Skip Navigation


Human Molecular Genetics Advance Access originally published online on September 25, 2006
Human Molecular Genetics 2006 15(21):3219-3228; doi:10.1093/hmg/ddl399
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
15/21/3219    most recent
ddl399v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (9)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Aggarwal, V. S.
Right arrow Articles by Morrow, B. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aggarwal, V. S.
Right arrow Articles by Morrow, B. E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dissection of Tbx1 and Fgf interactions in mouse models of 22q11DS suggests functional redundancy

Vimla S. Aggarwal1,{dagger}, Jun Liao1,{dagger}, Alexei Bondarev1, Thomas Schimmang2, Mark Lewandoski3, Joseph Locker4, Alan Shanske5, Marina Campione6 and Bernice E. Morrow1,*

1 Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, 2 Institute for Biology and Molecular Genetics, Superior Research Council and University of Valladolid, E-47003 Valladolid, Spain, 3 Genetics of Vertebrate Development Section, National Cancer Institute, Frederick Cancer Research and Development Center, PO Box B, Building 539, Frederick, MD 21702, USA, 4 Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, 5 Center for Craniofacial Disorders, CHAM, 1345 Bainbridge Avenue, Bronx, NY 10467, USA and 6 CNR-Institute of Neurosciences, Department of Biomedical Sciences, University of Padova, Padova, Italy

* To whom correspondence should be addressed. Tel: +1 7184304274; Fax: +1 7184308778; Email: morrow{at}aecom.yu.edu

Received August 2, 2006; Accepted September 18, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 22q11 deletion syndrome (22q11DS) is characterized by abnormal development of the pharyngeal apparatus. Mouse genetic studies have identified Tbx1 as a key gene in the etiology of the syndrome, in part, via interaction with the fibroblast growth factor (Fgf) genes. Three murine Fgfs, Fgf3, Fgf8 and Fgf10 are coexpressed in different combinations with Tbx1. They are all strongly downregulated in Tbx1–/– embryos, implicating epistatic interactions. Supporting this, Tbx1 and Fgf8 have been shown to genetically interact in the development of the fourth pharyngeal arch artery (PAA) and Fgf10 was identified to be a direct downstream target of Tbx1. To dissect the epistatic relationships of these genes during embryonic development and the molecular pathogenesis of the Tbx1 mutant phenotype, we generated Fgf10+/–;Tbx1+/– and Fgf3–/–;Tbx1+/– mice. Despite strong hypotheses that Fgf10 is the key gene downstream of Tbx1 in the development of the anterior heart field, we do not find evidence for genetic interaction between Tbx1 and Fgf10. Also, the Fgf3–/–;Tbx1+/– mutant mice do not show an additive phenotype. Furthermore, more severe defects do not occur in Fgf8+/–;Tbx1+/– mutants by crossing in the Fgf3 null allele. There is a possible additive effect only in PAA remodeling in the Fgf10+/–;Tbx1+/–;Fgf8+/– embryos. Our findings underscore the importance of potential functional redundancy with additional Fgfs in the development of the pharyngeal apparatus and cardiovascular system via Tbx1. This redundancy should be considered when looking at individual FGF genes as modifiers of 22q11DS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 22q11.2 deletion syndrome (22q11DS) [velo-cardio- facial syndrome (1) and DiGeorge syndrome (2) MIM 192430 [OMIM] / 188400], with an incidence of 1:4000 (3) is the most common microdeletion disorder in humans. It is associated with 1.5–3 Mb hemizygous 22q11.2 deletions and is characterized by multiple developmental anomalies, including cardiovascular [outflow tract (OFT) and aortic arch], ear, craniofacial (cleft palate, velo-pharyngeal insufficiency), thymic and parathyroid defects (4).

Mouse genetic studies (5–7) and mutational analysis in patients (8) have identified Tbx1, a gene on the deleted segment, as a key gene in the etiology of the syndrome. Tbx1 belongs to the T-box family of transcription factors, members of which have been shown to be important regulators of a number of tissues in development (8). Tbx1 null mutant mice show perinatal lethality and have defects in most of the structures affected in 22q11DS patients, including cardiovascular and craniofacial anomalies (7).

The pharyngeal apparatus is a transient vertebrate-specific embryonic structure, located lateral to the developing head, and is derived from all the three germ layers and the neural crest (9). The structures primarily affected in 22q11DS are derived from the pharyngeal apparatus, suggesting that abnormal development of this structure causes most features of this disease (10). Tbx1 is expressed in the pharyngeal pouch endoderm (PE), core and splanchnic mesoderm and transiently in the ectoderm (11–13).

The fibroblast growth factor (FGF) family of signaling molecules plays important roles in embryonic development by regulating cell proliferation, differentiation and migration (14). The Fgfs comprise a 22-member gene family encoding secreted proteins that signal through a family of tyrosine kinase receptors (FGFRs) which are encoded by only four genes. A variety of Fgf ligands [Fgf3 (15), Fgf4 (16), Fgf8 (17), Fgf10 (18), Fgf15 (19) and Fgf16 (20)] and receptors [Fgfr1 (20), Fgfr2 (20,21) and Fgfr3 (21)] are expressed in the developing pharyngeal apparatus.

In diverse organisms, Fgf signaling has been shown to genetically interact with the function of a number of T-box genes in development. Xenopus Brachyury (Xbra), a member of the T-box gene family, has been shown to directly activate expression of eFGF (22). Also, Xbra and eFGF are components of an indirect autoregulatory loop in which each maintains expression of the other (22,23). The gene, fgf8, genetically interacts with ntl and spt, two zebrafish T-box transcription factors in the development of the posterior mesoderm (24). Zebrafish tbx5 regulates fgf10, which in turn maintains tbx5 expression during limb outgrowth (25). Also, Tbx5 is upstream of Fgf signaling in limb bud development in mouse (26). Importantly, Fgf8 and Tbx1 genetically interact in the development of the fourth pharyngeal arch artery (PAA) (17).

The core mesoderm of the pharyngeal arches, derived from the paraxial mesoderm, forms the craniofacial muscles (27) and participates in the development of the PAAs (9). A subset of the pharyngeal mesoderm cells, termed the anterior heart field (AHF), has been shown to be important for the development of the cardiac OFT and the right ventricle (18,28–30). Tbx1 and Fgf10 are coexpressed in the AHF (17). Also, Fgf10 expression is lost in the AHF in the Tbx1–/– embryos (17). This suggests that Fgf10 may act downstream of Tbx1. In fact, Fgf10 has been shown to be a direct downstream target of Tbx1 in an in vitro cell culture system (12).

The endoderm of the pharyngeal apparatus gives rise to the thymus, parathyroid and part of the thyroid; organs which are frequently affected in 22q11DS. Tbx1 and another Fgf, Fgf3, are coexpressed, in the pharyngeal PE (15,17,31), and Fgf3 is downregulated in the PE of the Tbx1–/– embryos (32,33).

In view of these findings, we hypothesized that dosage reduction of either Fgf10 or Fgf3 would enhance the Tbx1 haploinsufficiency phenotype. We therefore generated Fgf10+/–;Tbx1+/– and Fgf3–/–;Tbx1+/– mice. Surprisingly, Fgf10+/–;Tbx1+/– and the Fgf3–/–; Tbx1+/– mutant mice do not show any additive phenotype.

