1 National Institutes of Health, NICHD, LMG, Bldg. 6B, 9000 Rockville Pike,
Bethesda, MD 20892, USA
2 University of Utah, Department of Neurobiology and Anatomy, 401 MREB, Salt
Lake City, UT 84132, USA
3 University of Chicago, Department of Organismal Biology and Anatomy, Chicago,
IL 60637, USA
4 University of California, Irvine, Department of Developmental and Cell
Biology, Irvine, CA 92697, USA
5 Princeton University, Department of Molecular Biology, Princeton, NJ 08544,
USA
6 Max-Planck-Institute for Developmental Biology, Spemannstr.35, 72076,
Tübingen, Germany
7 Howard Hughes Medical Institute, Division of Hematology/Oncology, Children's
Hospital, Boston, MA 02115, USA
Author for correspondence (e-mail:
piotrowski{at}neuro.utah.edu)
Accepted 25 June 2003
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SUMMARY |
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Key words: van gogh (vgo), tbx1, endothelin 1, Pharyngeal arch development, DiGeorge syndrome, Endodermal pouches, Aortic arches, Zebrafish
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Introduction |
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The phenotype of homozygous vgo mutants bears striking resemblance
to humans afflicted with DiGeorge syndrome (DGS). DGS is one of the most
common developmental diseases in humans, affecting approximately 1 in 4000
live births (Scambler, 2000).
Individuals with DGS are characterized by craniofacial defects, aortic arch
malformations, thymus hypoplasia, conotruncal heart defects and hearing loss
(Ryan et al., 1997
). The
majority of individuals carry a heterozygous deletion located on chromosomal
region 22q11.2. This region comprises
30 genes and is called the DiGeorge
critical region (DGCR). Combined studies of heterozygous deletions within the
DGCR and knockout experiments in mice have identified Tbx1 as a major
genetic determinant of aortic arch malformations in DGS
(Jerome and Papaioannou, 2001
;
Lindsay et al., 2001
;
Merscher et al., 2001
).
Tbx1 encodes a transcription factor belonging to a gene family
characterized by a highly conserved DNA-binding domain called the T-box. T-box
genes are important regulators of embryonic development, examples include the
founding family member T or Brachyury, which, when mutated
in mice or zebrafish, leads to the loss of notochord and tail structures
(Kispert et al., 1995
;
Schulte-Merker et al., 1994
).
Other examples are mutations in TBX5 and TBX3, which are
responsible for human Holt-Oram and Ulnar-mammary syndromes, respectively, in
which formation of limbs and cardiovascular system is disrupted
(Bamshad et al., 1997
;
Basson et al., 1997
), and the
spadetail mutation in zebrafish, which affects the formation of trunk
somites (Griffin et al.,
1998
).
We show that the zebrafish vgo mutant is defective in
tbx1, which is required for interactions between the cranial
mesendoderm and the neural crest cells that form the pharyngeal cartilages.
Transplantation of wild-type endoderm into vgo/tbx1 mutants induces
cartilage formation, which indicates that tbx1 acts autonomously in
the endoderm. Consequently, the neural crest defects are secondary to
endodermal defects in vgo/tbx1 mutants, as has been previously
suggested (Piotrowski and
Nuesslein-Volhard, 2000). The zebrafish vgo/tbx1 mutant
offers the opportunity to study pharyngeal arch development in cellular detail
and may contribute further to the understanding of DGS pathogenesis.
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Materials and methods |
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Sequence analysis of tbx1 in vgo
Primers were designed using the sequences flanking the exon-intron
boundaries of tbx1. Genomic DNA was extracted from a pool of
4-day-old homozygous mutant embryos. PCRs were performed separately for each
exon and the resulting products were cloned into the pGEM-T-Easy (Promega)
vector for sequencing. Mutations were confirmed independently by repeat PCR
reactions.
