1 Department of Organismal Biology and Anatomy, University of Chicago, Chicago,
IL 60637, USA
2 Department of Molecular Biology, Princeton University, Princeton, NJ 08544,
USA
3 Department of Cell Biology and Anatomy, Gautier Building, Room 517, 1011 NW
15th Street, University of Miami School of Medicine, Miami, FL 33136,
USA
Author for correspondence (e-mail:
abruce{at}uchicago.edu)
Accepted 30 July 2003
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SUMMARY |
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Key words: Zebrafish, T-box, eomesodermin, Maternal, VegT, Nodal, squint, bozozok, goosecoid, chordin, floating head, nieuwkoid/dharma
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Introduction |
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T-box genes belong to a highly conserved gene family that share a sequence
specific DNA-binding domain, called the T-box, that was first identified in
the mouse brachyury or T gene
(Herrmann, 1992) and
identifies these genes as putative transcription factors. Recently, maternally
expressed T-box genes have been identified in ascidians, newts and frogs, and
some show localized expression in the egg and early embryo. For example, the
maternal T-box gene, VegT, plays a critical role in germ layer
formation in the Xenopus embryo
(Horb and Thomsen, 1997
;
Lustig et al., 1996
;
Stennard et al., 1996
;
Zhang et al., 1998a
;
Zhang and King, 1996
). A
number of T-box family members have been identified previously in zebrafish
(Ahn et al., 2000
;
Begemann and Ingham, 2000
;
Dheen et al., 1999
;
Griffin et al., 1998
;
Griffin et al., 2000
;
Hug et al., 1997
;
Ruvinsky et al., 2000a
;
Ruvinsky et al., 1998
;
Tamura et al., 1999
;
Yonei-Tamura et al., 1999
);
however, all of these genes, including the VegT zebrafish ortholog
(tbx16/spadetail), are expressed zygotically and have no maternal
expression in the oocyte and early embryo. In view of the presence of
maternally expressed T-box genes in other species, and the importance of VegT
in Xenopus, we performed a screen for maternal T-box genes in the
zebrafish.
Here we describe the isolation and characterization of the first known
maternally expressed zebrafish T-box gene, a homolog of Eomesodermin
(Eomes). Eomes was identified originally in
Xenopus, where it is zygotically expressed immediately following
midblastula transition (MBT) in a dorsal to ventral gradient within the
marginal zone (Ryan et al.,
1996). Eomes has since been identified in other
vertebrates including mice and humans where it is also zygotically expressed.
However, in newts, Eomes is maternally and zygotically expressed
(Hancock et al., 1999
;
Sone et al., 1999
;
Yi et al., 1999
). Functional
analyses in Xenopus and mouse demonstrate common roles for
Eomes in mesoderm formation and early gastrulation movements
(Russ et al., 2000
;
Ryan et al., 1996
).
Although expression of zebrafish eomes in the nervous system of
segmentation-stage embryos has been described previously
(Mione et al., 2001), we
report its maternal and early zygotic expression, as well as its role during
early zebrafish development. The zebrafish eomes transcript localizes
cortically during oogenesis and to the vegetal region of the blastoderm during
early embryogenesis in a pattern reminiscent of VegT localization in
the frog embryo. Eomes protein is observed in nuclei on the dorsal side of the
embryo shortly after the MBT. Overexpression of eomes results in
Nodal-dependent ectopic expression of a subset of organizer specific genes,
which can lead to the formation of complete secondary axes. Loss-of-function
studies also support a role for eomes in induction of organizer-gene
expression.
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Materials and methods |
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Isolation of eomes
Degenerate PCR amplification, using standard conditions, was performed on a
zebrafish T3/T7-primer-amplified ovary cDNA library (ZapII, gift of
H.Takeda) using primers designed to amplify a 150-base pair (bp) fragment
within the T-box (Ruvinsky et al.,
2000b
). A 150 bp product was cloned into pGEM-T easy (Promega,
Madison, WI), labeled with [32P] and used as a probe to screen the
ovary library at high stringency (1.2x106 pfu). Filters were
hybridized in Church buffer overnight at 65°C, washed once in
2xSSC/0.1% SDS at room temperature and twice at 65°C in
0.1xSSC/0.1% SDS. Positive plaques (n=28) were purified and
cored. Dot-blot analysis revealed 19 of these positives to be the same gene.
Half of these were excised using the Rapid Excision Kit (Stratagene, La Jolla,
CA) and sequenced, including clone 2.5 (2.8 kb) which contained a complete
open-reading frame. Sequencing was carried out at the Princeton University
Syn/Seq facility. The GenBank Accession Number for zebrafish eomes is
AF329830.
Genetic mapping
Mapping was performed according to standard protocols (Hukreide et al.,
1999) using the LN54 radiation hybrid panel. Primers used were:
5'-ACAAAGTGGTGCGACCACCAAACTGG-3' (forward) and
5'-TGGTAGGAACTTCTGCTGCTCCATCC-3' (reverse).
Northern analysis
Total RNA from 20 dechorionated embryos at various stages was extracted
using the APGC RNA extraction method
(Chomczynski and Sacchi,
1987). RNA was separated on a 0.8% agarose/RNA borate/formaldehyde
gel, blotted overnight onto a nylon membrane, and hybridized in Church buffer
with either 32P-labelled eomes probe or
ß-actin probe (gift of I. Ruvinsky). Membranes were washed at
room temperature in 2xSSC/0.1% SDS and then in 0.1xSSC/0.1% SDS at
50°C. The blot was exposed for 7.5 and 34 hours at 80°C using
an intensifying screen.
Whole-mount in situ hybridization
In situ hybridization was performed as previously described
(Jowett and Lettice, 1994)
except anti-digoxigenin antibodies (Roche, Indianapolis, IN) were used at
1:10,000 to reduce background for eomes in situ hybridizations.