Fgf8, besides Fgf10, is expressed in the AHF (18). It may thus serve redundant functions with Fgf10 in this tissue. Fgf8, along with Fgf3, is expressed in the PE (17). As only about half of the Fgf8+/–;Tbx1+/– embryos present with aortic arch artery remodeling defects (17), we hypothesized that Fgf3 and Fgf8 may cooperate in the development of the PE. We thus generated Fgf10+/–;Tbx1+/–;Fgf8+/– and Fgf3–/–; Tbx1+/–; Fgf8+/– mice. We show that more severe defects do not occur in Fgf8+/–;Tbx1+/– mutants by crossing in the Fgf3 null allele. There is a possible additive effect only in PAA remodeling in the Fgf10+/–;Tbx1+/–;Fgf8+/– embryos. Our findings underscore the importance of potential functional redundancy with additional Fgfs in the development of the pharyngeal apparatus and cardiovascular system via Tbx1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Tbx1, Fgf10, Fgf3 and Fgf8
To compare the patterns of expression of Tbx1, Fgf10, Fgf3 and Fgf8 in the developing mouse embryo, we performed whole-mount RNA in situ hybridization studies. At E9.5, Tbx1 is expressed in the pharyngeal PE (Fig. 1B and I), core mesoderm of the pharyngeal arches and posterior otic vesicle (Fig. 1B). The endodermal expression domain of Tbx1 (Fig. 1I) overlaps with that of Fgf3 (Fig. 1C and H) and Fgf8 (Fig. 1D and J). Fgf10 is coexpressed with Tbx1 in the core mesoderm of the pharyngeal arches (Fig. 1A). Interestingly, Tbx1 (Fig. 1F), Fgf10 (Fig. 1E) and Fgf8 (Fig. 1G) are all coexpressed in the AHF cells, a subset of the pharyngeal mesoderm, which are thought to migrate and contribute to the wall of the cardiac OFT and the right ventricle (18,28,29).


Figure 3991
View larger version (109K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Whole-mount RNA in situ hybridization showing expression patterns of Fgf10 (A), Tbx1 (B), Fgf3 (C) and Fgf8 (D) at E9.5. *Core mesoderm of pharyngeal arches; ov, otic vesicle; pp, pharyngeal pouch; t, tail. Arrows (in A, B and D) point to the AHF. Sagittal sections of an Fgf10 (E), Tbx1 (F) and Fgf8 (G) in situ hybridization of E9.5 WT embryo show that expression of Fgf10, Tbx1 and Fgf8 overlaps in the AHF (arrows in E, F and G). Sagittal sections of an Fgf3 (H), Tbx1 (I) and Fgf8 (J) in situ hybridization of E9.5 WT embryo. Note that the three genes are coexpressed in the pharyngeal PE (arrows in H, I and J).

 
Tbx1 mutant mice have been described previously and found to model many aspects of 22q11DS (5). To determine whether the expression of these Fgfs is altered in Tbx1 mutants, we performed in situ hybridization of Tbx1–/– embryos at E9.5. In the Tbx1–/– embryos, Fgf3 (Fig. 2D) (32,33) and Fgf8 (17) expression in the pharyngeal endoderm (PE) is lost. The loss of expression is specific to these genes and not due to the lack of endodermal cells, as other endodermal markers such as Pax1 and Nkx2.5 are expressed in the endoderm of the null mutants (data not shown) (17). Ectodermal expression of Fgf10 is intact in the first arch of the Tbx1 homozygous null embryos (Fig. 2B). Diffuse Fgf10 expression is seen in the mesoderm of the first arch but is missing in the caudal arches of the Tbx1 homozygous null embryos (Fig. 2B). Also, the AHF domain of expression of Fgf10 (Fig. 2B) and Fgf8 (34) is not detected in these mutants, whereas expression of Nkx2.5 and Isl1, also present in the AHF, is however conserved (data not shown) (17). These results suggest that the Tbx1 function in OFT and pharyngeal pouch development, revealed by severe OFT and endoderm defects in the Tbx1–/– mutants, may be mediated by Fgf signaling in the AHF and the endoderm.


Figure 3992
View larger version (86K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Whole-mount RNA in situ hybridization reveals loss of Fgf10 expression in the AHF (arrows in A and B) in Tbx1–/– (B) when compared with WT (A) embryos at E9.5. Note the diffuse pattern of expression of Fgf10 in the core mesoderm of the first pharyngeal arch of the Tbx1–/– (B) when compared with WT embryos. cm, core mesoderm of first pharyngeal arch. Also, Fgf3 expression in the pharyngeal PE (arrows in C and D) is lost in Tbx1–/– (D) when compared with WT (C) embryos at E9.5.

 
Generation of Fgf10+/–;Tbx1+/– mice
Our in situ hybridization results show that the expression of Fgf10, which overlaps with that of Tbx1, is diminished in the Tbx1 null mutant mice in the core mesoderm and the AHF. Tbx1–/– mutants have missing and abnormal craniofacial muscles (35,36) and have a single cardiac OFT (5,7). Fgf10 has been shown to be a direct downstream target of Tbx1 in an in vitro cell culture system (12). Also, it has been proposed that Fgf10 may be the mediator of the cell non-autonomous function of Tbx1 in regulating cell contribution to the OFT (12). In view of these very important findings, we hypothesized that dosage reduction of Fgf10 would enhance the Tbx1 haploinsufficiency phenotype. We thus crossed the Tbx1+/– mice (5) with the Fgf10+/– mice (37) to generate Fgf10+/–;Tbx1+/– mice.

Genotyping of the litters from these crosses at P10 shows Mendelian distribution of 25% of Fgf10+/–, Tbx1+/–, wild-type (WT) and double heterozygous mice (Table 1). These proportions are in accordance with genes mapping to different chromosomes following an independent allelic segregation. Also, the presence of 25% double heterozygous mice demonstrates that double heterozygosity does not cause embryonic or perinatal lethality. External analysis of newborn double heterozygous pups did not reveal any significant abnormality. There was no difference in size observed between the double heterozygous and the WT pups, thus showing the absence of major growth defects.


