QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 February 2006
doi: 10.1242/dev.02264

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Arnold, J. S. Articles by Morrow, B. E. Search for Related Content PubMed PubMed Citation Articles by Arnold, J. S. Articles by Morrow, B. E. Social Bookmarking                         What's this?

Inactivation of Tbx1 in the pharyngeal endoderm results in 22q11DS malformations

¹ Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
² Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
³ Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

View larger version (39K): [in a new window]   Fig. 1. Conditional inactivation strategy. (A) Generation of Tbx1 wild-type (WT), Tbx1 hygro, Tbx1 flox and Tbx1 null alleles. (B) Correct targeting was confirmed through Southern blot hybridization with a probe distal to the targeting vector integration site, indicated by the red bar in A. The restriction enzymes used were Acc65I and NotI, and the expected sizes of the different alleles are indicated in A. (C) PCR amplification was used to genotype the Tbx1 hygro/+ (lane 6; PCR1), Tbx1 flox/+ (lanes 2 and 3; PCR2) and Tbx1 null/+ (lanes 4 and 5; PCR3) mice. The position of the primers is indicated in A.

View larger version (119K): [in a new window]   Fig. 2. Tbx1 expression compared with Cre expression. Tbx1 expression in wild-type (C,D,G,I,K) and homozygous conditional mutant mice (E,F,H,J,L) at E8.5 (C,E) and E9.5 (D,F-H), compared with Cre expression in Foxg1-Cre mice at E8.5 (A) and E9.5 (B). Coronal vibratome sections of whole mounts from D (wild type) and F (mutant) are shown in G and H, respectively. Note Tbx1 expression in the core mesenchyme (arrows) of both wild-type (G) and mutant embryos (H). The wild type also shows expression in the pharyngeal pouch endoderm (arrowhead in G). Immunohistochemistry with a polyclonal Tbx1 antibody on E11.5 sagittal sections confirms the presence of the Tbx1 protein in the splanchnic mesenchyme (arrow) of the mutant (J) and wild-type (I) embryos. Radioactive ISH on E11.5 sagittal sections shows Tbx1 expression in the otic vesicle (ov) and the adjacent splanchnic mesenchyme (K). The OV expression of Tbx1 is absent from the conditional null embryo (L), although the mesenchymal staining remains intact. (M-O) X-gal staining of E9.0 (O) and E9.5 (M,N) sagittal (M) and transverse (N,O) sections of ROSA26/Foxg1-Cre progeny. Cre activity is evident in the pharyngeal pouches (pp) and the otic vesicle (ov). (P-U) Tbx1 expression by ISH on coronal (P,Q) and sagittal (R-U) sections in control (P,R,T) and conditional null embryos (Q,S,U). Scale bars: 100 µm in E,H,N,Q,S,U; 0.2 mm in F; 0.5 mm in J,K. Asterisks indicate core mesoderm; ov, otic vesicle; pa, pharyngeal arch; pp, pharyngeal pouches.

View larger version (118K): [in a new window]   Fig. 3. The Tbx1 conditional mutant phenotype. (A,B) Tbx1 conditional mutant phenotype at E11.5 (B) compared with a wild-type littermate (A). PAs are indicated with a dashed red line and denoted with arrows. PA, pharyngeal arch. (C,D) Coronal sections through the same embryos show a hypoplastic pharynx in the mutant (D) compared with the wild-type littermate (C). PEKO/-, Tbx1 flox/null; Foxg1-Cre/+; WT, wild type. Scale bars: 0.5 mm in A; 300 µm in C.

View larger version (100K): [in a new window]   Fig. 4. ISH with Fgf3 and Fgf8. (A-D) ISH with Fgf3 (A,B) and Fgf8 (C,D) probes at E10.0 reveals expression of both genes in the pharyngeal pouches (asterisks) of wild-type embryos (A,C). Conditional null embryos of the same stage (B,D) display a loss of both Fgf3 and Fgf8 in the pharyngeal pouches. OV, otic vesicle; PEKO/-, Tbx1 flox/null; Foxg1-Cre/+; WT, wild type. Scale bars: 0.1 mm.

View larger version (104K): [in a new window]   Fig. 5. Craniofacial phenotype of Tbx1 mutants at E17.5, shown by transverse histological sections, and bone and cartilage staining. (A) Wild-type embryo. (B) The conditional mutant embryos at E17.5 are edematous and lack the pinna (p). (C,D) Histological analysis of the mutant (D) compared with wild-type offspring (C) reveals the presence of cleft palate in the mutants (D, star). (E-H) Bone and cartilage staining of E17.5 wild-type (E,G) and mutant (F,H) embryos. The mutant (F) has cleft palate (arrow), aplasia of the tympanic rings (arrowhead in E), and fused basisphenoid and basioccipital bones (asterisks). EC, ear capsule; PEKO/-, Tbx1 flox/null; Foxg1-Cre/+; WT, wild type; ZA, zygomatic arches. Scale bars: 0.8 mm in B; 300 µm in D; 0.5 mm in F,H.