Antisense riboprobes to gsc
(Stachel et al., 1993
),
chd (Miller-Bertoglio et al.,
1997
), no tail
(Schulte-Merker et al.,
1994
), nwk/dhm (Koos
and Ho, 1998
; Yamanaka et al.,
1998
), squint (Erter
et al., 1998
; Feldman et al.,
1998
), cyclops
(Rebagliati et al., 1998
;
Sampath et al., 1998
),
wnt8 (Kelly et al.,
1995b
), bmp2b
(Martinez-Barbera et al.,
1997
) and vega1/vox
(Kawahara et al., 2000
;
Melby et al., 2000
) were
synthesized as described previously.
The eomes riboprobe was transcribed from a 2.1-kb fragment of clone 2.5 that was generated by PCR amplification and ligated into pGEMT-easy. The forward primer was 5'-TGCTCACTGACTGTTTGAATG-3' and the reverse primer was 5'-CGGTGGTCATTTTTCTCT-3'. The plasmid was linearized with SpeI and transcribed with T7 polymerase. To distinguish endogenous eomes mRNA from injected eomes-VP mRNA (see below), a riboprobe to the C terminus of eomes (starting at nucleotide 1746) was generated, which produces a probe with a three nucleotide overlap with eomes-VP. A fragment was generated by PCR amplification using the forward primer 5'-CGCTACGCAATGCAGCCCTT-3' and the reverse primer 5'-GTTCTAGATTAAGGGCTGGTGTAGAAGGCG-3' and cloned into pGEMT-easy. To make riboprobe, the plasmid was digested with SpeI and transcribed with T7 polymerase.
Full-length template to synthesize forkhead7 (fkd7)
(Odenthal and Nusslein-Volhard,
1998) riboprobe was obtained by PCR amplification of a T3/T7
amplified 15-19 hour cDNA library (gift of B. Appel) using primers based on
the published sequence. The fkd7 riboprobe was generated using T7
polymerase following SalI digestion.
Sectioning and paraffin section in situ hybridization
Following whole-mount in situ hybridization and immunohistochemistry,
embryos were dehydrated in an alcohol series and embedded in either JB4
(Polysciences, Warrington, PA), according to the manufacturer's directions, or
araldite resin (Polysciences) through a graded series. Sectioning and paraffin
in situ hybridizations were performed as previously described
(Howley and Ho, 2000), except
the eomes probe was hybridized for 2 days at 65°C. Oocyte staging
was according to Selman et al. (Selman et
al., 1993
).
eomes expression constructs
For over-expression studies eomes was cloned into pCS2+ and
pCS2+MT (to produce a Myc epitope-tagged N-terminal fusion protein)
(Rupp et al., 1994). The
eomes ORF was amplified by PCR using the forward primer
5'-CGCCTCGAGACCGCCATGCAGTTAGAAAGCATCCTC-3' and the reverse primer
5'-GTTCTAGATTAAGGGCTGGTGTAGAAGGCG-3', and cloned into the
XhoI/XbaI site of pCS2+. To produce myc-eomes, the
eomes ORF was cloned into the StuI/XbaI site of
pCS2+MT, following PCR amplification with the forward primer
5'-GAGGCCTATGCAGTTAGAAAGCATCCT-3' and the reverse primer as above.
Plasmids were linearized with NotI and transcribed using the SP6
mMessage mMachine kit (Ambion, Austin, TX).
eomes activator and repressor constructs
To test whether eomes acts as a transcriptional activator or
repressor, N-terminal fusions of the eomes T-box to the
transcriptional activator domain of VP16 and the transcriptional repressor
domain from engrailed were made. The eomes-VP16 construct
(eomes-VP) was generated by cloning a PCR amplified fragment from
eomes-pCS2+ corresponding to amino acids 153-431 into the
ClaI site of pVP16-N (Kessler,
1997). The forward primer was eomesF-enR
(5'-CCATCGATTCCGCCATGGGTTCGGTTCTTCCACCCGCC-3') and the reverse
primer was eomesR-VP16 (5'-CCATCGATGCGGGCGCCGGGGACAATCTG-3'). As a
control, no tail-VP16 (ntl-VP) was made by cloning a
fragment encoding amino acids 1-232
(Schulte-Merker et al., 1994
)
into the ClaI site of pVP16-N vector. The forward primer was ntlF-enR
(5'-CCATCGATTCCGCCATGTCTGCCTCAAGTCCCGAC-3') and the reverse primer
was ntlR-VP16 (5'-CCATCGATAGATTGCTGGTTGTCAGTGCTGTG-3').
The engrailed (eng) repressor constructs were made by
cloning the same eomes fragment as above into the
ClaI/EcoRI site of pENG-N
(Kessler, 1997). To construct
eomes-eng the forward primer was eomesF-enR (described above) and the
reverse primer was eomesR-enR
(5'-CGGAATTCCGCGGGCGCCGGGGACAATCTG-3'). To generate the control
ntl-eng the forward primer was ntlF-enR (as above) and the reverse
primer was ntlR-enR (5'-CGGAATTCCAGATTGCTGGTTGTCAGTGCTGTG-3').
Plasmids were linearized with SacII (except eomes-VP and
ntl-VP, which were digested with NotI) and transcribed using
the SP6 mMessage mMachine kit. Results with the ntl constructs
differed from those observed using the eomes constructs, indicating
that the results obtained using the eomes constructs were not the
result of promiscuous T-box-binding activity. In addition, the defects caused
by overexpression of eomes-eng were rescued by co-injection
of myc-eomes RNA, indicating that the defects were specific.
Injection of eomes-eng alone resulted in abnormal phenotypes
in 63% (27/43) of injected embryos at 24 hours post fertilization (hpf). In
two separate experiments, 65% (34/52) of embryos co-injected with
eomes-eng and myc-eomes had normal
phenotypes at 24 hpf. Experiments using the VP16 and engrailed
constructs lacking any DNA-binding domain gave no phenotype.