Meiotic and radiation hybrid mapping
The vgotm208 allele was generated in the Tübingen
background in a large scale mutagenesis screen
(Haffter et al., 1996;
Piotrowski et al., 1996
). To
produce a polymorphic strain for genetic mapping, the fish carrying this
allele was crossed to wik fish (Knapik et
al., 1996
; Rauch et al.,
1997
). Homozygous and sibling embryos were phenotypically scored
at 4 dpf and fixed separately in 4% paraformaldehyde overnight. For extraction
of genomic DNA individual embryos were placed in 96-well PCR plates and
processed as described (Rauch et al.,
1997
). Bulk segregant analysis with SSLP markers was used to
identify the linkage group. Analysis of SSCP markers in individual embryos was
used to fine map the mutation. Radiation hybrid mapping of tbx1 was
performed as described (Hukriede et al.,
1999
), using forward primer
5'-AACATGTACTCTGCGACGAGTGCAC-3' and reverse primer
5'-CACGGATCTGCTAAAGGTGGTCTAG-3' on the LN54 panel. The PCR
conditions were 95°C for 4 minutes, 35 cycles of 94°C for 30 seconds,
65°C for 30 seconds, 72°C for 30 seconds each, followed by 72°C
for 7 minutes. The results were analyzed by interactive mapping software
available at
http://www.mgchd1.nichd.nih.gov:8000/zfrh/beta.cgi
Linkage analysis between tbx1 and
vgotm208
The A to T transition mutation in the vgotm208 allele
leads to the loss of an AlwN1 restriction site in the tbx1
gene, which co-segregated with the mutation. Genomic DNA was amplified by PCR
using the forward 5'-GCTCTGGAGTGAACTTGATTACCTG-3' and reverse
5'-AACGGTCAAGTAGGCCTGTAGCTAC-3' primers that flank the mutation.
The PCR product was digested with AlwN1 and analyzed on a 3% MetaPhor
agarose gel (BMA).
In situ hybridization
In situ hybridization experiments were performed as described
(Piotrowski and Nüsslein-Volhard,
2000). Probes used were for: hand2
(Angelo et al., 2000
),
edn1 (Miller et al.,
2000
), myod (Weinberg
et al., 1996
), crestin
(Luo et al., 2001
) and
fgf8 (Furthauer et al.,
1997
).
Confocal microangiography
Labeling of the blood vessels was performed essentially as described
(Weinstein et al., 1995) with
the following modifications: live, anesthetized 2.5 dpf larvae were mounted in
5% methylcellulose for fluorescent microsphere injections (Molecular Probes).
The microspheres were injected into the yolk blood stream that flows into the
heart. Images represent projections of z-series stacks that were
taken within 15 minutes of the injection.
Transplantation experiments
Wild-type donor embryos were injected with 5% rhodamine-dextran and 3%
lysine-fixable biotinylated-dextran (Molecular Probes) at the one- to two-cell
stage (diluted in 0.2 mM KCl). Cells were transplanted into the animal pole of
unlabeled embryos derived from heterozygous vgo parents at the late
blastula stage. Larvae were scored for neural crest and ear clones on day 3
using a BioRad confocal microscope.
edn1 rescue experiments and cartilage rescue
experiments
Donor embryos were injected with 2 pg activated Taram A/Alk4 (Tar*) at the
one-cell stage to convert most of the donor cells to an endodermal fate
(Peyrieras et al., 1998). As
lineage tracers, we used 2.5% Alexa rhodamine-dextran and 2%
biotinylated-dextran dissolved in 0.2 mM KCl. Donor cells were transplanted at
the late blastula stage into the margin of unlabeled embryos derived from
heterozygous vgo/tbx1 parents. For transplantation, we used an
air-filled standard 10 ml plastic syringe. At 24 hpf, the embryos were scored
for endodermal clones under a fluorescent microscope. Embryos with endodermal
clones were fixed with 4% paraformaldehyde overnight and processed for in situ
hybridization with edn1. The transplanted cells were visualized using
a avidin-biotinylated enzyme complex (ABC kit, Vector Laboratories)
(Moens and Fritz, 1999
). The
embryos were mounted ventral side upwards under bridged cover slips after the
yolk and the tail were removed. Subsequently the specimens were dehydrated in
100% methanol and cleared in a 1:2 solution of benzyl-benzoate:
benzyl-alcohol. They were then photographed on a Zeiss Axiophot using a
digital camera (Prog-Res/Kontron).
