Department of Biology, University of Rochester, Rochester, NY 14627, USA
Author for correspondence (e-mail:
langerer{at}mail.nih.gov)
Accepted 15 December 2004
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SUMMARY |
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Key words: Wnt, Cell fate specification, Animal-vegetal axis, Sea urchin, Embryo
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Introduction |
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Opposing this ß-catenin-dependent vegetal inductive cascade are the
ATFs, whose prototypical and most extensively studied member is SoxB1
(Kenny et al., 1999;
Kenny et al., 2003
). Maternal
SoxB1 protein is uniformly distributed among all nuclei through the eight-cell
stage, but then rapidly begins to disappear from micromeres soon after they
form at the fourth cleavage division. Beginning around the seventh cleavage,
SoxB1 also progressively clears from nuclei of macromere progeny. This
clearance is necessary because forced continued accumulation of SoxB1 protein
in vegetal blastomeres by injection of SoxB1 mRNA at the one-cell
stage blocks activation of genes in the endomesoderm GRN (L.M.A., R.C.A. and
E. Davidson, unpublished) and prevents all vegetal development
(Kenny et al., 2003
). This
phenotype is indistinguishable from that produced by loss of ß-catenin
nuclear function achieved through cadherin overexpression
(Howard et al., 2001
;
Logan et al., 1999
;
Wikramanayake et al., 1998
).
Conversely, morpholino antisense knockdown of SoxB1 protein leads to a large
increase in ß-catenin-dependent transcriptional activity, as measured by
a ß-catenin/TCF-Lef-dependent reporter transgene
(Kenny et al., 2003
). Thus, an
important function of SoxB1 in the early embryo is to limit the range of
ß-catenin activity along the AV axis.
An essential aspect of endomesoderm differentiation, in turn, is the
activation of mechanisms for the temporally and spatially controlled
downregulation of SoxB1 in vegetal blastomeres. Elimination of SoxB1 from
these vegetal lineages is completely dependent on nuclear ß-catenin
function (Wikramanayake et al.,
1998; Logan et al.,
1999
; Howard et al.,
2001
). In micromeres, the rapid disappearance of SoxB1 is most
likely to be regulated cell-autonomously, whereas in the overlying macromere
lineages, clearance at around seventh cleavage depends, at least in part, on a
signal from micromeres, because it is inhibited when they are removed
(Oliveri et al., 2003
).
Generation of this signal can be stimulated by Pmar1, a micromere-specific
transcription factor, because cells that lack nuclear ß-catenin, but are
supplied with Pmar1 mRNA, can induce clearance of SoxB1 protein in
neighboring cells (Oliveri et al.,
2003
). We have reported that SoxB1 protein levels also are
sensitive to the presence or absence of the transcriptional repressor Krl.
Like Pmar1, Krl is a ß-catenin target gene but, unlike
Pmar1, Krl is expressed in both micromere and macromere progeny
(Howard et al., 2001
;
Minokawa et al., 2004
).
Here, we show, using loss-of-function assays, that two different nuclear,
ß-catenin-dependent, mechanisms operate to downregulate SoxB1 by reducing
the level of its mRNA and by promoting turnover of the SoxB1 protein. Neither
of these mechanisms depends on Krl, as shown by morpholino knockdown of its
translation, suggesting that the previously demonstrated effects of Krl on
SoxB1 levels (Howard et al.,
2001) are quite indirect. The finding that SoxB1 is selectively
degraded in vegetal blastomeres was unexpected and demonstrates a new kind of
regulatory mechanism that mediates the antagonism between SoxB1 and
ß-catenin/Tcf-Lef activity in the endomesoderm GRN. This output may work
through Pmar1, as misexpression of this factor can promote SoxB1 turnover in
all cells of the embryo. Lastly, we provide evidence for at least two
different lineage-specific degradation mechanisms, as the turnover of SoxB1 in
macromere progeny, but not in micromere progeny, depends both on its nuclear
localization and on sequences in its C-terminal domain. The existence of these
two degradation pathways may reflect cell-autonomous regulation of SoxB1 in
the micromere lineage and non-autonomous regulation in macromere
descendants.
