1 Program in Cardiovascular Sciences, Baylor College of Medicine, Houston, TX
77030, USA
2 Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX
77030, USA
3 Departments of Pediatrics (Cardiology), Baylor College of Medicine, Houston,
TX 77030, USA
4 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030,
USA
* Author for correspondence (e-mail: baldini{at}bcm.tmc.edu)
Accepted 29 July 2005
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SUMMARY |
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Key words: Tbx1, Timed mutation, DiGeorge syndrome
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Introduction |
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Materials and methods |
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The following mouse mutant lines have been described previously:
TgCAGG-CreERTM (Hayashi and McMahon,
2002), Tbx1flox/lox and
Tbx1mcm/+ (Xu et al.,
2004
), Tbx1+/-
(Lindsay et al., 2001
), and
R26R (Soriano, 1999
). All
lines were backcrossed into the C57Bl6 genetic background for at least two
generations. Mice were genotyped using PCR as described in the original
reports. To induce nuclear translocation of the inducible Cre, including
CreERTM and MerCreMer encoded by the CAGG-CreERTM transgene
and the Tbx1mcm allele, respectively, pregnant mice were
treated with single intraperitoneal injection of tamoxifen (Sigma) with a dose
of 75 mg/kg body weight at the desired time points. Tamoxifen was dissolved in
absolute ethanol at the concentration of 100 mg/ml and then diluted 1:10 in
autoclaved sesame oil (Sigma) for injection. The excision of the loxP-flanked
exon 5 of the Tbx1 allele Tbx1flox was evaluated
as follows. Genomic DNA was extracted from whole E9.5 embryos exposed to TM
for 3, 6, 12 or 24 hours and it was used as template in PCR. The PCR primers
used to detect Tbx1flox allele
(Fig. 1A) are Tbx1loxP2-F
(5'-cgacccttctctggcttatg-3') and Tbx1loxP2-R
(5'-aaagactcctgcccttttcc-3'). PCR products were separated in 1.5%
agarose gel by electrophoresis and the intensity of the bands was measured
using NIH image 1.63 software on digital images of the gel. The percentages of
remaining Tbx1flox allele in TgCAGGCreERTM;
Tbx1flox/+ mutants were calculated as the ratio of the
intensity of Tbx1flox band to that of
Tbx1+ band, and the values were normalized with those of
Tbx1flox/+ embryos. A second PCR strategy was used to
evaluate the disappearance of the Tbx1flox allele and the
appearance of the Tbx1
E5 allele (see scheme in Fig.
S1A in the supplementary material). To do this, we used a primer pair
amplifying exon 5 and the flanking loxP sites, Tbx1E5-F
(5'-ggccctgcctaactcagatt-3') and Tbx1E5-R
(5'-aaagactcctgcccttttcc-3').
Reverse-transcription polymerase chain reaction (RT-PCR)
We have used RT-PCR to detect residual Tbx1 transcripts in
TgCAGG-CreERTM; Tbx1flox/- and control
(TgCAGG-CreERTM; Tbx1flox/+ or
Tbx1flox/+) embryos after exposure to TM for 24 hours (see
scheme in Fig. S1B in the supplementary material). Embryos at different stages
(E9.5-E12.5) were harvested 24 hours after TM injection and total RNA was
extracted from whole embryos using the Trizol reagent (Invitrogen). The
concentration of RNA samples was measured using a spectrophotometer and
adjusted to 100 ng/µl. cDNA was synthesized from mRNA using Superscript
first-strand synthesis system (Invitrogen) with random hexamers as the
primers, and then subjected to PCR amplification (30 cycles) using
Tbx1 and ß-actin-specific primer pairs. The PCR primers used to
examine Tbx1 mRNA level were Tbx1mRNA_F
(5'-TTTGTGCCCGTAGATGACAA-3') and Tbx1mRNA_R
(5'-AATCGGGGCTGATATCTGTG-3').