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Results |
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|
vgo represents a null allele of tbx1
Zebrafish tbx1 cDNA encodes an open reading frame of 460 amino
acids with extensive sequence similarity to mouse Tbx1 (AF326960)
(68.5% overall identity, 98.3% identity within the T-box;
Fig. 2A). In addition, the
exon-intron structure was identical to that of the mouse gene (data not
shown).
|
Zebrafish tbx1 is expressed in the ear and in mesendodermal
components of pharyngeal arches
Expression of zebrafish tbx1 starts at the beginning of
gastrulation (6 hpf) in the involuting cells within the hypoblast
(Fig. 3A). These
tbx1-expressing cells are likely to be the progenitors of the cranial
paraxial mesendoderm. Between 6- and 10-somite stages, tbx1
expression is found in parachordal mesendoderm as well as in a pair of
bilateral stripes on either side of the neural tube that correspond to the
cranial paraxial mesoderm, which extends from just posterior to the eyes
(midbrain level) to approximately the level of rhombomere 6
(Fig. 3B,C). At the 20-somite
stage, expression is detected in the primordia of the pharyngeal arches
(Fig. 3D, p1-p7). By 27 hpf,
expression is localized to the mesodermal core of the pharyngeal arches as
well as to the arch epithelium, but not to neural crest cells lying in between
(Fig. 3E). At 30 hpf,
pharyngeal expression marks elongated cell clusters in each arch, which
prefigure the forming arch muscles and possibly also mesenchyme cells
surrounding the aortic arches (Fig.
3F). These patches are separated by the endodermal pouches that
also express tbx1, which are more clearly observed in horizontal
sections (Fig. 3G; `e'). From
48 hpf onwards, tbx1 expression is seen in individual arch muscles,
which co-express myod at this stage
(Fig. 3H,I; arrows) and in
endodermal pouches separating individual arches.
|
vgo/tbx1 is required non-cell autonomously in neural crest
cells and cell autonomously in the ear
As vgo/tbx1 mutants exhibit defects in the neural crest-derived
head skeleton where tbx1 is not expressed, we tested whether
vgo/tbx1 is required within the surrounding mesendoderm. We attempted
to rescue vgo/tbx1 mutant cartilages (AB* background) by placing
wild-type cells into the pharyngeal mesendoderm. Transplantation of
unmanipulated wild-type cells usually results in small mesendodermal clones.
To achieve larger clone sizes in the pharynx, we injected the wild-type donors
prior to transplantation with Tar*, an activated version of the type 1
TGFß-related receptor Taram-A (Tar)
(David and Rosa, 2001;
David et al., 2002
;
Peyrieras et al., 1998
). Tar
is normally expressed in endodermal precursor cells and injection of its
activated form converts blastomeres to an endodermal fate. Tar*-injected cells
behave like endogenous endodermal cells in transplantation experiments
(David and Rosa, 2001
;
Peyrieras et al., 1998
;
Renucci et al., 1996
).
Relevant to our experiments is the fact that transplanted cells do not induce
ectopic expression of downstream nodal targets
(David and Rosa, 2001
). As
expected, donor cells in the mosaic host embryos contribute largely to the
pharynx, endodermal pouches of the pharyngeal arches and the digestive tract.
In 12 out of 43 vgo larvae (28%) in which transplanted wild-type
cells contributed to the mesendoderm of the pharyngeal arches, we observed
partial rescue of the neural crest-derived pharyngeal cartilages
(Fig. 4A,B). Rescue was
generally unilateral, corresponding to the side to which most transplanted
cells contributed, and in separate cases included restoration of cartilages in
the mandibular (Fig. 4B,
n=5), hyoid (Fig.
4A,B; n=3) and branchial arches
(Fig. 4A; n=4). These
results suggest that cartilage defects in vgo/tbx1 result from
defective signaling from the endoderm.
|
Regulatory interactions between vgo/tbx1, edn1 and
hand2
Edn1 and Hand2 (previously known as dHand) are
expressed in the pharyngeal arches and cause phenotypes similar to DGS if
knocked-out in mice (Kurihara et al.,
1995; Kurihara et al.,
1994
; Srivastava et al.,
1997
; Thomas et al.,
1998
) or mutated in zebrafish
(Miller et al., 2000
).
