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First published online 6 October 2004
doi: 10.1242/dev.01399

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Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors

Tonghuan Hu^1,2,*, Hiroyuki Yamagishi^1,2,3,*, Jun Maeda^1,2,*, John McAnally², Chihiro Yamagishi^1,2,3 and Deepak Srivastava^1,2,

¹ Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA
² Department of Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA
³ Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

View larger version (31K): [in a new window]   Fig. 1. Generation of Tbx1 hypomorphic and null alleles. (A) Targeting strategy. Tbx1+/+, wild-type allele; Tbx1tv, targeting vector; Tbx1neo, hypomorphic allele; Tbx1neo(–), null allele with neo; Tbx1null(–), null allele without neo. Exon numbers and restriction enzyme sites (C, ClaI; RI, EcoRI; Xb, XbaI) are indicated. White triangles represent Cre-recombinase sites; black triangles are FLP-recombinase sites. (B) Southern analysis of genomic DNA from ES cell clones after digestion with XbaI and EcoRI and hybridizing with 5' and 3' probes, respectively, which demonstrate targeted event (neo/+) compared with wild-type allele (+/+). (C) PCR genotyping analysis of various Tbx1 alleles with forward (f1,f2) and reverse (r1) primers. +/+, Tbx1 wild type; neo/neo, Tbx1 hypomorphic homozygous; –/–, Tbx1-null; neo/+, Tbx1 hypomorphic heterozygous; +/–, Tbx1 heterozygous; neo/–, Tbx1 compound mutant mice. (D) Quantification of Tbx1 mRNA transcripts detected by RT-PCR in heterozygous and hypomorphic E10.5 mouse embryos compared with wild type set at 100%.

View larger version (96K): [in a new window]   Fig. 2. Cardiovascular and thymic defects in Tbx1 hypomorphic mutant neonates. Frontal views of heart and vessels in Tbx1 hypomorphic heterozygous (Tbx1neo/+) neonates (B-D), and in Tbx1 hypomorphic homozygous (Tbx1neo/neo) neonates (E-H). (A) Wild-type neonate with thymus removed to illustrate normal septation of outflow tract into aorta (ao) and pulmonary trunk (pt), and patterning of the aortic arch with the right subclavian artery (rsa), right common carotid artery (rcc), left common carotid artery (lcc), left subclavian artery (lsa) and descending aorta (dao). (B) Tbx1neo/+ neonate had normal development of thymus (thy) and normal anatomy of great vessels with the aorta located to the right and dorsal to the pulmonary trunk. (C,D) Thymus was removed to visualize arch anatomy. (C) Tbx1neo/+ mice displayed aberrant origin of right subclavian artery (rsa) with it seen traversing behind the trachea (arrow) to connect to the dao. (D) Interruption of the aortic arch with no connection between the ascending and descending aorta (*).(E-H) Majority of Tbx1neo/neo neonates had persistent truncus arteriosus (ta) that resulted from failure of aorto-pulmonary septum formation. In addition, aplasia of the thymus occurred with complete penetrance. Exposure of the aortic arch in Tbx1neo/neo mice revealed a variety of aortic arch patterning defects in conjunction with persistent truncus arteriosus, including aberrant origin of the dao from the carotid (G), and abnormal origins of the carotid arteries and lsa (H).

View larger version (87K): [in a new window]   Fig. 3. Ear and craniofacial defects in Tbx1 hypomorphic (neo/neo) and null (–/–) mutants. (A-C) Left lateral views revealed that Tbx1neo/neo mutants (B) had smaller but normal shaped ears (arrows) compared with Tbx1neo/+ (A), which was similar to wild type. Tbx1-/- mutants (C) showed absent external ears. (D-F) Left-sided views of skeletal preparations of neonatal skulls focusing on ear region (blue or red indicates cartilage or bone, respectively). The malleus (ma) and incus (inc) middle ear structures were progressively hypoplastic in the Tbx1neo/neo (E) and Tbx1-/- (F) mutants compared with Tbx1neo/+ (D). The stapes (st), was hypoplastic in Tbx1neo/neo mutants and was absent in Tbx1-/- mutants. The cartilage primordium of petrous part of the temporal bone (t) also exhibited a gradient of hypoplasticity with progressive reduction in Tbx1 dose. (G-I) Ventral view of skull skeletal preparations demonstrated that both Tbx1neo/neo mutants (H) and Tbx1-/- mutants (I) had abnormal fusion of the basioccipital (bs) and basisphenoid (bo) bones. However, cleft palate was present only in Tbx1-/- mutants, caused by failure of fusion of the palatine shelves outlined by broken lines. The otic capsule (oc), an inner ear structure, also exhibited severe hypoplasticity in both Tbx1neo/neo mutants and Tbx1-/- mutants. (J-L) Left lateral views of skeletal preparations of the face and neck. Tbx1neo/neo (K) and Tbx1-/- (L) mutants failed to form the body of the hyoid bone (hy) in the neck. The rest of the neck cartilage structure (arrowheads), such as thyroid cartilage (tc) appeared normal in Tbx1neo/neo mutants, but was reduced to small and fragmentary structures in Tbx1-/- mutants (L). m, maxillary shelves; mx, maxilla; mb, mandible; tr, tympanic ring.

