Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
* Author for correspondence (e-mail: heabq9{at}chmcc.org)
Accepted 9 February 2004
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SUMMARY |
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Key words: Mixer, VegT, Endoderm, Mesoderm, Antisense oligo, Morpholino
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Introduction |
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The homeodomain transcription factor Mixer is a good candidate for a role
in boundary formation in the Xenopus gastrula
(Henry and Melton, 1998). Its
expression is dependent on maternal VegT and nodal related proteins and is
confined to an 8-hour period during gastrulation
(Henry and Melton, 1998
;
Xanthos et al., 2002
;
Xanthos et al., 2001
).
Mixer mRNA is expressed in the prospective endoderm cells of the
vegetal hemisphere in the early gastrula stage embryo, but is particularly
concentrated at the boundary between the equatorial, prospective mesoderm and
the vegetal, prospective endoderm regions
(Engleka et al., 2001
;
Henry and Melton, 1998
).
Previous functional studies show that Mixer is required for endoderm
development. Overexpression in animal caps leads to the ectopic induction of
endodermal molecular markers including endodermin, cerberus, DKK-1
and Xsox17 and their expression is blocked by a dominant inhibitory
form of Mixer, Mixer-ENR (Henry
and Melton, 1998
). Mixer-ENR mRNA-injected embryos
develop with gastrulation defects, as well as gut and head abnormalities, and
vegetal explants from such embryos lack the expression of late endodermal
markers IFABP and Xlhbox8
(Henry and Melton, 1998
).
However, the expression of Mixer mRNA in maternal VegT-depleted
embryos rescues the expression of Xsox17 to only a limited extent,
suggesting that its inducing activity relies on other VegT-dependent
co-factors (Xanthos et al.,
2001
). Mixer interacts with phospho-Smad2 through a Smad
interaction motif and acts as a transducer of Xnr signals
(Germain et al., 2000
).
There are six other Paired-like homeobox family members expressed
at the same time as Mixer in Xenopus gastrulae, including
Mix.1, the founding member, together with Mix.2,
Bix1/Mix4, Bix2, Bix3/Milk and Bix4
(Casey et al., 1999;
Ecochard et al., 1998
;
Henry and Melton, 1998
;
Latinkic and Smith, 1999
;
Mead et al., 1996
;
Rosa, 1989
). All have roles in
endoderm formation and gastrulation, and Mix.1 can also repress the
mesodermal marker Xbra (Latinkic
and Smith, 1999
). Two zebrafish Mix.1-related genes,
bon and mezzo have been described
(Kikuchi et al., 2000
;
Poulain and Lepage, 2002
).
Although neither are homologs of Xenopus Mixer, they have been shown
to act redundantly with each other in the formation of endoderm, as well as in
prechordal plate mesoderm formation
(Poulain and Lepage, 2002
).
Recently, Bon was also demonstrated to function in precursors of the axial
mesoderm to regulate anterior neural patterning
(Trinh et al., 2003
). In mice
and humans, only one family member, Mixl1 has been identified
(Pearce and Evans, 1999
;
Robb et al., 2000
). Mouse
Mixl1-/- mutants have defects in gastrulation and axial
mesoderm patterning, including increased expression of the mesodermal gene
Brachyury, and of the nodal signaling molecule
(Hart et al., 2002
). Relatively
normal early expression of endodermal markers Sox17 and Cer1
occurs, but the embryos die at the early somite stage (8.5 dpc), without
forming a heart or gut tube (Hart et al.,
2002
).
To establish the roles of Mixer in endoderm and mesoderm specification in Xenopus, we used antisense morpholino oligos (MixerMO) to block the translation of Mixer protein throughout the entire period of Mixer expression during gastrulation. We find that Mixer-depleted embryos develop with severe abnormalities of the head and gut, which are partially rescued by the expression of a non-complementary Mixer mRNA. The pattern of early zygotic gene expression is reproducibly altered at the gastrula stage, and the effects of Mixer-depletion are gene specific rather than germ layer specific. We show that Mixer loss-of-function results in overexpression of the mesodermal markers eomesodermin, Bix3 and Fgf8 in their usual equatorial location, as well as the spread of their expression into deeper endodermal territory. We also show that Mixer acts cell autonomously, and represses some genes (Bix3, Xnr5, Xnr1 and Fgf8) while activating others (cerberus, Xsox17). In functional assays of Mixer-depleted vegetal cells, we show that a major biological role of Mixer is to control the degree to which cells induce the formation of mesoderm.