View larger version (70K): [in a new window]   Fig. 6. Tbx1 conditional null embryos have abnormal musculature. (A-D) Transverse histological sections of E17.5 embryos. Tbx1 conditional null embryos have hypoplasia of the masseter (B) and pterygoid (D) muscles. Representative wild-type embryos are shown in A and C. mc, mandibular condyle; me, Meckel's cartilage; mt, masseter; pt, pterygoid. (E,F) Coronal sections through an E17.5 Tbx1 hygro/hygro embryo (H/H; F) reveal the presence of the masseter muscle (mt); a wild-type control embryo is shown in E. (G) ISH with Myod on an E10.5 wild-type embryo shows expression in the core mesenchyme of PAs (arrow). (H,I) Myod ISH reveals the lack of expression in the core mesenchyme of the first PA of homozygous conditional null (H) and Tbx1-/- (I) embryos. (J) Tbx1 expression in the core mesenchyme of PA1 is maintained in the homozygous conditional null mutant at E10.5 (arrow). WT, wild type; PEKO/-, Tbx1 flox/null; Foxg1-Cre/+; -/-, Tbx1-/-. Scale bar: 100 µm in B,D; 200 µm in F; 0.5 mm in I; 0.2 mm in J.

View larger version (139K): [in a new window]   Fig. 7. Thymus, thyroid and cardiovascular phenotype of Tbx1 conditional mutants shown by transverse histological sections. (A) An E17.5 wild-type embryo with the thymus gland (tm) and the location of the right subclavian artery (arrow) indicated. (B) A homozygous conditional null embryo of the same stage shows thymus aplasia (asterisks), as well as the presence of a retroesophageal right subclavian artery (RSA, arrow). (C,D) The thyroid glands (th) of wild-type (C) and mutant (D) embryos at E17.5. (E) Wild-type embryo shows normal separation of the aortic (a) and pulmonary (p) trunks. (F) Abnormal septation of the outflow tract results in persistent truncus arteriosus (PTA) in the mutant of the same stage. (G-I) Sections through the heart show a ventricular septal defect (VSD) in a Tbx1 homozygous conditional mutant (H) and a Tbx1-/- embryo (I), compared with a wild-type litter mate (G). (J-M) Transverse sections at E10.5 show normal development of atria (A) and ventricles (V), as well as the outflow tract in both wild-type (J,L) and conditional mutant (K,M) embryos. WT, wild type; PEKO/-, Tbx1 flox/null; Foxg1-Cre/+; -/-, Tbx1-/-. Scale bars: 300 µm in B,D,F; 100 µm in G-I; 50 µm in K,M.

View larger version (134K): [in a new window]   Fig. 8. Tbx1 heterozygous conditional phenotype. (A) Tbx1+/- mice have aplasia of the left fourth pharyngeal arch artery (PAA), as shown by sagittal histological sections at E10.5 (arrow indicates third PAA). (B) Conditional heterozygous mice appear normal at the same stage, with both the third and fourth PAA clearly present (arrows). (C,D) E17.5 Tbx1+/- embryos have RSA (arrow in C), whereas all conditional heterozygotes appear normal (D). The right subclavian artery in the conditional mutant (D) is indicated with an arrow. +/-, Tbx1+/-; PEKO/+, Tbx1 flox/+; Foxg1-Cre/+. Scale bars: 200 µm in B,D.

View larger version (37K): [in a new window]   Fig. 9. Model of Tbx1 function in pharyngeal development (E9.5-E11.5). (Top) In wild type, pharyngeal arches develop as bulges on either side of the pharyngeal endoderm (bold lines, coronal section; rostral, top; caudal, bottom). The arrows indicate approximate time in development; left, earliest, right, latest times. Tbx1, shown as red boxes on the endodermal lining, marks the position of the future pharyngeal pouches, which will extend outward to meet the surface ectodermal clefts. Tbx1 is expressed in the PE during pouch outgrowth but becomes downregulated once the pouches form. The gene is also expressed in the core mesenchyme of the developing arches, indicated by red boxes and circles. It is likely that Tbx1 in the endoderm is involved in epithelial-mesenchymal interactions with the core mesoderm (red arrows). (Bottom) Lack of distal pouch development in Tbx1-/- and conditional null embryos prevents the segmentation of the distal apparatus, altering NCC migration, growth and differentiation.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter