Development, Cell and Molecular Biology Group, Box 91000 LSRC, Duke
University, Durham, NC 27710, USA
* Present address: Department of Molecular and Cellular Biology, Harvard
University, BioLabs 2094, Cambridge, MA 02138, USA
Author for correspondence (e-mail:
david.mcclay{at}duke.edu)
Accepted 13 December 2002
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SUMMARY |
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Key words: Sea urchin, T-box, Tbx2/3, Oral/aboral, Morphogenesis
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INTRODUCTION |
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Several genes are expressed asymmetrically about the O/A axis in the
endoderm, mesoderm and ectoderm, but no genes have yet been identified that
reflect the oral/aboral polarity extending between all three. In the endoderm,
asymmetrically localized gene products include apical LvNotch protein, which
is enriched on the aboral side (Sherwood
and McClay, 1997). In the nonskeletogenic mesoderm, CyII actin is
distributed orally, and OrCT, CAPK and P1103 aborally
(Miller et al., 1996
;
Rast et al., 2002
). O/A
patterning has been studied most in the ectoderm and, thus, many gene products
with asymmetric distribution about the axis have been identified. Aboral genes
include Spec1 and Spec2
(Lynn et al., 1983
),
CyIIIa actin (Cox et al.,
1986
), arylsulfatase
(Sasaki et al., 1988
),
Hox8/Hbox1 (Angerer et al.,
1989
), SpMTA (Nemer
et al., 1995
) and P3A2
(Calzone et al., 1991
); whereas
oral genes include EctoV (Coffman and
McClay, 1990
) Otx (Li
et al., 1997a
; Yuh et al.,
2002
), SpCOUP-TF
(Vlahou et al., 1996
),
BMP2/4 (Angerer et al.,
2000
), PlOtp (Di
Bernardo et al., 1999
), Brachyury
(Gross and McClay, 2001
) and
goosecoid (Angerer et al.,
2001
).
Based on the expression patterns and likely function of members of the
family of T-box genes in regionalization of body plans in other organisms, we
hypothesized that they might play a similar role in the sea urchin embryo.
Members of the T-box family of transcription factors have been identified in
all metazoan organisms in which they have been sought (reviewed by
Papaioannou and Silver, 1998;
Smith, 1999
). T-box genes are
characterized by homology to the DNA-binding domain of Brachyury, the founding
member of the T-box gene family. The T-box encompasses
180 amino acids
and can be located anywhere in the protein
(Kispert and Hermann, 1993
).
T-box proteins share little homology outside this region and it is in the
T-box that the specificity for target promoters resides
(Conlon et al., 2001
). The
T-box family includes transcriptional activators such as Brachyury, Tbx5,
VegT and Eomesodermin
(Kispert et al., 1995
;
Horb and Thomsen 1997
;
Horb and Thomsen, 1999
;
Ryan et al., 1996
) as well as
transcriptional repressors such as Tbx2 and Tbx3 (Carreira
et al., 1998; He et al.,
1999
). The importance of T-box genes in development is underscored
by their involvement in a variety of human pathologies, including that of
Tbx5 in Holt-Oram syndrome (Basson
et al., 1997
; Li et al.,
1997b
), Tbx1 in DiGeorge syndrome
(Jerome and Papaioannou, 2001
;
Merscher et al., 2001
),
Tbx3 in ulnar-mammary syndrome
(Bamshad et al., 1997
) and,
possibly, Tbx2 in breast cancer
(Jacobs et al., 2000
).
Here, we report the identification and characterization of LvTbx2/3, a member of the Tbx2/3 subfamily of T-box genes, during development of the sea urchin embryo. LvTbx2/3 protein is concurrently expressed in the aboral territories of the endoderm, mesoderm and ectoderm. A series of perturbations to the molecular components that are thought to be involved in specifying the O/A axis revealed that the aboral distribution of LvTbx2/3 appears to be a common aspect of O/A specification in each of these tissues. Specifically, LvTbx2/3 expression is dependent on either ß-catenin or genes downstream of ß-catenin, and is prevented by ventralization with NiCl2, overexpression of LvBMP2/4 and disruption of the extracellular matrix (ECM). Thus, LvTbx2/3 is expressed downstream of, or relatively late in, the sequence of events that serve to specify this axis. That LvTbx2/3 expression can not be separated between the different tissues after perturbation indicates that O/A axis specification is linked in all three germ layers of the sea urchin embryo at the level of LvTbx2/3 and may occur in parallel to the distinct specification events that give rise to the ectoderm, endoderm and mesoderm of the embryo. Ectopic expression of LvTbx2/3 supports this conclusion in that universal expression of LvTbx2/3 profoundly affects the morphogenesis of ectoderm, endoderm and mesoderm without altering specification events of embryonic territories. Combined with the loss of expression of LvTbx2/3 after perturbation of O/A specification, these results indicate that LvTbx2/3 may be a downstream component of the O/A axis program that is involved specifically in morphogenesis of aboral territories in the embryo.
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MATERIALS AND METHODS |
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Cloning an LvTbx2/3 fragment
Degenerate primers were designed that corresponded to the amino acids
YIHPDSP (forward)/AVTAYQN (reverse) and used in a PCR reaction with cDNA
template prepared from mid gastrula poly(A)+ mRNA. PCR conditions
were 45 cycles of 96°C for 60 seconds, 40°C for 60 seconds, 72°C
for 2 minutes 45 seconds. The amplified, 234 bp products were gel purified,
cloned into the pGEMT vector (Promega) and sequenced bidirectionally (Duke
Sequencing Core). Clones were identified as PCR products of LvTbx2/3
by BLAST search.
cDNA library screens
Screens were performed essentially as described
(Gross and McClay, 2001) with
hybridizations performed at 55°C in 0.5 M NaHPO4 pH 7.2, 1 mM
EDTA, 7% SDS, after Church and Gilbert
(1984
). After rescreens, nine
clones were excised, sequenced and identified as LvTbx2/3 fragments.
A full-length open-reading frame was defined by overlapping individual
fragments.
Northern analysis
Northern blotting (RNA gel blot hybridization) for LvTbx2/3 was
performed as described (Gross and McClay,
2001). Blots were given two 5 minute washes with 6x SSPE,
0.5% SDS at room temperature, one 45 minute wash with 1x SSPE, 0.1% SDS
at 37°C, and one 45 minute wash with 1x SSPE, 0.1% SDS at 50°C.
After washing, the blot was wrapped in plastic wrap and placed on film for 72
hours at -70°C with an intensifying screen. It was then stripped in 50%
formamide, 6x SSPE for 30 minutes at 65°C and reprobed as above with
an L. pictus ubiquitin fragment as a loading control.
Antibody production
LvTbx2/3 fusion protein was expressed following PCR amplification of a
BamHI-XhoI fragment of LvTbx2/3 (encoding amino
acids 11-339) and subcloning into the pGEX4T-1 glutathione S-transferase (GST)
expression system (AmershamPharmacia Biotech). Expressed, affinity-purified
protein (80 µg) was mixed 1:1 with Freund's complete adjuvant and injected
into each of three guinea pigs (Charles River, Raleigh, NC). Animals were
boosted with 80 µg protein mixed 1:1 with incomplete Freund's adjuvant
after 21, 42 and 70 days. Bleeds were performed 31, 53 and 80 days after the
last injection and serum isolated as described
(Harlow and Lane, 1988).
Western analysis
1500 late-gastrula embryos were homogenized in the presence of protease
inhibitors, boiled and run on a 10% SDS-PAGE gel. Protein was blotted onto
nitrocellulose, blocked overnight at 4°C in 2% milk, 1% bovine serum
albumin (BSA) TBST and probed for 1.5 hours at room temperature with a 1:1000
dilution of either -Tbx2/3 or preimmune serum in 2% milk, 1% BSA TBST.
The blot was washed three times with TBS before applying goat
-guinea
pig HRP-tagged secondary antibody (Jackson Immunoresearch Laboratories) at
1:5000 for 1 hour at room temperature. Labeled proteins were visualized by ECL
(AmershamPharmacia Biotech).