View this table:
[in this window]
[in a new window]

  		Table 1. Mendelian ratio analysis at P10 of litters from Fgf10+/–xTbx1+/– and Fgf3+/–; Tbx1+/–xFgf3+/–; Tbx1 +/+ matings

 
Craniofacial malformations
The mesodermal core of the first pharyngeal arch gives rise to the muscles of mastication (38). Tbx1 (Fig. 1B) and Fgf10 (Fig. 1A) are coexpressed in this region at E9.5 and Fgf10 is more diffusely expressed in the first arch core of the Tbx1 null mutants (Fig. 2B) compared with WT embryos (Fig. 2A). The muscles of mastication are largely missing in the Tbx1–/– embryos (35,36). Also, an Fgf10 enhancer-trap nlacz transgene fails to be expressed in the proximal core region of the first arch in the same mouse model (36). We were interested in evaluating whether Tbx1 and Fgf10 co-operate in the development of the muscles of the first arch. Since the Fgf10+/–;Tbx1+/– double heterozygous mice survived in normal Mendelian ratios, we suspected that the muscles of mastication would be normal. To more carefully examine these muscles, we performed a detailed histological analysis of these muscles (temporalis, pterygoids and masseter) in the Fgf10+/–;Tbx1+/– double heterozygous embryos (n=5). All the muscles were present and appeared anatomically normal (Fig. 3A–C). It was found that most 22q11DS patients have palatal malformations and 10% have an overt cleft palate (39,40). Both the Tbx1 and the Fgf10 null mutants have a cleft palate (35,41). Thus, we analyzed the double heterozygous mice for cleft palate. The palate was normal in all the embryos examined (n=5) (Fig. 3D). This indicates that if any genetic interactions between Tbx1 and Fgf10 exist in craniofacial muscle or palate development, they are not disrupted by reducing the gene dosage of each gene by half. It is possible that further reduction in dosage will be necessary to elicit a phenotype.


Figure 3993
View larger version (108K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. (A–D) Normal craniofacial development in Fgf10+/–; Tbx1+/– mutants. Transverse sections of an Fgf10+/–; Tbx1+/– mutant at E17.5. Note that the muscles of mastication [massetter (A), pterygoids (B) and temporalis (C)] are normal in position and size. m, massetter; asterisk in (B) indicates the pterygoid muscles; t, temporalis muscle. (D) Palate is normal in the Fgf10+/–; Tbx1+/– embryos. (E and F) Transverse sections through the neck of an Fgf10+/–; Tbx1+/– mutant showing that the double heterozygous embryos do not have thyroid (E) and thymus (F) defects. Asterisk in (E) marks the lobes of the thyroid gland; {Delta} in F indicates the lobes of the thymus.

 
Thyroid and thymus anomalies
The phenotypic spectrum of 22q11DS may occasionally include hypothyroidism (40). The thyroid gland is missing in the Fgf10 null mutants (42) and the Tbx1 null mutants have a single lobed thyroid gland (35). Evaluation of the Fgf10+/–;Tbx1+/– mice did not reveal any thyroid anomalies (n=5) (Fig. 3E). Immune deficiencies are seen in a major subset of 22q11DS patients (40). This has been associated with hypoplasia or aplasia of the thymus gland. In fact, thymus gland aplasia was one of the hallmark features described in the first report of the syndrome (2). Tbx1–/– mice have thymus gland aplasia (7) and the Fgf10–/– mice have a hypoplastic thymus (42), raising the question if the two genes interact in thymic development. Analysis of the double heterozygous mice revealed a normally located thymus, which was appropriate in size (n=5) (Fig. 3F). This demonstrates that reduction of dosage of both Tbx1 and Fgf10 by half does not disrupt development of the thyroid and the thymus. As stated earlier, it is possible that further reduction in dosage may be necessary to observe a developmental defect.

Analysis of the cardiovascular system in Fgf10+/–;Tbx1+/– embryos
Cardiovascular defects, arising from the abnormal development of the OFT and the PAAs, occur in ~60–75% of 22q11DS patients (39,40). The AHF is a source of cells to lengthen the outflow end of the developing heart (18,28,29). Elongation of the OFT is necessary for wedging, which is responsible for the correct alignment of the aorta and the pulmonary trunk with the left and the right ventricles, respectively. Failure of this process leads to congenital heart malformations such as tetralogy of Fallot (TOF) and double outlet right ventricle (DORV) (43).

The distal OFT is hypoplastic in the Tbx1 homozygous mutant embryos (31), suggesting an important role of Tbx1 in regulating addition of cells from the AHF to the developing heart (44). Given the loss of AHF expression of Fgf10 in the Tbx1–/– mice, we postulated that this role of Tbx1 may be mediated by Fgf10. We thus investigated whether OFT development was impaired in the Fgf10+/–;Tbx1+/– mice.

We thus analyzed the phenotype of the Fgf10+/–;Tbx1+/– embryos at E17.5 (n=7) by standard histological techniques. All the embryos examined showed normal morphology of the heart and correct alignment of the aorta and the pulmonary trunk with the ventricles (Fig. 4A and B). The double heterozygous embryos did not show any alignment anomalies such as TOF or DORV. We additionally analyzed the double heterozygous embryos at E11.5 for OFT hypoplasia. Overall, the morphology of the heart chambers and the OFT region was normal in the Fgf10+/–: Tbx1+/– embryos (n=3) (Fig. 4C and D).


Figure 3994
View larger version (123K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Fgf10+/–; Tbx1+/– embryos do not show any cardiovascular anomalies. (A and B) Transverse sections of an Fgf10+/–; Tbx1+/– embryo at E17.5 show normal ventricular morphology and alignment of the aorta with the left ventricle and of the pulmonary trunk with the right ventricle. (C and D) Transverse sections through the heart of WT (C) and Fgf10+/–; Tbx1+/– (D) embryos at E11.5. The heart has normal morphology and the OFT (arrows in C and D) is normal in size and not hypoplastic in Fgf10+/–; Tbx1+/– (D) mutants with respect to the WT (C) embryos. (E–G) Transverse sections of an Fgf10+/–; Tbx1+/– mutant at E17.5 showing the normal progression (slides shown from caudal to rostral) of the head and neck vessels. ra, right atrium; a, aorta; rv, right ventricle; lv, left ventricle; la, left atrium; p, pulmonary trunk; aa, aotic arch; rbt, right brachiocephalic trunk; lcc, left common carotid; lsa, left subclavian artery.

 
The core mesodermal cells of the pharyngeal arches contribute to the development of the PAAs (9); Tbx1 and Fgf10 are coexpressed in the mesoderm. We thus examined the double heterozygous embryos for remodeling defects of the PAAs. None of the seven embryos examined had any aortic arch artery anomalies such as interrupted aortic arch (IAA), right aortic arch (RAA) or aberrant origin of the right subclavian artery (ARSA) (Fig. 4E–G).

We thus did not observe any defects in the cardiovascular system of the Fgf10+/–;Tbx1+/– embryos when compared with the WT (n=6), Tbx1+/– (n=4) and the Fgf10+/– embryos (n=5).

Fgf8 is coexpressed with Fgf10 in the AHF
Even though in situ expression analysis (Fig. 2B) and in vitro cell culture data (12) suggested that Fgf10 may be downstream of Tbx1 in the AHF; surprisingly, the Fgf10+/–;Tbx1+/– mice did not have any cardiovascular anomalies. This led us to hypothesize that other Fgfs in the AHF may compensate for the reduction of Fgf10 gene dosage. Also, the Fgf10 null mutants do not have any OFT alignment defects (37,45,46), suggesting that it is not the only Fgf ligand involved in the development of this tissue.