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Materials and methods |
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Construct preparation
SoxB1-GFP was constructed using SOE (splicing by
overlap extension) PCR
(Horton et al., 1989) to link
the SoxB1 protein-coding region (amino acid residues 1 to 344) in frame with
GFP. The SoxB1 fragment was generated by PCR with a forward primer containing
an XhoI site and N-terminal SoxB1 codons, and a reverse primer
containing C-terminal SoxB1 and N-terminal GFP codons. The GFP fragment was
generated from a pSP64T.clone construct containing GFP sequence originally
derived from Green Lantern (Promega), using an N-terminal GFP forward primer
and a reverse primer starting just downstream of the polyadenylation sequence.
After linking the SoxB1 and GFP fragments by three rounds of PCR, the fusion
was amplified with the outside primers and the amplimer was ligated into
pET-15b between the XbaI and EspI (blunted) sites. This
construct served as the parent plasmid for producing the series of SoxB1-GFP
variants described below, all of which lack 5' and 3' UTR
sequences. All constructs encoding SoxB1-GFP variants were verified by
sequencing across junctions and by translation of synthetic mRNAs in vitro;
translation of full-length proteins in vivo was verified by green
fluorescence.
SoxB1DBD (DNA-binding domain)-GFP was produced by synthesizing SoxB1DBD by
PCR as described previously (Kenny et al.,
2003). This fragment was then ligated in frame at the
EcoR1 site of GFP/pSP64T.clone. The template for production of
SoxB1GFP RNA was made by PCR using a 5' primer equipped with an
Sp6 promoter followed by N-terminal SoxB1 sequence (-12 to +9 with respect to
AUG; GCT CAG ACT GAC CAA AAT GTC TGT T), and a 3' primer downstream of
the polydT tract (CCG GAA TTC TGT CTT CTT CAA CAG GGT CTT; the
EcoR1 site is underlined). For the templates containing a partial
SoxB1 sequence C-terminal from the DNA-binding domain (NDBD SoxB1GFP and
3'NLS SoxB1GFP), the forward primers encoded an Sp6 promoter, followed
by a translation initiation site (Kozak,
1989
), and five SoxB1 codons, starting at either amino acid
residue 146 or residue 127, respectively. These constructs lacked any
SoxB1 5' or 3' UTR sequence. The construct encoding SoxB1
lacking the DNA-binding domain was made by deleting codons for 44 amino acid
residues from the DBD of the SoxB1GFP/pET-15b plasmid template (residues
83-126) using SOE PCR (Horton et al.,
1989
). The resulting fragment, retaining the two nuclear
localization signals, was amplified using the primers described above for
SoxB1GFP. SOE PCR was used to create 3' SoxB1-GFP deletions in the
C-terminal half of the protein: deletions 1, 2 and 1+3 lack amino acid
residues 138 to 252, 254 to 344 and 138 to 252 + 326 to 344, respectively. The
templates for producing Krl
(Howard et al., 2001
),
cadherin (Lee and Gumbiner,
1995
) and SoxB1 MO-immune
(Kenny et al., 2003
) mRNAs
have been described previously. SoxB1MO-immune mRNA contains no 5'
SoxB1 UTR sequence and approximately 1 kb of 3'UTR sequence.
The plasmid containing full-length Pmar1 cDNA sequence in pBK-CMV was
a gift from P. Oliveri and E. Davidson (California Institute of Technology,
USA).
Microinjection and confocal image analysis
Fertilized eggs were microinjected with synthetic mRNAs or SoxB1MO as
described previously (Kenny et al.,
2003). Capped, polyadenylated mRNAs were transcribed using the
mMessage mMachine kit (Ambion) and purified according to the manufacturer's
instructions with DNase digestion and LiCl precipitation. RNA concentrations
were chosen that generated sufficient GFP signals, but that were well below
the levels that inhibit ß-catenin function
(Kenny et al., 2003
), so that
the embryos developed normally. For all SoxB1 variants, approximately 250,000
mRNA molecules were injected. Injection solutions containing RNA, 10 mM
Tris-HCl (pH 8.0) and 30% glycerol were filtered by centrifugation through
0.22 µm filters. Shortly after hatching (20-23 hours post-fertilization),
at least 30 embryos were examined by fluorescence microscopy for SoxB1-GFP
distributions. They were then collected in a thin capillary pipette and
deciliated by passage sequentially through two 100-µl aliquots of cold,
hypertonic seawater (Stephens,
1997
) in a depression slide. The embryos were then equilibrated in
ASW, deposited into a drop of ASW underneath a coverslip and immediately
photographed using a Leica TS confocal microscope. Fluorescent GFP signals
were overlaid on DIC images using Adobe Photoshop.
