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First published online 1 September 2005
doi: 10.1242/dev.02018

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Timed mutation and cell-fate mapping reveal reiterated roles of Tbx1 during embryogenesis, and a crucial function during segmentation of the pharyngeal system via regulation of endoderm expansion

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

View larger version (27K): [in a new window]   Fig. 1. Cre-mediated recombination of the Tbx1flox allele and effects of TM on embryonic viability. (A) Schematic of the polymerase chain reaction (PCR) strategy used to evaluate Tbx1flox recombination. The primer pair (arrows) amplifies fragments of different lengths from the Tbx1flox and wild-type alleles of Tbx1flox/+ cells but not from the Tbx1E5 allele (which results from Cre-mediated recombination of Tbx1flox). (B) Representative PCR results from genomic DNA extracted from individual Tbx1flox/+ embryos (controls, Cre-) and TgCAGG-CreERTM;Tbx1flox/+ embryos exposed to TM in utero for the time indicated. There is progressive fading of the Tbx1flox amplicon. (C) The plot indicates the progressive decrease in the intensity (percentage) of the Tbx1flox amplicon compared with the Tbx1+ amplicon. Each point is the average of measurements from 12-17 embryos. Error bars indicate standard deviation. (D) Numbers of dead embryos at various harvest stages after TM injection at the time points indicated.

View larger version (28K): [in a new window]   Fig. 2. Time-course deletion of Tbx1 dissects the mutant phenotype. (A) Phenotypes scored in E18.5 TgCAGG-CreERTM;Tbx1flox/- or TgCAGG-CreERTM;Tbx1flox/+ (indicated by an asterisk) embryos after in utero exposure to TM at the stages indicated. The penetrance of defects was complete, unless indicated otherwise. Aortic arch defects included interrupted aortic arch type B and aberrant origin of the right subclavian artery. OFT, cardiac outflow tract; PTA, persistent truncus arteriosus. (B) Scheme of crucial time intervals of Tbx1 requirement for the structures or organs indicated, estimated from the results shown in A. The left extremity of each bar is set at the latest TM injection time that causes abnormalities, and the right end of the bar is set shortly after the next injection time. The gray areas in each bar indicate time required for full excision of the floxed allele (left) and possible extension of the time span (right). The arrow indicates that we do not know the end-point of the crucial time for the development of the secondary palate.

View larger version (77K): [in a new window]   Fig. 3. Tbx1 is required early for aortic arch patterning and later for cardiac outflow tract (OFT) morphogenesis. (A) Aortic arch patterning defects in an E18.5 TgCAGG-CreERTM;Tbx1flox/+ embryo exposed to TM at E7.5. The arrowhead indicates aberrant origin of the right subclavian artery; the arrow indicates interruption of the aortic arch, compared with normal arrangement in a Tbx1flox/+ control littermate (B). (C,D) Persistent truncus arteriosus (T) in an E18.5 TgCAGG-CreERTM;Tbx1flox/- embryo exposed to TM at E8.5 (C), compared with normal anatomy in a TgCAGG-CreERTM;Tbx1flox/+ littermate (D). (E-G) Intracardiac ink injection of E10.5 TgCAGG-CreERTM;Tbx1flox/+ embryos exposed to TM at E7.5 (E), E8.5 (F) and E9.5 (G). Numbers indicate the pharyngeal arch arteries (PAAs). The 4th PAA is absent in embryos exposed to TM at E7.5 (E) and exhibits very mild hypoplasia in embryos exposed to TM at E8.5 (F); normal morphology is present in the embryo exposed at E9.5 (G). (H-J) Timed fate mapping of Tbx1 expressing cells in E10.5 Tbx1mcm/+;R26R embryos exposed to TM at E7.5 (H), E8.5 (I) and E9.5 (J) stained with X-gal; coronal sections, cranial is upwards. Arrowheads indicate labeling of surface ectoderm, arrows indicate labeling of endothelial cells of the 4th PAA. (K-M,K'-M') Timed fate mapping of Tbx1-expressing cells in E12.5 Tbx1mcm/+;R26R embryos exposed to TM at E7.5 (K,K'), E8.5 (L,L'), and E9.5 (M,M') stained with X-gal; whole-mount preparation of the heart (K-M) and coronal section through the OFT (K'-M') at the truncal-conal transition. RSA and LSA, right and left subclavian arteries; BT, brachiocephalic trunk; Ao, aorta; PT, pulmonary trunk; RCA and LCA, right and left common carotid arteries; PA, pulmonary arteries; RV and LV, right and left ventricles. Scale bars: 0.5 mm in B,D,M; 0.2 mm in G; 50 µm in J; 100 µm in M'.