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Materials and methods |
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Explant culture
Mid-blastula (stage 8) wild-type and Mixer MO-injected embryos were
devitellined and dissected with tungsten needles on agar coated dishes in
1xMMR. After washing away dead cells, vegetal and animal pieces were
placed alone (Fig. 4A) or
together in the combinations described in
Fig. 6 and cultured on agar in
OCM for 2 hours. The recombinants were separated using tungsten needles and
stray vegetal cells were identified by their larger size and whitish, opaque
color and removed from the explants. Animal caps were then cultured in fresh
OCM on agar until sibling uninjected embryos were stage 11 and frozen in
batches of 10 caps for analysis
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Oligos and mRNAs
Three overlapping morpholino oligos were designed complementary to the
junction between the Mixer 5'UTR and the open reading frame.
Fig. 1A shows the reverse
complement of each oligo sequence aligned with Mixer, a
Mixer pseudo-allele from the EST sequencing project, the related
genes Mix.1 and Mix.2, and a morpholino-resistant mRNA
(Mixer MO-R) designed for the rescue experiments. Mixer MO-1 was most
efficient in blocking the in vitro translation of Mixer mRNA, but did
not block translation of Mix.1 mRNA
(Fig. 1B and data not shown).
However, it also caused cell disaggregation and apoptosis at the late gastrula
stage in doses above 15 ng. To determine whether this was a specific effect,
the oligo was injected into the four animal blastomeres of eight-cell stage
embryos to target cells that do not express Mixer mRNA. MO-1 caused
disaggregation and apoptosis of epidermal cells at the neurula stage, as
evidenced by TUNEL staining, indicating that disaggregation was not due to
blocking Mixer translation (data not shown). MO-2 was shifted eight
nucleotides 5' of the region targeted by Mixer MO-1 and caused
more severe apoptosis beginning at gastrula stage. Mixer MO-3
targeted a region four nucleotides 3' of Mixer MO-1. MO-3
caused no disaggregation effects (data not shown) and blocked translation of
Mixer mRNA, although with less efficiency than MO-1
(Fig. 1B). We confirmed by in
vitro translation that neither oligo blocked the translation of the most
closely related family member Mix.1 (data not shown). The experiments
described here were performed using both MO-1 and MO-3 oligos.
|
In vitro translation
Wild-type or Mixer MO-R mRNA was used in a Biotin in vitro
Translation kit (Roche). Reactions were assembled on ice and then incubated at
30°C for 1 hour. Aliquots of the reactions were separated in a 10%
Tris-HCl electrophoresis gel and blotted to PVDF. The membranes were blocked
then incubated with streptavidin-POD and developed with Chemiluminescence
substrate (Roche). Membranes were then exposed to X-ray film.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed on pigmented or albino
embryos as described (Harland,
1991) using BM Purple as substrate (Roche). After satisfactory
color development, embryos were fixed in MEMFA for 1 hour at room temperature,
washed and stored in 100% methanol. Embryos for half-mount in situ
hybridization were prepared by fixing whole gastrulae for 1 hour in MEMFA,
bisecting the embryos along the dorsoventral axis with a scalpel blade, fixing
1 additional hour in MEMFA, washing and storing in 100% ethanol. The in situ
hybridization were performed exactly as above with the exception of reducing
the proteinase K treatment to 5 µg/ml for 10 minutes to reduce damage to
embryos.
Analysis of gene expression using real-time RT-PCR
Total RNA was prepared from oocytes, embryos and explants using proteinase
K and then treated with RNase-free DNase as described
(Zhang et al., 1998).
Approximately 0.5 µg RNA was used for cDNA synthesis with oligo (dT)
primers followed by real-time RT-PCR and quantitation using the `LightCycler'
System (Roche) as described previously
(Kofron et al., 1999
). The
primers and cycling conditions used are listed in
Table 1. Relative expression
values were calculated by comparison to a standard curve generated by serial
dilution of uninjected control cDNA. Samples were normalized to levels of
ornithine decarboxylase (ODC), which was used as a loading control. Samples of
water alone or controls lacking reverse transcriptase in the cDNA synthesis
reaction failed to give specific products in all cases.
