1 Fox Chase Cancer Center, Philadelphia, PA 19111, USA
2 Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA
3 Department of Molecular Biology, UMDNJ, Stratford, NJ 08084, USA
* Author for correspondence (e-mail: mosseg{at}umdnj.edu)
Accepted 28 January 2004
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SUMMARY |
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Key words: C. elegans, Larval development, Hypodermis, Heterochronic genes, lin-14, lin-28, lin-42, lin-46, lin-57, let-7, Gephyrin, MoeA, moc-1, moc-2
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Introduction |
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The products of the heterochronic genes include at least two microRNAs
(lin-4 and let-7), an evolutionarily conserved protein with a unique pairing
of RNA-binding motifs (LIN-28), a protein related to the circadian rhythm
regulator period of Drosophila (LIN-42), and a nuclear
protein of unknown function (LIN-14)
(Ruvkun et al., 1989;
Ruvkun and Giusto, 1989
;
Lee et al., 1993
;
Moss et al., 1997
;
Jeon et al., 1999
;
Reinhart et al., 2000
).
Although insight has been gained into how the expression of certain
heterochronic genes is regulated, it is not yet known whether the identified
heterochronic genes constitute all the components of the pathway. Nor is it
known how the products of the heterochronic genes interact among themselves
and with other cellular components to effect the precise timing of
developmental events in worm larvae.
We have identified a new heterochronic gene, lin-46, which occupies a unique position in the larval developmental timing mechanism of C. elegans. Our analysis indicates that lin-46 functions at a step immediately downstream of lin-28, and acts at a branch-point in the heterochronic pathway. lin-46 encodes a protein that is homologous to bacterial and mammalian proteins, but as a developmental regulator, is novel.
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Materials and methods |
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Strains
The lin-28(lf) alleles used are null based on molecular analysis
(Moss et al., 1997;
Seggerson et al., 2002
). The
nature of the different lin-14 alleles used are indicated in the text
and tables. The lin-46 allele ma164 was used in all
experiments, except where indicated.
N2 (wild type), ME1 lin-28(ga54) I; lin-46(ma164) V, ME11
lin-42(n1089) II; lin-46(ma164) unc-76(e911) V, ME12
lin-46(ma164) unc-76(e911) V; lin-14(n360) X, ME13 aeEx1
(pJJ.1[lin-46:GFP] pRF4[rol-6(su1006)]), ME20
lin-46(ma164) V, ME21 lin-46(ma174) V, ME42
lin-46(ma164) unc-76(e911) V; lin-14(ma135)/szT1 X, ME58
lin-42(n1089) II; lin-46(ma164) unc-76(e911) V; lin-14(n179) X, ME63
lin-42(n1089) I; lin-14(n179ts) X, ME89 lin-28(n719) I;
wIs78, ME90 lin-28(n719) I; wIs78; lin-14(n536n540)/+ X, ME91
wIs78; lin-46(ma164) unc-76(e911) V, ME92 lin-28(n719) I; wIs78;
lin-46(ma164) unc76(e911) V, ME94 aeEx33 (pLT.18
[lin-46:GFP] pRF4[rol-6(su1006)]), ME97
lin-28(n719) I; wIs78; lin-46(ma164) unc-76(e911) V; lin-14(n536n540)/+
X, ME98 wIs78; lin-14(ma135)/szT1 X, ME99 wIs78;
lin-46(ma164) unc-76(e911) V; lin-14(ma135)/szT1 X, ME101
lin-41(ma104) I, lin-46(ma164) V, ME105 wIs78; lin-14(n179ts)
X, ME107 wIs78; lin-46(ma164) unc-76(e911) V; lin-14(n179ts) X,
CT8 lin-41(ma104) I, MT2257 lin-42(n1089) II, MT1848
lin-14(n360) X, PV5 lin-28(ga54) I, RG733 ced-1(e1735) I;
wIs78 and VT284 lin-14(ma135)/szT1 X. wIs78: [pDP#MM016B
(unc-119) + pJS191 (ajm-1::gfp + pMF1(scm::gfp) +
F58E10] (Abrahante et al.,
2003
).
