Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA
* Author for correspondence (e-mail: JL53{at}cornell.edu)
Accepted 6 July 2005
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SUMMARY |
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Key words: mls-2, HMX, Nkx5, Homeodomain, C. elegans, Mesoderm, Cleavage orientation, Cell proliferation, Cell fate specification, HLH-1, Myod, CYE-1
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Introduction |
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Previous studies have identified a number of regulatory factors that
function in the proper patterning and fate specification of the M lineage,
including the Hox factors MAB-5 and LIN-39, their cofactor CEH-20
(Kenyon, 1986;
Harfe et al., 1998b
;
Liu and Fire, 2000
), the
C. elegans Myod homolog HLH-1, the Twist ortholog HLH-8
(Harfe et al., 1998a
;
Harfe et al., 1998b
;
Corsi et al., 2000
;
Corsi et al., 2002
) and the
Notch receptor LIN-12 (Greenwald et al.,
1983
). MAB-5 and LIN-39, together with CEH-20, are crucial for
diversification of the M lineage, and they directly activate the expression of
HLH-8 (Liu and Fire, 2000
).
HLH-1 and LIN-12 have been implicated in cell fate decisions between
coelomocytes and sex myoblasts (Greenwald
et al., 1983
; Harfe et al.,
1998a
). These factors, however, do not account for all of the
mechanisms involved in the development of the M lineage.
We have carried out a genetic screen to identify mutations that affect the
proper patterning of the M lineage. In this report, we describe one of the
genes identified in this screen, mls-2 (mesodermal lineage
specification-2). mls-2 encodes a homeodomain protein of the HMX
family, also known as the Nkx5 family
(Pollard and Holland, 2000).
We report our analysis on the multiple and distinct functions of MLS-2 in the
M lineage. We further identify the bHLH transcription factor HLH-1 as a
downstream target of MLS-2 in regulating cell fate specification in the M
lineage.
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Materials and methods |
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Additional strains used in this work were as follows.
Mutagenesis screen and analysis of mls-2
PD4666(ayIs6) animals carrying an integrated hlh-8::gfp
reporter that labels all undifferentiated M lineage descendants were
mutagenized with ethylmethylsulfonate (EMS). Non-clonal F2 populations were
screened using a fluorescence stereomicroscope. Animals with abnormal M
lineage patterning were recovered for further analysis. We screened 15,000
haploid genomes and isolated 15 independent mutations with an altered M
lineage. One recessive mutation, cc615, showed 100% penetrance of M
lineage defects and defines the mls-2 locus. Three-factor mapping
using unc-2(e55) lon-2(e678) and dpy-8(e130) unc-124(hs10),
coupled with snip-SNP mapping (Wicks et
al., 2001), placed mls-2 between C07A12.2 and F43C9.2 on
the X chromosome. Among 13 likely candidates of the 40 predicted genes located
in this region, RNAi of C39E6.4 in wild-type animals phenocopied the
cc615 M lineage phenotype. Cosmid C39E6 rescued the cc615
mutant phenotype. The molecular lesions of cc615 were identified
through direct sequencing of PCR fragments encompassing the entire coding
region of C39E6.4.
Plasmid constructs and transgenic lines
mls-2 reporter constructs
5.5 kb of the mls-2 promoter sequence (5567 to 1),
the entire mls-2 coding region and 2.1 kb of the mls-2
downstream sequence were amplified through long-range PCR (Expand Long
Template PCR system, Roche), using cosmid C39E6 as template. The PCR products
were used to generate the following reporter constructs for analyzing the
expression pattern of mls-2:
Forced expression constructs
Detailed information on all the constructs is available upon request.
Plasmid pKM1110 (cye-1::gfp) was from Mike Krause
(Brodigan et al., 2003).
Transgenic lines were generated using the plasmid pRF4
(Mello et al., 1991
) or the
pha-1 rescuing plasmid pC1
(Granato et al., 1994
) as
markers.