In situ hybridization of WT embryos shows that Fgf8 (Fig. 1G) is coexpressed with Fgf10 (Fig. 1E) in the AHF at E9.5 (18). Fgf8 hypomorphs have cardiovascular anomalies including OFT septation, rotation and alignment defects (47,48), reminiscent of the defects seen in 22q11DS. Cre-mediated excision of an Fgf8 conditional allele in the Tbx1 expression domain gives rise to cardiovascular defects involving the OFT and proximal great vessels (49). Inactivation of Fgf8 in the AHF leads to OFT rotation/alignment defects (50), whereas ablation of Fgf8 in the pharyngeal ectoderm or ectoderm and endoderm causes aortic arch interruptions and not OFT defects (51). These results suggest that Fgf8 is required in the AHF for normal development of the OFT. We thus speculated that Fgf8 may compensate for the loss of Fgf10 in the AHF. Intact Fgf8–Tbx1 interaction in the AHF may be responsible for normal cardiovascular development in the Fgf10+/–;Tbx1+/– mice. We checked what could be the consequence of applying a dosage reduction of Fgf8 in the Fgf10+/–;Tbx1+/– mice with regard to the development of the OFT. We thus crossed the Fgf10+/–;Tbx1+/– mice with the Fgf8+/– mice (52) to generate Fgf10+/–;Tbx1+/–;Fgf8+/– mice.

Phenotypic analysis of Fgf10+/–;Tbx1+/–;Fgf8+/– mice
In order to evaluate the triple heterozygous embryos for cardiovascular defects, we performed a histological analysis at E17.5 (n=6). Analysis of the triple heterozygous embryos did not reveal any cardiac defects. The aorta and the pulmonary trunk were correctly aligned with the left and right ventricles, respectively. Alignment defects such as TOF or DORV were not seen in these mutants. This supports the idea that if any genetic interactions between Tbx1, Fgf8 and Fgf10 exist, they are not disrupted by reducing the gene dosage of each gene by half.

Tbx1 is required for the fourth PAA formation and growth. Hypoplastic fourth PAA is seen in 100% of the Tbx1+/– embryos at E10.5 (17). At term, depending on the genetic background, 3–27% of the mutants have fourth PAA-derived defects such as interrupted aortic arch-type B (IAA- B), RAA and ARSA (17,35). None of the Tbx1+/– mutants, in this study (in a C57BL/6xFVBxCD1 background) (n=5), showed aortic arch artery remodeling defects. Vascular anomalies such as ARSA (34%), RAA (17%) and IAA-B (17%) were seen in the triple heterozygous mutants (n=6) (Table 2) (Fig. 5B and C). The right subclavian artery arose from the aortic arch or from the descending aorta instead of the brachiocephalic trunk, in the mutants that had ARSA. We found that 17% of the Fgf8+/–;Tbx1+/– embryos had ARSA (n=6) (Table 2). Aortic arch remodeling defects described earlier were not seen in Fgf10+/–;Fgf8+/– embryos (n=5) as well as Fgf10+/–;Tbx1+/– embryos (n=7). This suggests that there is a possible genetic interaction between Tbx1, Fgf8 and Fgf10 in the development of the fourth PAA.


Figure 3995
View larger version (93K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. (A–F) Transverse sections of a WT (A and D), Fgf10+/–;Tbx1+/–;Fgf8+/– (B and C) and Fgf3–/–; Tbx1+/–; Fgf8+/– (E and F) embryo at E17.5. Note the RAA (B) and retro-esophageal right subclavian artery (arrow in C) in the Fgf10+/–; Tbx1+/–; Fgf8+/– mutant. aa, aortic arch. Note the hypoplastic thymus ({Delta} in E and F) in the Fgf3–/–; Tbx1+/–; Fgf8+/– mutant (E). Retro-esophageal right subclavian artery (arrow in F) and IAA-B (missing segment marked by an * in F) are also seen in the Fgf3–/–; Tbx1+/–; Fgf8+/– embryo (F).

 


View this table:
[in this window]
[in a new window]

  		Table 2. Defects in E17.5 embryos

 
Generation of Fgf3–/–;Tbx1+/– mice
Tbx1 (Fig. 1I) and Fgf3 (Fig. 1H) are coexpressed in the PE at E9.5. Also, Fgf3 (Fig. 2D) (32,33) and Fgf8 (17) expression are lost in the PE of the Tbx1–/– embryos. We hypothesized that Fgf3, besides Fgf8 (17), is another signaling molecule downstream of Tbx1 in the PE. We speculated that loss of Fgf3 would enhance the Tbx1 haploinsufficiency phenotype. To test this hypothesis, we crossed the Tbx1+/– mice (5) with the Fgf3–/– mice (53); in F1 generation, we then crossed Tbx1+/–;Fgf3+/– mice with Tbx1+/+; Fgf3+/– mice to obtain Fgf3–/–;Tbx1+/– mice. Genotyping of the F2 animals at P10 shows that mice of most of the genotypes are present in normal Mendelian ratios. Those not present in normal Mendelian ratios were the Fgf3–/– mice and Fgf3–/–;Tbx1+/– mice, which constituted 8.4 and 3.9% respectively, instead of the expected 12.5%, of the 178 pups genotyped (Table 1). Reduced viability of Fgf3–/– newborns has been reported before but defects that could result in lethality have not been described (54).

Phenotypic analysis of Fgf3–/– mice
Because of their decreased viability, Fgf3–/– embryos at E17.5 were histologically analyzed for possible developmental defects. After detailed histological analysis, it has not been possible to identify a specific cause of neonatal death for these mutants. Fgf3–/– embryos are present in the normal Mendelian ratios at E17.5 (data not shown), demonstrating that they survive embryogenesis. However, Fgf3–/– animals often appeared immature compared with their littermates and this may have compromised their survival. Interestingly, two of the three (67%) Fgf3–/– embryos examined at E17.5 had hypoplastic thymus glands (Fig. 6B) (Table 2), but this cannot account for neonatal lethality.


Figure 3996
View larger version (52K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. (A–C) Transverse sections of a WT (A), Fgf3–/– (B) and Fgf3–/–; Tbx1+/– (C) mutant at E17.5 through the thymus gland. Note the hypoplastic thymus ({Delta} in A–C) in the Fgf3–/– (B) and Fgf3–/–; Tbx1+/– (C) compared with the WT (A). Also, note the retro-esophageal right subclavian artey (arrow in C) in the Fgf3–/–; Tbx1+/– mutant.

 
Phenotypic analysis of Fgf3–/–;Tbx1+/– mice
Most of the physical malformations in 22q11DS arise from abnormal development of the pharyngeal apparatus. Fgf3 is lost in the PE of the Tbx1 null mutants (Fig. 2D). We thus analyzed the Fgf3–/–;Tbx1+/– embryos at E17.5 for defects arising from the pharyngeal apparatus. Histological analysis (n=10) revealed that seven embryos (70%) had hypoplastic thymus and two embryos (20%) had ARSA (Fig. 6C). About 10–27% (17,35) of the Tbx1+/– mice have abnormalities in the patterning of the aortic arch arteries but none of them have hypoplastic thymus (35). A total of 67% of the Fgf3–/– embryos have hypoplastic thymus (Table 2). We did not observe PAA or thymic defects in the Fgf3+/–;Tbx1+/– mice. These results suggest that Fgf3–/–;Tbx1+/– animals do not present with higher penetrance of thymus or PAA remodeling defects when compared with Tbx1+/– or Fgf3–/– animals.