Microscopy and phenotype analysis
Egg-laying ability was scored using a dissecting microscope. Gonad
development, seam cell number, and adult lateral alae formation were scored
using DIC and fluorescence microscopy at high magnification. Lateral
hypodermal cell lineages were deduced from counting seam cells of animals of
multiple ages from the L1 to the L4. These cells were identified by GFP
expression from wIs78, which contains scm-1:GFP and
ajm-1:GFP that mark seam cell nuclei and seam cell junctions,
respectively (Koh and Rothman,
2001). The stage of development was determined by extent of gonad
and germline development. Data were collected from animals both early and late
in each larval stage. Because cell division was ongoing near the beginning of
each stage, we present only seam cell numbers when most or all cell divisions
appeared to be complete.
Cold-sensitive period
lin-46(ma174) animals were raised at either the restrictive
(15°C) or permissive (25°C) temperature until specific stages in
development, when they were shifted to the other temperature for the remainder
of larval development. The animals were shifted when they were in the
lethargus period that precedes each molt. At adulthood, animals were then
scored for the presence of gaps in alae.
Genetic and physical mapping
Genetic mapping and physical mapping were performed using standard methods
(Fig. 3A; data available at
wormbase.org).
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Poly(A)+ RNA from a mixed stage population was subjected to RT-PCR using oligo(dT), SL1- and lin-46 specific primers. PCR products were cloned and sequenced to determine the structure of the gene.
A translational fusion reporter construct (pJJ.1) was made by introducing GFP in frame into the EagI site, nine codons from the stop codon, in the BglI-NgoMI subclone shown in Fig. 3A. A transcriptional fusion construct (pLT.18) was derived from the translational fusion by creating a 1.5 kb deletion within the gene, from BglII site in the second exon, 20 codons from the AUG, to the EcoRV site in the last exon, 15 codons from start of GFP. Both constructs were co-injected with pRF4(rol-6(su1006)). Four translational fusion lines and two transcriptional fusion lines showed consistent expression patterns as described in Results.
RNA interference
RNA-interference of lin-57 was achieved culturing worms on
bacterial strain HT115(DE3) containing a plasmid expressing lin-57
dsRNA (gift of A. Rougvie) in the presence of IPTG, ampicillin and
tetracycline (Timmons et al.,
2001; Abrahante et al.,
2003
; Lin et al.,
2003
).
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Results |
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Both mutant alleles of lin-46 completely suppressed the protruding
vulva (Pvul) and egg-laying defective (Egl) phenotype of three different
lin-28 null alleles (Table
1, lines 2 and 3, and data not shown)
(Moss et al., 1997). The Pvul
and Egl phenotypes of lin-28(lf) are due in part to precocious vulva
precursor cell (VPC) divisions and vulva development
(Euling and Ambros, 1996
). We
observed that lin-28(lf); lin-46(lf) animals had wild-type timing of
vulva development, with VPC divisions occurring in the L3 stage
(Fig. 1; data not shown).
lin-28 also governs the choice between L2- and L3-specific fates in
the lateral hypodermal seam cells (Ambros
and Horvitz, 1984
). Normally during the L1, L3 and L4 stages, most
seam cells undergo a single asymmetric cell division, after which the anterior
daughter joins the hypodermal syncytium and the posterior daughter remains a
seam cell (Fig. 1, wild type,
Vn). However, in the L2 of wild-type animals, the asymmetric division of
certain seam cells (V1-4, V6) is preceded by a symmetric division, which
increases the number of seam cells on each side of an animal by five;
additional seam cells are also added at this stage by descendants of other
hypodermal cells (H1 and T; Table
1, line 1; Fig. 1)
(Sulston and Horvitz, 1977
).
lin-28(lf) animals have fewer seam cells than wild type, because they
skip L2 lineage patterns that generate these additional seam cells;
lin-28(lf) animals also cease the molting after only three cycles
because of precocious execution of the L/A switch
(Table 1, line 2;
Fig. 1) (Ambros and Horvitz, 1984
). We
found that the lin-28(lf); lin-46(lf) animals had the normal number
of seam cells at every stage, as well as the normal four molts, indicating
suppression of the L2 lineage defects of lin-28(lf) by the
lin-46 mutation (Table
1, line 3; Fig. 1).
Furthermore, lin-46(lf) completely suppressed the precocious L/A
switch, as indicated by synthesis of adult alae, caused by lin-28(lf)
(Table 2, lines 3 and 4;
Fig. 1). These data indicate
that lin-46 affects all fate decisions governed by lin-28
and that the absence of lin-46 activity completely bypasses the
requirement of the animal for lin-28.
