi-1, two additional alleles of fozi-1,
cc607 and tm563, were made available to us. Like ot61
mutants, cc607 and tm563 mutants are viable, fertile and
display no obvious behavioral or morphological abnormalities. The
cc607 allele was retrieved from a screen for mesoderm lineage
differentiation defects (J.L., unpublished). cc607 is a putative
molecular null allele as it encodes a nonsense mutation causing premature
termination before both the Zn fingers and the FH2 domain
(Fig. 2B). Using rescue assays,
we show below that the Zn fingers are essential for fozi-1 function.
The other allele, tm563, kindly provided by a C. elegans
knockout consortium, completely deletes the Q-rich domain-containing exon 3
and a large part of exon 4, including the first Zn finger and the first half
of the second Zn finger (Fig.
2B). Owing to a premature stop codon introduced by the deletion,
tm563 can also be considered a molecular null allele. The
availability of these two molecular null alleles allowed us to examine whether
the variable phenotypic expressivity of fozi-1(ot61) mutants is
merely a reflection of a partial loss of gene function. Crossing the putative
null alleles cc607 and tm563 with the ASEL-specific
lim-6prom::gfp transgene, we observed gene expression
defects essentially identical to those seen in ot61 mutant animals
(Table 1). At least in the
context of the ASE subclass determination, ot61 may therefore
similarly be a null allele of fozi-1.
We examined the effect of the fozi-1(cc607)-null allele on several additional markers that define the ASEL fate, namely the two GCY genes gcy-6 and gcy-7 and the neuropeptide-encoding flp-4 gene. We found that all examined ASEL fate markers are affected in a similar manner in that they become partly de-repressed in a completely penetrant manner (Fig. 4A). The incomplete and variably expressive de-repression of ASEL fate markers in the ASER neuron of fozi-1-null mutants suggests that additional, fozi-1-independent mechanism(s) must exist to prevent complete de-repression of the ASEL fate markers.
In contrast to all previously defined ASE cell fate mutants, the de-repression of ASEL terminal fate markers in the ASER neuron of fozi-1 mutants is not accompanied by loss of ASER fate markers. Expression of the ASER-specific gcy-4, gcy-5 and hen-1 genes in fozi-1 null mutants is indistinguishable from wild-type expression (Fig. 4B). Taken together, the ASEL neuron appears completely unaffected in fozi-1 mutants in that ASEL markers are expressed and ASER fate markers are not de-repressed. By contrast, ASEL fate markers are de-repressed in ASER, but ASER fate markers remain unaffected. The ASER neuron therefore displays a novel `mixed' phenotype in fozi-1 mutants (Fig. 4C). Extending our previous mutant classification, we term this phenotype a `class IV' phenotype.
fozi-1 acts in a left/right asymmetric manner in ASER, but not ASEL
The phenotypic analysis of fozi-1 demonstrates that ASER, but not
ASEL, is affected in fozi-1 mutants. As fozi-1 appears to
encode a gene regulatory factor, the gene expression defects in ASER are most
easily explained by fozi-1 being expressed and acting in ASER. We
tested this prediction by a variety of means, including gene expression
pattern analysis, cell-specific rescue and mis-expression approaches.
In order to investigate fozi-1 expression, we generated a construct, fozi-1::gfp, in which the genomic fozi-1 locus is tagged with gfp. This construct efficiently rescues the fozi-1 mutant phenotype (Fig. 3A). Transgenic, adult animals display gfp expression in the nucleus of the AWC and ASE neuron class (Fig. 3B and data not shown). No other expression was observed in head ganglia. Three out of four lines displayed a bias in expression to ASER; by contrast, expression in the AWCL/R neurons is bilaterally symmetric (Fig. 3B,C). To corroborate that fozi-1 indeed functions in ASER, we generated a gcy-5prom::fozi-1 cDNA::gfp construct in which the gcy-5 promoter drives expression of fozi-1 fused to gfp specifically in postmitotic ASER neurons. The gfp moiety confirmed that fozi-1 was expressed and localized to the nucleus of transgenic animals. Four out of four gcy-5prom::fozi-1 cDNA::gfp transgenic lines rescued the lim-6prom::gfp de-repression defects in ASER observed in fozi-1(cc607)-null mutant animals. Taken together, expression and rescue experiments with the heterologous promoter demonstrate that fozi-1 acts specifically in ASER to repress ASEL cell fate.
Transgenic lines that contain multiple copies of genomic fozi-1 DNA show not only a rescue of the ASER defects of fozi-1 mutants (Fig. 3A, right bar) but also show a partial repression of lim-6 expression in ASEL (Fig. 3A, left bar). This ectopic activity of fozi-1 correlates with the degree of ASEL versus ASER bias of fozi-1::gfp expression (Fig. 3C). Transgenic line 3, which shows the most bias to ASER rescues the ASER defect but has little impact on ASEL fate determination, while transgenic lines 1 and 4, which show a larger degree of fozi-1::gfp in both ASER and ASEL (Fig. 3C), show suppression of the ASEL fate marker lim-6 in ASEL (Fig. 3A). These observations suggest that fozi-1 may be sufficient to repress ASEL fate if overexpressed in ASEL. To corroborate this notion, we generated a ceh-36prom::fozi-1 cDNA::gfp construct in which the ceh-36 promoter drives equal expression of gfp-tagged fozi-1 in both ASEL and ASER. Introduction of this construct into the fozi-1(cc607) mutant background showed equivalent repression of lim-6prom::gfp expression in both ASEL and ASER (Fig. 3A). fozi-1 is therefore sufficient to repress lim-6 when ectopically expressed in ASEL. Taken together, we conclude that fozi-1 expression is biased to ASER and that it acts autonomously to repress the expression of lim-6 and other ASEL-specific genes in ASER.
Last, as two transcription factors in the ASEL/R cell fate regulatory
network are repressed via 3'UTR dependent mechanisms (cog-1,
die-1), we tested whether the 3'UTR of fozi-1 contains
cis- regulatory information that may contribute to the left/right
asymmetric function of fozi-1. Using a sensor gene approach that
revealed the 3'UTR-dependent regulation of the cog-1 and
die-1 genes (Chang et al.,
2004; Johnston and Hobert,
2003), we found this not to be the case (data not shown).
Terminal differentiation genes are also controlled by pathways that act in parallel to fozi-1 and lim-6
The results presented so far can be summarized as shown schematically in
Fig. 4E. fozi-1 is
expressed in ASER and is required to repress ASEL-specific features in ASER.
The incomplete nature of de-repression of the ASEL markers suggests one of two
scenarios. In scenario 1, fozi-1 acts together with an unknown
repressor X to repress ASEL-specific features in ASER and only loss of both
fozi-1 and X causes complete de-repression of ASEL fate in ASER.
Alternatively, in scenario 2, de-repression of ASEL markers in ASER of
fozi-1 is incomplete as an activator Y is missing in ASER
(Fig. 4E).
The LIM homeobox gene lim-6 is expressed in ASEL and its loss
causes a mixed phenotype that is superficially the mirror image of the
fozi-1 loss-of-function phenotype. The ASER neuron is unaffected, but
the ASEL neuron displays a `mixed phenotype' characterized by a failure to
repress ASER features in ASEL (Hobert et
al., 1999; Johnston et al.,
2005; Ortiz et al.,
2006). Like in fozi-1 mutants, the defects in
lim-6-null mutants are also variably expressive
(Fig. 4D), thereby suggesting
the existence of lim-6- independent means to repress ASER fate
(Fig. 4E). Consistent with the
existence of a parallel pathway, repression of ASER fate markers induced by
ectopic expression of the lsy-6 miRNA in ASER does not absolutely
require lim-6 (Fig.
4D). We have previously shown that lim-6 is also required
to positively regulate the expression of ASEL-specific markers, namely the two
FMRFamide-encoding genes flp-4 and flp-20
(Johnston et al., 2005).
lim-6-null mutant animals show only a partially penetrant loss of
flp-4 and flp-20 expression, again indicating the presence
of a parallel pathway (indicated in by a factor Z in
Fig. 4E). Taken together, these
observations suggest that both lim-6 and fozi-1 require
parallel pathways to exert their function in ASEL and ASER, respectively.
fozi-1 acts downstream of the bi-stable feedback loop
We tested how fozi-1 gene function relates to the function of
components of the bi-stable feedback loop that controls ASEL and ASER fate
(Fig. 1A). We found that
disruption of this bi-stable loop causes a `symmetrization' of the normally
left/right asymmetric expression of fozi-1
(Fig. 5A). Specifically, in
animals in which the ASEL-inducers die-1 or the lsy-6 miRNA
are mutated, fozi-1 expression becomes de-repressed in ASEL. In
animals lacking the ASER-inducer cog-1, the bias of fozi-1
expression to ASER is also lost. These results are consistent with our
previous observations that the bi-stable feedback loop controls all features
of the ASEL/R fate decision (Johnston et
al., 2005).
