Department of Biochemistry and Biophysics, University of California, San Francisco, Mission Bay Genentech Hall, 600 16th Street, Room S312A, San Francisco, CA 94143-2200, USA
Author for correspondence (e-mail:
ckenyon{at}biochem.ucsf.edu)
Accepted 26 October 2004
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SUMMARY |
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Key words: Hox co-factor, ceh-20, unc-62, Cell migration, Vulva development, C. elegans
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Introduction |
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The C. elegans Q neuroblasts, QL and QR, and their descendants
migrate long distances along the anteroposterior body axis. QL and QR are born
as bilaterally symmetric cells in the posterior body region of the animal
(Fig. 1A)
(Chalfie and Sulston, 1981;
Sulston and Horvitz, 1977
).
The right Q cell (QR) and its descendants migrate towards the anterior,
whereas the left Q cell (QL) and its descendants migrate towards the
posterior. The stopping points of the Q descendants are not associated with
any obvious landmarks, and the mechanisms by which their final positions are
specified are not well understood.
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The egl-20 gene also influences the migrations of cells in the QL
lineage, but in a different way. In QL, EGL-20 switches on expression of the
Hox gene mab-5, the C. elegans ortholog of Drosophila
Antennapedia, via a canonical Wnt signaling pathway. mab-5 acts
cell-autonomously in the QL lineage, and is necessary and sufficient to
promote the posterior migrations of QL descendants
(Harris et al., 1996;
Salser and Kenyon, 1992
).
In addition to regulating neuroblast migration, the Hox gene
lin-39 also regulates vulva development. The vulva is generated by a
subset of the 12 ventral epidermal cells that are located in a row along the
ventral margin of the animal (Fig.
1B) (for reviews, see
Greenwald, 1997;
Shemer and Podbilewicz, 2003
;
Wang and Sternberg, 2001
).
These cells, called P(1-12).p, are born during the first larval stage. Soon
after their birth, all but the six central Pn.p cells, P(3-8).p, fuse with the
epidermal syncytium, hyp7 (Sulston and
Horvitz, 1977
). P(3-8).p are prevented from undergoing cell fusion
by LIN-39. CEH-20 also participates in cell fusion
(Shemer and Podbilewicz,
2002
). Together with LIN-39, CEH-20 represses eff-1, a
gene required for cell fusion (Mohler et
al., 2002
; Shemer and
Podbilewicz, 2002
). CEH-20 and LIN-39 also regulate the activity
of egl-18 and elt-6, GATA factors that redundantly inhibit
cell fusion (Koh et al.,
2002
). Furthermore, all of the Pn.p cells fused with hyp7 in most
ceh-20(ay42) animals (Shemer and
Podbilewicz, 2002
).
During the L3 stage, three of the six vulval precursor cells (VPCs),
P(5-7).p, are induced by the nearby gonadal anchor cell to generate the vulval
cell lineages. The other VPCs, P(3,4, and 8).p, divide once, and their
daughters then fuse with hyp7. Vulval cell-lineage specification is controlled
by several interacting signaling pathways. The `synMuv' pathway plays an
important role in determining whether a Pn.p cell generates vulval cell
lineages or not. This pathway prevents vulval cell division in the absence of
an inductive signal from the anchor cell (reviewed by
Fay and Han, 2000). Animals
defective in both class A and class B synMuv gene functions display a
multivulval phenotype because most of the vulval precursor cells adopt vulval
fates (Ferguson and Horvitz,
1989
). Many of the synMuv pathway components are members of the
nucleosome remodeling and histone deacetylation (NuRD) complex or proteins
that physically interact with histone deacetylases
(Brehm et al., 1998
;
Lu and Horvitz, 1998
;
Solari and Ahringer, 2000
). By
antagonizing the Ras pathway, the NuRD complex helps to limit the number of
cells adopting vulval fates.
Other signaling pathways involved in vulval lineage specification include:
(1) the Ras, Wnt and Rb pathways, and the SEM-4 transcription factor, which
upregulate lin-39 expression and allow the three Pn.p cells located
closest to the anchor cell to overcome the silencing effects of the synMuv
genes and generate vulval cell lineages
(Chen and Han, 2001a;
Eisenmann et al., 1998
;
Grant et al., 2000
;
Hoier et al., 2000
;
Maloof and Kenyon, 1998
); (2)
the Hox gene lin-39, which upregulates its own expression to permit
vulval induction and to instruct cells to generate vulval cell types instead
of different, posterior-specific, cell types
(Clandinin et al., 1997
;
Clark et al., 1993
;
Maloof and Kenyon, 1998
;
Wang et al., 1993
); and (3)
the forkhead-family transcription factor LIN-31, which prevents a
randomization of lineage patterns (Tan et
al., 1998
). Once the 22 vulval cells have been generated, they
undergo a complex pattern of vulval morphogenesis which includes the migration
of P5.p and P7.p descendants towards the descendants of P6.p during the final
stages of postembryonic development.
In this study, we show that the C. elegans Hox co-factor orthologs ceh-20 and unc-62 are required for two processes that are also regulated by Hox gene lin-39: (1) anterior migration of the QR neuroblast and its descendants; and (2) vulva formation. Surprisingly, we find that ceh-20 and unc-62, but not lin-39, are required for MIG-13 to promote anterior migration. To our knowledge, ceh-20 and unc-62 are the only two genes that have been implicated to function with mig-13 in regulating anterior migration. We also find that ceh-20 and unc-62 are required for several steps in vulva formation, and mutations in these genes result in phenotypes that are starkly different from those previously shown for lin-39 mutants. Thus, in both processes, these Hox co-factor orthologs may function, in part, independently of the Hox gene lin-39.