Heat-shock experiments
To examine the effects of forced expression of mls-2 on the M
lineage, animals carrying pYJ62[hsp16p::mls-2::mls-2 3' UTR]
were stage synchronized with respect to M lineage development using the
integrated hlh-8::gfp. Animals of the same stage were transferred to
the same plate, heat shocked at 37°C for 1 hour and analyzed at different
time points after recovery at 20°C. The M lineage phenotype was examined
using hlh-8::gfp, egl-15::gfp and CC::gfp markers, and
compared with that of control animals (LW0081) that had undergone the same
treatment.
To examine the consequences of forced expression of mls-2 on other cell types, animals carrying pYJ62 were stage synchronized and heat shocked at 37°C for 30 minutes to 1 hour, every 8 or 12 hours starting from late embryogenesis. The intestinal cells, seam cells and cells in the vulva were examined using cye-1::gfp, egl-17::gfp, DIC optics and DAPI staining.
RNAi
Plasmids yk276h9 (gift from Yuji Kohara, National Institute of
Genetics, Japan) and pVZ1200 (gift from Mike Krause, NIDDK, NIH, USA) were
used as templates for synthesizing dsRNA against mls-2 and
hlh-1, respectively, following the protocol of Fire and colleagues
(Fire et al., 1998). dsRNA was
injected into gravid adults of different genotypes. Progeny of injected
animals were scored for larval lethality and M lineage defects. Water-injected
animals were used as controls.
Antibodies and immunofluorescence staining
The N terminus of MLS-2 (amino acids 14 to 215) was cloned into pGEX-4T-1
(Smith and Johnson, 1988).
Plasmid pYJ67 was transformed into BL21(DE3)pLysS cells. Fusion proteins were
first purified under denaturing conditions using Glutathione sepharose 4B
beads (Amersham Biosciences) and further purified by SDS-PAGE. Gel slices
containing the purified fusion proteins were used to immunize rats (Cocalico
Biologicals, PA). The resulting antisera were tested by western blot analyses
using bacterially generated GST-MLS-2 fusion proteins. Antibodies were further
purified by pre-adsoption with extracts of mls-2(cc615) mutant worms
at 4°C overnight (Maloof and Kenyon,
1998
).
For immunostaining using anti-HLH-1 antibodies, animals were fixed
following the protocol of Harfe and colleagues
(Harfe et al., 1998a). For all
other antibodies, animals were fixed following the protocol of Hurd and
Kemphues (Hurd and Kemphues,
2003
). The following antibodies were used: preadsorbed rat
anti-MLS-2 (CUMC-R6; 1:400 to 1:1000), goat anti-GFP (Rockland
Immunochemicals; 1:5000), mouse anti-ß-galactosidase (Promega; 1:50) and
rabbit anti-HLH-1 (Krause et al.,
1990
) (gift of M. Krause; 1:400). All secondary antibodies were
from Jackson ImmunoResearch Laboratories and were used at a dilution of 1:100
to 1:400. Differential interference contrast and epifluorescence microscopy
were performed using a Leica DMRA2 compound microscope. Images were captured
by a Hamamatsu Orca-ER camera using the Openlab software (version 3.0.9,
Improvision). Subsequent image analysis was performed using Adobe Photoshop
7.0.
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Results |
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We analyzed the M lineage in cc615 mutants by following
post-embryonic development in live animals using molecular markers that label
different cell types of the M lineage. Using the hlh-8::gfp reporter
as a marker for undifferentiated cells in the M lineage
(Harfe et al., 1998b), the
first defect we observed was abnormal cleavage orientation in cc615
mutants. In wild-type animals, the M mesoblast always undergoes the first cell
division along the dorsoventral axis (Fig.
2A). However, among the 32 cc615 mutants examined, only
69% divided with this orientation, and the cleavage plane was sometimes tilted
(data not shown). In the remaining 31%, the first division of M was oriented
in the anteroposterior direction (Fig.
2B). Very rarely, we observed M division along the left-right axis
(data not shown). Subsequent cleavage orientations of the M lineage divisions
continued to be randomized in cc615 mutants
(Fig. 3B-D). The resulting M
descendants were located in the posterior of cc615 mutant animals in
variable patterns (see Fig. 2E
as an example), instead of being positioned in four quadrants, as in wild-type
animals (Fig. 2D).