We corroborated the downstream role of fozi-1 using two genetic
epistasis tests. In animals in which the lsy-6 miRNA is deleted, ASEL
displays the complete ASER gene expression profile including the repression of
lim-6 expression (Johnston and
Hobert, 2003). Repression of lim-6 in
lsy-6(ot71) null mutant animals requires fozi-1 activity as
fozi-1(ot61); lsy-6(ot71) double mutants display a de-repression of
lim-6prom::gfp in ASEL and ASER
(Fig. 5B). The same genetic
interaction is observed using die-1 mutants. In die-1(ot26)
mutant animals, lim-6 expression is repressed in ASEL
(Chang et al., 2004). This
repression requires fozi-1 as die-1(ot26); fozi-1(ot61)
double mutants display a de-repression of lim-6prom::gfp
in ASEL and ASER (Fig. 5B).
Together, these data demonstrate that fozi-1 acts downstream of
die-1, the output regulator of the bi-stable feedback loop
(Fig. 5C).
|
). The
fozi-1 parallel pathway that we evoked above
(Fig. 4E), therefore, depends
genetically on the activity of the bi-stable loop whose output regulator is
the die-1 gene. The most parsimonious way to present these regulatory
interactions is shown in Fig.
5C with an arrow from die-1 to lim-6 that is
parallel to the fozi-1-mediated regulation of lim-6. In
other words, lim-6 expression genetically requires both the presence
of die-1 and the absence of fozi-1.
Loss of die-1 also causes a completely expressive de-repression of
ASER fate in ASEL (Chang et al.,
2004), which contrasts the partially expressive de-repression of
ASER fate in the ASEL neuron of lim-6-null mutants. The
lim-6-parallel pathway that we evoked above
(Fig. 4E), therefore also
depends on the loop output regulator die-1. The most parsimonious
explanation of the genetic interactions is that the factor that acts in
parallel to lim-6 (factor Z in
Fig. 4E) is die-1
itself (Fig. 5C).
A loss of the lim-6 LIM homeobox gene, which acts downstream of
the bi-stable feedback loop (Fig.
1A) causes a partially penetrant defect in maintaining the
left/right asymmetric expression of loop components
(Johnston et al., 2005)
(broken line in Fig. 1A). As
lim-6 is de-repressed in ASER of fozi-1 mutants, we asked
whether a similar partially penetrant defect can be observed in
fozi-1 mutants, and we indeed find this to be the case
(Fig. 5D,E). Moreover, as would
be expected from a partial disruption of activity of bi-stable loop components
in lim-6-null mutants, asymmetric fozi-1 expression is also
partially affected in lim-6 mutants
(Fig. 5A).
Taken together, left/right asymmetric fozi-1 expression is controlled by components of the bi-stable feedback loop and asymmetric fozi-1 augments the maintenance of the asymmetric expression of loop components, probably through the regulation of lim-6 expression.
The Zn fingers but not the FH2 domain are essential for fozi-1 function
All factors previously known to play roles in the lateral ASE cell fate
decision are bona fide transcription factors or miRNAs. Therefore, we were
surprised to have identified with fozi-1 a gene that, on the one
hand, contains conventional signatures of a transcription factor (Zn fingers
and Q-rich domain), but on the other hand contains a FH2 domain, which has
been characterized as a cytoplasmic actinnucleation domain
(Zigmond, 2004). This prompted
us to learn more about the FH2 domain of FOZI-1 (henceforth termed
FH2FOZI-1).
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;
Xu et al., 2004), also allow a
potential dimeric interaction of FH2FOZI-1 domain
(Fig. 6A,B). However, the
similarity of FH2FOZI-1 with other FH2 domains is poor in those
regions that contact actin. In particular, two highly conserved amino acids,
equivalent to Ile1431 and Lys1601 in the Bni1 FH2 domain, which have been
shown to be essential for actin nucleation activity
(Otomo et al., 2005;
Xu et al., 2004), are altered
in FH2FOZI-1 (Fig.
6A). In addition, the linker between the lasso and knob regions,
which is also important for actin nucleation function, is severely shortened
in the FH2FOZI-1, thereby likely reducing its flexibility. The FH2
domains of all other nematode FH2 domain proteins (six besides FOZI-1;
http://smart.embl-heidelberg.de/)
look like standard FH2 domains in terms of conservation of individual key
residues and length of linker regions (data not shown). Taken together, our
sequence analysis makes two predictions about FOZI-1, namely that FOZI-1 may
still homodimerize via its FH2 domain but may have no role in controlling
actin polymerization as it lacks actin-binding surfaces and as the ring
structure made by FH2 domain dimers (Fig.
6B) may not be large enough to accommodate actin.
