Division of Developmental Biology MLC 7007, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
* Author for correspondence (e-mail: Christopher.Wylie{at}chmcc.org)
Accepted 26 March 2003
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SUMMARY |
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Key words: Adhesion, Ectoderm, Germ layer, LIM homeobox, VegT, Xenopus
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INTRODUCTION |
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In Xenopus, the primary germ layers are thought to be determined
by the cytoplasmic localization of maternal determinants and subsequent
cell-cell communication. Of the three germ layers, only the formation of
mesoderm and endoderm are understood, whereas very little is known about
specification of the ectoderm. Equatorial cells in the blastula form mesoderm
in response to signalling by zygotic TGF-ß-related growth factors
released by the vegetal cells. The T-box transcription factor VegT, which is
encoded by a vegetally localized RNA, is required for the specification of
endoderm in the vegetal hemisphere and is necessary for the generation of
mesoderm-and endoderm-inducing signals
(Xanthos et al., 2001;
Kofron et al., 1999
;
Zhang et al., 1998
). In
addition, both mesoderm and endoderm require cell-cell communication prior to
gastrulation for the formation of differentiated cell types
(Lemaire and Gurdon, 1994
;
Yasuo and Lemaire, 1999
).
In the absence of VegT, vegetal and marginal cells express
ectodermal genes (Zhang et al.,
1998). Once specified as ectoderm, cells differentiate either as
epidermis if BMP signalling is active or as neural tissue if BMP signalling is
absent; termed the `default state' model (reviewed by
Muñoz-Sanjuan and Brivanlou,
2002
). In contrast to mesoderm and endoderm, and consistent with
the default model, ectoderm can form in the absence of cell-cell contact
(Wilson and Hemmati-Brivanlou,
1995
), suggesting that maternal, cell autonomous factors are
responsible for ectoderm specification. Such factors must be present
throughout the embryo because ectoderm can be ectopically induced in vegetal
explants (which normally form endoderm) by depletion of maternal VegT
RNA (Zhang et al., 1998
) or by
overexpression of TGFß signalling antagonists
(Henry et al., 1996
).
In this work we focus on identifying factors involved in the genetic
program of ectoderm specification. We looked for transcription factors
upregulated in VegT-depleted vegetal explants as potential factors
downstream of the ectoderm specification pathway. We identified
Xlim5, a LIM-homeobox encoding gene as one such factor.
Xlim5 was originally identified by its close sequence similarity to
Xlim1 (Toyama et al.,
1995) and is expressed throughout the gastrula ectoderm before
becoming restricted to the anterior neural plate and later to the brain and
spinal cord. LIM-homeodomain proteins (LIM HD or Lhx proteins) have been
identified as important developmental regulators in many cell types and
contain two zinc-finger LIM domains followed by a homeodomain (reviewed by
Hobert and Westphal, 2000
). We
show that overexpression of Xlim5 in vegetal cells (prospective
endoderm) interferes with the ability of vegetal cells to segregate from
animal cells in cell-sorting assays without inducing ectoderm markers.
Xlim5 expression in whole embryos causes vegetal cells to relocate to
ectoderm-and mesoderm-derived regions and express late differentiation markers
of these tissues. Interference with Xlim5 function, using an Engrailed
repressor construct, or blockage of its translation with antisense morpholino
(MO) oligonucleotides, results in defects in ectoderm cell adhesion or neural
plate morphogenesis, respectively, without affecting the initial formation of
the ectoderm germ layer. These data provide evidence that Xlim5
regulates differential adhesion properties of animal cells in the blastula but
may not be required for other aspects of ectoderm fate specification.
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MATERIALS AND METHODS |
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Blastomere-sorting assays were performed essentially as described
(Turner et al., 1989). In the
VegT experiments, control uninjected or VegT-depleted
embryos were injected vegetally with 1 ng of rhodamine-conjugated dextran
(RLDX; Molecular Probes). At the early gastrula stage, embryos were
dissociated on agarose-coated dishes in 67 mM phosphate buffer (pH 7.4) and
transferred to Ca2+-Mg2+-free medium [CMFM; 7.5 mM Tris
(pH 7.6), 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3]. Various
combinations of blastomeres were mixed and reaggregated in OCM in small wells
in agarose dishes. Aggregates were incubated 4 hours to overnight before they
were fixed in MEMFA, dehydrated in methanol, cleared in Murray's clear (1:2
benzyl alcohol:benzyl benzoate) and visualized by confocal microscopy (Zeiss
LSM 510). The Xlim5 experiments were carried similarly except that
RNAs were injected vegetally and RLDX was injected animally. For timelapse
movies, cells were labelled in 10 µg/ml tetramethyl rhodamine
isothiocyanate (TRITC) as described
(Turner et al., 1989
) and
filmed using the Axiovision software (Zeiss) on an Axiovert 100M microscope
with a rhodamine filter set. Frames were collected every 20 seconds over a 1
hour period and assembled into movies using Quicktime software (Apple
Computer). Dissociated cells were stained with Sytox Green at a concentration
of 1 µM, according to the manufacturer's instructions (Molecular
Probes).
