1 Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth
Avenue, Pittsburgh, PA 15213, USA
2 Division of Biology, California Institute of Technology, Pasadena, CA 91125,
USA
* Author for correspondence (e-mail: ettensohn{at}andrew.cmu.edu)
Accepted 27 March 2003
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SUMMARY |
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Key words: Sea urchin embryo, Early development, Fate specification, Skeletogenesis, Primary mesenchyme cells, Alx1, Cart1, Biomineralization, Epithelial-mesenchymal transition, Alx3, Alx4
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INTRODUCTION |
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Recent studies have begun to elucidate the gene regulatory network that
underlies PMC specification. The initial specification of the large micromere
lineage is entrained by a patterning system linked to the animal-vegetal
polarity of the unfertilized egg
(Ettensohn and Sweet, 2000;
Brandhorst and Klein, 2002
;
Angerer and Angerer, 2003
). One
important component of this system is ß-catenin. ß-catenin is
localized in the nuclei of vegetal blastomeres during early cleavage and
becomes concentrated at the highest levels in the micromere lineage
(Logan et al., 1999
).
Nuclearization of ß-catenin is required for all known aspects of mesoderm
and endoderm formation, including large micromere specification
(Emily-Fenouil et al., 1998
;
Wikramanayake et al., 1998
;
Logan et al., 1999
;
Davidson et al., 2002
). This
indicates that ß-catenin, in combination with its LEF/TCF partner(s)
(Huang et al., 2000
;
Vonica et al., 2000
), provides
a very early input into the PMC gene network. One critical early target of
ß-catenin is the transcriptional repressor Pmar1
(Oliveri et al., 2002
). Pmar1
is transiently expressed in both large and small micromeres during early
development. In the large micromeres, Pmar1 is thought to block the expression
of an unknown, global repressor of PMC specification. As a consequence of
these early molecular events, other transcriptional regulators are activated
selectively in the large micromere lineage later in development, including
Ets1 (Kurokawa et al., 1999
)
and Tbr (Croce et al., 2001
;
Fuchikami et al., 2002
).
Presumably, these and other transcription factors control the expression of
downstream genes that regulate primary mesenchyme morphogenesis and
biomineralization (Zhu et al.,
2001
; Illies et al.,
2002
), although these links have not been established.
During normal embryogenesis, only the large micromeres give rise to
skeleton-forming cells. Under experimental conditions, however, the same
developmental program can be activated in other cell lineages. Removal of
micromeres at the 16-cell stage or PMCs at the early gastrula stage leads to
the transfating of macromere-derived cells (secondary mesenchyme cells, or
SMCs) to the PMC fate (Ettensohn,
1992; Sweet et al.,
1999
). The transfating cells exhibit all features of the PMC
phenotype that have been examined. They express PMC-specific molecular markers
(including MSP130, SM50, and SM30), migrate to PMC-specific target sites,
acquire PMC-specific signaling properties, and synthesize a normally patterned
skeleton (Ettensohn and McClay,
1988
; Ettensohn and Ruffins,
1993
; Guss,
1997
).
In this study, we have identified a new and essential component of the PMC gene network, Alx1. We show that Alx1 protein is required for an early step in the specification of the large micromere lineage and for the transfating of non-micromere-derived cells to a PMC fate. Alx1 is the first known invertebrate member of the Cart1/Alx3/Alx4 subfamily of Paired class homeodomain proteins. As these proteins have been shown to function in skeletogenesis in vertebrates, our findings have implications concerning the evolution of biomineralization within the deuterostomes.
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MATERIALS AND METHODS |
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Whole-mount in situ hybridization
In situ hybridization was carried out according to the method of Zhu et al.
(Zhu et al., 2001). SpAlx1
probe was generated from clone 16-I18 (a full-length clone) and LvAlx1 probe
was generated from a 3.2 kb cDNA that included the C-terminal third of the
protein-coding sequence and most of the 3'-UTR.