We tested these predictions using in vitro and in vivo assays. FH2 domains
affect actin polymerization by either inducing polymerization of actin
monomers or capping F-actin barbed ends
(Kovar et al., 2003;
Pruyne et al., 2002). We
tested the ability of purified FH2FOZI-1 to induce actin
polymerization in vitro using the pyrene actin assay. In contrast to the
control FH2 domain of the mouse FH2 protein Dia1, FH2FOZI-1 is
unable to induce actin polymerization (Fig.
6C). Similarly, FH2FOZI-1 is unable to cap barbed ends
(see Fig. S2A in the supplementary material) and does not bind actin filaments
in sedimentation assays (data not shown). These experiments confirm the
prediction that FH2FOZI-1 has lost its role in regulating actin
polymerization. The prediction that FH2FOZI-1 still maintains its
ability to dimerize is confirmed by our observation that purified
FH2FOZI-1 elutes from a Sephadex 200 gel filtration column as a
single peak of 
100 kDa, consistent with the formation of a
FH2FOZI-1 dimer (see Fig. S2B in the supplementary material). In
separate crosslinking experiments, the formation of FH2FOZI-1
oligomers could also be detected (Fig.
6D).
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Examining the functional relevance of the FH2 domain, we found that deletion of the FH2 domain diminished but did not completely eliminate the ability of the fozi-1::gfp construct to rescue the fozi-1 mutant phenotype (Fig. 3A). The FH2 domain of FOZI-1 therefore does not appear to be absolutely crucial for protein function, at least in the context of ASER differentiation.
In summary, FOZI-1 is unlikely to have a role in actin polymerization because: (1) there is no evidence for the existence of filamentous actin in the nucleus where FOZI-1 localizes; (2) detailed sequence analysis reveals that the FOZI-1 FH2 domain does not contain key features required for actin polymerization; (3) in vitro assays fail to reveal actin polymerization activity of the FOZI-1 FH2 domain; and (4) functional in vivo experiments indicate that the FH2 domain is not essential for gene function in the context of lateral ASE subclass specification. The only point that, at first sight, may argue for the importance of the FH2 domain is the ot61 allele, which prematurely terminates the FH2 domain. However, this mutation does not only disrupt the FH2 domain, but de-stabilizes the complete protein, as evidenced by a complete loss of anti-FOZI-1 antibody staining in ot61 mutant animals (J.L., unpublished).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The domain composition of FOZI-1 provides an example of domain rearrangement
The combination of FH2 and C2H2 Zn-finger domains in a single protein
appears to be unique to the nematode lineage. The fozi-1 gene
probably originated by gene arrangement that joined two initially independent
genes. What is the selective advantage of maintaining such a gene? The ancient
function of the FH2 domain is to control actin polymerization given that FH2
domains from yeast to mammals harbor such an activity
(Zigmond, 2004). However, our
sequence and biochemical analysis suggests that the FH2 domain of FOZI-1 has
lost this function. Nevertheless, the FH2 domain of FOZI-1 does harbor some
function as its deletion compromises the ability to rescue the fozi-1
mutant phenotype. Our analysis of the FH2 domain of FOZI-1 suggests that the
domain has retained its ability to homodimerize, another ancient property of
the domain that has been reported for all other characterized FH2 domain
proteins. Transcription factors often dimerize, which increases their
DNA-binding surface and enables cooperative DNA binding
(Harrison, 1991). It is
conceivable that the FOZI-1 FH2 domain serves the similar purpose of doubling
the DNA contact surface of FOZI-1 through homodimerization. This may also
explain why the FOZI-1 protein may still function, albeit only partially, upon
deletion of the FH2 domain. In our in vivo rescue assays, transgenic
array-induced overexpression of dimerization-defective FOZI-1 protein may
increase the cellular FOZI-1 protein concentration enough to alleviate the
need for dimerization-induced recruitment of proteins to DNA. Future
identification of FOZI-1 DNA target sites and biochemical assays will test the
validity of this hypothesis.
fozi-1 and lim-6 mutants define a novel, `mixed' ASE state
Previously, we have defined two states for ASEL/R gene expression profiles:
hybrid and stable (Johnston et al.,
2005). The hybrid state occurs in both ASEL and ASER during
embryonic and early larval stages in which most ASE markers (including the
ASEL-inducer lsy-6 and the ASER-inducer cog-1) are expressed
in both ASEL and ASER. Dependent on the activity of the bi-stable feedback
loop, this hybrid state of gene expression subsequently becomes restricted to
either the ASEL or ASER stable state. Genetic ablation of feedback loop
components causes both ASE cells to take on the complete ASEL cell fate
(`class I' mutants including cog-1) or the complete ASER cell fate
(`class II' mutants including lsy-6, die-1, lsy-2).