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Results |
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To confirm that these changes in gene expression were specific, we analyzed the effects of the introduction of the non-complementary Mixer mRNA (Mixer MO-R mRNA) into Mixer-depleted embryos. Fig. 3B shows that both upregulation and downregulation of target genes caused by the Mixer depletion were rescued by subsequent Mixer MO-R mRNA injection into the four vegetal blastomeres at the eight-cell stage.
As several mesodermal genes were upregulated in Mixer-depleted embryos, this suggested that a normal function of Mixer may be to keep these genes off in the developing endoderm. To test this, control and Mixer-depleted embryos were dissected at the mid-blastula stage into animal, equatorial and vegetal parts, which were cultured separately until the mid-gastrula stage and analyzed for the expression of eomesodermin, Fgf8, Bix1, Bix4 and Xsox17. Fig. 4A shows that Mixer depletion caused an increase in the expression of the mesodermal markers in vegetal explants, indicating that Mixer normally inhibits the ectopic expression of these mesodermal genes. By contrast, the amount of expression of Xsox17 is reduced in vegetal explants depleted of Mixer compared to control expression levels. This suggests that a normal role of Mixer is to repress mesodermal genes and activate endodermal genes in vegetal cells.
In order to study the effect of depleting Mixer on the regional expression of mesodermal and endodermal genes, wild-type and Mixer-depleted embryos were fixed and bisected at the early and mid-gastrula stages and the location of eomesodermin, Bix3, Fgf8, Xbra and cerberus mRNA was examined by in situ hybridization (Fig. 4B and data not shown). Although cerberus and Xbra expression was clearly reduced in Mixer-depleted embryos at both stages, the expression of eomesodermin, Bix 3 and Fgf8 was increased in equatorial cells and expanded into deeper cells. To examine the expansion of the domain of eomesodermin expression more closely, Mixer MO-1 was injected into single vegetal cells of eight-cell stage embryos, to block Mixer translation over only one quarter of the vegetal mass at the gastrula stage. In comparison with the control side, where eomesodermin mRNA is expressed in equatorial cells only, Fig. 4C shows an expansion of eomesodermin expression into the vegetal territory in the Mixer-depleted area (arrow). These data show clearly that a normal role of Mixer is to repress the expression of mesodermal genes in the endoderm.
Mixer regulates the capacity of the vegetal mass of the late blastula-stage embryo to induce mesoderm
Both gain- and loss-of-function experiments show that Mixer is required for
the increased expression of some genes and the decreased expression of others,
both in the mesoderm and endoderm. What is the biological role of Mixer? To
test this, we used recombination experiments
(Nieuwkoop, 1969) in which
wild-type animal caps from mid-blastula stage embryos were placed in contact
with vegetal explants from uninjected control or Mixer-depleted late blastula
stage embryos. After a 2 hour co-culture period, animal caps were peeled off
the vegetal masses, stripped of any adherent vegetal cells, cultured until
sibling embryos had reached the mid-gastrula stage and analyzed for mesodermal
markers, including Xbra, Xnr1, Fgf8, eomesodermin and the endodermal
marker Xsox 17. Fig. 5
indicates that Mixer-depleted vegetal masses induced higher levels of
expression of all the mesodermal markers in animal caps than did control
vegetal masses. By comparison, the endodermal marker Xsox 17 was not
significantly induced in caps by either control or Mixer-depleted vegetal
masses. The experiment was repeated with a similar result (data not shown).
This suggests that Mixer normally functions to control the amount of
mesoderm-inducing activity in the vegetal mass.
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Discussion |
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These studies show that the roles of Mixer are more complex than was
suggested previously. We find that Mixer negatively regulates several early
zygotic genes both in the mesoderm and endoderm, while positively regulating
others. Further experiments are required to determine whether these effects of
Mixer are direct or indirect. One possible scenario is that Mixer activates
genes such as cerberus directly and these then block Xnr5 signaling
activity, and thus the Xn5 autoregulatory loop reducing Xnr5 mRNA expression.
This seems unlikely because of the timing of expression of these two genes.
Xnr5 mRNA is already overexpressed in Mixer-depleted embryos at the
early gastrula stage when cerberus mRNA expression is only just
beginning [Fig. 3A; see Xanthos
et al. (Xanthos et al., 2002)
for a more extensive temporal series of cerberus mRNA expression].