Eomesodermin antisense morpholinos
Two antisense morpholinos to eomes were designed and synthesized
by Gene Tools, LLC (Philomath, OR): Eomes-MO1,
5'-CATTCTTCACTGTGCTGATAAAGGG-3'; and Eomes-MO2,
5'-CGCCAGGGAGGATGCTTTCTAACTG-3'. Morpholinos were injected at 7 ng
nl1 as described (Oates
and Ho, 2002).
Microinjections
Manually dechorionated embryos were immobilized in 2.5-3% methyl cellulose
or in an agarose mold and pressure injected according to Oates et al.
(Oates et al., 2000). All
embryos injected with RNA were co-injected with 33 ng µl1
of GFP RNA to trace the overexpressing cells and to score for proper
translation of injected RNAs. Fast green (Sigma, St. Louis, MO) was
co-injected (3-5 ng µl1) as a visual guide of injection
volume (approximately 0.5 nl of RNA was injected per embryo). eomes,
myc-eomes, eomes-VP and eomes-eng RNAs were injected into one or
two cells of eight- to 16-cell stage embryos. Two cell injections at the
eight- to 16-cell stage were done as described
(Koos and Ho, 1998
).
antivin RNA was injected into the yolk of one- to four-cell stage
embryos (Thisse et al., 2000
).
eomes and myc-eomes RNAs were injected at 200-250 ng
µl1, eomes-VP RNA was injected at 50 ng
µl1, eomes-eng RNA was injected at 15 ng
µl1 and antivin RNA was injected at 400 ng
µl1. Initial experiments revealed no differences in
activity between the eomes and myc-eomes constructs. We used
the Myc construct preferentially due to our ability to monitor the protein
distribution and to distinguish endogenous from exogenous protein. Control
embryos were injected with a mixture of GFP and lacZ RNAs or
GFP alone and exhibited no specific defects.
Animal pole microinjections
sqt and myc-eomes RNAs were injected alone or together
into a single cell at the animal pole of 64-256-cell stage embryos and fixed
at 50% epiboly (Chen and Schier,
2001). The sqt constructs used were either the coding
region alone (Rebagliati et al.,
1998
) or the coding region plus an additional
600 bp of
downstream sequence (gift from and M. Halpern). sqt was injected at
4-7 ng µl1 and myc-eomes RNA was injected at 250
ng µl1. Embryos were processed by in situ hybridization
for gsc and flh expression and by immunohistochemistry for
either GFP or Myc expression (see below). Embryos were included in the
analysis if they were successfully injected (as judged by antibody staining)
and displayed normal marginal expression of gsc or flh.
Interestingly, we found that injection of the sqt construct
containing the sqt 3' UTR sequence was not as potent in this
assay as the construct containing the coding region alone. Injection of the
construct containing the 3' UTR induced gsc expression less
frequently and 10-fold higher concentrations were necessary to induce a ring
of flh expression as opposed to a solid patch.
Generation of polyclonal antibodies to Eomes
A portion of the eomes cDNA, encoding amino acids L3-C165, was PCR
amplified as a 5' BamHI/3' HindIII fragment and
cloned into the pQE-30 vector containing a C-terminal tag of six histidines
(Qiagen, Valencia, CA). This construct was transformed into JM109 cells (Life
Technologies, Gaithersburg, MD) and recombinant protein was expressed and
Ni/NTA purified under native conditions using the Qiaexpress system according
to the manufacturer's instructions (Qiagen). The recombinant protein was used
to generate affinity purified polyclonal antibodies in rabbits (Zymed
Laboratories, San Francisco, CA).
Immunohistochemistry and western blots
Anti-Myc (9E10), -GFP (Molecular Probes, Eugene, Oregon) and -Eomes
antibody staining was performed according to Bruce et al.,
(Bruce et al., 2001). The 9E10
monoclonal antibody was developed by J. M. Bishop and obtained from the
Developmental Studies Hybridoma Bank under the auspices of the NICHD and
maintained at the University of Iowa, Department of Biological Sciences.
Anti-Myc, -GFP and -Eomes antibodies were used at dilutions of 1:100, 1:500
and 1:500, respectively. Fluorescent antibody staining was performed using
anti-rabbit Alexa 488 (Molecular Probes) at 1:500 and embryos were examined on
a Zeiss LSM 510 confocal microscope (Zeiss, Thornwood, NY). Western blots were
performed as described (Bruce et al.,
2001
), except that sphere-stage embryos were dissected from the
yolk prior to lysis. Approximately two embryo equivalents were loaded per
lane, and anti-Eomes and anti-Myc antibodies were used at 1:1000. To test the
specificity of Eomes-MO2, transcription and translation of
eomes-pCS2+ was carried out using the TnT Coupled Reticulocyte Lysate
System (Promega, Madison, WI). The reaction run with plasmid alone or in the
presence of 5 ng cyclops antisense morpholino as a control
(Karlen and Rebagliati, 2001
)
or 5 ng Eomes-MO2. Western blots were carried out as described above.
Xenopus oocyte and embryo manipulations
VegT depleted embryos were created as previously described by
injecting stage VI oocytes with 5 ng of antisense oligonucleotides to maternal
VegT (Zhang et al.,
1998a). The oligo used was an 18-mer: C*A*G*CAGCATGTACTT*G*G*C,
where * indicates a phosphorothioate bond. Oocytes were introduced into a
female host after maturation and vital dye labeling using the host-transfer
technique (Zuck et al., 1998
).
For rescue experiments, Xenopus VegT and zebrafish eomes
mRNAs were synthesized using the mMessage mMachine kit and were injected into
stage VI oocytes 24 hours after antisense injection or, in some cases, into
vegetal pole blastomeres of the eight-cell embryo. Embryos were obtained by in
vitro fertilization and were maintained in 0.1xMMR.
Imaging
Embryos were photographed on a Zeiss Axioplan microscrope (Zeiss) with a
Nikon D1 digital camera (Nikon, Melville, NY) or images were obtained using a
Zeiss LSM 510 confocal microscope (Zeiss). Figures were constructed in Adobe
Photoshop.