Fgf8 is coexpressed with Fgf3 in the endoderm
Even though the in situ expression analysis (Fig. 2D) suggested that Fgf3 may be downstream of Tbx1 in the PE; surprisingly, the Fgf3–/–;Tbx1+/– mice did not have increased penetrance of defects seen either in Tbx1+/– or Fgf3–/– mice. This led us to speculate that other Fgfs expressed in the PE may compensate for the loss of Fgf3.

In situ hybridization of WT embryos at E9.5 shows that Fgf8 (Fig. 1J) is coexpressed with Fgf3 (Fig. 1H) in the PE. Fgf8 hypomorphs have remodeling defects of the PAAs including IAA-B and RAA (47,48), reminiscent of the defects seen in 22q11DS. Thymic and parathyroid aplasia and hypoplasia was also in the Fgf8 hypomorphs (47). Also, Tbx1 and Fgf8 genetically interact in the development of the fourth PAA (17). These results suggest that Fgf8 is required for normal development of the derivatives of the PE. We thus speculated that Fgf8 may compensate for the loss of Fgf3 in the PE. To test this possibility, we generated Fgf3–/–;Tbx1+/–;Fgf8+/– mice by crossing Fgf3–/–;Tbx1+/– mice with Fgf3+/–;Fgf8+/– mice.

Phenotypic analysis of Fgf3–/–;Tbx1+/–;Fgf8+/– mice
We assessed the triple mutant embryos (Fgf3–/–;Tbx1+/–;Fgf8+/–) at E17.5 by the detailed histological analysis (n=4). Hypoplasia of the thymus was seen in three (75%) mutant embryos (Fig. 5E). Aortic arch remodeling defects such as ARSA and IAA-B were also observed (Fig. 5F) (Table 2) but were not increased in penetrance when compared with the Fgf8+/–;Tbx1+/– embryos (17). These data suggest that other Fgfs may interact with Tbx1 in the development of the PE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Genetic interaction between Tbx1 and Fgfs in the AHF
The AHF is a recently described population of pharyngeal mesodermal cells which forms the OFT and the right ventricle of the heart (18,28,29). The OFT is affected in one-third of the patients with congenital heart defects (55). Thus, it is important to understand the genetics and signals regulating the development of the OFT. 22q11DS is one of the most common genetic causes of OFT defects. Cells of the murine AHF express Tbx1 (the key gene implicated in 22q11DS) and Fgf10. Decreased contribution of the AHF cells to the OFT myocardium is seen in Tbx1 homozygous null mutant embryos (12). Also, targeted deletion of Tbx1 in the AHF causes a proliferation defect, which could explain the hypoplastic OFT seen in the null mice (12).

In situ hybridization analysis of the Tbx1 homozygous null mutant embryos, which model 22q11DS, shows downregulation of Fgf10 in the AHF. Also, it has been shown using an in vitro cell culture system that Tbx1 can bind to the Fgf10 promoter and activate a luciferase reporter (12). These results provided a compelling reason to test for an in vivo interaction between Tbx1 and Fgf10. Our results show that the Fgf10+/–;Tbx1+/– double heterozygous mice do not have any cardiac defects and do not appear to interact genetically, on their own.

A number of Fgfs are expressed in several specific tissues during development but seem to have no functional significance in the same. Even though Fgf2 is expressed by endothelial cells and is angiogenic, it does not appear to be essential for the development of the vascular system (56). Fgf3 is expressed in a variety of tissues during development, such as extraembryonic tissue, parietal endoderm, mesoderm in the primitive streak, hindbrain rhombomeres and the pharyngeal pouches. However, Fgf3 mice are viable as homozygotes and display a fully penetrant phenotype only with respect to the development of the tail (53,54). Similarly, although Fgf10 transcripts are present in many diverse tissues during development, including the AHF, Fgf10 null mutants do not have any OFT defects (37,45,46). Loss of Fgf10 causes defects at sites where it cannot be compensated by other Fgf genes. These developmental defects provide evidence that Fgf ligands can functionally compensate for the absence of Fgf normally expressed in the same tissue.

As Fgf8 has a similar expression pattern to Fgf10 in the AHF, we hypothesized that the two may act redundantly in the development of the OFT. We thus generated Fgf10+/–;Tbx1+/–;Fgf8+/– triple heterozygous mice. Surprisingly, these embryos do not have any cardiac defects. We believe that this could be due to the following reasons.

AHF development occurs normally in Fgf8 null heterozygotes (Fgf8+/–) but is disrupted at lower levels of Fgf8 that occur in Fgf8 hypomorphs (47,48); tissue-specific deletion of Fgf8 in the Tbx1 expressing domain or in the AHF (50) results in cardiac OFT defects (49), revealing the extreme sensitivity of the AHF to Fgf8 levels. Thus, it is possible that there is sufficient Fgf8 mRNA and protein in the Fgf10+/–;Tbx1+/–;Fgf8+/– triple heterozygous mice for normal development of the OFT.

Dosage sensitivity to Fgf8 is seen in the development of various organs including the salivary gland (57), cardiovascular system (47,48) and the telencephalon (58). These data support the hypothesis that the correct levels of Fgf8 are needed for normal development of numerous organs.

In addition to Fgf8 and Fgf10, Fgf15 is expressed in the murine AHF (19). Fgf15 homozygous null mutant embryos display cardiovascular anomalies primarily affecting the OFT, including alignment defects of the great vessels, overriding aorta and DORV (19). Fgf15+/–;Tbx1+/– compound heterozygous mice show no significant increase in the penetrance or severity of cardiovascular anomalies relative to either Tbx1+/– or Fgf15+/– mice (19). Consistent with these data, it is tempting to speculate that Fgf15 may also be redundant with Fgf8 and Fgf10 in the AHF.

Further evidence for our hypothesis comes from the analysis of heart defects in the Fgfr2-IIIb mutant mice (46). Fgfr2-IIIb is the major receptor for Fgf10. Most of the Fgfr2- IIIb mutant mice have OFT alignment anomalies such as overriding aorta and DORV (46). However, formation and alignment of the OFT are normal in the Fgf10 mutant mice. Thus, other Fgfs which act via Fgfr2-IIIb (Fgf3, Fgf7 and Fgf15) could be redundant with Fgf10 in the AHF.

An alternative explanation for our results is that Tbx1 and Fgfs do not interact in the AHF. This hypothesis is supported by the fact that the Fgf8+/–;Tbx1+/– (17), Fgf15+/–; Tbx1+/– (19), Fgf10+/–;Tbx1+/– and the Fgf10+/–;Tbx1+/–;Fgf8+/– (this report) embryos do not have AHF-related cardiovascular defects. Also, by driving Fgf8 expression in Tbx1 positive cells, it has been suggested that the function of Tbx1 in the AHF development is not mediated by Fgf8 (59). Conditional deletion of Fgfrs in the AHF in a Tbx1 mutant background will conclusively reveal genetic interaction between Tbx1 and the Fgf signaling pathway in the AHF.

Genetic interaction between Tbx1 and Fgfs in the pharyngeal PE
Defects in the thymus and parathyroid contribute to the morbidity of 22q11DS in the form of immunodeficiency and hypocalcemia, respectively (40). These glands are derivatives of the PE. Inactivation of Tbx1 in the PE results in 22q11DS malformations (33). However, the genetic pathways and signaling molecules downstream of Tbx1 in the PE are not known.