View larger version (104K): [in a new window]   Fig. 4. Tbx1 in thymic development and palatogenesis. (A-C) Mediastinum of E18.5 TgCAGG-CreERTM;Tbx1flox/- embryos exposed to TM at E8.5 (A) and E9.5 (B), compared with a Tbx1flox/- control (C). Asterisks indicate thymic lobes (absent in A). Cranial is upwards. (D,E) Histological sections of X-gal stained E12.5 Tbx1mcm/+;R26R embryos showing the thymic primordium (Th) after exposure to TM at E8.5 (D) and E9.5 (E). Coronal sections, cranial is up. (F,G) Secondary palate in a E18.5 TgCAGG-CreERTM;Tbx1flox/- embryo exposed to TM at E11.5 (F, cleft palate between the arrows), compared with that of a Tbx1flox/- control (G, normal fusion between the arrows). Cranial is upwards. Scale bars: 0.5 mm in C and G; 50 µm in E.

View larger version (75K): [in a new window]   Fig. 5. Tbx1 is required during pharyngeal segmentation. (A-C) Coronal sections of X-gal stained E10.5 Tbx1mcm/+;R26R embryos exposed to TM at E7.5 (A), E8.5 (B) and E9.5 (C); cranial is upwards. Arrowheads indicate the 3rd pharyngeal pouches, and arrows the 4th pharyngeal pouches. There is a progressive increase in number of labeled endodermal cells in the caudal segments. (D-F) External aspect of E10.5 TgCAGG-CreERTM;Tbx1flox/- embryos exposed to TM at E7.5 (D), E8.5 (E) and E9.5 (F). External abnormalities were only detected in embryos exposed to TM at E7.5 (D), which showed severe hypoplasia of the 2nd pharyngeal arch, II. (D'-F') Intracardiac ink injection of the embryos in D-F to visualize the pharyngeal arch arteries (PAAs). Numbers indicate the PAAs. The left 6th PAA in E' is absent, while the right one appears normal. (G-I) Coronal sections of E10.5 TgCAGG-CreERTM;Tbx1flox/- embryos exposed to TM at E7.5 (G), E8.5 (H) and E9.5 (I); cranial is upwards. Arrowheads, 3rd pharyngeal pouches; arrows, 4th pharyngeal pouches, black stain is residual ink injected into these embryos before paraffin embedding. (J-L) Left view of E10 TgCAGG-CreERTM;Tbx1flox/- embryos exposed to TM at E7.5 (J), E8.5 (K) and a Tbx1flox/- control (L) hybridized with a Pax1 probe to identify the 1st (I), 2nd (II) and 3rd (III) pharyngeal pouches. Scale bars: 0.5 mm in F; 0.2 mm in F' and L; 0.1 mm in C and I.

View larger version (42K): [in a new window]   Fig. 6. Tbx1 regulates endodermal cell proliferation. (A,B) Examples of immunohistochemistry with an anti-phospho H3 antibody used to evaluate endodermal cell proliferation in E10.0 TgCAGG-CreERTM;Tbx1flox/+ (control, A) and TgCAGG-CreERTM;Tbx1flox/- (test, B) embryos exposed to TM at E8.5. ec, ectoderm; en, endoderm. (C) Mitotic index (M.I.) count from at least 2000 cells per tissue per embryo; data refer to the average of three embryos per genotype; P values were calculated using the Student's t-test. Brain cells were counted as an internal control. Error bars indicate standard deviation. (D) The cartoon illustrates a model for Tbx1 role during pharyngeal segmentation. A cranial-to-caudal `wave' of Tbx1 gene expression causes cranial-to-caudal expansion of the pharyngeal endoderm. This is followed by invasion of neural crest-derived cells (NCC) and formation of a new pharyngeal arch. Tbx1 expression in pharyngeal mesoderm may also contribute to pharyngeal segmentation. PAA, pharyngeal arch artery. Scale bar: 50 µm in B.

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