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First published online 17 January 2007
doi: 10.1242/dev.02768
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1 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation, 3333 Burnett Avenue, Cincinnati, OH 45229, USA.
2 Physician Scientist Training Program, University of Cincinnati College of
Medicine, PO Box 670555, Cincinnati, OH 45267, USA.
* Author for correspondence (e-mail: christopher.wylie{at}cchmc.org)
Accepted 30 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: FoxI1e, Xema, Xenopus, Ectoderm
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Animal
cells are specified to become ectoderm, while equatorial and vegetal cells are
specified to form mesoderm and endoderm, respectively. The molecular
mechanisms of these events have been best studied in vegetal and equatorial
cells. Vegetal cells become specified to become endoderm by the vegetally
localized maternal T-box transcription factor VegT
(Zhang et al., 1998). These
cells release nodal-related factors, which act upon equatorial cells to induce
mesoderm. By contrast, little is known about how animal cells are specified to
become ectoderm. Ectoderm has long been considered a default pathway of
embryonic development, with cells not under the influence of the VegT pathway
adopting an ectodermal program of gene expression and development. Indeed, in
embryos that have been depleted of VegT, cells normally fated to become
endoderm and mesoderm instead become ectoderm
(Zhang et al., 1998).
Previous work has shown that the process of ectoderm specification begins
during the blastula stage. Dissociated cells from different regions of the
blastula that are mixed and allowed to reaggregate separate according to their
original regional identities (Turner et
al., 1989). The molecular mechanisms of this differential adhesion
are not yet known. However, it has been shown that the ability of animal and
vegetal cells to sort from one another is lost when VegT is depleted
(Houston and Wylie, 2003).
Further, singlecell transplantation experiments have shown that animal cells,
which are able to contribute to all three germ layers during the early and
mid-blastula stages, become gradually restricted to ectodermal fates from the
late blastula to gastrula stages (Snape et
al., 1987). Thus, specification of animal cells begins during the
late blastula stage.
Little is known about the initial events of ectoderm specification during
the blastula stage. The maternally and zygotically encoded Smad4 ubiquitin
ligase ectodermin (Dupont et al.,
2005) and the zygotic forkhead-box transcription factor Xema
(Suri et al., 2005), have both
been found to be important in this process. Both molecules have been reported
to be repressors of mesoderm formation in the ectoderm domain, rather than
activators of ectodermal gene expression. During the gastrula stage, the cells
of the animal region begin to subdivide into different ectodermal
subpopulations, with cells on the ventral side of the embryo giving rise to
epidermis in response to high levels of Bmp signaling, and those on the dorsal
side giving rise to the central nervous system (CNS) upon exposure to Bmp
antagonists released from the organizer. The cells at the border of the CNS
and the epidermis contribute to the neural crest population. It is clear that
all of the cells of the animal cap have the potential to contribute to either
neural structures (reviewed by De Robertis
and Kuroda, 2004; Stern,
2005) or epidermal structures
(Luo et al., 2002;
Tao et al., 2005). However,
not much is known about genes that act upstream of the decision to become
neural or epidermal, nor which signaling molecules and transcription factors,
present at the blastula stage, actively initiate ectodermal specification.
To identify candidate genes that may address these problems, we carried out Affymetrix chip analyses to identify mRNAs that are upregulated both in animal caps relative to vegetal masses of dissected blastulae, and in VegT-depleted embryos (in which the marginal and vegetal regions form ectoderm) relative to wild-type embryos. This identified several candidate genes, expressed from the mid-blastula stage onward, at high levels in the cells destined to form both epidermal and neural structures.
The gene most highly overexpressed in VegT-depleted blastulae was a
forkhead-type transcription factor previously identified as Xema
(Suri et al., 2005). Sequence
similarity places Xema in the FoxI1 class of genes, and so
we suggest the more systematic name FoxI1e, which we will use
throughout this paper. The forkhead transcription factor family includes a
large number of genes involved in a variety of developmental processes.