Oligos and mRNAs
The antisense oligodeoxynucleotides used were HPLC-purified
phosphorothioate-phosphodiester chimeric oligonucleotides (Sigma/Genosys) with
the base composition:VegT (VT9M): 5'-C*A*G*CAGCATGTACTT*G*G*C-3'
(Zhang et al., 1998).
Asterisks represent phosphorothioate bonds. Oligos were resuspended in
sterile, filtered water. An antisense MO against Xlim5 was obtained
from Gene-Tools: Xlim5-MO: 5'-TCATAGACTCCCCAACCAAAGACCC-3'.
This MO binds at nucleotide 491 of the full-length Xlim5 sequence (start codon is nucleotide 561).
Full-length Xlim5 was obtained by PCR from stage 10 cDNA, cloned
into pCRII-TOPO according to the manufacturer's instructions (Invitrogen) and
sequenced. A region containing the Xlim5-coding region was cloned
into pCS2+ by digesting with internal PvuII and HincII sites
and ligating to StuI digested pCS2+. This cDNA begins at nucleotide
545 and does not contain the MO-binding sequence. The Engrailed repressor
domain was fused to Xlim5 C-terminal to the homeodomain by subcloning a
BamHI-AfeI fragment of pCS2+Xlim5 into
BglII-SmaI digested pEnR/RN3P1.1
(Ryan et al., 1996).
Xlim5 mRNA was synthesized from NotI-linearized template
using the SP6 mMessage mMachine kit (Ambion). Xlim5-EnR and control
EnR RNA (pEnß1) were SfiI digested and transcribed with
a T3 kit. RNAs were LiCl precipitated and resuspended in sterile distilled
water.
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 one-sixth embryo equivalent of RNA was used for cDNA synthesis
with oligo(dT) primers followed by real-time RT-PCR and quantitation using the
LightCyclerTM System (Roche) as described previously
(Kofron et al., 2001
). The
primers and cycling conditions used are listed in
Table 1. Relative expression
values were calculated by comparison with a standard curve generated by serial
dilution of uninjected control cDNA. Samples were normalized to levels of
ornithine decarboxylase (ODC). Samples of water alone or
controls lacking reverse transcriptase in the cDNA synthesis reaction failed
to give specific products in all cases. The cDNA used in
Fig. 2B was generated
previously (Kofron et al.,
1999
) and was reassayed for Xlim5 expression.
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RESULTS |
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Ectopic expression of Xlim5 in VegT-depleted vegetal
explants could arise because either VegT or its downstream targets, such as
Xenopus nodal-related genes (Xnrs), are normally required to
repress ectoderm gene expression vegetally. Consistent with this idea, Toyama
et al. (Toyama et al., 1995)
showed that Xlim5 expression was inhibited by activin. To test
whether nodal-related genes could also inhibit Xlim5
expression, we examined Xlim5 expression in VegT-depleted
embryos and VegT-depleted embryos injected with a range of
Xnr2 RNA doses [60-600 pg; embryos from Kofron et al.
(Kofron et al., 1999
)]. We
found that high doses of Xnr2 could strongly inhibit Xlim5
expression in VegT-depleted embryos
(Fig. 2B), suggesting that the
nodal-related genes downstream of VegT in vegetal cells can
repress early ectoderm genes.