View larger version (93K): [in a new window]   Fig. 4. Pharyngeal mesoderm gene expression defects in Tbx1 mutants. (A-C) Section in situ hybridization at E9.5 demonstrated that Tbx1 expression was slightly reduced in the head mesenchyme and was absent in pharyngeal arch mesodermal core of Tbx1neo/neo mutants (B, compare with arrowheads in A), but the pharyngeal endoderm (pe) expression was conserved. Tbx1 transcript was absent in all cell types of Tbx1-/- mutants (C). Expression of Pax1, a pharyngeal endoderm marker (D-F), and Dlx2, a pharyngeal arch mesenchyme marker (G-I) remained unchanged in both Tbx1neo/neo mutants (E,H) and Tbx1-/- mutants (F,I). (J-L) Capsulin (Caps) was expressed normally in the pharyngeal mesoderm (pm) core in both Tbx1neo/neo mutants (K) and Tbx1-/- mutants (L) when compared with the wild-type littermates. (M-O) In adjacent sections from the same embryo, Foxa2 expression in pharyngeal endoderm was largely conserved, but expression in the pharyngeal mesoderm core was completely missing in both mutants (N,O). Results shown are representative of at least three separate experiments.

View larger version (52K): [in a new window]   Fig. 5. Fox cis element is necessary for pharyngeal mesoderm and cardiac outflow expression of Tbx1. (Upper panel) Genomic organization of the 5' mouse Tbx1 locus. Boxes indicate exons, and the translation start site (ATG) is designated as nucleotide number zero. Construct number is indicated on the left, and the corresponding expression pattern of lacZ is summarized on the right. Mutation of Fox site is indicated by star. (A,C,E,G,I), Right lateral view of representative embryos obtained with indicated constructs; (B,D,F,H,J) magnified right lateral views of above focused on pharyngeal arch and outflow tract region. Pharyngeal mesoderm (white arrowheads), pharyngeal endoderm (yellow arrowheads), cardiac outflow tract (oft) and head mesenchyme (hm) expression is evident.

View larger version (61K): [in a new window]   Fig. 6. Identification of regulatory regions for Fgf8 expression in pharyngeal arches and the outflow tract. (A) Genomic organization of the 5' mouse Fgf8 locus. Construct number is indicated on the left, and the corresponding expression pattern of lacZ in the outflow tract (OFT) or pharyngeal arch (PA) is summarized on the right. (B,C) Transverse sections of E9.5 transgenic mice (construct 6) stained for lacZ (B) or section in situ hybridization for Fgf8 transcripts (C) showing cardiac outflow tract (ot) and pharyngeal mesoderm (pm) expression. nt, neural tube. (D) Coronal section of transgenic mice made with construct 6 showing pharyngeal endoderm (pe) and ectoderm (ect) expression. (E) Right lateral view of representative E9.5 embryo obtained with stable transgenic line made with construct 6 showing pharyngeal arch (*) and cardiac outflow tract (black arrowhead) lacZ expression. (F,G) Fgf8-lacZ transgenic line from (E) in Tbx1neo/neo or Tbx1neo/– genetic background. Black arrowhead indicates progressive decrease in outflow tract expression. (H-J) Coronal section in situ hybridization of E10.0 embryos with Fgf8 probe indicates preserved ectodermal (white arrowheads) and pharyngeal endoderm (pe) expression of Fgf8 in Tbx1neo/neo or Tbx1neo/– embryos. Tbx1-/- embryos were identical to Tbx1neo/– in phenotype and gene expression.

View larger version (75K): [in a new window]   Fig. 7. Tbx1 and fibroblast growth factors in anterior heart field. (A) Sagittal section in situ hybridization of E9.5 embryos with Tbx1 (A), Fgf8 (B) or Fgf10 (C) riboprobes on wild-type (+/+, top row) or Tbx1neo/neo embryos (bottom row) focusing on pharyngeal arch region. Pharyngeal mesoderm that is probably in the anterior heart field dorsal to the heart is indicated by yellow arrowheads. Black arrowheads indicate pharyngeal core mesoderm expression; white arrowheads indicate pharyngeal endoderm; white arrows indicate pharyngeal ectoderm. Fgf8 and Fgf10 are downregulated in areas marked by yellow arrowheads in Tbx1neo/neo embryos. Bright-field images are shown. Right-most panels in C represent transverse sections through the cardiac outflow tract of E9.5 wild-type (+/+) embryos or transgenic embryos misexpressing Tbx1 in the heart under control of the ß-MHC promoter (ß-MHC-Tbx1). The domain of Fgf10 expression was expanded in transgenic mice, as detected by in situ hybridization. ov, otic vesicle; rv, right ventricle; lv, left ventricle; nt, neural tube.

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