Currently, over ten subclasses and 20 forkhead genes have been identified in
Xenopus. The FoxI1-class genes in Xenopus are
reportedly involved in ventral head specification (FoxI1a)
(Matsuo-Takasaki et al.,
2005), mesoderm formation (FoxI1b) and eye development
(FoxI1c) (Pohl and Knochel,
2005). In a previous study, FoxI1e was found to be
expressed only in the animal hemisphere of the embryo, starting at the
blastula stage, and when overexpressed, it inhibited the formation of mesoderm
and endoderm. It was therefore proposed to maintain the expression domain of
ectodermal genes by inhibiting the formation of other germ layers
(Suri et al., 2005).
In this work, we first confirm that FoxI1e is expressed in the animal half of the embryo at the blastula stage, but curiously, not in all of the cells, and that it is switched off in the CNS at the early neurula stage. We show that it remains on only in non-ciliated cells in the epidermis. We then show that FoxI1e expression is confined to the animal half of the blastula via inhibition by nodal signaling, downstream of VegT, in the vegetal half of the embryo. If this inhibition is overridden by injection of FoxI1e mRNA into vegetal cells, they adopt characteristics of ectodermal development, and express genes normally expressed in both the CNS and epidermis. This shows that FoxI1e is sufficient, when overexpressed ectopically, for activation of genes in both branches of ectodermal differentiation. This suggests that FoxI1e may be an activator of ectodermal differentiation, in addition to its previously identified role as an inhibitor of the other germ layers, and may act upstream of the separation of neural and epidermal cell fates in the ectoderm. To test this, we used a splice-blocking morpholino antisense oligodeoxynucleotide (SBMO) to block FoxI1e mRNA maturation, and hence expression of its gene product, in the early embryo. This caused defects in both epidermis and nervous system development, and the downregulation of both epidermal and neural mRNAs. Lastly, we show that FoxI1e is required for animal:animal cell adhesion at the mid-gastrula stage. In its absence, animal cells lose contact with the animal region of the embryo and fall into the blastocoel, where they mix with, and differentiate into, other lineages. These results show that FoxI1e is involved in the active initiation of ectodermal gene transcription, and probably plays a role before separation into epidermal and neural pathways.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) using
manually defolliculated oocytes injected with 7.5 ng VegT antisense
oligo before being fertilized via the host transfer technique. Staging was
according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967).
Dissections were performed at either stage 7 or stage 9 on agarosecoated
dishes in 1xMMR and then cultured in OCM. For the animal cap versus
vegetal mass microarray experiment, vegetal masses were explanted at stage 11.
Total RNA for Affymetrix experiments was isolated either by proteinase-K
treatment followed by phenol:chloroform extraction, or by Trizol (Gibco)
followed by purification through RNeasy columns (Qiagen). Each Affymetrix
experiment was performed once, and then confirmed by RT-PCR.
RT-PCR
Total RNA was extracted as previously described
(Zhang et al., 1998). Unless
otherwise indicated, input was two whole embryos, ten animal caps or five
vegetal masses per sample. cDNA was synthesized using oligo dT primers, and
semi-quantitative RT-PCR was carried out using the LightCycler system as
described by Kofron et al. (Kofron et al.,
2001). Ornithine decarboxylase (ODC) was used as
a loading control, and all values were normalized to ODC levels. In
all cases, water-only and reverse transcriptase-negative controls failed to
produce specific products. Each experiment was repeated a minimum of three
times in independent experiments to verify reproducibility of results.
Sequences for markerspecific primers are listed in Houston and Wylie
(Houston and Wylie, 2003). New
primers were generated for NRP-1 (U:
5'-TGATCCGCTCTGCACTCTTT-3', D:
5'-TGCTTTGAATTTGCAGTACATTG-3'), AP-2 (U:
5'-GCAGCCACCAACTCTTCTCT-3', D:
5'-CGTAGCTCCATTGCCTGTTC-3') and Sox-2 (U:
5'-TCTGCACATGAAGGAGCATC-3', D:
5'-CGTTCATGTGGGCATAAGTG-3').
Immunostaining and whole-mount in situ hybridization
-Tubulin staining was performed with 1:500 DMEM antibody (Sigma),
followed by HRP-(Jackson ImmunoResearch), Cy5-(Jackson), or
Alexa-488-conjugated (Molecular Probes) anti-mouse secondary antibody.