fozi-1 null mutant animals display an unusual phenotype. Rather than exhibiting a complete switch of cell fate in ASEL or ASER, fozi-1 mutants display a `mixed' fate phenotype in ASER. Whereas ASEL displays its ASEL-specific gene expression profile, ASER expresses ASEL terminal markers while maintaining expression of ASER terminal markers. In an almost mirror image of fozi-1-null mutant animals, lim-6-null mutant animals adopt an essentially normal ASER state in ASER but fail to repress specific ASER terminal markers in ASEL, again causing a `mixed' state, this time in ASEL. Taken together, both lim-6 (expressed in ASEL) and fozi-1 (expressed in ASER) are regulatory intermediaries that transduce an output from the bi-stable feedback loop. These factors thereby enable the progression of differentiation states from a hybrid precursor state to a terminally differentiated end state.
ASE subclass determination involves a complex regulatory architecture composed of several network motifs
Systematic analyses of gene regulatory networks in unicellular organisms
have revealed that transcription factors interact in numerous combinations of
simple network motifs (Lee et al.,
2002; Shen-Orr et al.,
2002). Here and in our previous work, we have extended the concept
of defined network motifs to cell fate determination in the nervous system of
metazoan organisms. We have shown (1) that a bi-stable feedback loop motif is
a key decision point in the ASEL/R fate determination process
(Johnston et al., 2005) and
(2) that multiple distinct network motifs can be intertwined into a multi-tier
regulatory architecture (this paper). The usefulness in considering these
motifs in the context of ASEL/R cell fate determination lies in the
well-defined properties of network motifs that can be mathematically modeled
(Fig. 7B) and which provide
clear predictions about the underlying logic of ASEL/R fate determination.
Specifically, the analysis of the fozi-1 gene and its relationship
with other regulatory network components suggests novel regulatory motifs that
act in conjunction with the previously described bi-stable feedback loop.
Emanating from the die-1 Zn finger transcription factor, and
including the fozi-1 Zn finger factor, all these motifs appear to be
variants of the feed-forward loop (FFL) network motif
(Fig. 7B,C), a motif commonly
found in transcription factor networks
(Lee et al., 2002;
Shen-Orr et al., 2002). A
defining feature of FFL motifs is that they provide a persistence detector
that measures whether a gene regulatory input persists long enough before
target genes are activated (Fig.
7B) (Mangan and Alon,
2003; Mangan et al.,
2003). In the cases described here, we infer the existence of FFL
network motifs based on the variable expressivity of null mutant phenotypes
(see legend to Fig. 7D for
detailed explanations). With the possible exception of hen-1, all
left/right asymmetrically expressed genes may be regulated through FFL motifs.
In each of these FFL motif configurations, die-1 and fozi-1
jointly regulate a target gene (Fig.
7C); expression of the target requires both the presence of
die-1 and the absence of fozi-1. The target gene is either a
terminal differentiation marker (gcy-7 etc.) or it is another
regulatory factor, lim-6, which then activates FLP genes and
represses ASER-specific GCY genes (Fig.
7D).
Connecting gene regulatory network motifs
Apart from being the starting point for several presumptive feed-forward
loops, the die-1 Zn-finger transcription factor is the output
regulator of a bi-stable, double-negative feedback loop, which contains
additional transcription factors and miRNAs, and was the first network motif
identified in ASE fate specification (Fig.
7A). die-1 therefore represents a crucial `node' in
connecting the bi-stable feedback loop to the feed-forward loops described
here. The theoretical behaviors of feedback and feed-forward motifs allow us
to speculate on how the ASEL/R fate decision may occur. The bistable feedback
loop may allow amplification of an initial, transient input into the system.
Such an input could be an intrinsic, lineage-derived cue or an externally
provided signal. After the reception of this input in a left/right asymmetric
manner, the feedback loop may increase and/or stabilize die-1 levels
in ASEL. As the feed-forward loops that emanate from die-1 may act as
a persistence detector, target genes will become activated only once the
feedback loop has ensured that die-1 levels are persistently above a
specific threshold level. The combined feedback and feed-forward regulatory
motifs finely tune the system, amplifying a crucial input and assessing the
stabilization and continuity of this amplification. We anticipate that the
overall logic of the ASEL/R cell fate choice may apply to other cell fate
decisions that have not yet been examined in extensive genetic detail.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/17/3317/DC1
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ACKNOWLEDGMENTS |
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