Edn1 is a small signaling peptide which regulates Hand2
(called hand2 in zebrafish), a bHLH transcription factor expressed in
the neural crest cells surrounding the mesodermal core of the arches
(Charite et al., 2001
;
Miller et al., 2000
). In both
mouse and zebrafish, edn1 is expressed in a similar pattern to
tbx1, in pharyngeal arch epithelia (both surface ectoderm and
pharyngeal endoderm) and in the mesodermal core
(Fig. 3E, Fig. 5A). This prompted us to
test whether vgo/tbx1, edn1 and hand2 interact in a
regulatory pathway. The existence of zebrafish mutants with mutations in all
three genes (vgo/tbx1, suc/edn1, and han/hand2) facilitates
the study of their epistatic relationships.
|
In vgo/tbx1 mutants, the expression of hand2 in neural
crest cells is reduced in the two anterior pharyngeal arches and absent in the
posterior arches (Fig. 5G,H).
However, expression in the cardiac mesoderm and fin buds is unaffected,
supporting studies showing that Hand2 expression is controlled by
different enhancers in these two regions
(Charite et al., 2001;
Thomas et al., 1998
). Defects
in hand2 expression in vgo/tbx1 mutants again are not simply
due to the absence of neural crest cells, as in situ hybridization experiments
with crestin, a pan-neural crest marker
(Luo et al., 2001
;
Rubinstein et al., 2000
) shows
the presence of neural crest cells even posterior to the otic vesicle of
mutant embryos (Fig. 5I,J,
arrows).
Therefore, reductions in edn1 and hand2 expression in
vgo/tbx1 mutants are probably due to gene regulation defects. A
simple regulatory cascade in which vgo/tbx1 is upstream of
edn1 and hand2 predicts that vgo/tbx1 expression is
unaffected in edn1/suc and hand2/han mutant
embryos, which is indeed the case (Fig.
6A,B). In addition, hand2 is downregulated in
edn1/suc mutants (Miller et al.,
2000), whereas in hand2/han mutants, edn1
expression is normal (Fig. 6C).
The results of these in situ hybridization experiments suggest that
vgo/tbx1 acts upstream of edn1 and hand2.
|
Endodermal vgo/tbx1-expressing cells induce
edn1
To confirm the result from the in situ hybridization experiments, we tested
if vgo/tbx1 regulates edn1 by transplanting wild-type
endodermal cells at the blastula stage into vgo/tbx1 mutants. In
three out of 20 vgo/tbx1 mutants that had transplanted cells in the
pharynx, we observed upregulation of edn1
(Fig. 7B). However,
transplantation of cells at this early stage leads to small mesendodermal
clones, because many cells also contribute to the ectoderm. The small number
of transplanted endodermal cells in vgo/tbx1 mutants might explain
why we only observed rescue of edn1 expression in a relatively low
number of mutant embryos. Therefore, we also transplanted Tar*-injected
wild-type cells into vgo mutants to ensure that the transplanted
cells contributed to the endoderm. In this experiment, 119 embryos received
transplanted cells in the pharyngeal region. Among these were 34
vgo/tbx1 mutants, out of which 32 showed an increase in the
expression level of edn1 close to the transplanted cells (94.7%,
Fig. 7C, arrows indicate
transplanted cells). In many embryos, edn1 expression was not only
detected in the transplanted cells themselves but also in several tiers of
cells adjacent to the transplanted cells. As vgo/tbx1 is a
transcription factor, an intermediate gene product must exist that transduces
the signal over several cell diameters. Consequently, our data suggests that
vgo/tbx1 is regulating edn1 indirectly, although we cannot
rule out that vgo/tbx1 also is regulating edn1 directly in a
subset of cells.
|
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Discussion |
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vgo/tbx1 function in the endoderm is required for normal
development of neural crest-derived pharyngeal structures
In our description of the vgo mutant phenotype, we hypothesized
that the neural crest defects are secondary to defects in the pharyngeal
endoderm (Piotrowski and
Nüsslein-Volhard, 2000). This suggestion is supported by the
expression pattern of tbx1, which is not expressed in neural crest
cells. Furthermore, we showed that transplantation of wild-type endodermal
cells can rescue cartilage formation in vgo/tbx1 mutants. The fact
that neural crest induction and migration appear normal in vgo
(Piotrowski and Nüsslein-Volhard,
2000
) also supports this conclusion. These three results suggest
that the mesendoderm is the source of a secreted signal required for the
proper differentiation of neural crest cells in the pharyngeal arches.