Blocking solution was 20% fetal calf serum and 4% bovine serum albumin in PBS
+ 0.1% Tween-20. For co-staining with FoxI1e antisense-mRNA probe,
embryos were first processed by the whole-mount in situ protocol modified from
Harland (Harland, 1991) and
then stained for -tubulin. The proteinase-K step was omitted to ensure
survival of the reactive epitope.
mRNA, morpholino oligos and injections
A coding-sequence-only FoxI1e clone was generated with primers
that included restriction sites for ClaI and NotI (U:
5'-TCGAATCGATATGAGTGCATTTGATCCACA-3', D:
5'-TCGAGCGGCCGCACCCATGTTAAACCCCACAG-3'), and then cloned into the
corresponding sites in pCS107. mRNA was synthesized using the SP6 mMessage
mMachine kit (Ambion). Three morpholinos targeting FoxI1e mRNA were
synthesized and rejected: two were designed to inhibit translation:
5'-GCTTGTGGATCAAATGCACTCATAG-3' (toxic) and
5'-CTAGCAGGTCACAAATACACCTGTA-3' (ineffective), and one was
designed against the splice donor site:
5'-TGAATGGTTATTACCTGGATCATCT-3' (only partially effective). A
splice acceptor-blocking oligo (SBMO) was found to be effective and non-toxic:
5'-ATAATTGCCCTTGCCTGTAAAGAAAA-3'. Intron junction sequence was
determined by designing primers against X. tropicalis genomic
sequence. PCR was performed using X. laevis genomic DNA as a
template, and the product was sequenced. Two pseudoalleles were identified,
and the morpholino was designed to target both. Intron-spanning primers were
designed to detect mature and immature forms of FoxI1e mRNA (U:
5'-CATGGAGCCCCAGATAAAAG-3', D:
5'-TTGGGTCCAAGGTCCAATAA-3'). For rescue experiments, mRNA and
SBMO, along with different lineage tracers, were injected sequentially rather
than mixed together in one injectate.
Lineage analysis
Four hundred picograms of mRNA encoding enhanced green fluorescent protein
(GFP) was injected to trace the descendents of individual D-tier cells at the
32-cell stage. For tracing injected animal cells, rhodamine-lysine dextran
(RLDX) or fluorescein-lysine dextran (FLDX) were co-injected: 15 ng was used
for eight-cell stage injections, and 5 ng was used for 32-cell stage
injections. For histology, embryos were fixed, dehydrated, cleared in xylenes,
embedded in paraplast and sectioned before imaging.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) embryos was compared. Second, expression was compared
between animal caps and vegetal masses at stage 11. We then looked for
candidates that were upregulated in VegT-depleted vegetal masses
relative to control vegetal masses and in wild-type animal caps relative to
wild-type vegetal masses. Normalized expression levels were compared and fold
changes were calculated. FoxI1e was upregulated approximately 40-fold
in isolated VegT-depleted vegetal masses, compared with wild-type
vegetal masses. In wild-type animal caps, FoxI1e mRNA was
overexpressed over 128-fold relative to wild-type vegetal masses
(Table 1). These microarray
results were confirmed by semi-quantitative RT-PCR. VegT-depleted whole
embryos, marginal zones and vegetal masses were compared with the
corresponding regions of control embryos at stage 10 to show that
FoxI1e mRNA is upregulated in the absence of VegT
(Fig. 1A). We then dissected
normal embryos at the late blastula stage (stage 9) into animal caps, equators
and vegetal masses, representing prospective ectoderm, mesoderm and endoderm,
respectively, and cultured them until the mid-gastrula stage (stage 11).
Comparative analysis of gene expression revealed that FoxI1e is
expressed primarily in the ectoderm, with lower levels in the mesoderm
(Fig. 1B). This result confirms
the previously reported expression pattern, which shows expression beginning
after the mid-blastula transition in the prospective ectoderm
(Suri et al., 2005).
Additionally, in situ hybridization for FoxI1e shows that although it
is expressed in the animal cap of the blastula, it is not present in all cells
(Fig. 2A), but instead, is
present in a salt and pepper arrangement. This pattern of expression is
maintained throughout early development. At later stages, expression is lost
from the developing nervous system, and maintained only in the epidermis. The
salt and pepper expression pattern confirms those shown in an in situ screen
of random cDNAs, by the Japanese National Institute of Basic Biology
(http://xenopus.nibb.ac.jp,
clone XL016g24), but not previously published.