Another possibility is that Mixer activates some genes and repress others
directly. As one dose of oligo upregulates some target genes, and
downregulates others in the same area, it seems unlikely that the type of
activity is concentration dependent. More likely, the behavior of Mixer is
context dependent and gene specific, its combinatorial activity with other
transcription factors, as well as with co-repressors and co-activators,
determining the final outcome of Mixer binding. Detailed analysis of the
regulatory regions of these target genes, and the identification of
Mixer-associated co-repressors and co-activators is necessary to determine if
Mixer acts as a transcriptional activator and repressor.
It is interesting that, although the T-box transcription factors eomesodermin, Xbra and Antipodean are all recognized as pan-mesodermal markers and have very similar expression patterns, they are not regulated in the same way by Mixer, as eomesodermin expression increases, while the other genes are downregulated after Mixer depletion. Thus, similarity of expression patterns is not necessarily indicative of similar mechanisms of gene regulation, and Mixer cannot be viewed as a simple mesodermal repressor.
Xnr1, Xnr5, Fgf3 and Fgf8, which are repressed
by Mixer, encode secreted proteins that act as mesoderm inducing signals in
Xenopus embryos. Xnr1 and Xnr5 have been shown to rescue mesoderm and
axis formation in VegT-depleted embryos
(Kofron et al., 1999;
Takahashi et al., 2000
), while
FGF proteins are implicated in muscle cell precursor formation
(Standley et al., 2001
). We
show in Nieuwkoop assays and by carrying out cell transplantation experiments,
that that the loss of Mixer activity results in excessive production of
mesoderm inducing signals. This suggests that one important role for Mixer is
in limiting the signaling that specifies mesodermal fates, and thus in
dictating the size of the mesodermal territory. One interesting aspect of this
possible role for Mixer is the timing of its expression. The temporal
expression of Mixer mRNA lags considerably behind that of Xnr and Fgf
genes. We and others have shown that Xnr1, Xnr2, Xnr4, Xnr5 and
Xnr6 are strongly expressed downstream of VegT at the late blastula
stage (Takahashi et al., 2000
;
Xanthos et al., 2002
), while
Mixer expression begins at this stage but peaks 4 hours later at the
gastrula stage (Xanthos et al.,
2002
). The onset of Mixer activity thus corresponds to
the time of `loss of competence' of the vegetal mass, as defined by its
ability to induce mesoderm in animal caps
(Jones and Woodland, 1987
),
and is preceded by a 3-4 hour time window when mesoderm induction is
uninhibited. It will be important to understand what, besides VegT, controls
the temporal pattern of expression of Mixer.
Interestingly, transplantation experiments suggest that Mixer expression is
not essential for endodermal fate specification. All individually transplanted
and most small groups of Mixer-depleted vegetal cells transplanted onto the
blastocoel floor of host embryos continued to develop in the endoderm germ
layer and did not redistribute to mesoderm, suggesting that Mixer-depletion is
insufficient to cause their relocation to mesoderm. Previous studies have
shown that other transcription factors including GATA5 and other Mix family
members have important roles and may act in parallel with Mixer in determining
endodermal fate (Casey et al.,
1999; Weber et al.,
2000
). It is also possible that the surrounding wild-type vegetal
cells release inductive signals that maintain the endodermal fate of the
Mixer-depleted cells.
A model for the role of Mixer in mesendoderm patterning
This and previous work suggest the following model for formation of
boundaries of expression of several VegT target genes
(Fig. 7). After MBT, VegT
activates the expression of Xnr genes, Xsox17, Gata4, Gata5 and
Gata6, and Fgf genes in vegetal cells. The secreted mesoderm-inducing
molecules induce adjacent marginal cells, which do not express VegT, to
express more of themselves as well as the presumptive mesodermal genes
including eomesodermin and Xbra. At the early gastrula
stage, Mixer is cell autonomously induced by VegT-expressing cells.
It represses further expression of Xnr1, Xnr5, Fgf3 and
Fgf8, and therefore prevents further mesoderm inducing signals being
released. Mixer also activates Xsox17 and cerberus
expression, consolidating endodermal fates for vegetal cells and further
limiting the range of signaling molecule activity by the antagonistic activity
of cerberus.
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ACKNOWLEDGMENTS |
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