The fgf3 gene has been shown to play a role in the zebrafish pharyngeal arch development (60,61). Fgf3 null mutant mice have been generated and defects in the development of the tail and inner ear have been described in these mutants (53,54). We show that 67% of the Fgf3 null mutants have a hypoplastic thymus. This is the first report to our knowledge describing PE-derived defects in these mutants.

Our results show that the PE domain of expression of Fgf3 is missing in the Tbx1 null mutants at E9.5. We had speculated that dosage reduction of Fgf3 would enhance the Tbx1 haploinsufficiency phenotype. Unexpectedly, the Fgf3–/–;Tbx1+/– mutant mice show no significant increase in the penetrance or severity of PE-derived defects relative to the Tbx1+/– or the Fgf3–/– mice alone.

Since Fgf8 is coexpressed with Fgf3 in the PE, we hypothesized that Fgf8 compensates for the lack of Fgf3 in the pharyngeal pouch. We thus generated Fgf3–/–, Tbx1+/–;Fgf8+/– mice. These mice too do not have increased penetrance or severity of either thymic or aortic arch artery remodeling defects when compared with Fgf3–/–, Tbx1+/– (5) or Fgf8+/–;Tbx1+/– (17) mice.

Fgf4 (16) and Fgf16 (20) are also expressed in the PE. A role for Fgf4 in the PE has yet to be established as the Fgf4 mutants are early embryonic lethal (62). The function of Fgf16 in the PE is yet to be described. It is thus possible that Fgf4 and/ or Fgf16 is functionally redundant with Fgf3 and Fgf8 in the PE.

A high degree of redundancy in Fgf signaling has been established during development. Simultaneous depletion of both FGF3 and FGF8 by injection of fgf3 and fgf8 morpholinos into WT zebrafish embryos or injection of fgf3 morpholinos into ace (Fgf8) mutants blocks otic vesicle formation in most treated embryos, indicating that these two FGFs have redundant roles in zebrafish otic placode induction (63–65). Mice double mutant for Fgf3 and Fgf10 have otic vesicles that are severely reduced in size, showing redundant roles of these Fgfs, acting in combination as neural signals for otic vesicle formation (53). In addition, fgf8 and fgf3 have redundant essential functions in pharyngeal cartilage development in zebrafish (66).

On the basis of this established functional redundancy in Fgf signaling in development, we propose that a number of Fgfs could be downstream of Tbx1 in the development of both the AHF and the PE. Conditional deletion of the AHF or endoderm-expressed Fgfs in the Tbx1 null background will reveal whether Fgf signaling is downstream of Tbx1 in the development of these structures.

Overall, our findings underscore the importance of functional redundancy of Fgfs in the development of the pharyngeal apparatus and the cardiovascular system via Tbx1. This redundancy should be considered when looking at individual FGFs as modifiers of 22q11DS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mouse strains and genotyping
Tbx1+/– (5), Fgf10+/– (37), Fgf8+/– (52) and Fgf3–/– (53) mice have been previously described. Genotyping was performed with the following PCR primer pairs: Tbx1 (35), Fgf3 (53) and Fgf8 (52) as previously described; Fgf10 mutant allele (Fgf10-F, 5'-CTT CCA GTA TGT TCC TTC TG-3'; Fgf10-R, 5'-TGC TGT CCA TCT GCA CGA GA 3-').

Histology
Mouse embryos were isolated in PBS and fixed in 10% neutral-buffered formalin (Sigma) overnight. Following fixation, the embryos were dehydrated through a graded ethanol series and then embedded in paraffin and sectioned at 7–10 µm. All sections were stained with hematoxylin and eosin using standard protocols. Serial sections were examined and representative sections are shown here.

Whole-mount in situ hybridization
Digoxigenin-labeled complementary RNA probes to mouse Tbx1 (13), Fgf8 (67), Fgf3 (32) and Fgf10 (41) were prepared by the standard methods. Whole-mount in situ hybridization was performed as previously described (32,68). Embryos were photographed, post-fixed in 4% PFA, dehydrated through a graded ethanol series, embedded in paraffin and sectioned at 10 µm.


   ACKNOWLEDGEMENTS
 
We thank Dr Radma Mahmood for technical assistance, Dr Douglas Epstein, Dr Gail Martin and Dr YiPing Chen for probes. This research was supported in part by research funds from the Center for Craniofacial Disorders at the Children's Hospital at Montefiore and by the generous support of the Fleisig Family Foundation. The Fleisig family has demonstrated their understanding of the importance of clinical and basic research by their generosity and their hope for future generations by personal example. This work was supported by grants from the March of Dimes (FY2005- 443) and NIDCD (R01DC05186-03) (B.E.M.).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Shprintzen R.J., Goldberg R.B., Lewin M.L., Sidoti E.J., Berkman M.D., Argamaso R.V., Young D. (1978) A new syndrome involving cleft palate, cardiac anomalies, typical facies, and learning disabilities: velo-cardio-facial syndrome. Cleft Palate J. 15:56–62.[Web of Science][Medline]

  2. DiGeorge A. (1965) A new concept of the cellular basis of immunity. J. Pediatr. 67:907–908.[CrossRef]

  3. Burn J. and Goodship J.A. (1996) Congenital heart disease. In Rimoin D.L., Connor J.M., Pyeritz R.E. (Eds.). Emery and Rimoin's Principles and Practice of Medical GeneticsChurchill Livingstone Vol. 1: pp. 767–828.

  4. Scambler P.J. (2000) The 22q11 deletion syndromes. Hum. Mol. Genet. 9:2421–2426.[Abstract/Free Full Text]

  5. Merscher S., Funke B., Epstein J.A., Heyer J., Puech A., Lu M.M., Xavier R.J., Demay M.B., Russell R.G., Factor S., Tokooya K., Jore B.S., Lopez M., Pandita R.K., Lia M., Carrion D., Xu H., Schorle H., Kobler J.B., Scambler P., Wynshaw-Boris A., Skoultchi A.I., Morrow B.E., Kucherlapati R. (2001) TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell 104:619–629.[CrossRef][Web of Science][Medline]

  6. Lindsay E.A., Vitelli F., Su H., Morishima M., Huynh T., Pramparo T., Jurecic V., Ogunrinu G., Sutherland H.F., Scambler P.J., Bradley A., Baldini A. (2001) Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice. Nature 410:97–101.[CrossRef][Medline]

  7. 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][Web of Science][Medline]

  8. Naiche L.A., Harrelson Z., Kelly R.G., Papaioannou V.E. (2005) T-box genes in vertebrate development. Annu. Rev. Genet. 39:219–239.[CrossRef][Web of Science][Medline]

  9. Graham A. (2003) Development of the pharyngeal arches. Am. J. Med. Genet. A 119:251–256.[Medline]

  10. Epstein J.A. (2001) Developing models of DiGeorge syndrome. Trends Genet. 17:S13–S17.[CrossRef][Medline]

  11. 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., Papaioannou V.E. (1996) Expression of the T-box family genes, Tbx1–Tbx5, during early mouse development. Dev. Dyn. 206:379–390.[CrossRef][Web of Science][Medline]