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-tubulin, which is expressed only in cells destined to develop
cilia on their surfaces (Deblandre et al.,
1999). To test the possibility that FoxI1e is expressed
specifically in ciliated cells, we co-stained tailbud embryos (stage 30) for
-tubulin protein and FoxI1e mRNA by both immunochemistry and
in situ hybridization, respectively. We found that, in fact, the reverse was
the case (Fig. 2B,C). Analysis
of multiple sections showed that these two markers were not expressed in the
same cells (Fig. 2D).
Expression of FoxI1e only in the animal half of the blastula could
be due to either localized activation, or to a combination of global
activation and localized inhibition in the vegetal cells. It is known from
previous work that VegT activates expression of nodal-class ligands
in vegetal cells, which, in turn, induce mesoderm expression in the marginal
zone. As FoxI1e is upregulated in the absence of VegT in both the
prospective mesoderm and endoderm, it is possible that its expression is
normally inhibited in the vegetal half of the embryo, either by VegT directly
(in the vegetal mass), or by nodal signaling downstream of VegT (in the
marginal zone), or both. To test this hypothesis, embryos were injected
vegetally at the two-cell stage with 1.5 ng of mRNA encoding CerS, a soluble
nodal inhibitor (Agius et al.,
2000), and cultured to the mid-gastrula (stage 11) and late
neurula (stage 18) stages. Embryos injected with CerS mRNA had
increased levels of FoxI1e mRNA, as shown by RT-PCR, relative to
controls (Fig. 1C). Therefore,
FoxI1e is an ectodermal gene with expression that is normally
suppressed by nodal signaling downstream of VegT.
FoxI1e is an activator of ectoderm differentiation
FoxI1e has been reported to be an inhibitor of mesoderm
differentiation (Suri et al.,
2005). Its overexpression in the marginal zone leads to a
downregulation of several mesodermal genes and upregulation of the epidermal
marker epidermal keratin. To test more extensively what properties
FoxI1e confers on cells that do not normally express it, we injected
vegetal cells with FoxI1e mRNA and tested their fates in two ways.
First, we excised vegetal masses at the late blastula stage and assayed them
for expression of ectodermal markers. As FoxI1e is normally expressed
in the animal cap, in cells that will form both epidermis and CNS, we tested
for markers of both of those tissues. Two-cell embryos were injected with 300
or 600 pg of FoxI1e mRNA, dissected at the late blastula stage (stage
9), and vegetal explants were cultured until the midgastrula stage (stage 11).
As determined by RT-PCR, FoxI1e upregulated mRNAs encoding the early
pan-ectodermal marker E-cadherin, the epidermal markers epidermal
cytokeratin and AP-2, the neural marker Sox-2 and the
neural crest marker Slug (Fig.
3A). Additionally, mRNAs encoding the endodermal markers
Xsox17 and endodermin were decreased
(Fig. 3B). To determine if
FoxI1e could further increase expression of these markers in the
ectoderm, where they are already expressed, we injected FoxI1e mRNA
into the animal cytoplasm at the two-cell stage and then examined gene
expression in animal caps at stage 11. Interestingly, high doses of
FoxI1e mRNA (600 pg) caused animal caps to dissociate, and lower
doses had no effect on ectodermal gene expression (data not shown).
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-tubulin revealed the presence of
cilia on the surfaces of the explants, which were absent from controls
(Fig. 3F,G). We conclude that
FoxI1e overexpression in vegetal cells leads to behavior and gene
expression characteristic of ectoderm.
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As shown above, FoxI1e can activate ectoderm, in addition to its
already known function of inhibiting mesoderm and endoderm formation. To test
the hypothesis that activation of ectoderm differentiation is a normal
function of FoxI1e, and if so, whether it is required for both neural
and epidermal differentiation, we carried out a loss-of-function analysis. We
designed a splice-blocking morpholino oligo (SBMO) that spans the
splice-acceptor site of the gene. RT-PCR analysis of random hexamer primed
cDNA from stage-14 embryos previously injected with the SBMO confirmed that it
inhibits FoxI1e mRNA maturation
(Fig. 5A). It has been
previously reported that the use of a morpholino that targets the
translational start site results in embryonic death by the gastrula stage
(Suri et al., 2005). To
determine if FoxI1e mRNA depletion using a splice-blocking morpholino
is toxic, we performed an animal cap assay to see if embryos depleted of
FoxI1e respond normally to mesoderm induction by activin. Embryos were
injected at the twocell stage with 40 ng SBMO and animal caps excised at the
late blastula stage (stage 9) were treated with activin. Animal caps depleted
of FoxI1e showed the same level of mesoderm induction as control animal caps
(Fig. 5C). Therefore, loss of
FoxI1e is not inherently toxic to embryonic cells.