Phenotypic analysis
E18.5 embryos were examined and photographed after manual dissection under
a stereomicroscope. After formalin fixation, the great arteries were
visualized by India ink injected into the left ventricle. Earlier embryos were
examined under the stereomicroscope, fixed and paraffin embedded for
histological analysis. Pharyngeal arch arteries were visualized at E10.5 using
intracardiac Ink injection. Embryos were then fixed and dehydrated in ethanol:
water: acetic acid: chloroform (95:3:1:1) solution and cleared in methyl
salicylate: benzyl benzoate (50:50) solution. ß-Galactosidase activity
was visualized by staining paraformaldehyde-fixed embryos with the X-gal
substrate, according to standard procedures. Stained whole-mount embryos were
photographed and then embedded in paraffin and cut into 10 µm histological
sections. Sections were counterstained with Nuclear Fast Red. Cell
proliferation was assessed by immunohistochemistry using an anti
phosphorylated histone H3 antibody (Upstate Biotechnology) on sections of
ethanol-fixed, paraffin-embedded E10.0 embryos. Whole-mount RNA in situ
hybridization was performed as previously described
(Albrecht et al., 1997) using a
Pax1 probe kindly provided by Dr R. Balling.
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Results |
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Of the phenotypic abnormalities assessed in this study, clefting of the
secondary palate is the only one that occurred after Tbx1 ablation at
E11.5 (Fig. 2A and
Fig. 4F,G). Thus, clefting in
these mutants is not due to early abnormalities of 1st pharyngeal arch
patterning, which are present in Tbx1-/- embryos
(Kelly et al., 2004), but most
likely to a role of Tbx1 in secondary palatogenesis, which occurs
from approximately E12.5.
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Tbx1 is required during segmentation of the pharyngeal system and regulates pharyngeal endoderm expansion
Timed cell fate mapping induced at E7.5, E8.5 and E9.5 revealed a
progressive `concentration' of labeled cells in the caudal region of the
pharyngeal endoderm (Fig.
5A-C). This is consistent with the described cranial-to-caudal
gradient of Tbx1 expression observed during the development of the
pharyngeal system (Vitelli et al.,
2002). These observations suggest a role of Tbx1 in the
process of cranial-to-caudal addition of pharyngeal arches and pouches that
occurs in the E8.0-E10.5 time interval. If Tbx1 expression is crucial
for progressive addition of pharyngeal arches and pouches over time, then
deletion of Tbx1 during this process should effectively arrest it
when the gene deletion occurs. To test this, we exposed
TgCAGG-CreERTM;Tbx1flox/- embryos to TM at E7.5 or
E8.5 and examined them at E10.5 when the entire complement of pharyngeal
arches and pouches should have formed. Embryos exposed at E7.5 exhibited
severe hypoplasia of the 2nd pharyngeal arch
(Fig. 5D) and lacked the
3rd-6th arches and intervening pharyngeal pouches
(Fig. 5D',G). This
phenotype is identical to that caused by germline Tbx1-/-
mutation. RNA in situ hybridization showed that Pax1, a marker of
pharyngeal pouch endoderm, is weakly expressed in the first pouch but does not
identify the caudal pouches of these embryos
(Fig. 5J, compare with 5L).
Embryos exposed to TM at E8.5 had normally segmented 1st, 2nd, and 3rd arches
and normal 1st and 2nd pouches (Fig.
5E,E',H), but the 3rd pharyngeal pouch was not identified by
Pax1 and consisted only of a small endodermal evagination that did
not approach the surface ectoderm (Fig.
5H,K). Thus, deletion of Tbx1 at the time of 3rd pouch
formation (between E9 and E9.5) resulted in developmental defects of the
segments caudal to and including the 3rd pouch, but did not affect the
development of segments cranial to the 3rd pouch. The addition of new arches
and pouches probably requires multiple morphogenetic events, the most basic of
which is the expansion of the endodermal cell population. Therefore, we tested
whether loss of Tbx1 during segmentation is associated with reduced
endodermal cell proliferation in
TgCAGG-CreERTM;Tbx1flox/- embryos exposed to TM at
E8.5. We have used immunohistochemistry with an anti-phosphorylated histone H3
antibody to detect proliferating cells in E10.0 embryos. Results showed that
the number of proliferating pharyngeal endodermal cells is reduced by almost
50% in TgCAGG-CreERTM;Tbx1flox/- embryos when
compared with TgCAGG-CreERTM;Tbx1flox/+ embryos
(P=0.03) (Fig. 6A-C).