Morpholino antisense oligonucleotides (MO) and mRNA injections
Fertilized L. variegatus eggs were injected as described
previously (Sweet et al.,
2002). S. purpuratus eggs were dejellied by incubating
for 10 minutes in pH 5.0 ASW before being placed in rows on protamine
sulfate-coated dishes. Eggs were fertilized on the dishes and injected within
5 minutes. LvAlx1 MO was complementary to the 5' end of the coding
region and had the sequence ACGGCATTGACGGGTAGAATAACAT. SpAlx1 MO was
complementary to the 5'-UTR and had the sequence
TATTGAGTTAAGTCTCGGCACGACA. For most experiments, MO injection solutions
contained 2 mM LvAlx1 or SpAlx1 MO, 20% glycerol (vol/vol), and 0.1% rhodamine
dextran (wt/vol).
Alx1 antibody production and immunostaining
A DNA fragment corresponding to amino acids 242-369 of LvAlx1 was subcloned
into pET32 (Novagen, Madison, WI) to create a histidine-tagged,
thioredoxin-fusion protein. The fusion protein was purified from E.
coli using a nickel column (Novagen) and then used to generate a rabbit
polyclonal antibody (Pocono Rabbit Farm and Laboratory, Canadensis, PA). For
Alx1 immunostaining, embryos were fixed in fresh 2% paraformaldehyde in ASW
for 20 minutes at room temperature. After rinsing once with ASW, the embryos
were postfixed/permeabilized with 100% methanol (5 minutes at -20°C). For
staining with 6a9, Endo1 or anti-myosin, embryos were fixed in methanol alone.
Embryos were washed 3x with PBS and incubated in 4% goat serum in PBS
(PBS-GS) for 30 minutes. They were transferred to flexible, round-bottom
96-well plates and staining was carried out as described by Hodor et al.
(Hodor et al., 2000). Primary
antibodies were crude LvAlx1 antiserum or preimmune serum (1:100 in PBS-GS),
mAbs 6a9 and Endo1 (full-strength tissue culture supernatants), and
anti-myosin (1:100 in PBS-GS). Secondary antibodies were
fluorescein-conjugated, goat anti-rabbit IgG or fluorescein-conjugated, goat
anti-mouse IgG+IgM (Cappel, ICN Biomedicals) (1:50 in PBS-GS). For double
staining experiments, embryos that had been stained with LvAlx1 antiserum and
fluorescein-conjugated secondary antibody were washed as described by Hodor et
al. (Hodor et al., 2000
) and
incubated overnight at 4°C with full-strength monoclonal antibody 6a9. The
embryos were washed again, incubated for 2-4 hours at room temperature in
affinity-purified, Texas red-conjugated goat anti-mouse IgG/IgM (H+L) (Jackson
Immunoresearch) (1:50 in PBS-GS), washed and mounted.
Quantitative PCR (QPCR)
Total RNA was isolated from control (uninjected) and experimental embryos
(injected with a 200 µM injection solution of MO or 2 mg/ml mRNA injection
solution) using RNAzol (Leedo Medical Laboratories, Houston, TX). DNA-Free
(Ambion) was used to eliminate contaminating DNA. First-strand cDNA was
synthesized with RNA extracted from 200-300 embryos using random hexamers and
the Taq Man kit (PE Biosystems). cDNA was diluted to a concentration of 1
embryo/µl. Specific primer sets for each gene were designed using the known
cDNA sequences and the program Primer3
(Rozen and Skaletsky, 2000)
(publicly available at
www.genome.wi.mit.edu/genome_software/other/primer3).