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Materials and methods |
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N2 wild-type var. Bristol,
tax-4::gfp,
mig-13(mu225) X; tax-4::gfp,
ceh-20(mu290) III; tax-4::gfp,
unc-62(mu232) V; tax-4::gfp,
mab-5(e2088) III; tax-4::gfp,
lin-39(n1760) III; tax-4::gfp,
ceh-20(mu290) III,
ceh-20(mu290) III; muIs32[mec-7::gfp] II,
unc-62(mu232) V,
unc-62(mu232) V; muIs32[mec-7::gfp] II,
unc-62(ku234) V,
unc-62(s472) V unc-46(e177);yDp1(IV;V;f),
mig-13(mu225) X, lin-39(n1760) III,
ceh-20(mu290) lin-39(n1760) III,
unc-62(mu232) V; lin-39(n1760) III,
mab-5(e2088) III,
ceh-20(mu290) mab-5(e2088) III,
unc-62(mu232) V; mab-5(e2088) III,
mig-13(mu31) X,
mig-13(mu225) X,
egl-20(n585) IV,
mig-13(mu31) X; egl-20(n585) IV,
egl-20(mu320) IV,
ceh-20(mu290) III; egl-20(mu320) IV,
ceh-20(mu290) III; egl-20(n585) IV,
ceh-20(mu290) III; mig-13(mu31) X,
unc-62(mu232) V; egl-20(mu320) IV,
unc-62(mu232) V; egl-20(n585) IV,
unc-62(mu232) V; mig-13(mu31) X,
lin-39(n1760) III; egl-20(mu320) IV,
lin-39(mu26) III; mig-13(mu31) X,
muEx89 [Punc-119::mig-13GFP],
mig-13(mu225) X; muEx89 [Punc-119::mig-13GFP],
lin-39(n1760) III; muEx89 [Punc-119::mig-13GFP],
ceh-20(mu290) III; muEx89 [Punc-119::mig-13GFP],
unc-62(mu232) V; muEx89 [Punc-119::mig-13GFP],
ceh-20(mu290) III;muEx261 [ceh-20::gfp + odr-1::rfp],
rrf-3(pk1426); muIs35 [mec-7::gfp],
nDf16/qC1[dyp-19(e1259) glp-1(q339)] III,
unc-62(s472) unc-46(e177) V; yDp1(IV;V;f),
wIs52[scm::gfp::lac-Z; unc-119(+)]
(Koh and Rothman, 2001),
ceh-20(mu290) III; wIs52[scm::gfp::lac-Z; unc-119(+)],
unc-62(mu232) V; wIs52[scm::gfp::lac-Z; unc-119(+)],
jcIs1[jam-1::gfp; rol-6(d)]
(Mohler et al., 1998),
ceh-20(mu290) III; jcIs1[jam-1::gfp; rol-6(d)],
unc-62(mu232) V; jcIs1[jam-1::gfp; rol-6(d)],
ceh-20(mu290) III; ayIs9[egl-17::gfp],
mu232; ayIs9[egl-17::gfp],
muIs3[mab-5::lacZ, rol-6(d)],
ceh-20(mu290) III; muIs3[mab-5::lacZ, rol-6(d)],
unc-62(mu232); muIs3[mab-5::lacZ, rol-6(d)],
ceh-20(mu290) III; unc-62(mu232) V
The positions of the Q.pa daughters were determined directly using Nomarski
optics at the end of L1. At this stage, the hypodermal V cells have divided
once and the P nuclei have all descended into the cord. The positions of the
Q.pa daughters were scored relative to the V cell daughters. The Q.ap cell
positions were determined using an integrated tax-4::GFP fusion
(Sym et al., 1999). Cell
positions of Q descendants were analyzed only in animals without additional
neurons in the body. In principle, this could introduce a selection bias;
however, we addressed this by extensive lineage analysis and other methods
(see Fig. S1 in the supplementary material). If the cell(s) of interest
migrated near a Vn.a cell that had divided, the `Vn.a position' was defined as
the distance bounded by a dorsoventral line through the center of the Vn.aa
nucleolus and a dorsoventral line through the center of the Vn.ap nucleolus.
Statistical analyses on the QR.pax distributions were performed using the
Mann-Whitney test on AVM and SDQR separately.
ceh-20 mutant isolation and characterization
The mu290 mutation was identified in a Nomarski screen for mutants
with misplaced Q.ap cells in mutagenized tax-4::gfp animals. Mutants
were generated with ethyl methanesulfonate (EMS). The QR.pax cell positioning
defect was assayed in mapping experiments. mu290 was mapped using STS
mapping (Williams et al.,
1992) to a 0.7 map unit region tightly linked to the physical
marker stP120 at the center of LG III. Chromosomal deficiencies were
then used to refine the map position. The QR.pax positioning defect was
generated when mu290 was placed in trans to the deficiency
nDf16. Transformation rescue of the QR.pax cell positioning phenotype
was obtained with pools of cosmids in this region. Ultimately, the rescuing
activity of the QR.pax cell positioning defect, as well as the multivulval and
egg-laying defects, were obtained with a single cosmid, F31E3. This cosmid
also rescued the extra ventral protrusion phenotype of mutant hermaphrodites.
A candidate open reading frame for mu290 was F31E3.1, which encodes a
C. elegans ortholog of Exd/Pbx
(Burglin and Ruvkun, 1992
). A
3.6 kb PCR fragment that encompasses this gene was injected at 1 ng/µl with
pPD93.97 (myo-3::gfp) at 100 ng/µl into mu290 animals.
Four out of four lines generated with this PCR fragment gave 48-75% rescue of
the QR.pax defect and over 85% rescue of the vulval defect. To identify the
molecular lesion in ceh-20(mu290), we isolated and sequenced
cDNA clones generated by RT-PCR (Frohman,
1993
) from wild-type and ceh-20(mu290) DNA. The
oligo(dT) primer was used to obtain the 3' end and the spliced leader
sequence SL1 primer to obtain the 5' cDNA end. Only a single isoform was
isolated from several clones. A single G to A transition was identified in the
cDNA product derived from ceh-20(mu290). This mutation
changes a conserved arginine (R) in the 3rd helix of the homeodomain to a
histidine. This R has been implicated as part of a nuclear localization signal
(NLS) in the third helix of the homeodomain that is less conserved than the
classical NLS RRKRR found at the N-terminal arm of the homeodomain. The R that
is mutated in ceh-20(mu290) is the second R in this less conserved
and weaker NLS, KRIRYKKNI (Abu-Shaar
et al., 1999
; Saleh et al.,
2000a
). ceh-20(mu290)/qC1[dyp-19(e1259) glp-1(q339) III;
him-5(e1490) was used for crosses since it was very difficult to mate
into ceh-20(mu290). Animals were outcrossed at least three times
before phenotypes were studied.