Overexpression of Xlim5 in vegetal cells inhibits sorting from animal
cells but does not alter germ-layer-specific gene expression
We next asked whether ectopic expression of Xlim5 was sufficient
to change vegetal cell adhesion properties to those of animal cells, and
whether it would also induce known ectoderm differentiation markers in vegetal
cells, thus mimicking VegT depletion. We assayed differential
adhesion in blastomere-sorting assays. Embryos were injected vegetally with
Xlim5 RNA (1-2 ng) at the two-cell stage. At the blastula stage
(stage 9), vegetal cells were dissociated, mixed with RLDX-labelled animal
cells and reaggregated for 4 hours to overnight before fixation. When
RLDX-labelled animal cells were mixed with unlabelled vegetal cells, they
rapidly sorted from each other and formed distinct populations within the
aggregate (Fig. 3A-A'',
Fig. 3C). By contrast, when
RLDX-labelled animal cells were mixed with Xlim5-expressing vegetal
cells, the animal cells failed to segregate from the vegetal cells
(Fig. 3B-B'', Fig. 3C). Instead, they formed
small clusters or remained as individual cells interspersed among
Xlim5-injected vegetal cells. As was the case for VegT
depletion, Xlim5 expressing vegetal cells did not sort out from
uninjected vegetal cells (data not shown).
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Xlim5 could alter adhesion either by converting vegetal cells to
an ectodermal fate or by acting in a more limited role to regulate
differential adhesion. To distinguish between these possibilities, we cultured
vegetal explants from Xlim5-expressing blastulae until the neurula
stage and assayed for expression of ectoderm and mesoderm-specific genes.
Real-time RT-PCR analysis of stage 22 vegetal explants
(Fig. 4) showed that, compared
with explants from uninjected embryos, Xlim5 did not upregulate the
expression of ectoderm markers Epidermal keratin, E-cadherin or
NCAM, nor markers for mesoderm (Muscle actin). The endoderm
markers Endodermin (Edd) and Xsox17 were not
significantly affected. Thus, at RNA concentrations that affect differential
adhesion, ectopic expression of Xlim5 did not induce ectoderm or
mesoderm genes in vegetal explants.
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To determine if the ectopic cells injected with Xlim5 were actually differentiating according to their new location, we performed co-immunostaining experiments. Embryos were injected as above, fixed at the tailbud stage and processed for cryosectioning and immunostaining. Sections were immunostained using mAb 12/101, a marker of mature somites, and an anti-ß-galactosidase antibody to identify progeny of the injected cells. In control embryos injected with ß-gal alone, the somite staining and the ß-gal staining were clearly separated (Fig. 6A), while embryos co-injected with Xlim5 and ß-gal, a population of cells in the somite were labelled with both antibodies (Fig. 6B, arrow). Thus, overexpression of Xlim5 in whole embryos is sufficient to cause endodermal cells to relocate to other germ layers and express a tissue-specific marker. This result differs from overexpression of Xlim5 in isolated endoderm where other germ layer markers were not induced.
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We further showed that Xlim5 was important for proper adhesion in the ectoderm by performing lineage analysis. Injection of ß-galactosidase (ß-gal) RNA into animal blastomeres at the 32-cell stage (A tier) labelled a scattered population of epidermal cells (Fig. 7D). However, when Xlim5-EnR RNA was co-injected with ß-gal, epidermal staining was lost and injected cells formed distinct clumps either in the pharyngeal cavity or in the endoderm (Fig. 7E). We next performed reaggregation assays to show that Xlim5-EnR specifically disrupts cell adhesion. Animal caps were dissected from control or Xlim5-EnR-injected embryos at stage 9 and dissociated in Ca2+ and Mg2+-free meduim. Dissaggregated blastomeres were then transferred into OCM and allowed to reaggregate until stage 11. Control cells formed either large aggregates or smaller clumps of two to four cells when incubated in Ca2+ and Mg2+--containing medium (Fig. 7G). Xlim5-EnR-injected cells in contrast remained dissociated or formed only small aggregates (Fig. 7H).
Because Xlim5-EnR-injected cells could dissociate due to cell death, we stained dissaggregated cells with Sytox Green to identify dead cells. Few control or Xlim5-EnR-injected cells showed any detectable Sytox Green staining (Fig. 7I,J). By contrast, cells killed by incubation in distilled water as a positive control were abundantly stained. These results argue that the cell dissociation seen in Xlim5-EnR-injected embryos is not due to abnormal cell migration or cell death. As Xlim5 is overexpressed in VegT-depleted vegetal cells, we next wanted to assess the contribution of Xlim5 to the overall phenotype of VegT-depleted embryos. We injected Xlim5-EnRRNA into the vegetal poles of VegT-depleted embryos to attempt to restore a normal phenotype. However, the VegT-depleted vegetal cells, which are converted to ectoderm, began to dissociate in a similar manner as Xlim5-EnR-injected animal cells (data not shown).