In addition to the cleavage orientation defects, cc615 mutants also exhibited defects in cell proliferation and cell fate specification in the M lineage. In wild-type hermaphrodites, the M mesoblast first undergoes four rounds of cell division to produce 16 descendants expressing hlh-8::gfp (Fig. 1). In cc615 mutant animals, M always completed the first three rounds of cell divisions, generating eight hlh-8::gfp expressing cells. However, zero to six of the eight cells divided for a fourth time in cc615 mutants, yielding a range of 8-14 M lineage descendants (Fig. 2E, Fig. 3B-D). Thus at late L1 stage, mls-2(cc615) mutant animals contained a reduced number of M lineage descendants when compared with wild-type animals at the same stage.
After the fourth round of cell division in the wild-type M lineage, two of
the 16 cells on the ventral side undergo an extra round of division, with the
M lineage producing 14 BWMs, 2 CCs and 2 SMs
(Fig. 1). Using Nomarski optics
in combination with the myo-3::gfp marker and the intrinsic
cc::gfp marker, which label BWMs and CCs, respectively
(Kostas and Fire, 2002), we
found that all cc615 mutants had the correct number of embryonically
derived CCs and BWMs, but lacked M-derived CCs, and most or all M-derived BWMs
(Table 1,
Fig. 2J,K,
Fig. 3B-D). Instead,
cc615 mutants contained more than two M-derived cells (three to nine
cells) that behaved like SMs (Table
1, Fig. 3B-D).
These SM-like cells were enlarged, expressed hlh-8::gfp and migrated
toward the vulva to various degrees (Fig.
2H). They divided two to three times to generate multiple sex
muscle precursors that differentiated into appropriate vulval and uterine
muscles, as verified by a number of sex muscle-specific GFP markers, including
egl-15::gfp, arg-1::gfp and NdE box::gfp
(Fig. 2K). The presence of
vulval muscles was also confirmed by observing the light diffraction of the
muscles using polarized light. Among the SM-like cells in cc615
mutants, two cells always migrated to the ventral side around the vulva and
generated the correct number of sex muscles that attached properly and
appeared functional. cc615 mutant animals did not have any egg-laying
defects (data not shown). Thus, cc615 mutant animals produce extra
SMs at the expense of M lineage-derived CCs and BWMs.
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The predicted MLS-2 protein is 341 amino acids in length and contains a
homeodomain between residues 201 and 260
(Fig. 4A,B). This homeodomain
is most similar to that of the Nkx5 family of homeodomain proteins, also known
as the HMX family. The HMX gene family is present in many organisms from sea
urchins to humans (Adamska et al.,
2000; Adamska et al.,
2001
; Bober et al.,
1994
; Deitcher et al.,
1994
; Herbrand et al.,
1998
; Martinez and Davidson,
1997
; Rinkwitz-Brandt et al.,
1995
; Rinkwitz-Brandt et al.,
1996
; Shaw et al.,
2003
; Stadler et al.,
1992
; Stadler et al.,
1995
; Stadler and Solursh,
1994
; Wang et al.,
1990
; Wang et al.,
2000
; Yoshiura et al.,
1998
). HMX proteins share homology in the homeodomain and in two
additional regions immediately C-terminal to the homeodomain
(Wang et al., 2000
;
Yoshiura et al., 1998
).
Alignment of MLS-2 with other HMX family members, and with a C.
elegans Nkx2.5 homolog, CEH-22
(Okkema and Fire, 1994
), is
shown in Fig. 4B,C. Similarity
between MLS-2 and other HMX proteins is limited to the homeodomain region.
MLS-2 is most similar to the chick HMX protein SOHo1
(Deitcher et al., 1994
).
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The GFP::MLS-2 fusion construct and antibody staining showed identical expression patterns (Fig. 5A-H). Expression of MLS-2 was first detectable in one or two cells in embryos at the 50-cell stage (data not shown) and is localized in the nucleus. MLS-2 continued to be expressed in proliferating cells that are primarily located at the anterior of the embryo and are presumably derived from the AB lineage (Fig. 5A-D). During morphogenesis, this expression became restricted to a small subset of head neuronal precursors. Expression persisted in six head neurons during postembryonic development (Fig. 5E,F). We also observed GFP::MLS-2 expression in unidentified cells near the vulva at the L2 and L3 stages (data not shown).