unc-62 mutant isolation and characterization
The mu232 mutation was identified in a screen for mutants with
misplaced Q.pax cells in mutagenized mec-7::gfp animals, as described
by Ch'ng et al. (Ch'ng et al.,
2003). In mu232/+ animals, the QR.pax cells were in
wild-type positions, indicating that mu232 is recessive and thus
likely to be a reduction or loss-of-function allele. We mapped mu232
to chromosome VL near stP3 and unc-62. mu232 failed to
complement s472, a null unc-62 allele, for the QR.pax
cell-positioning defect, suggesting that mu232 is an allele of
unc-62. mu232/s472 animals have a stronger QR.pax phenotype
than mu232 animals, consistent with mu232 being a hypomorph.
unc-62 exons were amplified from wild-type and mu232 using
primers around each predicted exon. Sequencing these genomic fragments
revealed that mu232 harbored a T
A transition that changed the
predicted initial Met(ATG) codon of exon 1b to a Lys(AAG) codon. We also
sequenced unc-62(ku234) (Van
Auken et al., 2002
) and discovered a G
A transition in the
same codon. mu232 failed to complement ku234 (gift from
Meera Sundaram) for the QR.pax positioning and Egl phenotypes. Because
mu232 and ku234 exhibited the lowest percentage of embryonic
and larval lethality among the known unc-62 homozygous alleles, we
examined larval phenotypes using mu232 after noting that
mu232 and ku234 exhibit very similar migration and vulval
phenotypes. mu232 was outcrossed more than three times before
phenotypes were studied.
Ectopic expression of mig-13
After verifying that the extrachromosomal array,
muEx89(Punc-119::mig-13GFP) (Q. Ch'ng, PhD
thesis, University of California, 2001), could create an anterior-promoting
environment mimicking that created by hs-mig-13 [see
Fig. 5 for rescued positions of
QR.pax and BDU in mig-13(-)], we crossed muEx89 into
ceh-20(mu290), unc-62(mu232), and lin-39(n1760). In animals
that were green, we believe that mig-13::GFP was ectopically
expressed because ALM (data not shown) and CAN (see Fig. S4 in the
supplementary material) were redirected to more anterior positions.
|
Immunofluorescence and ß-galactosidase detection
LIN-39 antibody staining was performed as described by Maloof and Kenyon
(Maloof and Kenyon, 1998).
Staged populations of animals were stained with both the LIN-39 antibody and
the monoclonal antibody MH27 (Francis and
Waterston, 1991
). Individual slides with animals of different
genotypes were stained simultaneously under identical conditions prior to
comparison, and the differences in staining intensity were repeatable. For
mab-5 expression, staged populations of muIs3[mab-5::lacZ,
rol-6(d)], ceh-20(mu290); muIs3[mab-5::lacZ, rol-6(d)],
and unc-62(mu232); muIs3[mab-5::lacZ, rol-6(d)] animals were
grown at 20°C. Larvae were fixed on slides and stained as described by
Maloof et al. (Maloof et al.,
1999
).
Lineage analysis and microscopy
Analysis of Q lineages and Vn.a lineages were performed from hatching to
late L1/early L2. Of the 27 wild-type Q cells whose lineages were analyzed,
Q.pp and Q.aa were still present by late L1/early L2 in only four (14.8%) and
three animals (11.1%), respectively, and none of the Q cell descendants
divided inappropriately. Vulval lineages were observed starting with the Pn.px
cell stage until the L4 molt by Nomarski optics.
RNA interference
rrf-3(pk1426) is more sensitive than wild-type animals to RNAi
(Simmer et al., 2002).
rrf-3(pk1426); muIs35[mec-7::gfp] animals in L4 were grown on
bacteria containing vector only or on bacteria expressing ceh-20
dsRNA, unc-62 dsRNA or lin-39 dsRNA. Their progeny were
analyzed for QR.pax positions in late L1, vulval induction in L4, and number
of ventral protrusions as young adults. Animals fed with the control plasmid
did not display the QR.pax positioning defect, vulval induction defects or
additional ventral protrusions.
Characterization of vulval phenotypes
Pn.p cell fusion status was scored by first crossing jam-1::gfp
(Mohler et al., 1998) into
ceh-20(mu290) and unc-62(mu232). The early L1
fusion event was scored in L2 when the adherens junctions of unfused cells
were visible as a circle. The later fusion event was scored in L3.
Vulval induction was scored at the L4 stage under Nomarski optics
(Sternberg and Horvitz, 1986).
Nuclei that were not hypodermal, neuronal or muscle were counted. The number
of vulval nuclei was used to extrapolate the number of induced Pn.p cells. A
Pn.p cell in which both daughter cells divide one more time, and both
granddaughters divide to generate seven or eight great granddaughters and no
hypodermal tissue was scored as 1.0 cell induction. A Pn.p, which generates
more than two but fewer than seven or eight cells was scored as 0.5 cell
induction. In wild-type, P5.p, P6.p and P7.p each undergo a 1.0 cell induction
whereas the other Pn.p cells are not induced, resulting in a total cell
induction of 3.0.
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Results |
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In 90% of ceh-20(mu290) animals, QR.ap was located between
the birthplace of QR and its wild-type location in the head
(Fig. 2). In addition, the
QR.pax cells were in the central body region rather than in the anterior
(Fig. 3), suggesting that cells
in the QR lineage stopped migrating prematurely. In
33% of
ceh-20(mu290) animals, QL.ap was not located in its normal position
in the tail (Fig. 2). In fact,
QL.ap could be found anterior to the birthplace of QL in
10% of
ceh-20 mutants, suggesting that this cell had migrated anteriorly
instead of posteriorly.
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To determine whether these phenotypes result from reduced ceh-20
activity, we first analyzed ceh-20(mu290)/+ heterozygotes. In
ceh-20(mu290)/+ animals, the QR.pax cells were mostly in wild-type
positions (see Fig. S1 in the supplementary material) and there were no extra
body neurons visible. We also found that the QR.pax phenotype was not enhanced
in ceh-20(mu290)/deficiency heterozygotes. Finally, as RNA
interference reduces gene activity, we fed ceh-20 dsRNA to an
RNAi-sensitive strain, rrf-3
(Simmer et al., 2002). The
progeny of these animals showed similar defects to those in
ceh-20(mu290) animals, including QR.pax cells in the mid-body region
and extra body neurons (see Fig. S2 in the supplementary material). Taken
together, these analyses indicate that ceh-20(mu290) is likely to
reduce gene function. Therefore, we conclude that in wild-type animals,
ceh-20 is required for the proper generation and migration of cells
in the Q lineage. ceh-20 may also have an earlier function in Q
development because, in stronger alleles of ceh-20 (obtained from M.