To determine the role of FoxI1e in embryonic development, we first examined the effects of global loss of FoxI1e function on early development, by injecting whole embryos at the two-cell stage with 20, 40 or 60 ng of the SBMO, into the animal region of each cell. FoxI1e-deficient embryos displayed dose-dependent abnormalities beginning at the gastrula stage. All injected embryos were delayed during gastrulation, but gastrulated completely by the time sibling controls reached mid-neurula stage (stage 16). Cement gland and neural-fold formation was also delayed dose-dependently (Fig. 5D). During early tailbud stages, FoxI1e-deficient embryos failed to elongate properly along the anteroposterior axis, had reduced head structures and began to develop gaps in their epidermis (Fig. 5E). The epidermal lesions in embryos injected with the highest does of the SBMO resulted in death of the embryos by osmotic lysis by the early tailbud stage. A morpholino oligonucleotide designed against the splice donor site of the FoxI1e mRNA caused similar but less severe effects (data not shown). RT-PCR analysis of embryos injected with this morpholino shows that FoxI1e mRNA maturation is not efficiently inhibited (Fig. 5B). We therefore used the splice acceptor site-targeting oligo for the rest of our experiments. Analysis of gene expression at stage 23 by RT-PCR in embryos injected with FoxI1e mRNA shows a downregulation of both neural and epidermal genes. mRNAs encoding the pan-epidermal genes such as E-cadherin and epidermal cytokeratin were reduced, as were those encoding pan-neural genes such as Sox-2, and the neural crest gene slug (Fig. 5F).
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From the tailbud stage (stage 22) onward, the Xenopus epidermis
contains cilia, the beating of which causes gliding movements of the larva
over the floor of the culture dish. We noticed that FoxI1e- deficient
embryos, unlike control embryos, failed to do this. To determine if this was
due to lack of cilia, we dissected out the role of FoxI1e in the
epidermis using the Xenopus fate map. We injected 10 ng SBMO,
together with RLDX as a lineage tracer, into one animal, ventral cell at the
eight-cell stage. In a normal embryo, this cell contributes to most of the
epidermis on one side of the embryo (Dale
and Slack, 1987). Embryos injected with RLDX alone had large
clones of fluorescent cells in the epidermis, which showed normal patterns of
cilia on their surfaces, as revealed by immunostaining for -tubulin (0%
abnormal, n=52). Embryos injected with RLDX, together with the SBMO,
also had clones of labeled cells in the epidermis. However, these had
dramatically decreased numbers of cilia, compared with cells derived from
noninjected cells (non-fluorescent clones) in the same embryo (67% abnormal,
n=66). To show that this effect is specific, we followed the
injection of SBMO and RLDX with another injection of FoxI1e mRNA with
GFP mRNA as a lineage label. Reintroduction of morpholino-resistant mRNA
rescues the loss of cilia (6% abnormal, n=68)
(Fig. 6A-F).
We next wanted to determine if FoxI1e is necessary for the activation of
ectoderm in the early embryo. To do this, we injected 60 ng SBMO into two-cell
stage embryos and dissected the animal caps at stage 7, before the onset of
zygotic transcription. Removal of the animal cap before the mid-blastula
transition ensures that the cells of the animal cap are never exposed to
mesoderm-inducing factors. If FoxI1e works only by repressing endoderm and
mesoderm in the animal cap, levels of early ectodermal marker expression would
be unchanged. If, however, there were a reduction in those markers, we would
conclude that FoxI1e actively promotes ectoderm formation. Animal caps
cultured alone from stage 7 would be expected to form only epidermal markers,
as they are not exposed to Bmp inhibitors from the organizer, which cause
neural specification. In this experiment, we therefore compared caps cultured
alone, to analyze their ability to form epidermis in the presence and absence
of FoxI1e, and injected with Bmp inhibitors, to analyze their ability to
express neural markers in the presence and absence of FoxI1e. In uninjected
animal caps, E-cadherin and epidermal cytokeratin mRNAs are
expressed, but not those of the neural markers Sox-2, NRP-1 or
NCAM. Injection of mRNA encoding cmBmp7, which inhibits all Bmp
signaling, causes a reduction in epidermal markers and an increase in neural
markers (Hawley et al., 1995).