Primer sets were chosen to amplify products 100-200 bp in length. Reactions
were carried out in triplicate using cDNA from 2 embryos/reaction as template
and SYBR green chemistry (PE Biosystems). Thermal cycling parameters were as
described previously (Rast et al.,
2000
) and data were analyzed using an ABI 5700 sequence detection
system. The average of data for the three cycles at the threshold
(CT) for each gene was normalized against the average CT
for ubiquitin mRNA, which is known to be expressed at constant levels during
the first 24 hours of development. Primer efficiencies (i.e., the
amplification factors for each cycle) were found to exceed 1.9. Relative folds
of difference between control and experimental embryos were calculated (see
Tables 1 and
2). In every experiment, a
no-template control was included for each set of primers. Each experiment also
included a control comparing levels of test mRNAs in uninjected embryos and
embryos injected with a control MO. Data were only included when no more than
one cycle of difference was observed for each mRNA tested.
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RESULTS |
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SpAlx1 and LvAlx1 also contain a perfectly conserved C-terminal OAR (otp,
aristaless, Rx) domain (Fig.
1A). Many Paired-class homeodomain proteins, including members of
the Cart1/Alx3/Alx4 subfamily, have an OAR domain at the C terminus (Gaillot
et al., 1999). The function of this domain is poorly understood, although it
may bind to and mask transactivation domains located elsewhere in the
proteins, a masking effect that can be relieved by the binding of other
proteins to the OAR domain (Amendt et al.,
1999; Norris and Kern,
2001
; Brouwer et al.,
2003
).
Except within the homeodomain and OAR domain, SpAlx1 and LvAlx1 show no significant similarities to other proteins by BLAST analysis. We noted, however, a 25-30 amino acid region immediately upstream of the homeodomain that contains a high number of charged residues (aspartic acid and lysine; Fig. 1A), a feature shared by the vertebrate Alx4 and Cart1 proteins. Moreover, SpAlx1 and LvAlx1 are proline-rich (12% proline residues outside the OAR domain and homeodomain), another characteristic of the vertebrate Alx3 and Alx4 proteins.
Developmental expression of Alx1 mRNA and protein
Northern blot analysis with a full-length probe complementary to
Spalx1 revealed a single major transcript with an apparent length of
5-5.5 kb (not shown). This corresponded well to the predicted size of the
Spalx1 mRNA based on the sequence of clone 16-I18 (5 kb).
Spalx1 mRNA was not detectable in the unfertilized egg or at very
early cleavage stages by northern analysis, but was strongly expressed by the
blastula stage.
Whole-mount in situ hybridization showed that Spalx1 mRNA was expressed specifically by cells of the large micromere-PMC lineage (Fig. 2). Expression was first detectable as one to two distinct intranuclear spots of staining in each of the four large daughter cells of the micromeres at the 56-cell stage. At this stage of development, most of the cells of the embryo have undergone the sixth cleavage division but the micromeres have divided only once, producing four large and four small daughter cells. Spalx1 mRNA accumulated only in the large daughter cells, the founder cells of the PMC lineage, beginning in the first interphase after these cells were born. After the next cell division, Spalx1 mRNA was detectable at higher levels in all eight large micromere progeny. Intense signal was evident during the blastula stage in presumptive PMCs. Spalx1 mRNA was expressed specifically by PMCs throughout gastrulation and later embryogenesis, although levels gradually decreased. Faint expression in PMCs was still evident at the early pluteus stage, the latest developmental stage examined.
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A polyclonal antiserum was generated against a 128-amino acid region in the C-terminal half of LvAlx1 (see Materials and Methods). This antibody cross-reacted with SpAlx1, probably because of the high degree of conservation between the two proteins. The anti-Alx1 antiserum specifically labeled the nuclei of cells of the large micromere-PMC lineage (Fig. 3). In L. variegatus, nuclear staining was evident at the blastula stage prior to PMC ingression and PMC nuclei were labeled throughout gastrulation. Double-labeling with the PMC-specific monoclonal antibody (mAb) 6a9 confirmed that the Alx1-expressing cells were PMCs (Fig. 3G). Nuclear staining of PMCs was still evident at the early pluteus stage, the latest developmental stage examined. In S. purpuratus, the earliest stage at which we could reliably detect nuclear localization of Alx1 was in the interphase following the first division of the large micromeres, i.e., when there were eight large micromere progeny (Fig. 6A). This was one cell division later than Spalx1 mRNA expression was first detectable by in situ hybridization and may reflect a lag between transcriptional activation of the Spalx1 gene and the accumulation of sufficient amounts of protein to be detected by immunostaining.