Immunostaining and whole-mount in situ hybridization
Embryos were fixed with 4% paraformaldehyde, 1xPBS
(phosphate-buffered saline), 0.2% Tween 20, for 15 minutes at room
temperature, and stained with a rabbit polyclonal SoxB1 antibody and a
CyIII-conjugated mouse anti-6e10 antibody, as described previously
(Kenny et al., 2003). Embryos
that were analyzed for both SoxB1 mRNA and protein were first
hybridized with digoxigenin-labeled SoxB1 antisense RNA. The template
consisted of a 415-bp BalI/NdeI fragment of the
SoxB1 coding sequence inserted into the EcoRV site of
pBluescript, which was digested with XhoI. RNA was synthesized with
T3 RNA polymerase in vitro. Hybridization and hybrid detection were carried
out according to Minokawa et al. (Minokawa
et al., 2004
) with the exception that the hybridization time was
shortened to one day. After staining with anti-digoxigenin linked to alkaline
phosphatase, embryos were immunostained by incubation for 2 hours with
anti-SoxB1antibody (1:1000) and 1 hour with goat anti-rabbit fluorescein
isothiocyanate (FITC)-conjugated antibody (1:500; Zymed).
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Results |
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A SoxB1GFP fusion protein is selectively degraded in vegetal cells
The mechanism that prevents accumulation of SoxB1 protein in vegetal cells
could be translational or post-translational. To begin to discriminate between
these alternatives, we injected mRNA encoding GFP-tagged SoxB1 lacking the
5' and 3' UTR sequences, in which translational control sequences
nearly always reside. In all experiments in which SoxB1 mRNAs were
injected, the dose was approximately 10-fold lower than the minimum dose that
interferes detectably with endomesoderm development, yet high enough to detect
GFP fluorescence. Live embryos accumulated this SoxB1-GFP fusion protein in a
very reproducible pattern: it was detectable in all nuclei of the embryo
beginning at about the eight-cell stage and was partitioned to nuclei of
mesomeres, macromeres and micromeres of the 16-cell embryo, in a manner
similar to endogenous SoxB1 protein (Fig.
2A). It was cleared from the descendants of micromeres and from
most of the macromere progeny by the early mesenchyme blastula stage. A
confocal image of a representative embryo is shown in
Fig. 2B. This result strongly
suggests that the post-transcriptional mechanism that clears SoxB1 from
vegetal cells is selective turnover, not translational control.
|
SoxB1-GFP degradation in vegetal cells does not depend on Krl, but can be promoted by Pmar1
We next examined the potential roles of two candidate genes, Krl
and Pmar1, in mediating SoxB1 turnover. Krl mRNA is
expressed in the cells in which SoxB1 is selectively degraded in response to
nuclear ß-catenin, and accumulates in a vegetal-to-animal wave like that
of nuclear ß-catenin. MO-mediated loss of Krl function causes endogenous
SoxB1 protein to accumulate to higher levels throughout most of the embryo,
including some cells within the endomesodermal territory that normally would
downregulate this protein. Embryos arrest at a stage that resembles a
mesenchyme blastula (Howard et al.,
2001). Conversely, mis/overexpression (MOE) of Krl by mRNA
injection eliminates SoxB1 protein from almost all cells of the embryo, with
the exception of a few near the animal pole
(Howard et al., 2001
). We
therefore tested the effect of altering Krl levels on SoxB1-GFP distributions
by injecting Krl mRNA or by blocking its translation with a KrlMO. As
shown in Fig. 2E,F, neither
perturbation detectably affected the clearance of SoxB1-GFP.