By contrast, we could not detect changes in proliferation of pharyngeal
mesenchyme in the same embryos (Fig.
6C). Thus, Tbx1, directly or indirectly, regulates the
expansion by cell proliferation of the pharyngeal endoderm during segmentation
(Fig. 6D).
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Discussion |
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Time-based phenotypic dissection of the DiGeorge syndrome model
The time course deletion of Tbx1 during embryogenesis dissected
most of the phenotypic abnormalities that we have tested. Data indicated that
Tbx1 is required throughout embryogenesis and that there are discrete
and distinct time intervals crucial for the development of the different
organs and structures affected in the model. The knowledge of these time
windows provides clues as to the role of Tbx1 in the development of
different pharyngeal derivatives. For example, thymic formation required
Tbx1 at E8.5-E9.5, this did not coincide with formation of the thymic
primordia (which occurs later) but with the formation of the 3rd pharyngeal
pouch, from which the thymus derives
(Gordon et al., 2004;
Manley and Blackburn, 2003
).
Thus, the role of Tbx1 in the formation of the thymus is secondary to
its role in the formation of the 3rd pouch. Surprisingly, however,
Tbx1 continues to be important for thymic development also after the
formation of the 3rd pouch, as demonstrated by organ dysmorphism caused by
Tbx1 deletion after E10.5. This late phenotype is more likely to be
the consequence of a role of Tbx1 in thymic organogenesis. We were
also surprised by the timing of secondary palate closure requirement. It has
been shown that the patterning of the first pharyngeal arch is abnormal in
Tbx1-/- mutants (Kelly
et al., 2004
), therefore we speculated that cleft palate may be a
consequence of these early defects. By contrast, timed-deletion data support a
role of Tbx1 in the secondary palatogenesis, which occurs between E12
and E14.5 (Kaufman and Bard,
1999
).
Tbx1 is required early for aortic arch patterning, late for outflow tract septation and identifies a subset of outflow tract-specific cardiomyocyte precursors
Tbx1 loss of function affects two segments of the cardiovascular
system, the aortic arch and the OFT
(Vitelli et al., 2002). We
have previously shown that tissue-specific deletion of Tbx1 can
separate these two groups of abnormalities
(Xu et al., 2004
) suggesting
distinct pathogenetic mechanisms. Here, we show that Tbx1 is required
in distinct time windows for the development of the aortic arch and OFT.
Counterintuitively, Tbx1 is required early for aortic arch patterning
(a relatively late process), and later for OFT growth and remodeling (a
relatively early process). Our data are consistent with a role of
Tbx1 that precedes or coincides with the formation of the 4th PAAs.
Our data also exclude a direct role of Tbx1 in the remodeling or
smooth muscle lining of the 4th PAAs, in contrast to hypotheses previously put
forward (Kochilas et al.,
2002
; Lindsay and Baldini,
2001
). Indeed, deletion of Tbx1 after the 4th PAAs are
formed, but before smooth muscle lining and remodeling occurs (TM injections
at E8.5, E9.5 and E10.5), resulted in no phenotypic consequences for aortic
arch patterning.
Timed-deletion revealed that the requirement for Tbx1 in OFT
development is restricted to a relatively late and narrow time-window
(E9.0-E9.5), which is not consistent with a crucial role in the early SHF cell
populations located medially to the cardiac crescent
(Kelly et al., 2001;
Meilhac et al., 2004
;
Mjaatvedt et al., 2001
;
Waldo et al., 2001
). Rather,
our findings are consistent with a crucial role of Tbx1 in regions
that provide precursors destined to the OFT at a later stage, such as the
splanchnic mesoderm (Kelly et al.,
2001
; Mjaatvedt et al.,
2001
; Waldo et al.,
2001
). Timed cell-fate mapping showed that the crucial time window
for the Tbx1 role in OFT development coincides with expression of
Tbx1 in cells destined to populate the OFT myocardium. It is also
possible that Tbx1-traced cells provide a cue for neural crest
infiltration or outflow cushion development, once they have entered the heart
tube. While several genes have been shown to mark cells of the SHF lineage
[e.g. Nkx2.5 (Stanley et al.,
2002
; Waldo et al.,
2001
; Xu et al.,
2004
), Isl1 (Cai et
al., 2003
), and Mef2c
(Dodou et al., 2004
)],
Tbx1 is the only gene known to mark a subpopulation of cardiomyocyte
precursors destined predominantly to the OFT, demonstrating the presence of
regionally specified cell populations within the SHF.