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The phenotypes of MO-injected S. purpuratus and L. variegatus embryos were essentially identical (Fig. 4). Development appeared normal during cleavage and blastula stages, and injected embryos hatched within 1 hour of sibling controls (L. variegatus, 23°C). At the late blastula stage, however, a striking phenotype became apparent. PMCs did not ingress in the MO-injected embryos and invagination of the vegetal plate was delayed by several hours relative to control sibling embryos. MO-injected embryos failed to form visible skeletal elements even after extended periods of culture (3 and 6 days for L. variegatus and S. purpuratus, respectively) (Fig. 4).
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Alx1 MOs interfered with normal PMC specification as assayed by the
expression of a battery of molecular markers
(Fig. 5,
Table 1). mAb 6a9 recognizes
PMC-specific cell surface proteins of the MSP130 family
(Ettensohn and McClay, 1988;
Illies et al., 2002
). Alx1
MO-injected S. purpuratus and L. variegatus embryos had
greatly reduced numbers of 6a9-positive cells
(Fig. 5G-H; and see below).
This effect was dose-dependent; high doses of MO completely blocked the
formation of 6a9(+) cells. The expression of SpMSP130-related 2 (a
MSP protein family member), SpP19 and SpFRP
(fibrinogen-related protein) (Zhu et al.,
2001
; Illies et al.,
2002
) was examined by in situ hybridization
(Fig. 5A-F). Most embryos
lacked detectable expression of these markers or had greatly reduced numbers
of mesenchyme cells that expressed the mRNAs. Finally, in S.
purpuratus we used QPCR to measure levels of expression of nine genes
expressed exclusively or selectively by cells of the large micromere-PMC
lineage (Table 1). SpAlx1 MO
had no detectable effect on levels of four of the mRNAs: tbr
(Fuchikami et al., 2002
),
ets1 (Kurokawa et al.,
1999
), delta (Sweet
et al., 2002
) and pmar1
(Oliveri et al., 2002
) when
assessed either at 18-20 hours or 23-24 hours post-fertilization. The level of
Spalx1 mRNA was slightly elevated in MO-injected embryos, suggesting
that Alx1 protein may act as a negative regulator of the alx1 gene.
Four of the mRNAs we tested, dri (G. Amore and E. Davidson, personal
communication), MSP130 (Parr et
al., 1990
), MSP130-related 2
(Illies et al., 2002
) and
sm50 (Katoh-Fukui et al.,
1991
), were down-regulated in MO-injected embryos.
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Control experiments showed that the MOs were effective in blocking the
expression of Alx1 protein and that the observed phenotype resulted
specifically from this inhibition. Alx1 MOs blocked nuclear accumulation of
Alx1 protein in the large micromere progeny as shown by immunostaining
(Fig. 3D). Injection of several
other MOs into S. purpuratus and L. variegatus eggs at the
same concentrations did not affect PMC formation or skeletogenesis. As an
additional control for specificity, we injected S. purpuratus Alx1 MO
into L. variegatus eggs. The LvAlx1 mRNA and SpAlx1 MO are mismatched
at 4/25 nucleotides, a degree of mismatching that has been shown to
significantly reduce the effectiveness of a MO directed against globin mRNA
(Summerton, 1999). We found
that concentrations of SpAlx1 MO that resulted in a robust phenotype in S.
purpuratus had no effect when injected into L. variegatus eggs.
Finally, as noted above, SpAlx1 and LvAlx1 MOs produced the same phenotype
although they were complementary to non-overlapping regions of the target
mRNAs.