Pmar1 is a second candidate for mediating ß-catenin-dependent SoxB1
turnover. Embryos mis/overexpressing Pmar1 are extremely vegetalized, with
most cells of the embryo being transformed to a PMC-like phenotype by the
mesenchyme blastula stage (Oliveri et al.,
2002). Micromeres lacking ß-catenin nuclear function but
supplied with Pmar1 do not accumulate SoxB1 and can induce its downregulation
in nearby cells (Oliveri et al.,
2003
), although it is not known whether this regulation operates
at the level of SoxB1 mRNA or protein. To test whether Pmar1 can
affect the stability of SoxB1 protein, mRNAs encoding Pmar1 and SoxB1-GFP were
co-injected. As shown in Fig.
2G, SoxB1-GFP is not stable in these embryos, indicating that
ectopic expression of Pmar1 can promote SoxB1 turnover. The effect of
eliminating Pmar1 was not tested because of Pmar gene redundancy (P. Oliveri
and E. Davidson, personal communication).
SoxB1 turnover in macromere derivatives requires functionally redundant sequences within its C-terminal region
SoxB1 contains a highly conserved HMG-box DNA-binding domain (DBD) that is
flanked by a short N-terminal (58 amino acid residues) and a much longer
C-terminal (207 residues) sequence. At the borders of the DBD are motifs
matching the two separate nuclear localization signals that are found in this
class of transcription factor (Fig.
3A). To begin mapping the sequences that are required for SoxB1
degradation, we injected mRNAs encoding mutated SoxB1 proteins, each tagged at
its C terminus with GFP. At least 30 embryos expressing each construct were
analyzed and the results were very reproducible. SoxB1-GFP chimeras lacking
most of the sequence C-terminal to the DBD were stable in all cells except the
PMCs (Fig. 3B). This suggests
that the essential destruction sequences in SoxB1 that are recognized in
macromere-derived endomesoderm reside C-terminal from the DBD, and that a
different, or additional, degradation mechanism functions in micromere
derivatives. Interestingly, each of several different peptides that were
partially deleted in the C-terminal region of SoxB1 was degraded
preferentially in both macromere and micromere progeny
(Fig. 3C-E). Although a number
of putative phosphorylation motifs are distributed throughout the C-terminal
region, the three regions that can mediate turnover do not share a common
candidate motif. These results suggest the existence of functionally redundant
sequences mediating SoxB1-GFP decay. Although these data do not rule out the
formal possibility that turnover is in some way compromised when the GFP and
SoxB1 DBD domains are closely spaced, such an effect would be restricted to
turnover in endomesoderm derived from macromeres and not from micromeres.
|
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SoxB1 may provide a negative feedback on its own mRNA accumulation
Previous studies showed that the distribution of SoxB1 mRNA
modulates from its uniform maternal pattern to a non-vegetal pattern during
blastula stages, around the seventh to eighth cleavage
(Kenny et al., 1999). If SoxB1
were a positive regulator of its own transcription, then its loss from vegetal
cells could account for reduced SoxB1 transcription in these cells.
To test this possibility, embryos were injected with SoxB1MO and
SoxB1 mRNA accumulation was analyzed semi-quantitatively by
whole-mount in situ hybridization. As shown in
Fig. 6, loss of SoxB1 did not
lead to a decrease in signal. Instead, the signal was strongly increased in
non-vegetal cells. This result corroborates real-time PCR measurements, which
indicate that SoxB1 mRNA levels in whole embryos increase about
tenfold in SoxB1MO embryos (C. Livi, L.M.A. and E. Davidson, unpublished).
Thus, these results show that SoxB1 does not positively regulate accumulation
of its mRNA and raise the possibility that it functions either directly or
indirectly in negative-feedback regulation.
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Discussion |
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Our results show that SoxB1 expression also is spatially regulated at the
level of protein turnover (Fig.
8B,C). When SoxB1 protein was synthesized from uniformly
distributed microinjected mRNA transcripts and detected by immunostaining, it
was found to accumulate in animal, but not vegetal blastomeres at the hatching
blastula stage (about 17 hours) (Fig.