Tbx1 regulates the expansion of the endoderm during pharyngeal segmentation
The embryonic pharyngeal system has a characteristic modular structure
resulting from progressive addition of new segments in a cranial-to-caudal
order. In Tbx1-/- embryos, the pharyngeal cavity is very
hypoplastic, pharyngeal pouches 2-4 are not recognizable, the first is
abnormal, the second pharyngeal arch is severely hypoplastic and the 3rd-6th
arches are not recognizable (Jerome and
Papaioannou, 2001; Kelly et
al., 2004
; Vitelli et al.,
2002
). The zebrafish Tbx1 mutant also has similar
pharyngeal defects (Piotrowski et al.,
2003
; Piotrowski and
Nusslein-Volhard, 2000
). These phenotypic observations led to
hypothesize a role of Tbx1 in pharyngeal segmentation
(Baldini, 2002
). The timed
deletion data reported here demonstrated that Tbx1 is required during
segmentation because elimination of Tbx1 while the segmentation is in
progress effectively stops it. Tbx1 timed deletion is followed by
downregulation of the proliferative activity of endodermal cells, indicating
that at least one role of Tbx1 in segmentation concerns regulation of
endodermal cell proliferation. We propose a model
(Fig. 6D) in which the
cranial-to-caudal wave of Tbx1 expression would cause a wave of
expansion of the pharyngeal endoderm that allows migrating neural
crest-derived cells to populate (and thus shape) the pharyngeal arches. This
model is also consistent with the hypothesis that the pharyngeal endoderm has
a primary role in pharyngeal segmentation
(Graham, 2001
;
Graham and Smith, 2001
).
However, because Tbx1 is also expressed in pharyngeal mesoderm and,
transiently, in the pharyngeal ectoderm, we cannot exclude a role for the gene
in these tissue during segmentation and (or) in triggering extracellular
signals required for endodermal cell proliferation.
Is downregulation of cell proliferation a general consequence of Tbx1 loss of function?
Tbx1 mutants and individuals with DGS have complex phenotypes. One
of the goals of our time-based dissection of the phenotype is to reduce
complexity so that individual abnormalities can be studied more effectively.
In addition, the availability of an inducible deletion system allows one to
test cellular phenotypes (e.g. proliferative activity) shortly after somatic
gene deletion, hence reducing the chance of possible adaptive changes that may
be effected in germline mutants. We speculate that Tbx1 may have a similar
role in different tissues. Our finding of reduced proliferation in the
pharyngeal endoderm is consistent with the finding that tissue-specific
deletion of Tbx1 in the precursors of OFT cardiomyocytes is
associated with reduced cell proliferation in the splanchnic
mesoderm/secondary heart field (Xu et al.,
2004). In addition, it has been shown that overexpression of
Tbx1 in the OFT causes expansion of cellularity in that organ
(Hu et al., 2004
).
Furthermore, the inner ear of Tbx1-/- embryos presents a
defect of otic epithelial cell expansion
(Vitelli et al., 2003
). Thus,
expansion of specific but different cell populations may be a general
mechanism of action of Tbx1. Future experiments should determine the molecular
effectors that mediate the role of Tbx1 in cell proliferation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/19/4387/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albrecht, U., Eichele, G., Helms, J. A. and Lu, H. C. (1997). Visualization of gene expression patterns by in situ hybridization. In Molecular and Cellular Methods in Developmental Toxicology (ed. G. P. Daston), pp.23 -48. New York: CRC Press.
Baldini, A. (2002). DiGeorge syndrome: the use
of model organisms to dissect complex genetics. Hum. Mol.
Genet. 11,2363
-2369.