Stern), the Q cell failed to complete its lineage (3/3 animals; data not
shown).
unc-62 encodes a homothorax-like gene also required for Q descendant migration
We also screened for Q migration mutants using
mec-7::gfp, which is expressed in QR.paa and QL.paa
(Ch'ng et al., 2003). In this
screen, we isolated mu232, a mutant with QR.pax cells located
posterior to their normal stopping points
(Fig. 3). Subsequent genetic
and molecular analyses (Materials and methods) indicated that mu232
is a reduction-of-function allele of the C. elegans homothorax
(hth) ortholog unc-62, a member of the Meis family of Hox
co-factors.
In Drosophila, hth and exd mutants have similar
phenotypes. Likewise, we found that unc-62 mutants share the cell
positioning defects exhibited by ceh-20 mutants. In unc-62
mutants, 70% of QR.ap cells and 100% of QR.pax cells stopped posterior to
their wild-type positions, in distributions similar to those in
ceh-20 mutants (Figs 2
and 3). Likewise, in 70%
of unc-62 mutants, QL.ap was located anterior to the birthplace of QL
(Fig. 2). Finally, QL.pax cells
were in wild-type positions in unc-62 mutants, as in ceh-20
mutants (data not shown). The one Q phenotype that ceh-20(mu290)
animals exhibited that unc-62(mu232) animals did not was the Q
lineage defect.
Mutations in ceh-20 and unc-62 also affected the
anteriorwards migration of BDU, a neuron whose cell body migrates a short
distance anteriorly during embryogenesis. In 70% of animals with a
mutation in ceh-20(mu290) or unc-62(mu232), this migration
was incomplete (see below and Fig.
5).
ceh-20 and unc-62 guide migrating cells
In theory, the shortened anterior migration of cells in mutants could
simply be due to a requirement for CEH-20 and UNC-62 activity in the basic
mechanisms of cell motility. However, we found that a mutation in
ceh-20 or unc-62 could also change the direction of cell
migration. First, direct observation of cell movements by Nomarski optics
revealed that QR.a or QR.p migrated towards the posterior rather than the
anterior direction in two out of nine ceh-20(mu290) animals. In
addition, these ceh-20 or unc-62 mutations could redirect
the anterior-bound cells of the QR lineage towards the posterior when combined
with a mutation in a Wnt homolog egl-20 (see below and
Fig. 4). Although we cannot
exclude the possibility that ceh-20 and unc-62 are required
for some aspect of cell motility, these findings indicate that CEH-20 and
UNC-62 activities are required for the guidance or positioning of cells along
the AP axis.
|
ceh-20 and unc-62, but not lin-39, are required for MIG-13 to position cells along the A/P axis
QR descendants may migrate further anteriorly in lin-39 mutants
than they do in ceh-20 or unc-62 mutants because cells in
lin-39 mutants, but not in ceh-20 or unc-62
mutants, retain the ability to respond to MIG-13. Consistent with this
hypothesis, the distribution of QR.pax cells in the mig-13(mu31);
lin-39(n1760) double mutant was shifted further posteriorly than in
either single mutant (data not shown) (Sym
et al., 1999). This distribution nearly overlapped with the
distribution seen in ceh-20 or unc-62 single mutants,
raising the possibility that ceh-20 and unc-62 may have a
function in the mig-13 pathway. A mig-13(mu31null) mutation
can cause cells in the QR lineage to reverse direction and migrate toward the
posterior in an egl-20(-) background
(Fig. 4A)
(Harris et al., 1996
;
Sym et al., 1999
). Similarly,
in ceh-20 or unc-62 double mutants with a null or
haploinsufficient allele of egl-20, QR.pax cells could be posterior
to the birthplace of QR (Fig.
4B,C). However, combining null mutations in lin-39 and
egl-20 did not cause cells in the QR lineage to migrate posteriorly
(Fig. 4D). In addition,
removing mig-13 activity from ceh-20(mu290) or
unc-62(mu232) did not result in the posterior migration of QR
descendants (Fig. 4B,C).
Finally, in ceh-20(mu290); unc-62(mu232) double mutants, there were
QR.pax cells posterior to the birthplace of QR (see Fig. S3 in the
supplementary material). Collectively, these results suggest that
ceh-20 and unc-62, but not lin-39, may act in the
same pathway as mig-13 to promote anterior migration.
To test directly whether ceh-20 and unc-62 were required for cells to respond to the anterior-promoting activity of mig-13, we asked whether expression of mig-13::gfp in our ceh-20(mu290) and unc-62(mu232) mutants using the pan-neural unc-119 promoter (see Materials and methods) could rescue the shortened anterior migrations of cells in the Q lineage. In mig-13(null) and lin-39(null) animals, pan-neuronal mig-13 expression could rescue the QR.pax positioning defect; however, in ceh-20(mu290) or unc-62(mu232) animals it could not (Fig. 5). In addition, Punc-119::mig-13 failed to fully rescue a different anterior cell migration, that of BDU, in ceh-20 and unc-62 mutants (Fig. 5). In these mutant backgrounds, pan-neuronal expression of mig-13 could not fully restore anterior migration of BDU or cells in the QR lineage but could shorten the posterior migration of another cell, CAN (see Fig. S4 in the supplementary material). Thus, we believe that mig-13 was appropriately misexpressed in these mutants. Together, these results suggest that ceh-20 and unc-62, but not lin-39, are required for QR descendants and BDU to respond to the anterior-promoting activity of mig-13.
Shortened anterior migration defect of QR descendants in ceh-20 and unc-62 mutants is not due to decreased lin-39 or increased mab-5 expression in these cells
Another possible explanation for the truncated anterior migration of QR
descendants in ceh-20 and unc-62 mutants was that cells in
the QR lineage inappropriately express the Hox gene mab-5. If so,
then removing mab-5 activity in our ceh-20 or
unc-62 mutants should allow QR descendants to migrate further
anteriorly. However, the cell distributions of QR.p descendants in the
mab-5(-) double mutant with either ceh-20(mu290) or
unc-62(mu232) were nearly indistinguishable from those of
ceh-20(mu290) or unc-62(mu232) single mutants, respectively,
indicating that ectopic expression of mab-5 was unlikely to underlie
the migration shortening in these mutants
(Fig. 3). In concordance with
this finding, mab-5::lacZ was not ectopically expressed in
QR or its descendants in these mutants (data not shown).