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Results |
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Expression of eomes mRNA
Northern blot and in situ hybridization analyses confirmed that
eomes mRNA is expressed maternally in both oocytes and early
cleavage-stage embryos, as well as zygotically for a short period following
MBT (3 hpf) at approximately the 1000-cell stage. Northern analysis revealed
that an eomes transcript of 4.1 kb was present through the
1000-cell stage, whereas a slightly smaller transcript was found during the
sphere and dome stages (Fig.
1A). Maternal eomes transcript levels decreased
dramatically just prior to the onset of zygotic transcription at MBT
(
512-cell stage) and immediately following MBT, eomes mRNA
levels transiently increased, presumably due to new zygotic transcription
(Fig. 1A, compare 512-cell lane
with 1000-cell lane). Thereafter, eomes transcript levels gradually
decreased, persisting to the shield stage at 6 hpf
(Fig. 1A and data not
shown).
|
In the one-cell stage embryo after fertilization eomes mRNA was detected both in the yolk cytoplasmic streams and at the junction between the yolk and the blastoderm (Fig. 1E arrow). During early cleavage stages, the maternal eomes transcript was distributed in a vegetal to animal gradient in the cells of the blastoderm, with the highest concentration of mRNA detected vegetally (Fig. 1F-H). This graded pattern of eomes expression was maintained until just prior to MBT when transcripts became difficult to detect by in situ hybridization, in agreement with our northern analysis (Fig. 1A).
Immediately following MBT, a burst of zygotic eomes expression was
observed in a pattern similar to the maternal distribution because staining
was most intense in the vegetal cells located closest to the yolk
(Fig. 1I-K). Sectioning of
embryos confirmed the vegetal to animal gradient of expression at the
oblong/sphere stage (Fig. 1J).
Furthermore, eomes mRNA was not detected in the yolk syncytial layer
(YSL), an extraembryonic tissue that forms below the blastoderm
(Fig. 1J arrow)
(Kimmel et al., 1995).
Although low levels of transcript were still detected at the dome
(Fig. 1A) and shield stages
(data not shown) by northern analysis, eomes levels were undetectable
by in situ hybridization by the dome stage (4.3 hpf, data not shown). As
previously described (Mione et al.,
2001
), zebrafish eomes has a later zygotic expression
domain in the forebrain beginning at the 4-5 somite stage (11.5 hpf). This is
similar to the previously described pattern of Eomes expression in
the brain of frogs, mice and chicken
(Bulfone et al., 1999
;
Ciruna and Rossant, 1999
;
Hancock et al., 1999
;
Ryan et al., 1996
).
Eomes protein is localized to nuclei on the dorsal side of the
embryo
An affinity purified polyclonal antibody was generated against the
N-terminal portion of Eomes, excluding the highly conserved T-box. Eomes is an
approximately 94 kDa protein, as shown by western blotting of sphere-stage
embryos (4 hpf), which is somewhat larger than the 73 kDa predicted by the
eomes sequence (Fig.
2A lane 1). Preimmune serum did not detect any proteins
(Fig. 2A lane 2). Injection of
myc-eomes RNA into embryos, which produces a Myc-epitope tagged
fusion protein, followed by western blotting, further demonstrated the
specificity of the antibody because both anti-Eomes and anti-Myc antibodies
recognized the same protein (data not shown). Staining of 24 hpf embryos
revealed nuclear expression in the brain, in agreement with the in situ
hybridization pattern at this stage (Fig.
2B arrowheads) (Mione et al.,
2001). In a further control experiment, no staining was observed
in 24 hpf embryos incubated with the pre-immune serum
(Fig. 2C).
|
Eomes, a putative transcription factor, is predicted to function in the nucleus after MBT, when zygotic transcription begins. In accordance with this, reproducible nuclear expression of Eomes protein was first detected in most nuclei of the embryo at 3 hpf, just around the time of MBT (data not shown). A strikingly asymmetric pattern of nuclear staining was observed beginning at the sphere stage (4 hpf). In addition to cytoplasmic expression in most cells of the blastoderm, the protein was detected in nuclei predominantly on one side of the embryo at this stage (Fig. 2F arrow). To determine the region of the embryo that correlated with the nuclear staining, we performed double-labeling studies. Antibody staining for Eomes and in situ hybridization for two different dorsal markers, gsc and flh, revealed that Eomes localized to nuclei on the dorsal side of the embryo (Fig. 2G,H). Eomes protein appeared to be co-expressed with most flh-expressing cells at the sphere (4 hpf) and dome stages (4.3 hpf) (Fig. 2G-I). Eomes was also co-expressed with a subset of gsc-expressing cells at the sphere stage (Fig. 2J). The asymmetric expression pattern of nuclear Eomes was detected through the dome stage (4.3 hpf). In addition, we detected Eomes in nuclei of the leading edge of the enveloping layer (Fig. 2K arrowheads). By 50% epiboly (5.3 hpf), Eomes was no longer detected.
Zebrafish eomes cannot fully rescue VegT-depleted
Xenopus embryos
We noted that the embryonic expression pattern of maternal eomes
transcript was strikingly similar to that of maternal VegT in
Xenopus, which is also distributed in a vegetal to animal gradient
within the early embryo and appears to be required for the generation of
vegetal signals involved in endoderm and mesoderm formation
(Casey et al., 1999;
Kofron et al., 1999
;
Zhang et al., 1998b
). By
contrast, the subcellular distribution of Eomes is more similar to
Xenopus Eomes, which is expressed zygotically in a D/V gradient
(Stennard et al., 1999
).