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Interestingly, the suppression of the lin-28(lf) phenotype was quite complete: whereas 100% of lin-28(lf) animals have precocious adult alae, the lin-28(lf); lin-46(lf) double mutant animals were devoid of adult alae at the L4 stage (Table 2, lines 3 and 4). By contrast, the suppression of lin-14 hypomorphic alleles was substantial, albeit incomplete. For example, for lin-14(n360), the percentage of animals with full-length precocious adult alae was reduced from 77% to 5%; however, 95% of double mutant animals had some amount of precocious alae (Table 2, lines 9 and 10).
Because lin-14 and lin-28 act together to govern the cell fates of the L2 and later, our findings suggest that lin-46 has different relationships with these two genes. The ability of lin-46(lf) to suppress lin-14 hypomorphic alleles but not a null allele may reflect that at least some lin-14 activity is required for suppression, and, furthermore, that lin-46(lf) suppresses the phenotype of lin-28(lf) by raising lin-14 activity to a compensatory level. Importantly, a requirement for lin-14 in the suppression would suggest that lin-14 can act independently of lin-28 to control late stage fates. Because of its implications for how the heterochronic pathway functions, we further investigated this possibility.
Suppression of lin-28 does not require lin-14
To determine whether lin-14 is indeed required for the suppression
of lin-28(lf) phenotype by lin-46(lf), we assessed
hypodermal development in strains bearing a null allele of lin-14.
lin-14(0) animals skip L1-specific lineage patterns, and precociously
execute the L2-specific division patterns that increase the seam cell number
(Ambros and Horvitz, 1987). It
has been previously shown that a lin-28(lf); lin-14(0) strain
executes the L/A switch one stage earlier than a lin-14(0) strain;
however, the effect of this mutant combination on seam cell number was not
assessed (Ambros, 1989
). We
observed that the increase of 10 to 15 seam cells that occurs abnormally early
during the L1 in lin-14(0) animals did not occur in the absence of
lin-28, consistent with an L3 lineage pattern occurring two stages
early (Table 3, lines 1 and 3).
This observation indicates the hypodermal lineage patterns executed in the L1
in lin-14(0) animals are controlled by lin-28, and is
evidence that lin-28 acts independently of lin-14 to govern
these cell fate choices.
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Interactions of lin-46 with other heterochronic mutants
To further establish the position of lin-46 in the developmental
timing pathway, we examined its interactions with other precocious
heterochronic mutants. In addition to lin-14 and lin-28,
several other genes have been identified that result in precocious expression
of the L/A switch when mutated: lin-41, lin-42 and lin-57
(Z. Liu, PhD thesis, Harvard University, 1990)
(Abrahante et al., 1998;
Abrahante et al., 2003
;
Lin et al., 2003
). For this
analysis, we again examined the ability of a lin-46(lf) to suppress
the precocious execution of the L/A switch that is characteristic of these
mutants.
lin-46(lf) partially suppressed the precocious synthesis of adult
alae of a lin-42(lf), whereas it had little or no effect on the
precocious alae caused by loss of the activities of lin-41(lf) and
lin-57(lf) (Table 2,
lines 11-16). Thus, lin-46 appears to function upstream of
lin-41 and lin-57 and downstream or in parallel with
lin-42 in controlling the L/A switch. These results are consistent
with lin-46 acting early in the heterochronic pathway, because both
lin-41 and lin-57 are thought to act late in development,
downstream of lin-28 in controlling the L/A switch (see
Fig. 5)
(Slack et al., 2000;
Lin et al., 2003
). The
earliest time of action of lin-42 has not been determined, but
previous genetic analysis, as well as these data, are consistent with it
acting early in the heterochronic pathway (Z. Liu, PhD thesis, Harvard
University, 1990).
lin-46 mutations cause retarded development at two stages
In examining the effects of lin-46(lf) in the absence of other
heterochronic mutations, we observed an increase in the number of seam cells
after the L2 stage when the animals were grown at 15°C
(Table 1, line 8). This
increase probably reflects a reiteration of the L2-specific lineage pattern at
the L3 stage, in which the five seam cells that divide twice in the L2, do so
again in the L3 (Fig. 1). This
retarded lineage defect was not observed when animals were raised at 20°C,
indicating a strict cold-sensitivity for this defect
(Table 1, line 7). Because the
mutant alleles of lin-46 are likely to result in the complete loss of
function of the gene (see below), a reiteration of L2 lineages in
lin-46(0) would indicate that the normal role of lin-46 is
to promote the expression of L3-specific cell fates at the appropriate
time.