Immunolocalization and image analysis
Embryos were fixed in 2% paraformaldehyde, 60% artificial sea water (ASW)
for 10-12 minutes at room temperature, before being permeablized for 60
seconds with ice cold, 100% methanol. They were then washed three times with
PBS, blocked 10-20 minutes in PBS, 4% normal goat serum (NGS; GibcoBRL) and
incubated overnight at 4°C in primary antibody, 4% NGS. After washing four
times in PBS, they were blocked as above and incubated for 60 minutes at room
temperature in secondary antibody, 4% NGS (either Cy3 or Cy5-conjugated;
Jackson Immunoresearch Laboratories). Embryos were then washed four times in
PBS and mounted in 70% glycerol. LvTbx2/3 and LvBrac sera were diluted 1:500
for all images. Undiluted supernatants of monoclonal antibodies (mABs) 5a7
(EctoV), 5c7 (Endo1) and 295 were used with the above fixation and incubation
conditions. All images were obtained using a 40x Plan-Neofluar
oil-immersion objective (NA=1.3) on a Zeiss laser-scanning confocal microscope
(Carl Zeiss, Thornwood, NY) mounted on a Zeiss Axiovert inverted microscope.
Where necessary, 1 µm sections from single label images were rendered into
3D projections using Zeiss confocal software. Double labeled images were taken
sequentially using appropriate filters and subsequently overlayed using Adobe
Photoshop 5.0.
Chemical treatments
Treatment of embryos with either NiCl2 or
ß-aminopropionitrile (ßAPN) were performed as described
(Hardin et al., 1992;
Wessel and McClay, 1987
).
Generation of LvTbx2/3 constructs
Full-length LvTbx2/3 was generated by subcloning fragments from
individual excised cDNA clones obtained in library screening (details
available on request). For ectopic overexpression studies, an SpOtx
5' UTR plus the first five amino acids of SpOtx was cloned in
frame, 5' to the LvTbx2/3 translation-start site. This leader
sequence has been demonstrated to provide an excellent translation start site
for mRNA constructs in the sea urchin
(Sherwood and McClay, 1999).
All clones were sequenced bidirectionally to verify fidelity.
mRNA preparation and injection
LvG-cadherin and LvBMP2/4 were linearized and
injected as described (Logan et al.,
1999
; Angerer et al.,
2000
). LvTbx2/3 was linearized with XhoI and
used as a template to generate in vitro-transcribed 5' capped mRNA using
the T3 mMessage mMachine kit (Ambion). Concentrations of mRNA were determined
by spectrophotometry, and by comparison to known amounts of RNA using both gel
electrophoresis and dotting onto a 0.6% agarose gel.
Quantitative PCR (QPCR)
RNA was isolated using Trizol (Invitrogen). Reverse transcription reactions
were performed using oligo dT priming and MMLV-reverse transcriptase (Gibco).
Reactions were purified using a PCR-purification kit (Qiagen). QPCRs were
performed using Roche LightCycler Fast Start Master SYBR as manufacturers
instructions. Primers used were ubiquitin
(Rast et al., 2000) and
LvTbx2/3. A Tbx2/3 plasmid was used to generate a standard curve for
quantification, and ubiquitin was used to normalize the cDNA samples. Each
time point was determined from two independent batches, and each reaction was
confirmed by gel electrophoresis.
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RESULTS |
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The ectoderm, endoderm and mesoderm are all specified prior to LvTbx2/3 expression. Because LvTbx2/3 was distributed in a subset of cells in each of these tissues, we next characterized the temporal details of LvTbx2/3 protein expression (Fig. 5). LvTbx2/3 was localized to the nucleus at all stages examined. At mesenchyme blastula stage, LvTbx2/3 protein was observed in cells of the presumptive endoderm and ectoderm but not the mesoderm, as neither the ingressed skeletogenic nor the presumptive nonskeletogenic mesoderm expressed LvTbx2/3 protein when these territories were defined by marker genes (data not shown). A view of the vegetal surface of an early-gastrula stage embryo is shown in Fig. 5D. LvTbx2/3 was present at high concentrations in the presumptive endoderm and the ectoderm that surrounds the blastopore, whereas invaginated tissues contained much less protein. Asymmetric distribution in the endoderm and ectoderm continued through mid-gastrula stage (Fig. 5E,F). Between mid-gastrula and late-gastrula stages, LvTbx2/3 started to be expressed in cells of the skeletogenic mesenchyme lineage and the asymmetric localization in the invaginated endoderm became more apparent (Figs 5, 6). LvTbx2/3 protein in early and late-plutei embryos is shown in Fig. 5I-K. From an animal view of an early pluteus embryo that has been optically sectioned and projected so that the animal-most ectoderm is removed, asymmetric distribution of LvTbx2/3 was observed in the ectoderm of the embryo and in the archenteron (Fig. 5I). A vegetal projection of a similarly staged pluteus embryo revealed that LvTbx2/3 is present in the ectoderm that surrounds the anus and was very strong in the distal-most portions of the extending embryonic arms (Fig. 5J). Although the concentration of LvTbx2/3 began to decline at the late pluteus stage, it was still observed asymmetrically in the ectoderm, endoderm and skeletogenic mesenchyme (Fig. 5K).