Because eomes and VegT are the only known maternal T-box
genes that are expressed in zebrafish and Xenopus, respectively, we
hypothesized that maternal Eomes function in the fish might be analogous to
maternal VegT function in the frog. As one test of this hypothesis, zebrafish
eomes was assayed for its ability to rescue Xenopus oocytes
that had been depleted of VegT by injection of antisense
oligonucleotides. Xenopus embryos depleted of maternal VegT
failed to form endoderm and induce mesoderm, with the result that the
morphogenetic movements of gastrulation, including blastopore formation and
epiboly, did not occur (Kofron et al.,
1999
; Zhang et al.,
1998a
). In three experiments, injection of 300 pg of VegT
mRNA into VegT-depleted embryos rescued 83% (40/48) of embryos (data
not shown). By contrast, injection of eomes RNA [300 pg
(n=13), 150 pg (n=18), 50 pg (n=19), 10 pg
(n=38), and 2 pg (n=48)] failed to fully rescue
VegT-depleted Xenopus embryos. Injection of eomes
at concentrations of 300 pg or higher resulted in exaggerated
gastrulation-like movements (invagination) that initiated equatorially, a
position that is significantly higher than in normal embryos, and resulted in
very large abnormal blastopores that failed to close. Such results indicate
that zebrafish eomes is much more potent than VegT at
inducing cellular movements in Xenopus. VegT-depleted embryos
injected with eomes failed to form a normal blastopore lip and did
not gastrulate, but a limited degree of ectodermal streaming characteristic of
epiboly was observed in a small percentage of cases (21%). Our results
indicate that zebrafish Eomes is unable to functionally replace maternal VegT
in the frog and that these two genes have different activities in early
embryos. This raises the possibility that the function of zebrafish
eomes might be more similar to the zygotic activity of its ortholog,
Xenopus Eomes.
Overexpression of eomes induces secondary axes
Localization of Eomes to nuclei on the dorsal side of the zebrafish embryo
indicated that Eomes might play a role in patterning the organizer. To
investigate the role of eomes during early development, synthetic
mRNAs containing either the coding region (eomes) or a coding
region-Myc-epitope fusion (myc-eomes) were used to evaluate the
molecular and morphological effects of overexpressing eomes in early
embryos. Injection of either eomes or myc-eomes RNA (and
GFP RNA as a tracer) into early zebrafish embryos led to the
formation of secondary axes (Table
1), some of which possessed fully formed heads, including eyes
(Fig. 3C). These secondary axes
were examined during somitogenesis for expression of ntl/Brachyury, a
marker of the notochord (Schulte-Merker et
al., 1994), and at 24 hpf for expression of fkd7, a
marker of ventral neural tube and endoderm
(Odenthal and Nusslein-Volhard,
1998
). Ectopic patches of ntl/Brachyury expression were
observed in 55% of injected embryos (12/22). Although in most cases the two
axes were separate and distinct, at least to the level of the tail, in some
cases fkd7 staining revealed double axes that were side by side
(Fig. 3D). Analysis of
transverse sections of these embryos revealed two axes that were well
patterned, containing two neural tubes which both expressed fkd7
(Fig. 3E) as well as two
regions of separate notochords (data not shown). Importantly, secondary axes
consisted of cells that expressed either eomes or myc-eomes
(confirmed by GFP expression) and unlabeled cells, indicating that
eomes-expressing cells could recruit their non-expressing neighbors
into the duplicated axis.
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Normally, gsc is expressed in cells that give rise to the
prechordal plate (Stachel et al.,
1993) whereas chd is expressed more broadly on the dorsal
side of the embryo (Miller-Bertoglio et
al., 1997
). Ectopic expression of gsc and chd
was detected in eomes- and myc-eomes-injected embryos along
the margin at shield stage (6 hpf, Fig.
4A-F), although anti-Myc antibody staining revealed that nuclear
expression of Myc-Eomes was not confined to the margin
(Fig. 4C). Immunostaining with
the anti-Myc antibody also revealed that the cells that ectopically expressed
gsc (Fig. 4C, Myc and
gsc staining are in separate focal planes) were primarily adjacent to
cells that expressed Myc-Eomes. Thus, overexpression of eomes
appeared to induce gsc expression non-cell autonomously. Ectopic
chd was usually adjacent to and partially overlapped with the
Myc-Eomes expressing cells. Cells expressing ectopic chd and Myc were
observed (white Fig. 4F
outline) as were cells that expressed chd alone
(Fig. 4F red outline). Thus,
our experiments indicate that eomes induced ectopic chd
expression in myc-eomes expressing cells as well in neighboring
non-myc-eomes expressing cells, indicating both cell autonomous and
non-cell autonomous induction.
Early notochord precursor cells express flh
(Talbot et al., 1995).
Injection of myc-eomes, induced the ectopic expression of
flh in scattered marginal cells in a pattern unlike the uniform
domains of ectopic expression of gsc and chd seen under
similar experimental conditions (Fig.
4H, arrowhead; Table
2). Immunostaining of myc-eomes injected embryos with the
anti-Myc antibody revealed that ectopic flh expression was confined
to cells that expressed Myc-Eomes (Fig.
4I). Thus, ectopic expression of myc-eomes induced
flh expression cell-autonomously, in contrast to the
non-cell-autonomous induction of gsc and chd.
We next examined the timing of induction of these three genes by comparing
the onset of ectopic expression with the normal temporal expression profile of
each gene. Endogenous gsc expression is first detected just after the
MBT at 3.5 hpf (A.E.E.B., unpublished) but the earliest stage at which ectopic
gsc expression was observed was the dome stage (4.3 hpf, 5/9
embryos). The chd transcript is first expressed at the oblong stage
(3.7 hpf) (Miller-Bertoglio et al.,
1997), whereas ectopic chd was first detected at the dome
stage (8/14 embryos). Initially, flh is expressed at the dome stage
(4.3 hpf) (Talbot et al.,
1995
) and ectopic expression of flh was first detected at
this stage also (4.3 hpf, 4/8 embryos) in myc-eomes injected embryos.
Thus, unlike gsc and chd, the first ectopic expression of
flh corresponded to the onset of endogenous expression.