Because the phenotype of Alx1 MO-injected embryos resembled in some
respects the phenotype of embryos in which early cleavage divisions have been
equalized (Langelan and Whiteley,
1985), we performed one experiment to directly test the
possibility that the MO might perturb the pattern of early cleavage and
micromere formation. We allowed MO-injected S. purpuratus embryos to
develop to the 16-cell stage, then removed from the dish any that showed signs
of abnormal cleavage. The remaining embryos exhibited the same PMC()
phenotype described above. Although we did not follow the pattern of cell
division in the MO-injected embryos after fourth cleavage, these observations
suggest that the MO does not affect PMC specification by altering the spatial
pattern of early cleavage.
Upstream regulators of Alx1
Beta-catenin function is required for the expression of all
mesendoderm-specific mRNAs that have been analyzed to date. This observation,
and the fact that the micromeres and their progeny have high levels of nuclear
ß-catenin (Logan et al.,
1999), suggested that activation of Alx1 expression might be
dependent on ß-catenin. To test this hypothesis, Alx1 expression was
assayed in embryos that had been injected with mRA encoding full-length
Xenopus C-cadherin (experiments in L. variegatus), or the
transmembrane/cytoplasmic domain of LvG-cadherin (experiments in S.
purpuratus). Embryos injected with these cadherin mRNAs lack detectable
levels of nuclear ß-catenin and exhibit an animalized phenotype
(Wikramanayake et al., 1998
;
Logan et al., 1999
). In such
embryos, we could detect no nuclear LvAlx1 protein
(Fig. 3H) and expression of
Spalx1 was dramatically reduced as assayed by QPCR
(Table 2). These experiments
demonstrate that zygotic activation of LvAlx1 is dependent on
ß-catenin.
Pmar1 is a critical early transcriptional regulator in the gene network
that controls PMC specification (Oliveri
et al., 2002). The first expression of Spalx1, detectable
by in situ hybridization analysis, followed that of pmar1 by one cell
cycle. These observations raised the possibility that expression of
Spalx1 might be regulated by pmar1. Consistent with this
hypothesis, we found that overexpression of Pmar1 or an engrailed-pmar1 fusion
protein (EnHD) (Oliveri et al.,
2002
) resulted in a striking increase in levels of Spalx1
mRNA expression as assayed by QPCR (Table
2). The fact that overexpression of wild-type Pmar1 and EnHD
produced similar effects supports the view that Pmar1 normally acts as a
repressor (Oliveri et al.,
2002
). Moreover, we observed that overexpression of Pmar1 (or
EnHD) could activate Spalx1 expression to high levels even in
cadherin mRNA-injected embryos (Table
2).
Alx1 and transfating of cells to a skeletogenic fate
In undisturbed embryos, SMCs do not express detectable levels of Alx1 mRNA
or protein (Figs
2,3).
We tested whether the transfating of SMCs following PMC removal was associated
with activation of Alx1 expression. The entire complement of PMCs was removed
microsurgically from mesenchyme blastula stage embryos and the resultant
PMC() embryos were double-immunostained with Alx1 antibody and mAb 6a9
at various times after the operation. Early in the transfating process (i.e.,
at the late gastrula stage), we observed numerous Alx1-expressing cells at the
tip of the archenteron, many of which also stained with mAb 6a9
(Fig. 6). We also observed
Alx1-positive cells that were not stained with mAb 6a9. This is consistent
with the finding that in normal embryos, expression of Alx1 precedes that of
the MSP130 proteins.
As noted above, embryos injected with Alx1 MOs typically failed to form skeletal elements even after prolonged periods in culture. This suggested that Alx1 MOs blocked not only the initial specification of PMCs but the transfating of other cell lineages to a skeletogenic fate. We investigated this further by counting numbers of 6a9(+) cells in L. variegatus embryos at different developmental stages after MO injection. In one experiment, separate batches of zygotes from a single fertilization were injected with either 5 or 10 pl of MO injection solution. The relative amounts of injection solution were carefully controlled by delivering either one or two pulses (100 mseconds each) with the picospritzer, using the same microneedle to inject both sets of eggs.