The above observations provide evidence that Xlim5 is required for proper cell adhesion within the ectoderm. However, active repression of Xlim5-regulated genes via the Engrailed repressor domain may produce more severe effects than if Xlim5 were simply not present. We therefore attempted to block Xlim5 function using morpholino antisense oligos (MO). Xlim5-MO-injected (40-50 ng Xlim5 MO) embryos appeared to develop normally through the gastrula stage before showing a profound delay in the formation of the neural plate and subsequent neural folds. The delay is particularly evident at the neural tube closure stage when the majority of MO-injected embryos have failed to close the anterior part of the neural tube. To test the specificity of the Xlim5-MO we co-injected Xlim5 RNA along with the MO and assayed neural tube closure at stage 18. Xlim5-MO-injected embryos failed to complete neural fold closure (Fig. 7L, middle row, 9/50 normal closure) at the same time as controls (Fig. 7L, top row, 60/60 normal closure); however, the majority of rescued embryos had closed their neural folds (Fig. 7L, bottom row, 28/50 normal closure).
To determine if the delay in neural plate development was due to a loss of neural fate, we assayed molecular markers at the gastrula stage by real time RT-PCR (Fig. 7M). We found that both epidermal and neural markers Msx1 and Sox2 were slightly reduced in expression, while Xslug, a neural crest marker, was severely reduced in expression. Xbra, a marker for posterior mesoderm and notochord at this stage, was unaffected. Surprisingly Xlim5 itself was increased, suggesting that Xlim5 might regulate its own expression by a negative-feedback mechanism. At later stages (stage 18), ectoderm markers (Epidermal keratin, NCAM, E-cadherin) were still slightly affected but returned to near normal levels (data not shown). Exogenous Xlim5 RNA rescued the effects of the Xlim5-MO on gene expression, confirming the specificity of these effects. This RNA does not contain the MO-binding site, thus rescue is by replacement of Xlim5 and not by MO competition. Overall, the loss of animal cell adhesion in Xlim5-EnR-injected embryos and the abnormal neural fold morphogenesis in Xlim5-MO-injected embryos further suggest that Xlim5 is important in the proper development, although not the initial specification, of the ectoderm.
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DISCUSSION |
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Role of Xlim5 in germ layer development
The T-box transcription factor VegT is required in Xenopus for the
specification of endoderm in vegetal cells and for the expression of molecules
that induce mesoderm in equatorial cells. In previous work, we have found that
ectoderm-specific genes were ectopically expressed in equatorial and vegetal
cells of VegT-depleted embryos
(Zhang et al., 1998). Here, we
extend those observations by showing that ectoderm genes are activated during
gastrula stages in the vegetal cells of VegT-depleted embryos. In
addition, we find that uninjected animal cap cells do not sort from
VegT-depleted vegetal cells. These results argue that loss of
VegT (and subsequent Nodal signalling) is sufficient to activate
ectoderm differentiation in vegetal cells. However, in vegetal:vegetal sorting
assays VegT-depleted cells or vegetal cells expressing Xlim5
remained randomly mixed with control vegetal cells. Surprisingly, in timelapse
movies of cell sorting, we found that vegetal cells are inherently non-motile
during the early gastrula stage, while animal cells are highly motile. This
lack of motility could be due to the presence of a vegetally localized
molecule that blocks cell movement. Alternatively, the large size of vegetal
cells at this stage could prevent efficient cell motility despite the
expression of ectoderm adhesion molecules.
In current models for germ layer determination, high levels of
VegT/TGF-ß signalling specifies endoderm, medium levels induce mesoderm
and the absence of TGF-ß signalling results in ectoderm. Our results are
consistent with this model in general, as we find that the LIM-homeodomain
gene Xlim5, an early marker for ectoderm
(Toyama et al., 1995), is
activated in the absence of VegT and is repressed by Xnr2.
However, overexpression of Xlim5 in vegetal cells does not
recapitulate VegT depletion with regard to activation of
differentiated ectoderm markers. Other transcription factors upregulated in
VegT-depleted embryos, as yet unknown, must be responsible for
inducing ectoderm-specific gene expression in vegetal cells. Unfortunately, we
were unable to determine whether blockage of ectopic Xlim5 in
VegT-depleted vegetal cells could restore normal development because
of the subsequent dissociation of these cells. In this work, both gain- and
loss- of-function approaches support an alternate role for Xlim5
specifically in regulating cell adhesion.