To investigate whether eomes acted as a transcriptional activator
or repressor to induce ectopic organizer markers, we fused the putative
DNA-binding region of eomes (the T-box) to the VP16
transcriptional-activator domain (eomes-VP) and to the
transcriptional-repressor domain of engrailed (eomes-eng).
These domains have been shown to impart transcriptional activation and
repression when fused to a heterologous DNA-binding domain in several
organisms, including flies, frogs and zebrafish
(Conlon et al., 1996;
Han and Manley, 1993
;
Kessler, 1997
;
Koos and Ho, 1999
). We then
compared the overexpression phenotypes of eomes-VP and
eomes-eng with that of native eomes. Overexpression of
eomes-VP produced phenotypes identical to those seen following
eomes and myc-eomes overexpression (Tables
1,
2), indicating that
eomes acts as a transcriptional activator to induce ectopic
expression of organizer markers and induce the formation of secondary axes.
This is consistent with the fact that many characterized T-box genes,
including frog VegT and Eomes, function as transcriptional
activators (Tada and Smith,
2001
). In contrast, overexpression of eomes-eng failed to
induce ectopic expression of gsc, chd and flh (data not
shown). When eomes-eng expressing cells were located
dorsally, expression of gsc and flh was inhibited
(Fig. 5B,D), which was opposite
to the effect seen after overexpression of eomes. Co-injection of
eomes and eomes-eng resulted in normal embryos at
24 hpf, demonstrating the specificity of the eomes-eng
construct (see Materials and methods for details). These results indicate that
eomes might be required during normal development to induce the
expression of a subset of organizer genes.
|
|
Zebrafish eomes can regulate its own expression
Transcriptional induction of gsc, chd and flh occurs
during the zygotic phase of embryonic development. To establish a link between
the maternal and zygotic phases of eomes expression, we explored the
possibility that eomes regulates its own expression. Embryos were
injected with eomes-VP and fixed at the shield stage, when endogenous
eomes is undetectable by in situ hybridization. Embryos were
processed by in situ hybridization using an eomes riboprobe against
the C-terminal region, which was not contained in the eomes-VP mRNA.
Expression of eomes was detected in shield-stage embryos injected
with eomes-VP (83%, 30/36, Fig.
7B), indicating that Eomes-VP and, by inference, Eomes can
activate and possibly maintain its own transcription. In addition, we found
that injection of eomes-VP into MZoep embryos (see below)
resulted in induction of eomes expression (93%, 50/54), indicating
that this autoregulation is Nodal-independent.
|
|
By contrast, injection of myc-eomes into MZoep mutants often expanded the expression of chd. In MZoep mutants, two cells at opposite corners of an eight-cell-stage embryo were injected with myc-eomes. After processing for both chd and Myc expression at the shield stage, we observed expanded expression of chd on the dorsal side (7/22, 32%) (Fig. 8J, arrowhead). However, we never saw ectopic expression of chd on the ventral side, despite the presence of ventral Myc-staining cells (Fig. 8J arrow). Thus, overexpression of myc-eomes in MZoep embryos led to expanded chd expression on the dorsal side of MZoep embryos, but failed to induce ectopic expression of gsc or flh. Interestingly, no ectopic expression of chd was detected in MZoep mutants injected with eomes-VP (0/35). Double axes were never observed in injected MZoep embryos raised to 24 hpf (0/11). Similarly, no secondary axes were detected in wild-type embryos co-injected with antivin and myc-eomes (0/13).
These findings indicated that Nodal signaling was required for eomes to induce ectopic expression of the organizer genes gsc and flh, and to induce secondary axes. It also appeared that eomes acted through a non-Nodal pathway to induce chd expression on the dorsal side of the embryo, but required an intact Nodal pathway to induce ectopic chd expression on the ventral side of the embryo.
eomes can modulate sqt activity
To further investigate the interaction between eomes and Nodals,
we performed overexpression experiments at the animal pole. Injection of RNAs
into a single cell at the animal pole allows specific interactions to be
examined in isolation from marginal signals. Previous work
(Chen and Schier, 2001)
demonstrated that injection of sqt into the animal pole leads to the
local induction of gsc in sqt-expressing and immediately
adjacent cells. This work led to the proposal that the prospective shield is
patterned by a Sqt morphogen gradient, which normally acts from the margin to
activate gsc expression at high levels and flh expression at
lower levels within the marginal region of the embryo
(Chen and Schier, 2001
). Our
experiments revealed that sqt induced gsc locally but
eomes induces gsc at a distance. Thus, we examined the
consequences of injecting both sqt and eomes into a single
cell in the animal pole of embryos at the 64- to 256-cell stage. In agreement
with earlier work (Chen and Schier,
2001
), we found that injection of sqt alone led to
induction of gsc in a small patch (31/31 embryos,
Fig. 9A). By contrast,
injection of myc-eomes alone did not induce gsc expression
at the animal pole (0/15 embryos). However, co-injection of sqt and
myc-eomes led to the induction of a ring-like domain of gsc
expression (30/34 embryos, Fig.
9B) in which gsc expression was induced around the
injected cells (revealed by Myc antibody staining) but not in the central
region where the majority of Myc-Eomes stained cells were located
(Fig. 9C). From these results,
we concluded that eomes appears able to modulate Sqt induction of
gsc.
|
Thus, it appears that Eomes might modulate Sqt signaling, possibly by dampening it to a level that permits flh expression in the central region but is insufficient to induce gsc. This intriguing results needs to be confirmed by additional experiments in order to understand the nature of this interaction and how eomes and Nodals interact at the dorsal margin.