If CEH-20 and UNC-62 do not repress mab-5 expression in QR
descendants, then do they regulate migration of these cells by controlling
lin-39 expression? By staining wild-type animals with an antibody
against LIN-39, we show that QR could express lin-39 in its nucleus
before it delaminates from the epithelium, but expression at this early time
is usually not high (see Fig. S5A in the supplementary material, Table 1). After delamination
but before division, more QR cells expressed lin-39 strongly (see
Fig. S5B in the supplementary material). After QR divides, lin-39
expression persisted and continued throughout the lineage. In
unc-62(mu232) animals, lin-39 expression in QR and its
daughters resembled that of wild-type
(Table 1). In
ceh-20(mu290) animals, however, lin-39 expression was often
stronger than that of wild-type in QR before delamination, after delamination
and after division (see Fig. S5D-F in the supplementary material,
Table 1). Neighboring V and P
cells in ceh-20(mu290) also had increased LIN-39 staining. These
differences were validated in many repeated staining comparing wild-type and
ceh-20(mu290) animals under identical staining conditions. Because
lin-39 overexpression does not inhibit anterior cell migration in the
QR lineage (Hunter and Kenyon,
1995), the altered lin-39 expression we observed in
ceh-20 mutants was unlikely to contribute to the shortened migrations
of these cells.
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Mutations in ceh-20 or unc-62 disrupt several aspects of vulva formation
In addition to having Q lineage defects, all ceh-20(mu290) and
unc-62(mu232) hermaphrodites were egg-laying defective (Egl).
Furthermore, all ceh-20(mu290) hermaphrodites formed `bags of worms'
as their progeny hatched internally. Instead of having a single, centrally
located vulval protrusion, as in wild-type, 94% (n=100) of
ceh-20(mu290) mutants have multiple vulval protrusions (Muv
phenotype) on the ventral surface. We tested whether this phenotype was a
loss-of-function phenotype, using RNA interference and found that over 20%
(n=50) of the progeny of animals fed bacteria expressing
ceh-20 dsRNA had more than one ventral protrusion. This finding,
together with our finding that mu290/+ animals have no ventral
protrusions (100%, n=132), suggest that mu290 reduces the
level of an activity of ceh-20 required for normal vulva
formation.
Extra ventral protrusions can be caused by ectopic induction of Pn.p cells
that do not normally contribute to vulva formation, or by defective
morphogenesis (Chen and Han,
2001b; Sternberg and Han,
1998
). Examination of vulva development using Nomarski optics
revealed that in over 95% of ceh-20(mu290) hermaphrodites, Pn.p cells
that do not normally undergo vulva development, that is, P3.p, P4.p and/or
P8.p, are often ectopically induced to form a vulval invagination
(Fig. 6B;
Table 2). In addition, anterior
and posterior Pn.p cells that normally fuse with the hypodermal syncytium,
P(1,2,9-11).p, could also be induced so that their descendants formed a small
invagination (Fig. 6C). In the
six ceh-20(mu290) hermaphrodites lineaged, P1.p divided in two
animals, P1.px divided in one animal, P2.p divided in four animals, P2.px
divided in one animal, P3.px divided in three animals and P8.px divided in one
animal. In one animal, not only were the great-granddaughters of P4.p
inappropriately generated, but one even divided despite the fact that even the
great-granddaughters of P(5-7).p do not normally divide. In contrast to these
ectopic inductions, vulval precursor cells that are normally induced,
P(5-7).p, were sometimes not induced in ceh-20(mu290) hermaphrodites
(Table 2). When they were
induced, P(5 or 7).p descendants could form invaginations separate from the
one formed by the P6.p descendants (Fig.
6B), suggesting that P5.p or P7.p descendants sometimes failed to
migrate toward P6.p descendants during morphogenesis (48%, n=50).
Curiously, P7.p descendants formed a separate invagination or failed to fully
migrate towards P6.p descendants in
40% of unc-62(mu232)
(Fig. 6F) and
50% of
unc-62(ku234) hermaphrodites (n=25 per strain). This defect
probably contributes not only to their Egl phenotype but also to the 8% of
animals of each strain which had an extra ventral protrusion (n=100
per strain). In ceh-20(RNAi) animals, we also observed ectopic
induction of P3.p and P4.p (Fig.
6D, Table 2) as
well as separate invaginations formed by P5.p or P7.p (10%, n=50).
Thus, both ectopic induction and defective morphogenesis contribute to the
multiple ventral protrusions in hermaphrodites with reduced CEH-20 activity,
and UNC-62 activity is also required for proper morphogenesis.
|
|
Because the extreme anterior and posterior Pn.p cells, (P1,2,9-11).p, often
divided in ceh-20(mu290) mutants, we hypothesized that these cells
failed to fuse to the hypodermal syncytium in mutant animals. To characterize
this defect better, we used the jam-1::GFP marker, which labels cell
adhesion junctions of unfused, but not fused, Pn.p cells
(Mohler et al., 1998). In
wild-type hermaphrodites, only P(3-8).p remain unfused after the early fusion
event during L1. P3.p fuses by L3 in
50% of wild-type animals. In
ceh-20(mu290) hermaphrodites, as in wild-type, the six central Pn.p
cells remained unfused, but P1.p, P2.p and P(9-11).p sometimes also failed to
fuse during the early fusion event (Table
3A). Some anterior and posterior Pn.p cells, as well as P3.p,
still remained unfused in the L3 stage
(Table 3B), suggesting that
CEH-20 activity is required to generate the proper number of VPCs. Like
ceh-20(mu290) animals, unc-62(mu232) hermaphrodites also
exhibited the Pn.p cell fusion defect, although with lower penetrance
(Table 3).
|
ceh-20 and unc-62 also pattern V cells
A row of six lateral hypodermal cells (V1-V6) runs along each side of newly
hatched wild-type animals. During the first larval stage, each V cell divides
asymmetrically along the AP axis, generating an anterior daughter (Vn.a) that
fuses with hyp7 and a posterior daughter (Vn.p) that adopts the seam cell fate
and continues to divide (Sulston and
Horvitz, 1977).