Moreover, we observed that two mutant alleles of lin-46 each caused a similar failure of the L/A switch at the end of larval development, resulting in gaps in adult alae (Table 4, lines 4 and 7; Figs 1, 2). The penetrance and expressivity of this phenotype are significantly enhanced by growth at low temperature. For example, the retarded L/A switch defect of lin-46(ma164) showed 6% penetrance at 25°C, 32% at 20°C, and 86% at 15°C (Table 4, lines 4-6). Because this cold sensitivity was true of mutant alleles of lin-46, which, as described below, are likely to be null by molecular criteria, it appears to be a property of a strain lacking lin-46 activity, rather than a property of the molecular lesions themselves.
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Interestingly, the retarded phenotype of lin-46(lf) is nearly completely suppressed by lin-28(lf). For example, the penetrance of the L/A switch defect in lin-28(lf); lin-46(ma164) was 8% at 15°C, which is significantly reduced relative to 86% for lin-46(ma164) alone (Table 4, lines 4 and 13). Likewise, we observed no early cell lineage defects in the double mutant grown at 15°C (Table 1, line 4; Fig. 1). This mutual suppression of lin-46(lf) and lin-28(lf) suggests that lin-46 is not in a simple linear pathway downstream of lin-28, but rather that their activities converge, possibly on common targets.
By contrast, the retarded phenotype of lin-46(lf) was not suppressed by a lin-14 null mutation. Rather, lin-46(lf) causes a severe retarded defect in a lin-14 null mutant background (67%), as it does in an otherwise wild-type background (86%; Table 4, lines 4 and 17). This result further indicates that lin-14 is not required for the effect of lin-46 on developmental timing.
lin-46 encodes a homologue of bacterial MoeA and vertebrate gephyrin.
lin-46 was cloned by genetic mapping and transformation rescue
(Fig. 3A; see Materials and
methods). The smallest rescuing fragment encodes one complete gene, annotated
as R186.4 in the genome sequence. Northern analysis and cDNA library screening
failed to detect a transcript from this gene (data not shown). RT-PCR analysis
revealed that the gene is expressed, has four introns, and its mRNA is
trans-spliced, polyadenylated, and 1.4 kb long
(Fig. 3A). The sequence of
amplified genomic DNA from three lin-46 mutant strains revealed
mutations in all three that would affect the protein encoded by this gene
(Fig. 3B). Two of these
lesions, from alleles ma164 and ma174, probably result in
severe truncations of the protein, strongly suggesting they are molecular
nulls. Furthermore, frameshift mutations introduced into the rescuing clone
rendered it unable to complement the lin-46 mutant (data not shown).
A clone of the orthologous gene from the related species C. briggsae
rescued the C. elegans lin-46 mutant phenotype, indicating that its
function is conserved between these species (data not shown). We therefore
concluded that lin-46 corresponds to R186.4.
lin-46 has the potential to encode a 391 amino acid protein with
homology along its entire length to MoeA of bacteria and the C-terminal
domains (referred to as the E-domain) of the mammalian protein gephyrin; other
related proteins are Cinnamon of Drosophila and CNX1 of
Arabidopsis (Fig. 3C)
(Kamdar et al., 1994;
Stallmeyer et al., 1995
).
Gephyrin is a submembraneous scaffolding protein that aids in clustering
glycine and GABA receptors at postsynaptic neurons
(Kneussel and Betz, 2000
).
MoeA is involved in the last step of the biosynthesis of molybdenum co-factor,
a metal coordinating molecule in many molybdo-enzymes, although the exact
function of MoeA in this process is not known. We discuss possible molecular
functions of the LIN-46 protein below.