Alx1 MO-injected embryos showed a dose-dependent suppression of the
transfating response. In L. variegatus, microsurgical removal of
micromeres or PMCs leads to the appearance of 60-70 6a9-positive cells 24
hours postfertilization at 23°C
(Ettensohn and McClay, 1988;
Sweet et al., 1999
). Embryos
injected with the lower dose of LvAlx1 MO showed greatly reduced numbers of
6a9-positive cells, even after 29 hours of development (mean no. of
6a9-positive cells=26.1, s.d.±9.7, n=20). Embryos injected
with the higher dose of MO showed almost complete suppression of transfating
at 29 hours (mean no. of 6a9-positive cells=3.3, s.d.±4.1,
n=17). The reduced numbers of 6a9-positive cells in MO-injected
embryos could not be attributed simply to a delay in transfating.
Immunostaining of MO-injected embryos at earlier times showed that transfating
began at about 19 hours, as judged by the earliest appearance of faintly
stained, 6a9-positive cells in association with the archenteron. This
corresponds closely to the time at which transfating is first detectable
following microsurgical removal of micromeres [see
table 1 in Sweet et al.
(Sweet et al., 1999
)] or PMCs
(Ettensohn and McClay, 1988
)
when embryos are cultured at 23°C.
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DISCUSSION |
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Alx1 MOs produced very limited effects on cell types other than
skeletogenic mesoderm. Embryos injected with Alx1 MO gastrulated and
subsequently formed a compartmentalized gut and polarized ectoderm. Alx1
MO-injected embryos also formed large numbers of pigment cells. This is in
clear contrast to embryos lacking micromeres, which are almost devoid of
pigment cells (<5 pigment cells/embryo)
(Sweet et al., 1999). Pigment
cell specification requires an inductive signal from the large micromeres, a
signal recently shown to be the protein Delta
(Sweet et al., 2002
). The
formation of large numbers of pigment cells in Alx1 MO-injected embryos is
consistent with QPCR data showing that Alx1 MO has little effect on
Delta mRNA expression.
Alx1 MO-injected embryos showed a consistent delay in invagination and in
the subsequent differentiation of the archenteron. Multiple factors probably
contributed to these phenotypic effects. Surgical removal of micromeres at the
16-cell stage delays invagination in both S. purpuratus
(Ransick and Davidson, 1995)
and L. variegatus (Sweet et al.,
1999
). This may reflect an early inductive interaction between
micromeres and overlying veg2 cells or a mechanical potentiation of
invagination resulting from PMC ingression (see
Ransick and Davidson, 1995
).
It appears that Alx1 protein is expressed too late in cleavage to activate the
putative early inducing signal (Ransick
and Davidson, 1995
). It is more likely, therefore, that the delay
in invagination in MO-injected embryos is due to mechanical effects, or
possibly to interference with yet another micromere-derived signal, distinct
from both Delta and the putative early signal. With respect to the later
differentiation of the archenteron, there is evidence that stimulatory signals
are produced by PMCs during gastrulation. Removal of PMCs at the mesenchyme
blastula stage delays archenteron differentiation without affecting the timing
of invagination (Hamada and Kiyomoto,
2000
). PMCs normally secrete a variety of proteins into the
blastocoel matrix during gastrulation (Zhu
et al., 2001
) and archenteron differentiation may be regulated by
these factors.
The PMC gene network
Based on the present study and other recent work
(Kurokawa et al., 1999;
Zhu et al., 2001
;
Davidson et al., 2002
;
Fuchikami et al., 2002
;
Illies et al., 2002
;
Oliveri et al., 2002
), a
framework of the gene network that controls PMC specification can now be
constructed (Fig. 7). Maternal
inputs into this network lead ultimately to the activation or repression of
genes that control the complex morphogenetic behaviors of PMCs (ingression,
migration and cell fusion) and their terminal differentiation into
biomineral-forming cells.