DGS often has been described as being caused by autonomous defects in the
development of neural crest cells, an explanation supported by the fact that
neural crest ablation in chicks results in very similar phenotypes, as have
been reported for DGS. By contrast, similar to our findings in
vgo/tbx1 mutant fish, a study in
Tbx1/ mice suggests that neural crest
defects are indirect (Kochilas et al.,
2002). However, as TBX1 is not the only gene responsible
for causing DGS, downstream targets of TBX1 that are expressed in
neural crest cells may also represent candidate genes for causing DGS. In
contrast to what we found for neural crest cells, we have demonstrated that
vgo/tbx1 is required cell autonomously in the outgrowing semicircular
canals of the ear. Therefore, TBX1 may not only be responsible for
the pharyngeal arch defects characteristic of DGS but also for the ear defects
found in these individuals (Reyes et al., 1999;
Funke et al., 2001
).
Genetic components of the tbx1 pathway in the pharyngeal
arches
A large number of genes are expressed in the pharyngeal arches,
underscoring the genetic complexity of their development
(Garg et al., 2001).
vgo/tbx1 appears to be one of the earliest genes involved in this
process as disruption of its function affects the segmentation of the
endodermal pouches (Piotrowski and
Nüsslein-Volhard, 2000
), which is one of the earliest events
in arch morphogenesis. In mice and chickens, Shh
(Garg et al., 2001
;
Yamagishi et al., 2003
),
Fgf8 (Frank et al.,
2002
; Vitelli et al.,
2002
) and Vegf
(Stalmans et al., 2003
) have
been suggested to be part of the Tbx1 pathway.
Among genes implicated in DGS and in pharyngeal development, zebrafish
mutations have been isolated in fgf8, tbx1, edn1 and hand2,
and therefore we focused our study on these loci. Our experiments suggest
regulatory interactions between the tbx1 and the edn1
pathways. In mice, such a relationship might not exist, as Edn1
expression has been reported to be normal in the aortic arch endothelial cells
of Tbx1/ animals
(Kochilas et al., 2002).
However, expression of Edn1 in the pharyngeal arches of
Tbx1/ mice has not been directly tested. In
zebrafish, transplantation of wild-type cells into vgo/tbx1 mutant
embryos showed a clear increase in edn1 expression in the pharynx in
cells close to the transplanted cells (Fig.
7B,C). The fact that edn1 expression was also induced in
cells up to several cell diameters away from the transplanted cells implies
that tbx1 regulates one or several intermediate factors that in turn
regulate edn1, possibly in addition to direct regulation in certain
cell types.
We found that gene regulation within the anterior and posterior arches
differs slightly (Fig. 8C).
This result is not surprising, given that most jaw mutants mainly affect
either the anterior or the posterior arches
(Piotrowski et al., 1996;
Schilling et al., 1996
). A
model for genetic interactions between vgo/tbx1, edn1 and
hand2 in the pharyngeal arches and a summary of expression data are
presented in Fig. 8. Within the
first arch of the mouse, Edn1 has been shown to be regulated by
Fgf8 (Trumpp et al.,
1999
; Tucker et al.,
1999
), and Tbx1 and Fgf8 have been shown to
interact genetically (Funke et al.,
2001
; Vitelli et al.,
2002
). In zebrafish, fgf8 does not appear to regulate
edn1, as fgf8/ace mutants
(Reifers et al., 1998
) do not
show defects in edn1 gene expression (data not shown). Likewise,
vgo/tbx1 does not appear to be upstream of fgf8, as
fgf8 expression is normal in vgo/tbx1 mutants
(Fig. 6F). Owing to gene
duplication events, the zebrafish genome contains more redundant genes than
mice, and it is possible that other Fgfs play a role in zebrafish pharyngeal
arch development. This is likely because, in contrast to mice and chicks, in
which fgf8 is expressed in all endodermal pouches, fgf8 is
only expressed in the mandibular arch in zebrafish. Regulation of
edn1 by another member of the Fgf family may be responsible for the
remaining, although reduced, edn1 and hand2 expression still
present in the first arch of vgo/tbx1
(Fig. 5B,H; see
Fig. 8C).
|
In summary, the identification of tbx1 as the gene responsible for the vgo mutation allows for mechanistic studies of pharyngeal development in zebrafish and adds a potentially useful model system to the available tools for understanding the etiology of DGS.
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ACKNOWLEDGMENTS |
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Footnotes |
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