![]() |
Discussion |
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The zebrafish T-box gene eomes is maternally expressed
The vertebrate Eomes orthologs in humans, mice and chicken
(Papaioannou, 2001) are
expressed zygotically, but Eomes is maternally and zygotically
expressed in zebrafish and newts. Genes related to Eomes have also
been identified in invertebrates. Amphioxus Eomes/Tbr1/Tbx21, which
is considered an Eomes-like precursor gene, is maternally and
zygotically expressed (Horton and
Gibson-Brown, 2002
; Ruvinsky
et al., 2000b
), whereas the ascidian genes Ci-VegTR and
As-mT are expressed strictly maternally
(Erives and Levine, 2000
;
Takada et al., 1998
). Recent
work indicates that As-mT might be an Eomes/Tbr1/Tbx21
ortholog but the orthology of Ci-VegTR is unclear
(Horton and Gibson-Brown,
2002
). Therefore, with the exception of Xenopus VegT, it
appears that most, if not all, maternally expressed T-box genes described to
date are closely related to Eomes. Thus, it is likely that the last
common ancestor of the chordates possessed a maternally expressed
Eomes-like gene, and that maternal expression was subsequently lost
in some vertebrate species. This hypothesis also indicates that maternal
expression of VegT might be unique to Xenopus and,
therefore, is unlikely to represent the ancestral condition of VegT
or a VegT-precursor gene. Consistent with this, the zebrafish
homologue of the Xenopus VegT gene, called tbx16/spadetail,
is only expressed zygotically (Griffin et
al., 1998
; Ruvinsky et al.,
1998
).
Because of the similarity in expression patterns of Xenopus VegT and zebrafish eomes, we were wondered if zebrafish Eomes had an analogous functional role to that of Xenopus maternal VegT during germ-layer specification. One possible evolutionary scenario is that VegT has assumed the functional role previously filled by a maternally expressed Eomes gene. Our initial experiments involving injection of the zebrafish eomes gene into VegT-depleted frog embryos did not completely rescue the depletion phenotype, indicating that zebrafish eomes might be involved in different processes to that of frog VegT. However, because a library screen is not exhaustive, we cannot rule out the possibility that another maternal T-box gene, which is yet to be identified, functions like VegT in zebrafish to establish the primary germ layers. It will be interesting to determine how the presence or absence of maternal Eomes influences early developmental programs in different organisms, and if a maternally expressed VegT homologue is identified in organisms other than amphibians.
eomes transcript is localized maternally
As described in this paper, zebrafish eomes transcript was
expressed in a localized pattern during oogenesis and early embryogenesis. In
zebrafish, most maternal transcripts that have been examined are either
localized to the future animal pole of the oocyte or remain ubiquitously
expressed throughout the cytoplasm. In the early embryo, these transcripts are
found evenly distributed throughout the entire blastoderm
(Howley and Ho, 2000). To
date, only three maternally expressed mRNAs have been identified that exhibit
localized patterns of expression in the zebrafish embryo, namely daz1,
brul and vasa (Maegawa et
al., 1999
; Suzuki et al.,
2000
; Yoon et al.,
1997
). Although the specific embryonic localization patterns of
eomes and vasa differ, they share some characteristics. Both
eomes and vasa mRNAs localize to the junction between the
yolk and the blastomeres, and both are distributed in a vegetal to animal
gradient in one-cell-stage embryos (Braat
et al., 1999
; Howley and Ho,
2000
). Furthermore, eomes and vasa mRNAs are the
only transcripts that localize cortically in the oocyte
(Howley and Ho, 2000
). Thus,
the mRNA localization patterns of vasa and eomes indicates a
possible relationship between cortical localization during oogenesis and
localization along the yolk/blastomere interface in the early embryo.
Eomes protein is present maternally
Maternal Eomes protein is present in oocytes and preMBT embryos, but does
not localize to nuclei until immediately before MBT, which raises the
possibility that any function for Eomes as a transcription factor is held
latent until the onset of zygotic expression. Overexpression studies revealed
that exogenous Eomes localizes to nuclei and induces expression of the
endogenous eomes gene. Thus, one crucial function of maternal Eomes
might be to activate zygotic transcription of eomes.
Role of eomes in patterning of the organizer
Overexpression of eomes resulted in the formation of secondary
axes that arise from sites of ectopic expression of the zygotic organizer
markers gsc, chd and flh in domains close to the margin of
the zebrafish gastrula. These results indicated that eomes is
sufficient to induce a functional organizer, but only from cells situated in
or close to the margin. Overexpression of eomes induced the gsc,
chd and flh genes at different times and through different
mechanisms. Ectopic induction of gsc occured non-cell autonomously
and was first detected at the dome stage, 1 hour after gsc
expression is initiated normally. Ectopic chd expression occured both
cell-autonomously and non-cell autonomously at the dome stage, which is 30
minutes after endogenous expression begins. These observations indicate that
eomes induced chd and gsc by an indirect mechanism
that is likely to involve the production of a signaling factor that is
diffusible or acts in a cell-cell relay. Furthermore, they indicate that the
embryo cannot respond to exogenous Eomes until a distinct time point during
the dome stage of development, presumably reflecting a requirement for a
competency supplied by some other factor or factors. Importantly, flh
is induced by eomes cell autonomously, and is first detected at the
dome stage, when flh expression is normally initiated. This timing is
consistent with a direct regulation of flh expression by
eomes.
Although several early genes involved in organizer formation, such as nwk/dhm and gsc, are expressed in spatial domains that closely prefigure the organizer, neither the eomes transcript nor the initial distribution of nuclear-localized Eomes protein at MBT were limited to the future organizer region. However, beginning at the sphere stage, Eomes protein was rapidly excluded from most nuclei in the embryo and was retained in the nucleus only on the dorsal side of the embryo. This could be the result of a ventral factor that prevents Eomes entering the nucleus. Alternatively, a dorsal factor might retain Eomes in the nucleus. Combining this expression analysis and the timing of organizer gene induction described above, we propose that the timing of dorsal nuclear localization of endogenous Eomes marks the onset of its activity in organizer patterning.