Gephyrin, as well as Cinnamon and CNX1, are essentially fusions of
homologues of the bacterial proteins MoeA and MogA. Indeed, all three of these
proteins from higher eukaryotes are believed to participate in molybdenum
cofactor biosynthesis, as the bacterial proteins do
(Kamdar et al., 1994;
Stallmeyer et al., 1995
;
Feng et al., 1998
). We noted
that C. elegans is unusual among multicellular eukaryotes in having
its MoeA and MogA homologues encoded separately, and, furthermore, having two
MoeA paralogues, one of which is encoded by lin-46
(Fig. 3C). We have named the
genes encoding the other MoeA homologue and the MogA homologue of C.
elegans moc-1 and moc-2, respectively (for molybdenum cofactor
biosynthesis related; Fig. 3C).
The biological roles of these genes in C. elegans are not yet
known.
lin-46 is expressed in hypodermal cells during larval development
Green fluorescent protein (GFP) reporter constructs indicated that
lin-46 is expressed in hypodermal cells, particularly the lateral and
ventral hypodermis, in brief periods during larval development
(Fig. 4 and data not shown).
Animals bearing a transcriptional fusion (see Materials and methods) showed
intense fluorescence in lateral hypodermal cells around the time of each molt,
especially during the L2- and L4-lethargus
(Fig. 4A,B). In L2 animals, not
all seam cells fluoresced simultaneously, and often only a cluster of an even
number of cells fluoresced in a given animal, suggesting the expression of the
fusion is related to seam cell division, which occurs somewhat asynchronously
during larval development (Sulston and
Horvitz, 1977). A translational fusion reporter showed a similar
expression pattern, although it was less intense
(Fig. 4C). The translational
fusion also showed strong constitutive expression in the cell bodies and axons
of the bilateral motor interneurons AVB, but we do not know the significance
of this expression. The LIN-46:GFP fusion protein localized to both cytoplasm
and nucleus, but mostly in the non-nucleolar part of the nucleus
(Fig. 4D). Thus, LIN-46 is
present in cells whose fates it governs near the time they execute
stage-specific fates. Its appearance at multiple times suggested the
possibility that lin-46 functions more than once during larval
development.
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Discussion |
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Interactions governing the L2/L3 fate choice
The heterochronic pathway is characterized by dynamic interactions among
regulators that make binary choices in stage-specific cell fates
(Ambros, 1989). We previously
presented a model for the interactions governing the choice of L2 versus L3
cell fates (Seggerson et al.,
2002
). In this model, lin-14 and lin-28 are
repressed by the microRNA lin-4 at the same time they support each other's
expression via a lin-4-independent feedback circuit. This positive regulatory
circuit is proposed to occur by the repression of two negative regulators,
x and y, whose identities are not known.
Here we extend this model by proposing that lin-46 acts specifically to promote the activity of y, and furthermore, that y acts both to regulate lin-14 expression and to control developmental timing at two larval stages (Fig. 5A). Therefore, y represents both the point of convergence of the lin-28 and lin-46 activities, as well as a branch-point in the pathway that affects lin-14 expression and L3 cell fates. We have no reason yet to believe that lin-14 expression and L3 cell fates are regulated by distinct factors. Therefore, we continue to indicate this as a single activity, the hypothetical factor y, which may consist of multiple factors.
In our model, we have placed lin-46 outside of the feedback
circuit based on the relative penetrance of the lin-28(lf) and
lin-46(lf) phenotypes and their mutual suppression (Tables
1 and
4). Based on molecular
analysis, the alleles of lin-28 and lin-46 used in these
experiments are null (Fig. 3)
(Moss et al., 1997). Thus,
their mutual suppression implies that lin-28 and lin-46
affect a common process, but do not act in a simple linear pathway. If it were
a simple pathway, then the lin-28(lf); lin-46(lf) animals would
display the same degree of retarded development as lin-46(lf)
animals. Furthermore, the difference in penetrance of the lin-28(lf)
and lin-46(lf) phenotypes suggests they have different degrees of
influence on cell fates. Specifically, whereas a lin-28(lf) is always
fully precocious, the lin-46(lf) retarded phenotype is incomplete and
conditional. For these reasons, we propose that lin-46 is not
directly part of the lin-14/lin-28 positive feedback circuit, and
therefore is not itself y, but rather acts to promote the activity of
y (Fig. 5A).