To characterize the M lineage expression pattern of mls-2, we
performed double-labeling experiments using anti-MLS-2 antibodies and the M
lineage-specific hlh-8::gfp or hlh-8::lacZ markers
(Harfe et al., 1998b).
mls-2 expression in the M lineage was first detectable in the M
mesoblast (Fig. 5P-R), and was
retained during the first three rounds of cell divisions, such that
mls-2 expression was still detectable in eight M descendants
(designated 8-M stage, n>200,
Fig. 5M,N). However, after one
more round of cell division (at the 16-M stage), no MLS-2 signal was detected
either by anti-MLS-2 antibodies or by the gfp::mls-2 fusion construct
(n>50, data not shown). Although we cannot rule out the
possibility that a low level of MLS-2 protein is present after the 8-M stage,
the loss of MLS-2 signal at the 16-M stage appears to be due to the
instability of the MLS-2 protein, because the mls-2 promoter is still
active in M lineage descendants after the fourth round of cell division, as
detected by a transcriptional mls-2p::gfp::mls-2 3' UTR
construct (Fig. 5O). Neither
the mls-2 promoter activity nor the MLS-2 protein was detected in the
SM lineage (Fig. 5S-U) or the
differentiated BWMs and CCs (data not shown). Thus mls-2 is expressed
in the proliferating cells of the early M lineage
(Fig. 5V).
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In order to gain insight into the nature of the tm252 mutation, we examined the localization of the mutant MLS-2 protein in tm252 animals. Antibody staining showed that the mutant MLS-2 protein in tm252 animals was stably made in both the M lineage (Fig. 5K,L) and the head neurons (data not shown), identical to that of wild-type MLS-2 protein. However, the mutant MLS-2 protein perdured longer and was still detectable at the 16-M stage in tm252 mutants (Fig. 5K,L), unlike wild-type MLS-2, which is absent at this stage. Perdurance of the mutant protein was also detected in head neurons in tm252 larvae (data not shown). This perdurance appeared to be transient, as we did not detect any mutant MLS-2 protein in the SM lineage and the signal in the additional neurons eventually faded away at the L2 larval stage (data not shown).
Several lines of evidence showed that tm252 behaved as a semi-dominant, gain-of-function allele. First, tm252/+ animals exhibited similar, although less severe, M lineage defects compared with tm252/tm252 animals (Table 1). Second, as described above, the mutant MLS-2 protein in tm252/tm252 animals perdured longer after the 8-M stage in the M lineage (Fig. 5K,L). Finally, removal of the mutant MLS-2 protein in tm252/tm252 animals by mls-2(RNAi) led to abnormal division orientations, reduced cell proliferation and complete loss of CC fates, phenocopying the M lineage phenotypes of cc615 mutants.
Mis- and overexpression of mls-2 cause a variety of defects in the M lineage
Since loss or gain of mls-2 function caused proliferation defects
in the M lineage, we next examined the consequences of mis- and overexpression
of mls-2 inside and outside of the M lineage. We introduced a
heat-shock inducible hsp::mls-2 transgene (pYJ62) into the strain
LW0081 (see Materials and methods). We then applied heat shock at various
stages of larval development and examined the consequences. As expected,
anti-MLS-2 antibody staining revealed high levels and global expression of
MLS-2 in these transgenic animals after heat shock (data not shown).
Endogenous mls-2 expression was detected in the M lineage from 1-M to 8-M stages. When heat shocked during these stages, a small fraction of transgenic animals died as larvae (data not shown). The remaining animals developed to fertile adults, but with various M lineage defects, including randomized cleavage orientations, increased cell proliferation and abnormal numbers of CCs and SMs (Table 2). Thus, the level of mls-2 gene product in the M lineage is critical for its proper development.
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All M lineage-derived cells are fully differentiated at late L4 and adult stages. When heat shock was applied to transgenic animals at these stages, no M lineage defects were observed, suggesting that forced expression of mls-2 in differentiated cells of the M lineage had no effect.