In Xenopus, Eomes transcript and protein are expressed in a dorsal
to ventral gradient in the mesoderm, and overexpression studies demonstrate
that Eomes can induce the expression of mesodermal markers in a
dose-dependent manner (Ryan et al.,
1996). High levels of ectopic Eomes induce dorsal
mesodermal markers, such as gsc and chd, and lower levels
induce ventral mesodermal markers (Ryan et
al., 1996
). Thus, the distribution and function of zebrafish Eomes
appears broadly similar to that of Xenopus, with the exception that
the zebrafish gene was not observed to induce ventral mesodermal fates.
Reduction-of-function experiments also support a role for eomes in the establishment of organizer-gene expression. Overexpression of a dominant-negative eomes construct, as well as injection of an antisense morpholino oligonucleotide, led to either loss or reduction of gsc and flh expression. These results are consistent with a requirement for eomes in the induction of a subset of organizer genes.
Eomes and Nodals
Although the mechanism by which eomes regulates organizer gene
expression is unclear, Nodal signaling appears to be required and, in the case
of at least one gene (chd) the requirement for an intact Nodal
pathway might be limited to one side of the embryo only. Induction of gsc,
chd (on the ventral side) and flh by eomes
overexpression required Nodal signaling because these genes were not induced
when eomes was injected into MZoep embryos. Nodals are
secreted signaling molecules. Thus, it was surprising to find that the
cell-autonomous induction of flh by eomes depended on Nodal
signaling. A likely possibility is that transcription factors activated
downstream of the Nodals act in combination with eomes to induce the
expression of flh. In this case, eomes and Nodals would act
in parallel to induce flh expression.
The role of Nodal signaling in the induction of gsc and
flh expression has been examined previously in studies that have
demonstrated that induction of gsc requires high levels of Nodal
signaling whereas low levels are sufficient for flh induction
(Gritsman et al., 2000). It
has been hypothesized that Nodal signaling acts as a classical morphogen in
patterning the organizer: cells close to the sqt-expressing margin
receive high levels of Sqt and express gsc, and cells at a distance
from the margin are exposed to lower Sqt concentrations and express
flh (Chen and Schier,
2001
). The gsc-expressing cells later give rise to the
prechordal plate and flh expressing cells give rise to the notochord.
Our finding that overexpression of eomes resulted in induction of
flh cell-autonomously and gsc at a distance indicates that
eomes overexpression might have effects on flh and
gsc expression that are the reciprocal of Nodal overexpression.
This intriguing possibility was investigated further by animal pole injections, which indicated that eomes could modulate the induction of gsc and flh by Sqt. Injection of sqt alone led to a solid patch of ectopic expression of gsc. However, in embryos co-injected with sqt and eomes, gsc was not expressed in a central region in which the density of sqt and eomes overexpressing cells is highest. One possible explanation for these results is that Eomes dampens Nodal function locally. Additionally, injection of sqt alone at the animal pole led to induction of flh in a ring, at a distance from the sqt source, but co-injection of sqt and eomes led to expression of flh in a solid patch. Thus, we suggest that these results are consistent with eomes reducing Nodal function to a level that is sufficient to induce flh but insufficient to induce gsc.
Additional experiments are required to verify this result and demonstrate such an interaction between eomes and sqt occurs at the dorsal margin. One possibility that we are currently investigating is that eomes may act in concert with Nodals at the dorsal margin to distinguish the notochord and prechordal plate territories, defined by flh and gsc expression, respectively.
In contrast to gsc and flh, the ability of eomes
to induce ectopic expression of chd is not entirely Nodal dependent.
Overexpression of myc-eomes in MZoep embryos resulted in an
enlarged domain of chd expression on the dorsal side of injected
embryos, but ectopic expression of chd on the ventral side of
MZoep embryos injected with myc-eomes was not observed. This
indicates that eomes can interact with a factor that is active in
MZoep embryos and confined to the dorsal side of the embryo to induce
chd expression. One possible candidate is nwk/dhm, which,
with the Nodals, has been shown to regulate chd expression
(Koos and Ho, 1998;
Shimizu et al., 2000
).
Although induction of chd by eomes occurs in
boz-mutant embryos, this does not rule out an interaction between
eomes and nwk/dhm that is only apparent in a background in
which Nodal signaling is absent. We also observed that injection of
myc-eomes, but not eomes-VP, caused expanded expression of
chd on the dorsal side of MZoep embryos. This indicates that
the VP16 construct lacks a domain necessary for the Nodal-independent
interaction, resulting in chd induction.
Loss-of-function data in the mouse and frog points to a conserved
evolutionary role for Eomes in early cell movements
(Graham, 2000). Injection of a
putative dominant-negative Eomes construct into Xenopus
embryos leads to the formation of exogastrulae, which is indicative of
abnormal cell movements (Ryan et al.,
1996
). Aberrant cell movements are also implicated in gastrulation
defects in mice that lack Eomes, in which cells fail to migrate into
the primitive streak (Russ et al.,
2000
). The phenotypes we observed when eomes-eng was
over-expressed globally in early embryos included defects in organizer
formation, but also indicated that eomes might play a role in the
cellular rearrangements of early gastrulation (A.E.E.B., C.H. and R.K.H.,
unpublished). This indicates a conserved evolutionary role of Eomes
in regulating cell movements. A detailed analysis of these defects will be
presented elsewhere.
eomes in early zebrafish development
In summary, we have shown that the maternal T-box gene eomes has
both Nodal-dependent and Nodal-independent activities in the early zebrafish
embryo. Overexpression of eomes resulted in the Nodal-dependent
activation of a subset of organizer genes. By contrast, overexpression of a
dominant-negative construct prevented the expression of these genes. Reducing
Eomes protein levels by injection of an antisense oligonucleotide morpholino
also reduced the expression of these genes. We also demonstrated that
eomes can induce its own expression by a Nodal-independent mechanism.
Thus, Eomes appears be involved in establishing the expression of a subset of
dorsal-organizer genes. It will be interesting to determine how the nuclear
localization of Eomes to the dorsal side is controlled and which downstream
genes Eomes regulates.
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ACKNOWLEDGMENTS |
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Footnotes |
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