The difference in the penetrance between the suppression by lin-46(lf) of lin-28(lf) and the retarded L2 phenotype of lin-46(lf) also suggests that changes occur in how the L2/L3 fate decision is made at different times. In other words, cell fates at the L2 stage in a lin-28(lf) are more sensitive to the loss of lin-46 than are fates at the L3 in wild type (Table 1; Fig. 1). Perhaps the decision to execute L3 fates is more robust at the third larval stage in wild type because of additional activities that are not present at the second larval stage in a lin-28 mutant. Furthermore, in no situation does the absence of lin-46 cause L2-specific cell fates to be reiterated indefinitely, further suggesting that additional factors come to influence the L2/L3 fate decision, ultimately in favor of L3 fates.
The role of lin-14 in late stage cell fates
Our observations help to further illuminate the relationship between
lin-14 and lin-28 in controlling the execution of cell fates
after the first larval stage. Two models have been proposed to explain the
activities of lin-14, lin-14A and lin-14B that independently
affect L1 and later developmental events
(Ambros and Horvitz, 1987). One
is that different isoforms of the LIN-14 protein possess these distinct
activities (Wightman et al.,
1991
; Reinhart and Ruvkun,
2001
). However, this model is not supported by the finding that a
molecular clone of a single isoform of LIN-14 can fully rescue
lin-14(lf) (Hong et al.,
2000
). A second model is that the LIN-14 protein forms a temporal
gradient through larval development, analogous to morphogen gradients in the
Drosophila embryo (Ruvkun and
Giusto, 1989
; Reinhart and
Ruvkun, 2001
). However, our findings indicate that lin-14
affects L2 developmental events only through lin-28, which requires a
genetic circuit between these genes that engages late in the first larval
stage (Table 3)
(Seggerson et al., 2002
). In
other words, the lin-14B activity essentially reflects the
participation of lin-14 in a positive feedback circuit with
lin-28.
This model explains why lin-46(lf), which bypasses the requirement
for lin-28, suppresses the L2 defects of lin-14(lf) but not
its L1 defects. Hypomorphic alleles of lin-14, such as
lin-14(n360) and lin-14(n179ts) used in these studies,
reduce the activity of lin-14, but do not eliminate it. At the end of
the first larval stage, when the positive feedback circuit between
lin-14 and lin-28 is engaged, a mutation in lin-14
would lead to a reduction in lin-28 expression, and then a further
reduction in lin-14. We have previously proposed that y in
our model mediates the reduction of lin-14 in this circuit
(Fig. 5A, left)
(Seggerson et al., 2002).
However, if lin-46 is absent, the activity of y would be
attenuated and the reduction of lin-14 would not be further
amplified. Therefore, lin-46(lf) suppresses a lin-14
hypomorph by essentially upregulating the existing lin-14 activity.
As a corollary, we propose that part of the reduction of lin-14
activity of hypomorphic alleles is due to further downregulation via the
positive feedback circuit.
Importantly, the reason a lin-46 mutation does not suppress a
lin-14 null allele is twofold: first, lin-14 normally acts
through the feedback circuit and lin-28 to control the L2/L3 fate
choice, as we have shown (Table
3); and second, the lin-28-repressive component of the
feedback circuit is not engaged early in the first larval stage of a
lin-14 mutant, as has been shown previously
(Seggerson et al., 2002).
Therefore, the effects of lin-28 and lin-46 on the L2/L3
fate decision are independent of the feedback circuit because they occur in
the early L1 when lin-14 is absent. Supporting this conclusion is our
observation that lin-46(lf) causes a severe retarded phenotype in a
lin-14(0) background at 15°C
(Table 4). Thus, the different
genetic interactions lin-46 has with lin-14 and
lin-28 reflect the underlying change in the relationship between
lin-14 and lin-28 that occurs at the end of the L1 stage
with the engagement of the lin-4-independent feedback circuit
(Fig. 5A) (Seggerson et al., 2002
).
lin-46 affects L3 and adult fates independently
Our data also indicate that the role of lin-46 in regulating the
L/A switch appears to be independent of its role in the L2/L3 fate decision.