We found that the nuclei of the anterior daughters of some V cells were
sometimes replaced by two small nuclei in ceh-20(mu290) animals, and
more often in unc-62(mu232) animals (see Fig. S7 in the supplementary
material, Table 4). We
determined that the two small nuclei were indeed daughters of Vn.a cells by
lineage analysis (data not shown). These cells did not express a seam-specific
GFP marker (Koh and Rothman,
2001) and thus were not transformed into Vn.p-like seam cells
(data not shown). Instead, these cells appeared to fuse with hyp7: first, the
adherens-junction marker jam-1::GFP failed to label these cells at
the L2 stage (data not shown); and second, we determined the lineage of a
dividing Vn.a cell in an unc-62(mu232) animal harboring
jam-1::GFP and found that there was only a short period of time when
Vn.ax cells were outlined in green (see Fig. S8 in the supplementary
material). Because the cell outlines of Vn.ax can be observed, albeit
transiently, Vn.a division occurs before fusion. Whether ceh-20 and
unc-62 actively inhibit cell division or simply promote fusion of
Vn.a cells so that cell division cannot occur is currently unknown.
|
ceh-20 is expressed in Q, P, and V cells and their descendants
We constructed a ceh-20::gfp translational fusion gene
(pLY11) to investigate where and when ceh-20 is expressed. Transgenic
ceh-20(mu290) animals bearing pLY11 were rescued for the QR.pax
positioning phenotype, the Muv/Egl, and the Vn.a division phenotype,
suggesting that ceh-20::gfp was expressed in cells that require its
function for these processes. In all cells expressing ceh-20, the
expression was stronger in the nucleus than in the cytoplasm. We detected
ceh-20::gfp expression in QR and QL
(Fig. 7A,B) and their
descendants throughout their migrations to the end of L1. V cells and their
daughters also expressed ceh-20::gfp
(Fig. 7A-C). Expression
persisted in the descendants of the V cells through the adult stage (data not
shown). At hatching, all P cells expressed ceh-20::gfp
(Fig. 7A,B). Before the
anterior and posterior Pn.p cells fuse, they also expressed ceh-20.
In L3 hermaphrodites, expression was maintained in P(3-8).p
(Fig. 7D). We identified
ceh-20::gfp expression in several other cell types. These
included M, BDU, ALM, HSN, body wall muscle cells, I4, all L1 ventral cord
neurons and a few unidentified neurons in the head behind the posterior bulb
of the pharynx. ceh-20::gfp expression and nuclear localization did
not change in the unc-62(mu232) background (data not shown).
|
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Discussion |
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CEH-20 and UNC-62 are required for a step in Q cell migration that involves the transmembrane protein MIG-13
Our interest in these ceh-20 and unc-62 mutants was
initially stimulated by the fact that their cell migration phenotypes are
quite similar to those produced by loss-of-function mutations of the
transmembrane protein MIG-13 (Sym et al.,
1999). Mutations in all of these genes prevented the anteriorwards
migration of BDU and severely shortened the anteriorwards migration of the QR
descendants. Moreover, we found that our ceh-20 and unc-62
mutations, in combination with other Q migration mutants, produced phenotypes
similar to those we had seen previously with mig-13. For example,
combining a unc-62 or ceh-20 mutation with a lin-39
null allele abolished nearly all anteriorwards migration of the Q descendants.
Combining an unc-62 or ceh-20 mutation with an
egl-20/Wnt null mutation could cause cells in the QR lineage to
reverse direction and migrate posteriorly rather than anteriorly. Finally, we
found that global overexpression of MIG-13 was unable to stimulate
anteriorwards migration in ceh-20 or unc-62 mutant
backgrounds. Together, these findings suggest that, in Q cell migration,
ceh-20 and unc-62 may function in the same regulatory step
as MIG-13. The possibility that ceh-20 and unc-62 are simply
required for mig-13 expression is ruled out by the finding that these
ceh-20 and unc-62 mutations do not affect the pattern of
MIG-13::GFP (data not shown). Because CEH-20 and UNC-62 are known to regulate
transcription, one possibility is that they regulate the expression of
another, as yet unidentified, molecule that functions in the same process as
MIG-13 to promote anteriorwards migration. Alternatively, CEH-20 and UNC-62
may act in a pathway that is independent of and parallel to other pathways
involving LIN-39, MIG-13, and EGL-20. Our data would also be consistent with
such a model in which CEH-20 and UNC-62 act in a pathway that is dominant over
these other pathways, increasing the difficulty with which mig-13
overexpression rescues the shortened anterior migration in ceh-20 or
unc-62 mutant animals. Determining which cells require
ceh-20 and unc-62 for proper Q descendant
migration and identifying ceh-20 and unc-62 effector(s) may
facilitate the understanding of mechanisms that regulate cell migration along
the anteroposterior axis in C. elegans.
Roles of ceh-20 and unc-62 in vulva development
Our genetic studies of ceh-20 and unc-62 demonstrate that
they play multiple roles in regulating vulva development. First, in
ceh-20 and unc-62 single mutants, cells anterior and
posterior to the VPCs sometimes fail to fuse with the syncytial hypodermis.
Although ectopic lin-39 expression could explain this phenotype, it
does not, because, as in wild type, lin-39 is expressed in only the
six central Pn.p cells in ceh-20 and unc-62 mutants even
when some anterior and posterior Pn.p cells are unfused (data not shown).