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Injection of pmar1 mRNA causes mesomeres to adopt a PMC-like fate
(P. Oliveri and D. McClay, personal communcation). In contrast, overexpression
of alx1 mRNA is insufficient to convert other cell lineages to a
skeletogenic fate (data not shown). Pmar1 is therefore likely to
control key regulators of PMC fate specification other than alx1,
probably including other transcription factors expressed selectively or
exclusively by the large micromere lineage. Zygotic expression of
ets1 is restricted to the large micromere lineage in
Hemicentrotus pulcherrimus
(Kurokawa et al., 1999),
although in L. variegatus this gene is expressed by both PMCs and
SMCs (X. Zhu and C.A.E., unpublished observations). Zygotic expression of
tbr is restricted to the large micromere lineage
(Croce et al., 2001
;
Fuchikami et al., 2002
).
Alx1 is expressed earlier than either ets1 or tbr
but our QPCR studies suggest it is not required for the expression of either
gene (Table 1 and
Fig. 7). Dri, another
transcriptional regulator expressed by PMCs (G. Amore and E. Davidson,
personal communication) is regulated positively by alx1
(Fig. 7).
Alx1 is required for at least two distinct morphogenetic processes in the
large micromere lineage: (1) ingression (epithelial-mesenchymal transition)
and (2) skeletogenesis. The molecular changes required for ingression have not
yet been identified, although this process is associated with changes in cell
shape, protrusive activity, adhesive properties and cell surface architecture
(Fink and McClay, 1985;
Miller and McClay, 1997
). In
contrast, a large number of terminal differentiation gene products have been
identified that function in the formation of the biomineralized skeleton (see
Illies et al., 2002
;
Wilt, 2002
)
(Fig. 7). These gene products
are expressed specifically in the large micromere lineage beginning at the
mid-late blastula stage, prior to PMC ingression. We examined the expression
of four such markers in this study, SpMSP130, SpMSP130-related 2, SpP19 and
SpSM50, and found that all four are regulated positively by Alx1. This
suggests that Alx1 is a key regulator of the molecular subprogram that
controls skeletogenesis.
Activation of the PMC gene network through a regulative pathway:
different upstream inputs lead to the same output
Our MO studies show that Alx1 is essential not only for normal PMC
specification but also for the transfating of non-micromere lineages to a
skeletogenic fate. The most likely explanation is that Alx1 is required in the
transfating cells. In support of this view, we have shown that Alx1
(Results) and several of its downstream targets
(Guss, 1997) are selectively
activated in the transfating cells. There are other possible interpretations
of our transfating experiments that would involve a role for Alx1 in the
micromeres, but these can be excluded based on other experimental data. For
example, an early, Alx1-dependent signal from large micromeres might be
required for veg2-derived cells to become competent to transfate. This is
clearly not the case, however, as a robust transfating response is observed
when micromeres are removed from 16-cell stage embryos, prior to the onset of
Alx1 expression (Sweet et al.,
1999
). Another possibility is that in MO-injected embryos, the
large micromere progeny continue to provide the signal that suppresses
transfating, either because the signal is independent of Alx1 or because
levels of the MO are too low to effectively block the signal. Neither scenario
is consistent with the dose-dependent effect of the MOs, however. For example,
if MOs were only partially blocking the PMC-derived signal then higher
concentrations would be more effective and lead to greater numbers of
transfating cells. In fact, the opposite was observed. We conclude that Alx1
is required in the transfating cells where it functions to regulate key
subprograms within the PMC gene network.
The PMC gene network is activated in different ways in transfating cells
and large micromeres. Transfating cells activate the network by a mechanism
responsive to cell signaling, whereas in large micromeres the pathway is
activated via a signal-independent, maternal mechanism that concentrates
ß-catenin in micromere nuclei
(Ettensohn and Sweet, 2000;
Brandhorst and Klein, 2002
;
Angerer and Angerer, 2003
). Our
findings show that the divergence in the normal and regulative pathways lies
upstream of alx1 (Fig.