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LvTbx2/3 in the sequence of O/A axis specification and
patterning
The striking asymmetry of the distribution of LvTbx2/3 about the O/A axis
in the endoderm, ectoderm and mesoderm raises the possibility that O/A
polarity might be either established or maintained by the same molecular
component(s) in all three germ layers of the embryo. To place LvTbx2/3 in the
framework of specification pathways and patterning events that impinge on the
formation of the O/A axis, and to gain further insights into the mechanisms of
O/A axis specification, the distribution of LvTbx2/3, 5a7
(Fig. 7A) and LvBrac
(Fig. 7B) were examined under a
variety of perturbations to this axis.
|
Pharmacological perturbation of O/A patterning
Treatment of embryos with NiCl2 at any point between the hatched
blastula and early gastrula stages disrupts O/A patterning events
(Hardin et al., 1992). Embryos
perturbed in this manner are oralized, displaying defects in ectodermal
patterning manifested by the formation of a circumferential stomodaeum around
the animal pole, rather than at a localized site, and the formation of ectopic
spicule clusters. These animals express EctoV and LvBrac around their entire
circumference except the vegetal plate
(Hardin et al., 1992
;
Gross and McClay, 2001
).
Treatment of embryos with 1 mM NiCl2 resulted in expression of
LvBrac throughout the entire ectoderm (Fig.
7E) and elimination of LvTbx2/3 expression in all tissues
(Fig. 7F).
BMP2/4 in O/A specification
Recent evidence indicates that an animally derived BMP2/4 ortholog affects
O/A specification (Angerer et al.,
2000). In situ analysis localizes BMP2/4 mRNA to
presumptive oral ectoderm at the hatching blastula stage. Ubiquitous
overexpression of BMP2/4 mRNA animalizes the embryo, causing it to
form a ball of squamous epithelium whereas lower concentrations radialize the
ectoderm of the embryo, as indicated by the formation of multiple clusters of
spicules. At concentrations of BMP2/4 mRNA that radialize the
spicules, oral expression of LvBrac was prevented but vegetal LvBrac
expression was normal (Fig.
7G). Aboral expression of LvTbx2/3 was not observed in the
ectoderm, endoderm or skeletogenic mesoderm under such conditions
(Fig. 7H). This indicates that
ectopic expression of BMP2/4 prevented the normal expression of
LvTbx2/3 in all three germ layers, and that O/A polarity in the
ectoderm, mesoderm and endoderm is linked by some common genetic or molecular
mechanism that is likely to be sensitive to changes in BMP2/4 levels.
The failure to observe stomodael LvBrac protein after ectopic expression of
BMP2/4 indicates that BMP2/4 signals prevent the expression
of a subset of genes in the oral ectoderm and do not uniformly oralize the
embryo. Expression of LvBrac is normal in the vegetal blastopore region,
indicating that, unlike in the stomodaeum, LvBrac regulation in this
region is refractory to ectopic BMP2/4 injected at this level.