Role of Xlim5 in regulating differential adhesion
The role of LIM-HD proteins in regulating cell adhesion independently of
cell fate has several parallels in development
(Hukriede et al., 2003;
Kania et al., 2000
;
Zhao et al., 1999
). Gene
targeting of the Xlim5 homologue, Lhx5, in mice causes a failure in
the migration and differentiation of hippocampal cell precursors
(Zhao et al., 1999
). Although
the nature of the defect in this case is not clear, the abnormal migration
indicates that adhesion may be affected in these cells. Recently, in
Xenopus, the closely related Xlim1 gene was shown to be
required for cell movements during gastrulation mediated by regulation of
paraxial protocadherin (PAPC)
(Hukriede et al., 2003
).
Interestingly, expression of organizer genes and neuroectoderm markers were
essentially normal in Xlim1-depleted embryos, suggesting that the
major defect is in cell movement or adhesion. In addition, disruption of
Lim1, a gene highly similar to Lhx5, in a subset of
motoneurones caused inappropriate axon targeting to dorsoventral compartments
of the limb muscle (Kania et al.,
2000
). In this case, the mistargeting of
Lim1-/- axons occurred without overall loss of motoneurone
identity. In dissociated cells and explants, we show that Xlim5 can
alter adhesion without inducing ectoderm markers. By contrast,
lineage-labelling experiments in intact embryos show that ectopic
Xlim5-injected vegetal cells go on to express markers of the
surrounding tissue. One explanation for this apparent discrepancy could be
that Xlim5 causes inappropriate adhesion of vegetal cells to other
germ layers early in development. Subsequently, injected cells might come
under the influence of germ layer-specific or tissue-specific inducing
molecules and then differentiate according the surrounding cells. Our
lineage-labelling experiments in late gastrula and early neurula embryos
support this idea. We do not find evidence for any ectopic cells at the
gastrula stage, after regional specification of germ layers has taken place.
However, we do find ectopic Xlim5-injected cells by the neurula stage
after significant morphogenetic events have taken place. The most likely
explanation for these observations is that Xlim5-injected cells
adhere to an inappropriate germ layer and then are carried with that tissue
during morphogenesis.
In Xlim5 loss-of-function experiments, we also find evidence for altered cell adhesion in the ectoderm without overall loss of ectoderm fate. Injection of the Xlim5-EnR construct causes animal cells to dissociate. By contrast, inhibition of Xlim5 translation with a MO causes a delay in neural fold morphogenesis. Without a specific antibody to determine the extent of protein depletion in MO-injected embryos it is difficult to say whether these two effects are qualitatively different. In addition, because the Xlim5-MO increases Xlim5 expression, the efficiency of the MO is likely to decrease during development and allow normal morphogenesis to occur. Alternatively, an Xlim5 pseudoallele (X. laevis is allotetraploid) may exist that is not targeted by the MO. Although we have not found another Xlim5 allele either by PCR or through database searches, we cannot rule out its existence. It may be preferable to carry out these experiments in the diploid Xenopus tropicalis to avoid problems with pseudoalleles. The effects of MO injection were rescued by injection of Xlim5 RNA, showing that the defects, however subtle, are in fact specific to inhibition of Xlim5. Overall, we have shown through two different methods that interfering with Xlim5 function impairs normal ectoderm adhesion without a loss of ectoderm marker expression.
Some key questions arising from this work are: what adhesion molecules are
regulated by Xlim5 and what role could the uncoupling of initial cell fate
specification from adhesion play in normal development? To address the second
question, activation of a germ layer-specific adhesion program independent of
specification could serve to sharpen germ layer boundaries by allowing animal
cells that receive low doses of inducing factors to still migrate to the
correct tissue. Adhesion factors potentially regulated by Xlim5
include members of the cadherin and protocadherin families as well as Eph
receptors and ephrin ligands, which have been implicated in mediating cell
adhesion and migration (Holder and Klein,
1999). Interestingly, inhibition of NF-protocadherin, an
ectoderm-specific protocadherin (Bradley et
al., 1998
), or activation of Ephrin B1 signalling
(Jones et al., 1998
) both
produce cell dissociation effects similar to Xlim5-EnR injection.
This possibility is intriguing given the recent demonstration that
Xlim1 regulated expression of PAPC
(Hukriede et al., 2003
).
Finally, it will be important to identify maternal factors involved in
regulating Xlim5 expression in order to establish a genetic hierarchy
of ectoderm development.
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ACKNOWLEDGMENTS |
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