Cai, C. L., Liang, X., Shi, Y., Chu, P. H., Pfaff, S. L., Chen, J. and Evans, S. (2003). Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877-889.[CrossRef][Medline]
Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (1996). Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206,379 -390.[CrossRef][Medline]
Dodou, E., Verzi, M. P., Anderson, J. P., Xu, S. M. and Black,
B. L. (2004). Mef2c is a direct transcriptional target of
ISL1 and GATA factors in the anterior heart field during mouse embryonic
development. Development
131,3931
-3942.
Gordon, J., Wilson, V. A., Blair, N. F., Sheridan, J., Farley, A., Wilson, L., Manley, N. R. and Blackburn, C. C. (2004). Functional evidence for a single endodermal origin for the thymic epithelium. Nat. Immunol. 5,546 -553.[CrossRef][Medline]
Graham, A. (2001). The development and evolution of the pharyngeal arches. J. Anat. 199,133 -141.[CrossRef][Medline]
Graham, A. and Smith, A. (2001). Patterning the pharyngeal arches. BioEssays 23, 54-61.[CrossRef][Medline]
Hayashi, S. and McMahon, A. P. (2002). Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244,305 -318.[CrossRef][Medline]
Hu, T., Yamagishi, H., Maeda, J., McAnally, J., Yamagishi, C.
and Srivastava, D. (2004). Tbx1 regulates fibroblast growth
factors in the anterior heart field through a reinforcing autoregulatory loop
involving forkhead transcription factors. Development
131,5491
-5502.
Jerome, L. A. and Papaioannou, V. E. (2001). DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat. Genet. 27,286 -291.[CrossRef][Medline]
Kaufman, M. H. and Bard, J. B. L. (1999). The Anatomical Basis of Mouse Development. San Diego: Academic Press.
Kelly, R. G., Brown, N. A. and Buckingham, M. E. (2001). The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev. Cell 1,435 -440.[CrossRef][Medline]
Kelly, R. G., Jerome-Majewska, L. A. and Papaioannou, V. E.
(2004). The del22q11.2 candidate gene Tbx1 regulates
branchiomeric myogenesis. Hum. Mol. Genet.
13,2829
-2840.
Kochilas, L., Merscher-Gomez, S., Lu, M. M., Potluri, V., Liao, J., Kucherlapati, R., Morrow, B. and Epstein, J. A. (2002). The role of neural crest during cardiac development in a mouse model of DiGeorge syndrome. Dev. Biol. 251,157 -166.[CrossRef][Medline]
Lindsay, E. A. and Baldini, A. (2001). Recovery
from arterial growth delay reduces penetrance of cardiovascular defects in
mice deleted for the DiGeorge syndrome region. Hum. Mol.
Genet. 10,997
-1002.
Lindsay, E. A., Botta, A., Jurecic, V., Carattini-Rivera, S., Cheah, Y.-C., Rosenblatt, H. M., Bradley, A. and Baldini, A. (1999). Congenital heart disease in mice deficient for the digeorge syndrome region. Nature 401,379 -383.[CrossRef][Medline]
Lindsay, E. A., Vitelli, F., Su, H., Morishima, M., Huynh, T., Pramparo, T., Jurecic, V., Ogunrinu, G., Sutherland, H. F., Scambler, P. J. et al. (2001). Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410,97 -101.[CrossRef][Medline]
Manley, N. R. and Blackburn, C. C. (2003). A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from? Curr. Opin. Immunol. 15,225 -232.[CrossRef][Medline]
Meilhac, S. M., Esner, M., Kelly, R. G., Nicolas, J. F. and Buckingham, M. E. (2004). The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell 6,685 -698.[CrossRef][Medline]
Merscher, S., Funke, B., Epstein, J. A., Heyer, J., Puech, A., Min Lu, M. M., Xavier, R. J., Demay, M. B., Russell, R. G., Factor, S. et al. (2001). TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104,619 -629.[CrossRef][Medline]
Mjaatvedt, C. H., Nakaoka, T., Moreno-Rodriguez, R., Norris, R. A., Kern, M. J., Eisenberg, C. A., Turner, D. and Markwald, R. R. (2001). The outflow tract of the heart is recruited from a novel heart-forming field. Dev. Biol. 238,97 -109.[CrossRef][Medline]
Moraes, F., Novoa, A., Jerome-Majewska, L. A., Papaioannou, V. E. and Mallo, M. (2005). Tbx1 is required for proper neural crest migration and to stabilize spatial patterns during middle and inner ear development. Mech. Dev. 122,199 -212.[CrossRef][Medline]
Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S. and Itoh, N. (2000). FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochem. Biophys. Res. Commun. 277,643 -649.[CrossRef][Medline]
Piotrowski, T. and Nusslein-Volhard, C. (2000). The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio). Dev. Biol. 225,339 -356.[CrossRef][Medline]
Piotrowski, T., Ahn, D. G., Schilling, T. F., Nair, S.,
Ruvinsky, I., Geisler, R., Rauch, G. J., Haffter, P., Zon, L. I., Zhou, Y. et
al. (2003). The zebrafish van gogh mutation disrupts tbx1,
which is involved in the DiGeorge deletion syndrome in humans.