We also subjected the transgenic animals to multiple rounds of heat-shock during larval development and examined other cell types outside of the M lineage, including intestinal cells, seam cells and cells in the vulva (see Materials and methods). Although we observed slower growth and a 10% penetrance of the Pvl phenotype in the transgenic animals, we found no evidence of increased cell proliferation in the cell types examined (data not shown).
mls-2(tm252) requires cye-1 to promote extra cell divisions in the M lineage
To better understand the mechanism of how MLS-2 regulates cell
proliferation in the M lineage, we looked for cell cycle regulators that might
be involved in the hyper-proliferation in tm252 mutants. One cell
cycle regulator is CYE-1, a primary trigger of S phase
(Moroy and Geisen, 2004;
Fay and Han, 2000
;
Brodigan et al., 2003
).
There are two strong loss-of-function alleles of cye-1, ar95 and
eh10 (Fay and Han,
2000; Brodigan et al.,
2003
). We found that both alleles showed similar M lineage
defects: a subset of cells generated from the first three rounds of cell
divisions failed to divide for a fourth time, and one or both M-derived CCs
were missing (Table 1). Similar
to the findings of Brodigan et al.
(Brodigan et al., 2003
), most
cye-1 mutant animals contained two SMs (
95%, n>100),
and the SMs exhibited variable division patterns ranging from no division to
the wild-type number of divisions. A subset of the SM descendants
differentiated into cells expressing egl-15::gfp
(Table 1).
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HLH-1 is a downstream target of MLS-2 in regulating cell fate specification
In addition to reduced cell proliferation, cc615 mutants exhibited
randomized cleavage orientation and cell fate transformation from CCs and BWMs
to SMs in the M lineage. Several previously described mutants share similar M
lineage defects. mab-5(e1239) and hlh-8(nr2061) mutants show
abnormal cleavage orientation in the M lineage, whereas mab-5(e1239)
and hlh-1(cc561ts) exhibit fate transformation from CCs to SMs
(Kenyon, 1986;
Harfe et al., 1998a
;
Harfe et al., 1998b
;
Corsi et al., 2000
;
Corsi et al., 2002
). Both
mab-5 (encoding a Hox factor) and hlh-8 (encoding CeTwist)
are expressed in the early M lineage, with hlh-8 being a direct
target of mab-5 and lin-39 (S. J. Salser, PhD thesis,
University of California, San Francisco, 1995)
(Harfe et al., 1998b
;
Liu and Fire, 2000
).
hlh-1 encodes CeMyoD and is expressed in both the embryonic and
M-derived BWMs, as well as in the early M lineage from the 2-M stage to 16-M
stage, prior to differentiation (Krause et
al., 1990
; Harfe et al.,
1998a
).
We investigated the regulatory relationship between mls-2 and mab-5, and hlh-8 and hlh-1. Normal levels and patterns of MLS-2 expression were detected by anti-MLS-2 antibodies in lin-39(n1760) mab-5(e1239), hlh-8(nr2061) and hlh-1(cc561ts) mutants at the non-permissive temperature (Fig. 6A,B, data not shown). Similarly, expression of mab-5::gfp and hlh-8::gfp was detected in the M lineage of cc615 mutants (Fig. 2B-H, Fig. 6C). By contrast, the M lineage-specific expression of HLH-1 was not detectable by anti-HLH-1 antibodies in cc615 mutants, even though the same animals showed positive staining of HLH-1 in the embryonic BWMs (Fig. 6D-F). These results suggest that the M lineage expression of hlh-1 requires MLS-2 activity. Consistent with this, hlh-1(RNAi) in cc615 mutants did not produce any additional phenotypes in the M lineage. Furthermore, hlh-1(cc561ts); mls-2(tm252) double mutants completely lacked M-derived CCs (data not shown), resembling the phenotype of hlh-1(cc561ts) single mutants. Additional cell proliferation in the M lineage was still detected in the hlh-1(cc561ts); mls-2(tm252) double mutants, indicating that HLH-1 is not required for MLS-2 function in cell proliferation. Thus MLS-2 acts through HLH-1 to regulate coelomocyte fate specification in the M lineage. Forced expression of hlh-1 in the M lineage could not rescue the loss of CC fates in cc615 mutants (data not shown), suggesting that at least one other target of MLS-2 exists in regulating CC fate specification in the M lineage.