1C), thus demonstrating post-transcriptional control. When mRNA
encoding a SoxB1-GFP protein chimera was injected, fluorescence initially was
detectable in all cells of eight- to 16-cell embryos
(Fig. 2A), but the protein
turned over in both micromere and macromere progeny at the early mesenchyme
blastula stage. Analysis of the behavior of partial SoxB1 peptides similarly
tagged with GFP provided evidence for a separate protein turnover mechanism
that functions only in micromeres: turnover in macromere derivatives requires
signals in the C-terminal region and the NLS sequences, whereas neither of
these is required for selective degradation in micromere derivatives
(Fig. 3B,
Fig. 4). Either of these
mechanisms could account for the observation by Kenny et al.
(Kenny et al., 1999) that the
amount of SoxB1 DNA-binding activity per microgram of total protein is about
5-fold lower in whole-cell extracts of micromeres, when compared with extracts
of macromeres plus mesomeres. Together, these observations suggest that the
micromere-specific mechanism may be activated as early as the 16-cell stage.
However, we have not been able to demonstrate this directly by the available
assays because turnover of GFP-tagged peptides was not observable until the
early mesenchyme blastula stage (Figs
3,
5). Potential explanations for
this delay include the possibility that the level of SoxB1-GFP that is
required for detection overloads the turnover mechanisms, or that the
conformation of the SoxB1-GFP fusion proteins somehow slows the rate of
turnover. As misexpression of SoxB1 blocks all vegetal development
(Kenny et al., 2003
), an early
micromere-specific mechanism for removal of SoxB1 could be a crucial feature
of activating the endomesoderm GRN.
In normal embryos, SoxB1 mRNA begins to be downregulated in
macromere progeny between seventh and eighth cleavages (10-15 hours)
(Fig. 8D). This presumably
reflects regulation at the level of transcription, as there is no evidence at
the present time for a lower stability of SoxB1 mRNA in vegetal
blastomeres. At the same time, SoxB1 protein begins to disappear from the
presumptive secondary mesenchyme and endoderm, starting within the more
vegetal blastomeres (Kenny et al.,
1999). The exact timing of transcriptional repression versus
selective protein turnover, and the relative contributions of these mechanisms
to the establishment of polarized SoxB1 distributions are not yet clear.
However, it is important to note that the protein turnover mechanism is active
and robust in the early blastula. We showed that SoxB1 protein is cleared from
embryos that have uniformly distributed microinjected SoxB1 MO-immune
mRNA that is
3-fold higher than the level of endogenous mRNA in the egg
and throughout cleavage (Fig.
1C). The functional significance of SoxB1 protein turnover is
suggested by the fact that normal vegetal differentiation proceeds on schedule
in these embryos, despite the persistence of elevated levels of uniformly
distributed SoxB1 mRNA. Thus, the embryo appears to have an excess
capacity to downregulate SoxB1 in vegetal blastomeres at the level of protein
turnover. These observations also can explain why even higher levels of
microinjected SoxB1 message, i.e. about 10-fold above normal
endogenous levels, are required to evoke the animalized misexpression
phenotype (Kenny et al.,
2003
). Together, the available data strongly suggest that the
post-translational mechanism serves to clear SoxB1 protein from endomesoderm
more rapidly than could be achieved by downregulating mRNA abundance. However,
definitive evaluation of the relative importance of the transcriptional and
post-translational mechanisms in regulating SoxB1 distributions and normal
patterning of fates along the AV axis will require specific inhibition of
SoxB1 turnover, which is not yet possible.
Our previous work has shown that reduction of SoxB1 mRNA levels in
vegetal blastomeres is dependent on the function of nuclear ß-catenin. In
the present work, we have demonstrated that differential turnover of SoxB1-GFP
is blocked in embryos in which ß-catenin function is blocked by
overexpressing C-cadherin. As SoxB1 turnover follows entry of ß-catenin
into nuclei of macromere progeny, this process could be cell-autonomous.
However, available evidence suggests that the link between nuclear
ß-catenin function and SoxB1 turnover in macromere derivatives is
indirect and, at least partially, non cell-autonomous. Oliveri et al.