Development 130,5043
-5052.
Raft, S., Nowotschin, S., Liao, J. and Morrow, B. E.
(2004). Suppression of neural fate and control of inner ear
morphogenesis by Tbx1. Development
131,1801
-1812.
Revest, J. M., Suniara, R. K., Kerr, K., Owen, J. J. and
Dickson, C. (2001). Development of the thymus requires
signaling through the fibroblast growth factor receptor R2-IIIb. J.
Immunol. 167,1954
-1961.
Robinson, S. P., Langan-Fahey, S. M., Johnson, D. A. and Jordan,
V. C. (1991). Metabolites, pharmacodynamics, and
pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer
patient. Drug Metab. Dispos.
19, 36-43.
Sadek, S. and Bell, S. C. (1996). The effects of the antihormones RU486 and tamoxifen on fetoplacental development and placental bed vascularisation in the rat: a model for intrauterine fetal growth retardation. Br. J. Obstet. Gynaecol. 103,630 -641.[Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain [letter]. Nat. Genet. 21,70 -71.[CrossRef][Medline]
Stanley, E. G., Biben, C., Elefanty, A., Barnett, L., Koentgen, F., Robb, L. and Harvey, R. P. (2002). Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5. Int. J. Dev. Biol. 46,431 -439.[Medline]
Taddei, I., Morishima, M., Huynh, T. and Lindsay, E. A.
(2001). Genetic factors are major determinants of phenotypic
variability in a mouse model of the DiGeorge/del22q11 syndromes.
Proc. Natl. Acad. Sci. USA
98,11428
-11431.
Verrou, C., Zhang, Y., Zurn, C., Schamel, W. W. and Reth, M. (1999). Comparison of the tamoxifen regulated chimeric Cre recombinases MerCreMer and CreMer. Biol. Chem. 380,1435 -1438.[CrossRef][Medline]
Vitelli, F., Morishima, M., Taddei, I., Lindsay, E. A. and
Baldini, A. (2002). Tbx1 mutation causes multiple
cardiovascular defects and disrupts neural crest and cranial nerve migratory
pathways. Hum. Mol. Genet.
11,915
-922.
Vitelli, F., Viola, A., Morishima, M., Pramparo, T., Baldini, A.
and Lindsay, E. (2003). TBX1 is required for inner ear
morphogenesis. Hum. Mol. Genet.
12,2041
-2048.
Waldo, K. L., Kumiski, D. H., Wallis, K. T., Stadt, H. A., Hutson, M. R., Platt, D. H. and Kirby, M. L. (2001). Conotruncal myocardium arises from a secondary heart field. Development 128,3179 -3188.[Medline]
Xu, H., Morishima, M., Wylie, J. N., Schwartz, R. J., Bruneau,
B. G., Lindsay, E. A. and Baldini, A. (2004). Tbx1 has a dual
role in the morphogenesis of the cardiac outflow tract.
Development 131,3217
-3227.
Yagi, H., Furutani, Y., Hamada, H., Sasaki, T., Asakawa, S., Minoshima, S., Ichida, F., Joo, K., Kimura, M., Imamura, S.-i. et al. (2003). Role of TBX1 in human del22q11.2 syndrome. Lancet 362,1366 -1373.[CrossRef][Medline]