The ECM in O/A patterning
Disruption of the ECM with ßAPN, a drug that prevents collagen
crosslinking implicates the ECM in O/A specification or maintenance. Embryos
treated with ßAPN do not gastrulate and do not express the
aboral-ectoderm-specific Spec1 gene
(Wessel et al., 1989). The
effects of ECM disruption on LvBrac and LvTbx2/3 expression was assayed
(Fig. 7I,J). Neither stomodael
LvBrac (Fig. 7I) nor aboral
LvTbx2/3 (Fig. 7J) were
expressed following treatment with ßAPN. This indicates that an intact
ECM is necessary for specification and/or maintenance of gene expression in
both the oral and aboral territories of the ectoderm, not solely in the aboral
territory as previously thought. Normal expression of LvBrac in the vegetal
blastopore region indicates that this perturbation did not affect LvBrac
regulation in this region.
Functional characterization of LvTbx2/3
The results of the perturbation studies detailed above place aboral
LvTbx2/3 expression downstream of several signals and specification
events that are known to be involved in the formation of the O/A axis. To
determine the role of LvTbx2/3 in the formation of this axis, ectopic
mRNA expression studies were performed. Ectopic LvTbx2/3 expression
produced drastic morphological defects in derivatives of all germ layers,
suggesting that LvTbx2/3 functions in each germ layer
(Fig. 8). Between 60-75% of
embryos that ubiquitously express LvTbx2/3 mRNA displayed severe
morphological abnormalities 24-48 hours post-fertilization (three- to fivefold
overexpression obtained following injection of 0.75-1 pg/pl of mRNA amounting
to 600-1000 copies of LvTbx2/3 mRNA per cell).
|
At 48 hours post-injection, the skeletons of embryos injected with LvTbx2/3 lacked a consistent pattern, with each embryo elaborating a different, abnormal skeletal phenotype. Two such embryos are presented in Fig. 8, and it is clear that, when compared to a normal pluteus-stage embryo (Fig. 8A,B), patterning of the skeletogenic mesoderm was grossly perturbed (Fig. 8E-H). Embryos that expressed LvTbx2/3 ectopically also had severe endodermal defects. In a few cases, exogastrulae were observed following ectopic LvTbx2/3 expression (data not shown) but, most often, defects were manifest in an archenteron that had multiple `chambers' rather than a typical tripartite structure. Despite their abnormal morphology embryos stained positively for the Endo1 antigen (5c7), which is normally expressed in the midgut and hindgut (Fig. 8I). Vegetal (blastopore) LvBrac expression in embryos that ectopically express LvTbx2/3 was also normal, indicating that the endodermal defect was independent of LvBrac in the vegetal plate. In other words, it occurred after gastrulation (Fig. 8J).
It is well established that, in the sea urchin embryo, the skeletogenic
mesoderm uses spatial and temporal patterning cues that are localized to the
ectoderm to form appropriate skeletal structures (reviewed by
McClay, 1999). The
morphological skeletal abnormalities observed in embryos that express
LvTbx2/3 ectopically could result from inappropriate expression of
either oral-specific or aboral-specific genes in the ectoderm that are induced
by ectopic LvTbx2/3 expression. Thus, downstream patterning cues
would also be misexpressed or absent. Embryos were stained either 24 hours
(data not shown) or 48 hours after ectopic expression of LvTbx2/3
using antibodies against the two markers of oral ectoderm, EctoV and LvBrac
(Fig. 8K,L). EctoV expression
was confined to one region of the embryo, which indicates that the ectoderm
contained an oral territory (Fig.
8K). LvBrac was expressed in a stomodael domain, indicating that
substructures in the oral ectoderm were also specified
(Fig. 8L). mAb 295 is an
antibody that recognizes the ciliary band, a neurogenic region composed of
both oral and aboral cells (Cameron et al.,
1989
). In embryos injected with LvTbx2/3, mAb 295 stained
an amorphous region around the embryo, indicating that although oral and
aboral territories have been specified and subdivided in the ectoderm, the
boundary is not tightly localized (Fig.
8M).