Specifically, lin-46(lf) animals grown at 20°C display normal
cell lineage patterns early, but at the same temperature there is significant
failure of the L/A switch at the L4 stage (Tables
1 and
4;
Fig. 1). Thus, the failure of
the L/A switch in lin-46(lf) animals can occur without a prior
lineage defect. Because lin-46:GFP reporters are expressed in the L4
stage (Fig. 4B), this
independence of early and late retarded defects may reflect that
lin-46 acts twice, once to promote L3 fates and again later to
promote adult-specific fates. However, we found that the cold-sensitive period
of lin-46(lf) strain for the adult fates is prior to the L3 stage.
This finding suggests that lin-46 normally acts only once at a
branch-point in the heterochronic pathway where some of its targets influence
L3 fates immediately, and other targets, which are more sensitive to its
absence, govern adult fates (Fig.
5A). This latter possibility is consistent with lin-46
acting at a step immediately downstream of lin-28. In our model,
therefore, y may represent multiple factors, some of which affect the
L2/L3 fate decision and the regulation of lin-14, and others which
affect the L/A switch through the other known heterochronic genes
(Fig. 5A). One possibility is
that lin-28 and lin-46 directly affect the microRNA let-7,
which then regulates other genes that control the L/A switch
(Fig. 5A). This is consistent
with the failure of lin-46(lf) to suppress the precocious phenotypes
of the later-acting genes lin-41 and lin-57
(Table 2). Further analysis
will determine whether lin-46 and lin-28 indeed affect the
expression or activity of let-7. Because let-7 or let-7-like
microRNAs have been hypothesized to regulate lin-14, it is an
intriguing possibility that some of factors that constitute y are
microRNAs.
LIN-46 may form a scaffold for a multiprotein assembly
The homology of the LIN-46 protein to MoeA and gephyrin further provides a
basis for explaining its function. The crystal structure of MoeA shows it to
exist as a homodimer, and it is likely that proteins with sequence similarity
to MoeA do so as well (Schrag et al.,
2001; Xiang et al.,
2001
). We have observed that the LIN-46 protein strongly interacts
with itself in a yeast two hybrid assay, further suggesting LIN-46 forms
multimers (L. Tang and E.G.M., unpublished). Gephyrin is believed to dimerize
with itself via its C-terminal MoeA-like region, and trimerize with its
N-terminal MogA-like region (see Fig.
3). The ability of gephyrin to multimerize and form a scaffold is
the basis for its ability to cluster and aid the activity of interacting
proteins (Schwarz et al.,
2001
). Although gephyrin is known to act both in synaptic
transmission and in molybdenum co-factor biosynthesis, LIN-46 does not appear
to have a role in either of these processes in C. elegans; possibly
its paralog, MOC-1, has these roles. We speculate that a general feature of
proteins related to gephyrin, therefore, may be their ability to form
scaffolds that avail them to participating in diverse biological roles,
including development.
Based on several lines of evidence, including the homology to gephyrin, we
propose a molecular model for LIN-46 in which it acts as a scaffolding protein
for a multiprotein complex that determines cell fates
(Fig. 5B). Supporting this
model is the striking cold sensitivity of lin-46 null alleles
(Table 2). Cold sensitivity can
be a feature of mutations affecting the dynamics of multiprotein structures,
such as microtubules and bacteriophage capsids
(Huffaker et al., 1988;
Bazinet et al., 1990
). LIN-46
may potentiate the activity of a complex that includes cell fate regulators or
other developmental timing factors. LIN-46 is not essential for the activity
of this complex, because development of a lin-46 null mutant is
nearly normal at 20°C, but the absence of LIN-46 protein may substantially
reduce the activity of the complex at low temperature, possibly by causing the
retention of a negative regulator of the complex. Because the LIN-28 protein
appears to be a specific mRNA-binding protein
(Moss et al., 1997
), the
expression of some of the components of the complex may be repressed when
lin-28 is normally active (Fig.
5B). The suppression of the loss of lin-28 activity would
therefore result from balance between the overexpression of components of the
complex, and reduced efficiency of their function.
In summary, we believe lin-46 occupies a unique position in the heterochronic pathway and furthers our understanding of the complex genetic network that governs the temporal regulation of developmental events. The fact that the LIN-46 protein resembles gephyrin expands the biological function of this protein family to development. LIN-46 is likely to interact with other proteins in controlling cell fate choices, and the identities of these proteins, which may also be regulatory targets of LIN-28, will provide further insight into how the temporal components of cell fates are determined.
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ACKNOWLEDGMENTS |
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