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Discussion |
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Previous studies have shown that the G1 cyclin CYE-1 is present in many
postembryonic blast cells, and is required for vulval and M lineage
development (Fay and Han,
2000; Brodigan et al.,
2003
). Fay and Han (Fay and
Han, 2000
) have also reported that only the last rounds of cell
divisions during vulval development are affected in cye-1 mutants.
Consistent with their findings, we observed that cye-1(eh10) mutants
exhibited proliferation defects only after the first three rounds of cell
divisions in the M lineage, suggesting that maternal CYE-1 is sufficient for
those cell divisions. Because mls-2(cc615) mutants and
cye-1(eh10) mutants showed very similar proliferation defects, MLS-2
may be required to regulate either the expression or the activity/stability of
CYE-1. We found that zygotic expression of cye-1 was not affected in
cc615 mutants. However, a cye-1 null mutation,
cye-1(eh10), suppressed the over-proliferation defects of
mls-2(tm252) mutants. Thus MLS-2 might regulate the stability and/or
activity of CYE-1 in the M lineage.
MLS-2 controls cell fate specification through HLH-1 in the M lineage
In addition to regulating cell proliferation, MLS-2 also plays a role in
regulating cell fate specification. mls-2(cc615) mutants exhibit cell
fate transformation from BWMs and CCs to SMs, suggesting that MLS-2 function
is required for the CC and BWM fates. One of the known factors required for
these fates is the C. elegans Myod homolog HLH-1
(Harfe et al., 1998a). Our
data suggest that HLH-1 is a specific target of MLS-2 in regulating cell fate
specification. We have found that HLH-1 is not present in the M lineage in
cc615 mutants (Fig.
6), and that reducing HLH-1 activity in tm252 mutants
suppresses the CC cell fate without affecting the hyper-proliferation defects.
Currently it is not clear whether hlh-1 is a direct target of MLS-2
and whether other targets of MLS-2 also exist in regulating cell fate
specification in the M lineage.
MLS-2 has multiple and distinct functions in regulating cleavage orientation, cell proliferation and cell fate specification
MLS-2 is a cell type-specific homeodomain protein that regulates cleavage
orientation, cell proliferation and fate specification in the M lineage.
Several lines of evidence support the model that MLS-2 exerts its multiple
functions through distinct mechanisms. First, the gain-of-function
tm252 mutants only exhibited defects in cell proliferation and fate
specification without affecting cleavage orientation, suggesting that the
function of MLS-2 in regulating cleavage orientation can be uncoupled from its
other functions. Second, lack of CYE-1 activity specifically suppressed the
hyper-proliferation defects of tm252 mutants, whereas removal of
HLH-1 activity specifically affected CC fate specification. These results
suggest that MLS-2 has distinct sets of downstream target genes regulating
cell proliferation and cell fate specification.
Previous studies in a number of systems have shown that cell fate
specification is tightly coupled to cell division and cell cycle progression
(Cremisi et al., 2003;
Malicki, 2004
). In particular,
progression through the cell cycle is required for the expression of cell fate
determinants, as shown in the fly nervous system
(Weigmann and Lehner, 1995
)
and the C. elegans vulval precursor cells
(Ambros, 1999
). We failed to
detect HLH-1 expression even in proliferating cells in the M lineage of
mls-2(cc615) mutants, suggesting that the expression of HLH-1 was not
dependent on cell cycle progression in the M lineage. At this point, we cannot
rule out the possibility that other cell fate determinant(s) are not expressed
in mls-2(cc615) mutants because of reduced cell proliferation.
Developmental control of MLS-2 activity and coordination between cell proliferation and differentiation
Our studies suggest that mls-2 expression can be regulated at both
the transcriptional level and the post-transcriptional level. At the
transcriptional level, the mls-2 promoter is specifically active in
the early M lineage (1-M to 16-M stage). At the post-transcriptional level,
MLS-2 protein can only be detected up to the 8-M stage
(Fig. 5). Interestingly, it is
at the 16-M stage when most M-derived cells exit the cell cycle and become
differentiated (Fig. 1).