  12. Xu H., Morishima M., Wylie J.N., Schwartz R.J., Bruneau B.G., Lindsay E.A., Baldini A. (2004) Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract. Development 131:3217–3227.[Abstract/Free Full Text]

  13. Nowotschin S., Liao J., Gage P.J., Epstein J.A., Campione M., Morrow B.E. (2006) Tbx1 affects asymmetric cardiac morphogenesis by regulating Pitx2 in the secondary heart field. Development 133:1565–1573.[Abstract/Free Full Text]

  14. Bottcher R.T. and Niehrs C. (2005) Fibroblast growth factor signaling during early vertebrate development. Endocr. Rev. 26:63–77.[Abstract/Free Full Text]

  15. Mahmood R., Mason I.J., Morriss-Kay G.M. (1996) Expression of Fgf-3 in relation to hindbrain segmentation, otic pit position and pharyngeal arch morphology in normal and retinoic acid-exposed mouse embryos. Anat. Embryol. (Berl.) 194:13–22.[Medline]

  16. Niswander L. and Martin G.R. (1992) Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114:755–768.[Abstract]

  17. Vitelli F., Taddei I., Morishima M., Meyers E.N., Lindsay E.A., Baldini A. (2002) A genetic link between Tbx1 and fibroblast growth factor signaling. Development 129:4605–4611.[Abstract/Free Full Text]

  18. Kelly R.G., Brown N.A., 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][Web of Science][Medline]

  19. Vincentz J.W., McWhirter J.R., Murre C., Baldini A., Furuta Y. (2005) Fgf15 is required for proper morphogenesis of the mouse cardiac outflow tract. Genesis 41:192–201.[CrossRef][Web of Science][Medline]

  20. Wright T.J., Hatch E.P., Karabagli H., Karabagli P., Schoenwolf G.C., Mansour S.L. (2003) Expression of mouse fibroblast growth factor and fibroblast growth factor receptor genes during early inner ear development. Dev. Dyn. 228:267–272.[CrossRef][Web of Science][Medline]

  21. Trokovic N., Trokovic R., Partanen J. (2005) Fibroblast growth factor signalling and regional specification of the pharyngeal ectoderm. Int. J. Dev. Biol. 49:797–805.[CrossRef][Web of Science][Medline]

  22. Casey E.S., O'Reilly M.A., Conlon F.L., Smith J.C. (1998) The T-box transcription factor Brachyury regulates expression of eFGF through binding to a non-palindromic response element. Development 125:3887–3894.[Abstract]

  23. Isaacs H.V., Pownall M.E., Slack J.M. (1994) eFGF regulates Xbra expression during Xenopus gastrulation. EMBO J. 13:4469–4481.[Web of Science][Medline]

  24. Draper B.W., Stock D.W., Kimmel C.B. (2003) Zebrafish fgf24 functions with fgf8 to promote posterior mesodermal development. Development 130:4639–4654.[Abstract/Free Full Text]

  25. Ng J.K., Kawakami Y., Buscher D., Raya A., Itoh T., Koth C.M., Rodriguez Esteban C., Rodriguez-Leon J., Garrity D.M., Fishman M.C., Izpisua Belmonte J.C. (2002) The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129:5161–5170.[Medline]

  26. Agarwal P., Wylie J.N., Galceran J., Arkhitko O., Li C., Deng C., Grosschedl R., Bruneau B.G. (2003) Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo. Development 130:623–633.[Abstract/Free Full Text]

  27. Trainor P.A., Tan S.S., Tam P.P. (1994) Cranial paraxial mesoderm: regionalisation of cell fate and impact on craniofacial development in mouse embryos. Development 120:2397–2408.[Abstract/Free Full Text]

  28. Waldo K.L., Kumiski D.H., Wallis K.T., Stadt H.A., Hutson M.R., Platt D.H., Kirby M.L. (2001) Conotruncal myocardium arises from a secondary heart field. Development 128:3179–3188.[Web of Science][Medline]

  29. Mjaatvedt C.H., Nakaoka T., Moreno-Rodriguez R., Norris R.A., Kern M.J., Eisenberg C.A., Turner D., Markwald R.R. (2001) The outflow tract of the heart is recruited from a novel heart-forming field. Dev. Biol. 238:97–109.[CrossRef][Web of Science][Medline]

  30. Buckingham M., Meilhac S., Zaffran S. (2005) Building the mammalian heart from two sources of myocardial cells. Nat. Rev. Genet. 6:826–835.[CrossRef][Web of Science][Medline]

  31. Vitelli F., Morishima M., Taddei I., Lindsay E.A., Baldini A. (2002) Tbx1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways. Hum. Mol. Genet. 11:915–922.[Abstract/Free Full Text]

  32. Raft S., Nowotschin S., Liao J., Morrow B.E. (2004) Suppression of neural fate and control of inner ear morphogenesis by Tbx1. Development 131:1801–1812.[Abstract/Free Full Text]

  33. Arnold J.S., Werling U., Braunstein E.M., Liao J., Nowotschin S., Edelmann W., Hebert J.M., Morrow B.E. (2006) Inactivation of Tbx1 in the pharyngeal endoderm results in 22q11DS malformations. Development 133:977–987.[Abstract/Free Full Text]

  34. Hu T., Yamagishi H., Maeda J., McAnally J., Yamagishi C., 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.[Abstract/Free Full Text]

  35. Liao J., Kochilas L., Nowotschin S., Arnold J.S., Aggarwal V.S., Epstein J.A., Brown M.C., Adams J., Morrow B.E. (2004) Full spectrum of malformations in velo-cardio-facial syndrome/DiGeorge syndrome mouse models by altering Tbx1 dosage. Hum. Mol. Genet. 13:1577–1585.[Abstract/Free Full Text]

  36. Kelly R.G., Jerome-Majewska L.A., Papaioannou V.E. (2004) The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis. Hum. Mol. Genet. 13:2829–2840.[Abstract/Free Full Text]

  37. Sekine K., Ohuchi H., Fujiwara M., Yamasaki M., Yoshizawa T., Sato T., Yagishita N., Matsui D., Koga Y., Itoh N., Kato S. (1999) Fgf10 is essential for limb and lung formation. Nat. Genet. 21:138–141.[CrossRef][Web of Science][Medline]

  38. Keith L. and Moore T.V.N.P. The Developing Human: Clinically Oriented Embryology 6th ed. W.B. Saunders Company.

  39. Emanuel B.S., McDonald-McGinn D., Saitta S.C., Zackai E.H. (2001) The 22q11.2 deletion syndrome. Adv. Pediatr. 48:39–73.[Medline]

  40. Ryan A.K., Goodship J.A., Wilson D.I., Philip N., Levy A., Seidel H., Schuffenhauer S., Oechsler H., Belohradsky B., Prieur M., Aurias A., Raymond F.I., Clayton-Smith J., Hatchwell E., McKeown C., Beemer F.A., Dallapiccola B., Novelli G., Hurst J.A., Ignatius J., Green A.J., Winter R.M., Brueton I., Brondum-Nielsen K., Scambler P.J., et al. (1997) Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study. J. Med. Genet. 34:798–804.[Abstract/Free Full Text]