Thus, UNC-62 and CEH-20 appear to regulate cell fusion independently of
LIN-39. Second, ceh-20 is required to prevent cells outside of the
vulval equivalence group from generating vulval cell lineages and also for
ensuring that the central VPCs do generate vulval cell lineages. Consistent
with this, 26% of P6.p cells in L3 ceh-20(mu290) hermaphrodites
(versus 0% in wild-type) fail to express egl-17::gfp, an early marker
for the primary fate (data not shown). Although we did not see induction
and/or lineage defects in unc-62(mu232) mutants, it is probable that
unc-62 is also involved in these processes since non-vulval VPC
descendants and vulval-like descendants of Pn.p cells outside of the vulval
equivalence group have been observed when a weak unc-62 allele has
been placed over a unc-62(null) or a deficiency encompassing the
unc-62 gene (Van Auken et al.,
2002
). In addition, cell divisions of P(5-7).p were often
prematurely terminated or misoriented in ceh-20(mu290)
hermaphrodites, suggesting that CEH-20 is required for the proper execution of
the primary and secondary vulval fates. Finally, in ceh-20 and
unc-62 mutants, the descendants of P5.p and P7.p sometimes failed to
migrate towards the descendants of P6.p
(Fig. 6)
(Van Auken et al., 2002
),
indicating that proper morphogenesis of the VPC descendants requires CEH-20
and UNC-62 activity. Thus, both ceh-20 and unc-62
participate in multiple steps in vulva formation.
The vulval phenotypes exhibited by ceh-20(mu290) animals are
reminiscent of those in NuRD complex mutants such as lin-40 MTA
(metastasis associated factor) and hda-1 HDAC (histone deacetylase).
Like ceh-20(mu290) animals, lin-40 or hda-1 mutants
also have disrupted axes of transverse divisions, and their P5.p and P7.p
descendants may form a separate invagination
(Chen and Han, 2001b;
Dufourcq et al., 2002
).
Animals doubly mutant for lin-40 and a class B synMuv gene also
exhibit underinduction of P(5-7).p and overinduction of P(3,4,8).p
(Chen and Han, 2001b
). In
addition, mutating lin-40 increases the frequency of unfused P3.p
cells, and, in an egl-27 MTA mutant background, a lin-40
mutation causes posterior P(9-11).p cells to also remain unfused
(Chen and Han, 2001a
).
We note that whereas the VPCs remained unfused in our
ceh-20(mu290) animals, these cells have been reported to fuse with
hyp7 in ceh-20(ay42) mutants
(Shemer and Podbilewicz,
2002). One possible explanation for this discrepancy is that,
although both mutations alter residues in the conserved homeodomain, they may
disrupt different functions of ceh-20: mu290 changes a
residue that is predicted to form hydrogen bonds with the guanine of the TGAT
core in the major groove of the DNA, whereas ay42 changes a residue
that is predicted to contact the Hox protein (E. Chen, PhD thesis, Yale
University, 1996) (Passner et al.,
1999
; Piper et al.,
1999
).
How might ceh-20 and unc-62 interface with known
regulators of vulva development? It has been shown that CEH-20, together with
LIN-39, participates in Pn.p fusion by regulating the expression of fusion
effector eff-1 and GATA factors egl-18 and elt-6
(Koh et al., 2002;
Shemer and Podbilewicz, 2002
).
Our findings suggest that CEH-20 and UNC-62 may have additional functions
during vulva formation. The C. elegans NuRD complex components
repress transcription during vulval fate specification by regulating chromatin
structure (Ahringer, 2000
). A
recent study demonstrated that in mammalian cells, the ceh-20 homolog
PBX1 can interact with histone deacetylase HDAC
(Saleh et al., 2000b
). By
analogy to their mammalian counterparts, CEH-20 might interact with HDA-1
(HDAC). Thus, in general terms, our findings raise the possibility that CEH-20
and UNC-62 are required either for the expression of specific NuRD complex
proteins, or for the proper function of the NuRD complex during vulva
development (see L. Yang, PhD thesis, University of California, San Francisco,
2003). Future work analyzing the relationships between ceh-20,
unc-62, and NuRD complex components may provide a more comprehensive view
on how vulva development is controlled in C. elegans.
CEH-20 and UNC-62 may act independently of Hox protein LIN-39 in some processes regulated by all three genes
Mutations in ceh-20 and unc-62 affect processes that Hox
gene lin-39 also affects, such as cell migration and vulva
development. Because ceh-20 and unc-62 have been shown to
act as Hox co-factors in the C. elegans mesoderm and embryo
(Liu and Fire, 2000;
Van Auken et al., 2002
), we
considered the possibility that the ceh-20(mu290) and
unc-62(mu232) phenotypes could be caused by altered target-site
specificity of LIN-39. However, based on the many phenotypic differences we
observe between ceh-20(mu290) or unc-62(mu232) and
lin-39(n1760) animals, it is probable that ceh-20 and
unc-62 have functions that are at least partly independent of
lin-39 in the ectoderm.
Some of the Ceh-20 and Unc-62 phenotypes we observed were initially
suggestive of a LIN-39-co-factor role for CEH-20 and UNC-62. For example, in
ceh-20(mu290), unc-62(mu232) and lin-39(null) mutants,
anterior migrations of QR descendants are shortened. Consistent with this,
ceh-20 is expressed in the nucleus of QR and its descendants where
lin-39 acts autonomously (Clark
et al., 1993; Wang et al.,
1993
). In vulva development, our observation that P(5-7).p cell
divisions sometimes terminated prematurely in ceh-20(mu290) animals
is reminiscent of that seen in lin-39 mutants under conditions in
which VPC fusion to the hypodermis was prevented and in animals with reduced
activity of a LIN-39 target, egl-18
(Koh et al., 2002
;
Shemer and Podbilewicz,
2002
).
Other phenotypes, however, suggest that ceh-20 and unc-62
may function somewhat independently in processes that lin-39 also
regulates. For example, QR descendants migrate further anteriorly in
lin-39(null) mutants than in ceh-20(mu290) or
unc-62(mu232) animals. In addition, unlike ceh-20(mu290) or
unc-62(mu232) animals, lin-39(null) mutants do not share the
shortened BDU migration phenotype with mig-13 mutants or exhibit QR
descendants migrating in the wrong direction in the absence of egl-20
(Harris et al., 1996;
Sym et al., 1999
).
Furthermore, ectopic mig-13 expression can rescue the anterior
migration defect of QR descendants in lin-39(null) animals but not in
ceh-20(mu290) or unc-62(mu232) animals. For this reason, the
hypothesis that ceh-20 and unc-62 act in a mig-13-
dependent process rather than a strictly lin-39-dependent process
seems reasonable. Although it is theoretically possible that another C.
elegans homolog of exd, ceh-40, is also involved in this
process, we have observed no migration phenotypes upon RNA interference of
ceh-40 in the rrf-3 background, and no enhancement of
migration phenotypes in the ceh-20; rrf-3 background (data not
shown).