In this experiment, embryos were injected with the SBMO at the two-cell stage,
and some were further injected with cmBmp7 mRNA at the four-cell
stage. Animal caps were excised at stage 7 and cultured until stage 14. We
found by RT-PCR that animal caps depleted of FoxI1e expressed reduced levels
of epidermal markers relative to uninjected animal caps, and that those
injected with cmBmp7 mRNA and depleted of FoxI1e expressed reduced
levels of neural genes relative to those injected with cmBmp7 mRNA
alone (Fig. 7A-E). As expected
in caps excised at stage 7, mesodermal gene transcripts were almost
undetectable (Fig. 7F). From
these data, we conclude that FoxI1e actively promotes ectodermal fates, both
neural and epidermal, in animal cap cells.
FoxI1e controls the localization of ectodermal cells within the embryo
One of the gain-of-function effects of FoxI1e was that vegetal cells
expressing it moved to the ectoderm rather than the endoderm. This raises the
possibility that one of the functions of FoxI1e might be to control positions
of cells expressing it in the embryo. It is known that regional cell
localization is strictly controlled in the blastula. There is limited cell
mixing, as indicated by the existence of a fate map, and cells from different
regions that are mixed will rapidly sort out again
(Turner et al., 1989).
However, very little is known about the molecular mechanisms of this process,
or whether it is part of the ectodermal specification that takes place during
the blastula stage. If FoxI1e were required for maintaining animal cells in
the animal hemisphere, then removing it would cause them to relocate to other
regions of the embryo. To test this hypothesis, we injected RLDX alone or RLDX
+ 15 ng SBMO into a single ventral animal cell at the 32-cell stage (cell A4).
Embryos were fixed at the tailbud stage (stage 40) and stained for cilia with
-tubulin antibody. The descendents of this cell are normally restricted
to the epidermis and are therefore located at the surface of the tailbud stage
embryo. This was the case in embryos that were injected with RLDX alone (92%,
n=60, Fig. 8A). Of the
remaining 8%, the most frequent location of extra-epidermal cells was the CNS.
However, even in these embryos, the vast majority of signal is in the
epidermis, with only occasional cells elsewhere. In embryos co-injected with
15 ng SBMO, there was a dramatic decrease in the amount of RLDX signal at the
surface of the embryos, indicating fewer descendents of the injected cell had
reached their normal position within the embryo
(Fig. 8B). Instead, there was a
collection of RLDX-positive cells in the abdomen of the embryo (72%,
n=60). We performed similar experiments for cells that normally
contribute to the nervous system. We injected RLDX alone or RLDX + 15 ng SBMO
into a single dorsal animal cell at the 32-cell stage (cell A1)
(Dale and Slack, 1987) and
cultured the embryos to the tailbud stage (stage 35-37). This cell would
normally give rise to descendents in the nervous system. Eighty-seven percent
of RLDX-injected embryos had signal restricted to neural structures
(n=45). As with the ventral injection, there were a small number of
embryos that had cells in other structures, primarily the epidermis. In
embryos co-injected with SBMO, signal was found both in the nervous system and
in the gut (25%, n=32).
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), some cells were found scattered in other germ layers
(data not shown). In embryos co-injected with the splice-blocking morpholino,
large groups of RLDX-positive cells were clearly visible along loops of the
intestinal epithelium and contributed to the brush border along with
RLDX-negative cells (Fig. 8D).
Experiments in which we injected the A1 blastomere (which gives rise
predominantly to the CNS), rather than A4, gave corresponding results. In
RLDX-only embryos, the majority of cells contributed to the CNS, but when
co-injected with SBMO, they also contributed to normal gut epithelium (data
not shown).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The data also show that FoxI1e controls cell position in the early embryo.