  41. Alappat S.R., Zhang Z., Suzuki K., Zhang X., Liu H., Jiang R., Yamada G., Chen Y. (2005) The cellular and molecular etiology of the cleft secondary palate in Fgf10 mutant mice. Dev. Biol. 277:102–113.[CrossRef][Web of Science][Medline]

  42. Ohuchi H., Hori Y., Yamasaki M., Harada H., Sekine K., Kato S., 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][Web of Science][Medline]

  43. Yelbuz T.M., Waldo K.L., Kumiski D.H., Stadt H.A., Wolfe R.R., Leatherbury L., Kirby M.L. (2002) Shortened outflow tract leads to altered cardiac looping after neural crest ablation. Circulation 106:504–510.[Abstract/Free Full Text]

  44. Kelly R.G. (2005) Molecular inroads into the anterior heart field. Trends Cardiovasc. Med. 15:51–56.[CrossRef][Web of Science][Medline]

  45. Min H., Danilenko D.M., Scully S.A., Bolon B., Ring B.D., Tarpley J.E., DeRose M., Simonet W.S. (1998) Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev. 12:3156–3161.[Abstract/Free Full Text]

  46. Marguerie A., Bajolle F., Zaffran S., Brown N.A., Dickson C., Buckingham M.E., Kelly R.G. (2006) Congenital heart defects in Fgfr2-IIIb and Fgf10 mutant mice. Cardiovasc. Res 71:50–60.[Abstract/Free Full Text]

  47. Frank D.U., Fotheringham L.K., Brewer J.A., Muglia L.J., Tristani-Firouzi M., Capecchi M.R., Moon A.M. (2002) An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome. Development 129:4591–4603.[Abstract/Free Full Text]

  48. Abu-Issa R., Smyth G., Smoak I., Yamamura K., Meyers E.N. (2002) Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse. Development 129:4613–4625.[Abstract/Free Full Text]

  49. Brown C.B., Wenning J.M., Lu M.M., Epstein D.J., Meyers E.N., Epstein J.A. (2004) Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse. Dev. Biol. 267:190–202.[CrossRef][Web of Science][Medline]

  50. Park E.J., Ogden L.A., Talbot A., Evans S., Cai C.L., Black B.L., Frank D.U., Moon A.M. (2006) Required, tissue-specific roles for Fgf8 in outflow tract formation and remodeling. Development 133:2419–2433.[Abstract/Free Full Text]

  51. Macatee T.L., Hammond B.P., Arenkiel B.R., Francis L., Frank D.U., Moon A.M. (2003) Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development. Development 130:6361–6374.[Abstract/Free Full Text]

  52. Meyers E.N., Lewandoski M., Martin G.R. (1998) An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18:136–141.[CrossRef][Web of Science][Medline]

  53. Alvarez Y., Alonso M.T., Vendrell V., Zelarayan L.C., Chamero P., Theil T., Bosl M.R., Kato S., Maconochie M., Riethmacher D., Schimmang T. (2003) Requirements for FGF3 and FGF10 during inner ear formation. Development 130:6329–6338.[Abstract/Free Full Text]

  54. Mansour S.L. (1994) Targeted disruption of int-2 (fgf-3) causes developmental defects in the tail and inner ear. Mol. Reprod. Dev. 39:62–67 Discussion 67–68.[CrossRef][Web of Science][Medline]

  55. Srivastava D. and Olson E.N. (2000) A genetic blueprint for cardiac development. Nature 407:221–226.[CrossRef][Medline]

  56. Ortega S., Ittmann M., Tsang S.H., Ehrlich M., Basilico C. (1998) Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2. Proc. Natl Acad. Sci. USA. 95:5672–5677.[Abstract/Free Full Text]

  57. Jaskoll T., Witcher D., Toreno L., Bringas P., Moon A.M., Melnick M. (2004) FGF8 dose-dependent regulation of embryonic submandibular salivary gland morphogenesis. Dev. Biol. 268:457–469.[CrossRef][Web of Science][Medline]

  58. Storm E.E., Rubenstein J.L., Martin G.R. (2003) Dosage of Fgf8 determines whether cell survival is positively or negatively regulated in the developing forebrain. Proc. Natl Acad. Sci. USA. 100:1757–1762.[Abstract/Free Full Text]

  59. Vitelli F., Zhang Z., Huynh T., Sobotka A., Mupo A., Baldini A. (2006) Fgf8 expression in the Tbx1 domain causes skeletal abnormalities and modifies the aortic arch but not the outflow tract phenotype of Tbx1 mutants. Dev. Biol. 295:559–570.[CrossRef][Web of Science][Medline]

  60. David N.B., Saint-Etienne L., Tsang M., Schilling T.F., Rosa F.M. (2002) Requirement for endoderm and FGF3 in ventral head skeleton formation. Development 129:4457–4468.[Abstract/Free Full Text]

  61. Nissen R.M., Yan J., Amsterdam A., Hopkins N., Burgess S.M. (2003) Zebrafish foxi one modulates cellular responses to Fgf signaling required for the integrity of ear and jaw patterning. Development 130:2543–2554.[Abstract/Free Full Text]

  62. Feldman B., Poueymirou W., Papaioannou V.E., DeChiara T.M., Goldfarb M. (1995) Requirement of FGF-4 for postimplantation mouse development. Science 267:246–249.[Abstract/Free Full Text]

  63. Phillips B.T., Bolding K., Riley B.B. (2001) Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction. Dev. Biol. 235:351–365.[CrossRef][Web of Science][Medline]

  64. Maroon H., Walshe J., Mahmood R., Kiefer P., Dickson C., Mason I. (2002) Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle. Development 129:2099–2108.[Abstract/Free Full Text]

  65. Leger S. and Brand M. (2002) Fgf8 and Fgf3 are required for zebrafish ear placode induction, maintenance and inner ear patterning. Mech. Dev. 119:91–108.[CrossRef][Web of Science][Medline]

  66. Crump J.G., Maves L., Lawson N.D., Weinstein B.M., Kimmel C.B. (2004) An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning. Development 131:5703–5716.[Abstract/Free Full Text]

  67. Crossley P.H. and Martin G.R. (1995) The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121:439–451.[Abstract]

  68. Epstein J.A. (2000) Pax3 and vertebrate development. Methods Mol. Biol. 137:459–470.[Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cereb CortexHome page
T. R. Kiehl, E. W. C. Chow, D. J. Mikulis, S. R. George, and A. S. Bassett
Neuropathologic Features in Adults with 22q11.2 Deletion Syndrome
Cereb Cortex, January 1, 2009; 19(1): 153 - 164.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
J. Zhang, Y. Lin, Y. Zhang, Y. Lan, C. Lin, A. M. Moon, R. J. Schwartz, J. F. Martin, and F. Wang
Frs2{alpha}-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis
Development, November 1, 2008; 135(21): 3611 - 3622.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
15/21/3219    most recent
ddl399v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (9)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Aggarwal, V. S.
Right arrow Articles by Morrow, B. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aggarwal, V. S.
Right arrow Articles by Morrow, B. E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?