7). Despite different upstream inputs, the gene regulatory network
downstream of Alx1 in transfating cells and large micromeres appears to be
identical.
The Cart1/Alx3/Alx4 subfamily and the evolution of
biomineralization
The primary sequence of the Sp/LvAlx1 homeodomain indicates that this
protein is the first invertebrate member of the Cart1/Alx3/Alx4 family of
Paired-class homeodomain proteins. In our molecular phylogenetic analysis, the
Sp/LvAlx1 homeodomain clustered with this gene family even when only the most
closely related Paired-class homeodomain subfamilies were included
(Fig. 1B)
(Galliot et al., 1999). There
are also similarities between Sp/LvAlx1 and members of the Cart1/Alx3/Alx4
family outside the homeodomain; viz., the presence of a charged domain
upstream of the homeodomain, a C-terminal OAR domain, and an overall abundance
of proline residues.
In vertebrates, the Cart1/Alx3/Alx4 proteins have been implicated in the
formation of the limb skeleton and the neural crest-derived skeleton of the
face and neck. These three genes are expressed in similar patterns by neural
crest-derived mesenchyme of developing craniofacial regions and by the
mesenchyme of developing limbs (Zhao et
al., 1994; Qu et al.,
1997
; ten Berge et al.,
1998
; Beverdam and Meijlink,
2001
). Mice with compound mutations in Alx3 and Alx4 have severe
defects in neural crest-derived skeletal elements
(Beverdam et al., 2001
).
Alx4/Cart1 double mutants have a similar phenotype
(Qu et al., 1999
) (see
Beverdam et al., 2001
). In
humans, mutations in ALX4 have been shown to cause defects in skull
ossification (Wu et al., 2000
;
Mavrogiannis et al., 2001
).
Alx4 and Cart1 appear to recognize identical palindromic sites on DNA and bind
to these sites as homo-or heterodimers (Qu
et al., 1999
). In vertebrates, it is not yet clear whether these
proteins function primarily in fate specification, cell death, division, or
other developmental processes.
The fact that similar proteins are involved in skeletogenesis in sea
urchins and vertebrates raises the possibility there might be an evolutionary
link between certain features of skeleton formation in these two groups of
deuterostomes. It has been proposed that the ancestral deuterostome (the most
recent common ancestor of echinoderms, hemichordates and chordates) may have
had an extensive calcitic skeleton much like that of modern sea urchins
(Jefferies et al., 1996;
Dominguez et al., 2002
). This
controversial hypothesis is based partly on the interpretation of paleozoic
fossils known as mitrates bilaterally symmetrical organisms that
possessed gill-slits and large, calcitic tests. Similarities among proteins
associated with the biominerals of echinoderms and other animals suggest that
certain features of biomineralization may have even more ancient origins,
i.e., predating the deuterostome-protostome split. For example, C-lectin
domain-containing proteins have recently been shown to be associated with
biominerals of echinoderms, molluscs and vertebrates
(Mann and Siedler, 1999
;
Mann et al., 2000
;
Illies et al., 2002
). One
hypothesis is that biological calcifying systems in various metazoans (e.g.,
corals, crustaceans, molluscs, echinoderms and vertebrates) all originated
from a common, pre-existing system that initially functioned to suppress
mineral deposition in the Neoproterozoic marine environment, which was
probably saturated with CaCO3
(Marin et al., 1996
;
Westbroek and Marin, 1998
).
Our findings support the view that the ancestral deuterostome possessed a
mesenchymal cell lineage that engaged in biomineralization, and that an
Alx1-like protein was involved in the specification these cells. Such a
primordial cell lineage may have been utilized in a variety of ways during
deuterostome evolution to contribute to biomineralized structures in different
animals.
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ACKNOWLEDGMENTS |
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