It has been known for many years that there is a good fate map in the early
Xenopus embryo. In fact, this is one of the attractive features of
using Xenopus as a model for early vertebrate development. However, a
fate map requires limited cell mixing during early development, a fact that
has also been shown by blastomere sorting experiments
(Turner et al., 1989) and
tissue recombination experiments
(Nieuwkoop, 1973;
Smith and Slack, 1983).
Although it is generally understood that as germ-layer specification occurs
genes are expressed that somehow control differential adhesion in the embryo,
and maintain the separation of these nascent germ layers in the blastula, its
mechanism is not known. The data presented here show that molecules that
control regional identity of animal cells are controlled by FoxI1e. This
represents the first developmental system in which it has been shown that a
forkhead-class transcription factor controls cell position in the embryo.
It is interesting to note that animal cells lacking FoxI1e that lose contact with the rest of the animal cell population do not die or differentiate as ectopic ectoderm, but instead become specified according to their new positions. This suggests that the normal function of FoxI1e is to switch animal cells from a pluripotent state to a more specified state, and in its absence, animal cells remain pluripotent.
The expression pattern of FoxI1e is interesting because in situ hybridizations do not show expression in every cell of the tissues in which it is expressed. In fact, this information is present on a website that documents the results of an expression screen (http://xenopus.nibb.ac.jp), but has not been previously published. This variegated pattern of expression could be due to cell cycle control of expression, in which case it would be expressed in all cells, but not at the same time, once cell divisions have become asynchronous. Alternatively, intercellular signaling could restrict expression, by mutual inhibition mechanisms, to one cell surrounded by non-expressing cells. The mechanisms underlying this expression pattern, as well as its function, require further analysis.
A previous functional analysis of FoxI1e
(Suri et al., 2005) suggested
that its primary role was to act as an inhibitor of mesoderm specification.
This work used an antisense morpholino oligo that caused the embryos to die at
the gastrula stage, so that analysis past this point could not be carried out.
Before the publication of the Suri et al. paper, we had started a functional
analysis using a sequence in the same region of the mRNA. However, its
toxicity made us look for alternative sequences that we could use. After
screening a number of sequences, we chose a morpholino oligo that blocks
maturation of the mRNA, by binding to a splice acceptor site. This oligo did
not cause any general toxicity of the embryo, and thus allowed a more detailed
functional analysis of FoxI1e, which revealed the fact that it plays essential
roles in the activation of ectodermal genes as well as regional identity of
the animal cells.
The identification of the roles of FoxI1e allows a more complete model to
be proposed of the cell interactions that lead to the division of the blastula
into three cell populations (Fig.
10). The maternal activators of FoxI1e expression must be
present throughout the early embryo, because, in the absence of VegT,
molecular markers of both neural and epidermal lineages appear in marginal and
vegetal cells (Xanthos et al.,
2001; Zhang et al.,
1998). Alternatively, in the absence of VegT, the influence of
ectodermdetermining molecules could reach further vegetally due to a reduction
in ectoderm-inhibitory factors. In areas of high levels of nodal expression,
downstream of VegT, FoxI1e expression is inhibited. In the most
animal region, where nodal expression is lowest, FoxI1e expression is
activated. Once FoxI1e is expressed, a mutual inhibition is set up
that generates a progressively more precise boundary between the ectoderm and
the other two germ layers. Cell mixing across this boundary is prevented by
expression of surface proteins that control cell position by differential
adhesion. Many elements of this model require further testing, including the
functional relationship between FoxI1e and other factors known to be important
in ectoderm differentiation such as ectodermin
(Dupont et al., 2005) Xoom
(Hasegawa et al., 2001) and
Xlim5 (Houston and Wylie,
2003), as well as the epidermal proteins AP-2
(Luo et al., 2002) and
grainyhead-like1 (Tao et al.,
2005), and the many early CNS factors including Sox2 and Sox3. It
is also likely that there will be signaling pathways in addition to nodals
that control the spatial and temporal expression of FoxI1e, and we know little
detail concerning its downstream targets. However, the outline of a global
mechanism of germ-layer formation is beginning to emerge.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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