(Oliveri et al., 2003) have
reported that efficient SoxB1 clearance from endomesoderm requires a signal
from micromeres, as it is diminished when micromeres are removed after the
fourth cleavage. This signal was reported to be mediated by Pmar1 function, as
micromeres carrying C-cadherin mRNA could downregulate endogenous SoxB1, both
in themselves and in nearby blastomeres, if supplied with exogenous
Pmar1 mRNA. Although these experiments demonstrate a possible
function for Pmar1 upstream of a signal that promotes loss of SoxB1 in
macromere progeny, tests of loss of Pmar1 function will be required to confirm
that it is necessary in the normal embryo; to date such tests have been
confounded by the multiplicity of Pmar1-related genes. Interestingly,
injection of Pmar1 mRNA also conferred on mesomeres the same
capacity, although mesomere derivatives never express Pmar1 in normal embryos.
This observation suggests a cell-autonomous role for Pmar1 in the clearance of
SoxB1 from micromeres. Our experiments do not reveal whether the early
micromere-specific protein turnover mechanism depends on nuclear
ß-catenin and Pmar1, because over accumulation of SoxB1 mRNA in
cadherin-expressing embryos could overwhelm this mechanism by the early
mesenchyme blastula stage, when we carried out our analyses.
Finally, SoxB1 appears to exert a potent negative feedback on its own
transcription because SoxB1 mRNA levels are elevated about 10-fold in
SoxB1MO-knockdown embryos (Fig.
6, Fig. 8E) (C.
Livi, E. H. Davidson and L.M.A., unpublished). As SoxB1 is a DNA-bending
`architectural factor' without detectable ability to activate transcription
independently (Kenny et al.,
2001), it might interact with other regulatory factors to directly
repress its own expression, or to activate production of an intermediary
repressor. The fact that SoxB1 mRNA increases dramatically in
abundance in presumptive ectoderm of SoxB1MO-knockdown mesenchyme blastulae,
but is undetectable in all endomesoderm
(Fig. 6B,
Fig. 8E), suggests that this
feedback loop is restricted to ectoderm, whereas other,
ß-catenin-dependent, mechanisms downregulate SoxB1 transcription
in vegetal blastomeres. We think it likely that this feedback loop serves to
limit SoxB1 accumulation in presumptive ectoderm after cleavage, as the rate
of cell division decreases. Transcription of several ectoderm-specific genes
has been found to decrease significantly during this same period
(Gagnon et al., 1992
;
Lee et al., 1992
).
Modulation of transcription factor concentrations via regulated turnover is
a relatively unusual developmental mechanism. An example closely related to
the selective degradation in early blastomeres described here is provided by
the early C. elegans embryo, in which germline regulatory protein
degradation is activated in somatic blastomeres during the first few
asymmetric cleavages (Pellettieri et al.,
2003). An example analogous to the antagonism between SoxB1 and
nuclear ß-catenin has been described in Xenopus embryos: in that
system selective proteolysis of the homeodomain repressor Xom is activated on
the future dorsal side of the embryo, thus preventing it from inhibiting
activation of dorsal-specific genes, including goosecoid
(Zhu and Kirschner, 2002
).
Targeted protein turnover has also been shown to be a response to various
signals, as is probably the case for SoxB1 degradation in macromere progeny.
For example, the dorsal/Nf
b cytoplasmic tether cactus/I
b is
degraded in response to signaling through Toll/cytokine receptors (reviewed by
Belvin and Anderson, 1996
) and
the stability of ß-catenin itself is regulated through Wnt signaling (for
a review, see Nelson and Nusse,
2004
). Our understanding of ß-catenin-dependent pathways has
expanded dramatically in recent years with most emphasis placed on regulatory
target genes, encoding either transcription factors or signaling molecules. To
our knowledge, this is the first description of a ß-catenin-dependent
pathway that promotes the turnover of a key developmental regulator of
transcription, the SoxB1 protein of the sea urchin embryo.
Note added in proof
Using GFP-tagged ß-catenin, Weitzel et al.
(Weitzel et al., 2004)
recently demonstrated a pattern of protein stability along the animal-vegetal
axis of sea urchin embryos that is reciprocal to that of SoxB1.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: School of Medicine and Dentistry, University of Rochester,
Rochester, NY 14642, USA
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