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DISCUSSION |
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O/A polarity in the sea urchin embryo
That LvTbx2/3 is expressed in the aboral territories of the
endoderm, ectoderm and mesoderm provides a point of entry to examine the
regulation of gene expression along the O/A axis in each of these tissues. To
this end, we perturbed several events that are thought to be involved in
patterning this axis and examined the effects on the expression of LvTbx2/3
and LvBrac, markers of aboral and oral gene expression, respectively. Results
of these experiments suggest that either ß-catenin or the expression of
genes downstream of ß-catenin is necessary for the expression of both
proteins and, thus, gene expression along both the A/V and the O/A axes of the
sea urchin embryo. Pharmacologically blocking formation of the aboral axis
with NiCl2 prevented LvTbx2/3 expression in all tissues, suggesting
that gene expression along this axis is uniformly sensitive to this
perturbation. In addition, it is possible that gene expression of axial
information is controlled by mechanisms common to each germ layer rather than
through different pathways in each.
The results on protein distribution after ectopic expression of
BMP2/4 are particularly interesting because previous experiments in
Strongelocentrotus purpuratus embryos demonstrated that oral
expression of EctoV was blocked by ectopic expression of BMP2/4 but
the aboral domain of Spec1 expression increased
(Angerer et al., 2000). In our
study, ectopic expression of BMP2/4 in L. variegatus
prevented the expression of both aboral LvTbx2/3 and oral LvBrac
(Fig. 7G,H). Thus, two aboral
genes, Spec1 and LvTbx2/3 differ in their response to
ectopic expression of BMP2/4. The ectoderm of these embryos may be a
locked in some sort of pre-aboral ectoderm state in which some aboral proteins
are expressed but others are not because the signals necessary for their
expression are inhibited by increased concentrations of BMP2/4. The most
obvious explanation is that BMP2/4 might be a component of aboral
specification, but, given the different Spec1 and LvTbx2/3
responses to BMP2/4 perturbation, other, aboral-specification
mechanisms must also exist. Evidence for a veg1-derived signal to overlying
animal tissues has been observed recently (D.R.M. and J.M.G., unpublished
observations), and this signal is sensitive to ectopic BMP2/4 expression.
Thus, the defects in O/A gene expression described here might result from a
perturbation to this signal. Further characterization of the BMP2/4 pathway,
and the identification of more markers for O/A-axis formation in the ectoderm
will likely clarify this issue. It is also possible that species-specific
differences in specification of the oral and aboral axes might explain this
discrepancy. S. purpuratus embryos at least partially differentiate
aboral ectoderm autonomously, whereas L. pictus embryos require
vegetal signaling to do so (Wikramanayake
et al., 1995
). Therefore, the loss of LvTbx2/3 expression
in embryos of L. variegatus following overexpression of
BMP2/4 could reflect a slightly different role of this pathway in
specifying structures along the O/A axis in Lytechinus species of
urchins than that in S. purpuratus.
LvTbx2/3 patterning and morphogenesis in the sea urchin
embryo
Based on perturbation studies, LvTbx2/3 expression is downstream
of the specification of endoderm, mesoderm and ectoderm, including initial O/A
specification events in these tissues. When ectopically expressed,
LvTbx2/3 consistently produces abnormal morphological phenotypes and
patterning deficiencies in derivatives of each tissue. Nevertheless, markers
for specific germ layers and axial regions are expressed
(Fig. 8). Thus, what is the
role of LvTbx2/3 in the aboral territories? Genes downstream of
LvTbx2/3 may be involved directly in patterning and morphogenesis, as
suggested by the skeletal and endodermal phenotypes that result from the
ectopic LvTbx2/3 expression studies presented here. Several other
T-box genes have also been noted to have distinct functions during
morphogenesis. These include Brachyury in gastrulation movements
(Kimmel et al., 1989;
Conlon et al., 1996
;
Wilson and Beddington 1997
;
Gross and McClay, 2001
),
Eomesodermin in the formation of bottle cells and initiation of
gastrulation (Ryan et al.,
1996
; Russ et al.,
2000
), spadetail in paraxial mesoderm migration
(Griffin et al., 1998
;
Yamamoto et al., 1998
), and
Tbx24 in somite segmentation
(Nikaido et al., 2002
). It
will be of great interest to refine the position of LvTbx2/3 in a
network of O/A specification when more genes are identified in this
gene-regulatory network. Also, using the differential screening technologies
that are available currently, it should be possible to identify downstream
targets of LvTbx2/3 and determine their roles in patterning and morphogenesis
along this axis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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