Because mutants lacking MLS-2 fail to undergo the fourth round of cell
division after the 8-M stage, MLS-2 needs to be present in the eight
proliferating M lineage descendants to allow them to divide. Once these cells
divide, MLS-2 activity needs to be promptly removed to allow these cells to
exit the cell cycle and differentiate. Consistent with this, we observed
excessive proliferation in tm252 mutants where the mutant MLS-2
protein perdured beyond the 8-M stage. Thus MLS-2 appears to be a
pro-proliferation and/or anti-differentiation factor whose activity needs to
be tightly regulated to allow the transition from cell proliferation to
differentiation. Because the tm252 allele deletes the entire exon 3
and part of exon 4, those regions may contain the regulatory element(s) that
are required for regulating the stability of the MLS-2 protein.
The HMX protein family: conserved and divergent sequences and functions
HMX family of homeodomain proteins shares a highly conserved homeodomain
and two additional conserved regions located immediately C-terminal to the
homeodomain (Fig. 4). These two
additional regions do not appear to be conserved in MLS-2 and the chick
protein SOHo1 (Deitcher et al.,
1994) (Fig. 4).
Outside of these conserved regions, sequences are highly diverged among the
HMX family members.
Previous studies have shown that the vertebrate and the Drosophila
HMX proteins share a common expression in the nervous system, with the
vertebrate HMX proteins also being expressed in a number of sensory organs
(Adamska et al., 2000;
Adamska et al., 2001
;
Bober et al., 1994
;
Deitcher et al., 1994
;
Hadrys et al., 1998
;
Herbrand et al., 1998
;
Kiernan et al., 1997
;
Rinkwitz-Brandt et al., 1995
;
Rinkwitz-Brandt et al., 1996
;
Stadler et al., 1995
;
Stadler and Solursh, 1994
;
Wang et al., 1990
;
Wang et al., 1998
;
Wang et al., 2000
;
Yoshiura et al., 1998
;
Adamska et al., 2001
). Mice
lacking either Hmx2 or Hmx3, or both, have various defects
in the CNS and the inner ear (Wang et al.,
1998
; Wang et al.,
2001
; Wang et al.,
2004
). Significantly, Drosophila Hmx can rescue the CNS
defects, as well as the inner ear defects, of the Hmx2; Hmx3 double
knockout mice (Wang et al.,
2004
). These observations suggest a conserved function of the HMX
proteins in the nervous system. We have also observed neuronal expression of
mls-2 (Fig. 5). Thus,
MLS-2 might share with other HMX family members their conserved functions in
the nervous system.
We have found that MLS-2 plays crucial roles in the development of the
postembryonic mesoderm in C. elegans. Expression of HMX proteins
outside of the nervous system and the sensory organs has been reported, and
mice lacking Hmx3 show inner ear defects and maternal reproductive
defects (Martinez and Davidson,
1997; Shaw et al.,
2003
; Stadler and Solursh,
1994
; Wang et al.,
1998
). Therefore, like mls-2, the Hmx genes in
other organisms might also be expressed and functioning in additional cell
types, especially during postembryonic development.
We have shown that MLS-2 is required for cell proliferation and fate
specification in the M lineage. Mice that lack Hmx2, or both
Hmx2 and Hmx3, exhibit reduced proliferation in the otic
epithelium and the adjacent mesenchyme, as well as alteration of cell fates in
the otic vesicle (Wang et al.,
2001; Wang et al.,
2004
). Interestingly, although Drosophila Hmx can
functionally replace Hmx2 and Hmx3 in the murine inner ear
and hypothalamus, it appears to be more effective in rescuing the decreased
cell proliferation defects of the Hmx2; Hmx3 double mutants
(Wang et al., 2004
). We
therefore propose that one of the most conserved functions of HMX proteins is
to regulate cell proliferation. We have identified one cell cycle regulator,
CYE-1, whose activity/stability is regulated by MLS-2. Future studies of HMX
proteins in other systems will shed light on whether this and other functions
of MLS-2 are conserved for all HMX proteins.
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ACKNOWLEDGMENTS |
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