In vulva development, we observed that many ceh-20(mu290) and
ceh-20(RNAi) hermaphrodites, and some unc-62(mu232) animals,
are multivulval, whereas lin-39(null) mutants are vulvaless. Removing
lin-39 activity causes all Pn.p cells, including P(3-8).p, to fuse in
late L1 (Clark et al., 1993;
Wang et al., 1993
). By
contrast, in ceh-20(mu290) and unc-62(mu232) mutants,
P(3-8).p remain unfused as in wild type, and Pn.p cells that normally fuse
sometimes fail to fuse. In lin-39(null) animals rescued for the early
fusion defect, P(5-7).p often fail to complete their lineages, but induction
of P(3,4,8).p has not been observed
(Maloof and Kenyon, 1998
). In
ceh-20(mu290) and ceh-20(RNAi) animals, however, P(3,4,8).p
could also be induced, and, for ceh-20(mu290), this ectopic induction
could occur even in a lin-39(null) background
(Table 2). In addition,
ceh-20 and unc-62 have a role in vulval morphogenesis
(Fig. 6), whereas
lin-39 has been shown to have no role in promoting cell migration
during vulva formation (Shemer and
Podbilewicz, 2002
).
Although it has previously been shown that CEH-20/LIN-39 heterodimers bind
enhancers of GATA factors that regulate P(3-8).p fusion
(Koh et al., 2002) and that
CEH-20 and LIN-39 together repress the fusion effector eff-1
(Shemer and Podbilewicz,
2002
), we hypothesize that ceh-20 and unc-62
have some functions that are independent of lin-39 for vulva
formation based on the stark differences in their phenotypes. We speculate
that there are partners for CEH-20 and UNC-62 besides LIN-39 in the Pn.p
lineages. As vulval phenotypes have not been shown for other C.
elegans Hox gene loss-of-function mutants, it is possible that CEH-20
pairs with a non-Hox homeodomain protein. There is precedence for this,
because a Drosophila ortholog of CEH-20, EXD acts as a co-factor for
the non-Hox homeodomain protein Engrailed
(Peifer and Wieschaus, 1990
;
van Dijk and Murre, 1994
).
Comparison of ceh-20 and unc-62 with their Drosophila and vertebrate homologs
Do ceh-20 and unc-62 function in similar ways as their
homologs? First, previous studies in Drosophila and vertebrates have
demonstrated that the phenotypes produced by mutations in the homologs of
ceh-20 and unc-62 resemble one another. For example, in
Drosophila, exd and hth mutants both exhibit posterior
transformations of embryonic segments
(Peifer and Wieschaus, 1990;
Rieckhof et al., 1997
). In
zebrafish, mutations in either lazarus or meis (the
exd and hth homologs, respectively) cause defects in
hindbrain segmentation (Choe et al.,
2002
; Popperl et al.,
2000
; Waskiewicz et al.,
2001
; Waskiewicz et al.,
2002
). Here, we show that this property of these genes is
conserved: mutations in C. elegans ceh-20 and unc-62 both
disrupt, in similar ways, the migration of Q descendants, Pn.p fusion, vulval
morphogenesis and the patterning of V cell descendants.
Second, as mentioned above, in many situations, mutations in Hox co-factor
genes produce phenotypes that mimic those of Hox mutants. In fact,
Drosophila exd and hth were identified based on their
sharing embryonic segmentation defects with Hox mutants
(Peifer and Wieschaus, 1990;
Rieckhof et al., 1997
). In
C. elegans, although there are some similarities between the Q
migration defects of unc-62, ceh-20 and Hox gene mutants, the details
of their phenotypes are very different. The same holds true for vulva
development. Thus, ceh-20 and unc-62 may have functions
independent of lin-39 in anteriorwards migration and vulva
development. In Drosophila, Hox-independent functions of exd
and hth have been demonstrated for antennal formation
(Casares and Mann, 1998
;
Yao et al., 1999
). However, in
contrast to the C. elegans situation in which lin-39 is
required and expressed in the cells with defects in migration, fusion or
division, Drosophila Hox genes are neither required nor expressed in
the eye-antennal disc from which the antenna forms
(Casares and Mann, 1998
;
Clark et al., 1993
;
Wang et al., 1993
;
Yao et al., 1999
).
Third, Hox gene expression is regulated, in part, by Hox co-factors. In
Drosophila, exd and hth are required to maintain
Sex-combs reduced expression in the salivary gland
(Henderson and Andrew, 2000).
In zebrafish, Hox genes interact with their co-factors to cross-regulate the
expression of other Hox genes (Maconochie
et al., 1997
; Popperl et al.,
1995
; Studer et al.,
1998
). As a result, reducing the function or altering the
intracellular localization of zebrafish lazarus or meis/prep
genes resulted in decreased expression of various Hox genes.
(Choe et al., 2002
;
Popperl et al., 2000
;
Waskiewicz et al., 2001
). In
C. elegans, although ceh-20 does not activate mab-5
or lin-39 expression in the mesoderm, it is required for the
autoregulatory expression of ceh-13, the C. elegans ortholog
of Drosophila Hox gene labial
(Liu and Fire, 2000
;
Streit et al., 2002
). We found
that lin-39 expression was upregulated, instead of downregulated, in
the Q cells of some animals with reduced ceh-20 or unc-62
function. Although this was somewhat unexpected, it is possible that
ceh-20 and unc-62 downregulate Hox gene expression in
conjunction with non-HOX proteins downstream of signaling pathways. Although
the wingless and the TGFß pathways have not been shown to inhibit Hox
gene expression, they do influence Hox gene expression in C. elegans
(Stoyanov et al., 2003
;
Streit et al., 2002
). Because
lin-39 upregulation was not restricted to the Q cells in
ceh-20 or unc-62 mutants, we raise the possibility that
ceh-20 and unc-62 could function with repressors of
lin-39 expression such as SAM domain proteins
(Zhang et al., 2003
).
Taken together, these comparisons suggest that C. elegans ceh-20 and unc-62 share many properties with their homologs, but the degree to which the Hox-independent and Hox-dependent roles of these genes and their homologs dominate may differ in different organisms and even between different tissues within the same organism.
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Supplementary material |
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Carolina Center for Genome Sciences, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599-7264, USA
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