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

This Article Summary Full Text Full Text (PDF) 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 Liu, C. Articles by Martin, J. F. Search for Related Content PubMed PubMed Citation Articles by Liu, C. Articles by Martin, J. F. Social Bookmarking                         What's this?

Pitx2c patterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions

Chengyu Liu^1,*, Wei Liu^1,*, Jennifer Palie¹, Mei Fang Lu¹, Nigel A. Brown² and James F. Martin^1,

¹ Alkek Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Blvd, Houston, TX 77030, USA
² Department of Anatomy and Developmental Biology, St. George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 ORE, UK

View larger version (91K): [in a new window]   Fig. 1. Expression of Pitx2c in the branchial arch mesoderm and aortic sac. (A,B) Whole-mount images of 9.5 dpc wild-type embryos hybridized to a Pitx2c-specific probe showing Pitx2c in left branchial arch mesoderm (arrow in B). (C-E) Transverse serial sections through a 9.5 dpc wild-type embryo hybridized to Pitx2c probe. Pitx2c expression is denoted by arrows. (F,G) Coronal sections through a 9.5 dpc embryo hybridized to a Pitx2c probe showing Pitx2c expression in left branchial arch mesoderm near junction of the aortic sac with the branchial arch artery (arrow in F). Pitx2c expression within left aortic sac myocardium is denoted by arrowhead. In G, arrow designates Pitx2c expression in dorsal branchial arch mesoderm in proximity to forming branchial arch arteries. (H,I) Parasagittal section through a 10.5 dpc embryo hybridized to Pitx2c probe. Arrow denotes ventral Pitx2c expression while arrowhead in I indicates less abundant dorsal Pitx2c expression. (J-M) Transverse sections through hearts of 10.5 dpc embryos hybridized to Pitx2c probe. Arrows in J,K,L indicate Pitx2c expression in dorsal mesocardium, while arrowhead in L indicates Pitx2c expression in left atrium. Arrows in M indicate Pitx2c expression in right ventricular and interventricular myocardium. as, aortic sac; ba, branchial arch; baa, branchial arch artery; da, dorsal aorta; oft, outflow tract.

View larger version (31K): [in a new window]   Fig. 2. Gene targeting strategy to generate the Pitx2 c allele. (A) Summary of exon use by Pitx2 isoforms. (B) Pitx2 genomic structure and Pitx2c-specific targeting strategy. Boxes represent exons and straight lines introns. P1 and P2 indicate two promoters that regulate expression of different isoforms. (C) Pitx2 c targeted allele before and after PGKneomycin removal. At the bottom, the Pitx2 null allele (abcnull) and cre knock-in alleles (abccreneo) also used in this study are shown. (D) Southern blot with flanking probes: left panel shows a Southern blot of tail DNA probed with 5' flanking probe and center panel shows Southern blot probed with 3' flanking probe. Right panel shows a Southern blot probed with 3' flanking probe after crossing to CMV cre recombinase deletor strain to generate Pitx2 c+/- mice. After recombination, the 3' flanking probe hybridizes to an 8 kb fragment and a 10 kb fragment in mice retaining PGKneomycin. The `+' above lanes denotes mice retaining PGKneomycin and `-' indicates a mouse that deleted PGKneomycin. (E) Lateral view of wild-type and Pitx2 c-/- 18.5 dpc embryos.

View larger version (61K): [in a new window]   Fig. 3. Pitx2c in remodeling the great vessels and outflow tract. (A) Remodeling of aortic arch arteries and derivation of mature aortic arch vessels (adapted from Moore, 1982). (B-D) Corrosion cast of wild-type (B) and Pitx2 c-/- embryos with correct direction of aortic arch (C) and reversed orientation (D). (E-G) Corrosion cast of a Pitx2 c-/- embryo with double aortic arch showing ventral (E), ventral oblique (F) and dorsal (G) views. (H-J) Diagrams of the corrosion cast to more clearly show changes in vessel morphology. Each diagram is associated with the cast directly above. (K-N) India ink injection into wild-type and Pitx2 c-/- embryos at 11.5 dpc. Right and left oblique views are shown. (O,P) Casting dye injection into 18.5 dpc wild-type and Pitx2 c-/- embryos showing DORV in mutant (P). ao, aorta; baa, branchial arch arteries; cc, common carotid; d, ductus arteriosus; in, innominate artery; lcc, left common carotid; lpa, left pulmonary artery; lsa, left subclavian artery; pa, pulmonary artery; pt, pulmonary trunk; rcc, right common carotid artery; rpa, right pulmonary artery; rsa, right subclavian artery; s, subclavian artery.

View larger version (60K): [in a new window]   Fig. 4. Fate mapping with Wnt1 cre transgenic and the Rosa26 reporter. (A,B) Ventral view of whole-mount lacZ staining of 12.5 dpc wild-type and Pitx2 c-/- mutant embryo. (C-F) Rostral (C,D) and caudal (E,F) transverse section through the conotruncus of lacZ stained embryos showing lacZ-labeled cardiac crest derivatives contributing to valves and cushions of OFT (denoted by arrows). (G-J) Parsagittal sections through 10.5 dpc wild-type (G,I) and Pitx2 c-/- (H,J) mutant embryos at different mediolateral planes of section. Branchial arch arteries are numbered. ao, aorta; pt, pulmonary trunk.

View larger version (40K): [in a new window]   Fig. 5. Whole-mount in situ with markers of outflow tract myocardium. (A,B) 10.5 dpc wild-type (A) and Pitx2 c-/- (B) embryos hybridized with semaphorin 3c probe. (C,D) 11.5 dpc wild-type (C) and Pitx2 c-/- (D) embryos hybridized with semaphorin 3c probe showing expression in outflow tract myocardium (arrows). (E,F) 12.5 dpc wild-type (E) and Pitx2 c-/- (F) embryos hybridized with semaphorin 3c probe showing that expression of semaphorin 3c is reduced in the mutant (n=3) (arrows). (G,H) Whole-mount views of 12.5 dpc wild-type (G) and Pitx2 abccreneo;abcnull (H) null mutant embryos. Pitx2 abccreneo;abcnull embryos demonstrate embryonic rotation, anterior body wall closure defects and eye anomalies typical of Pitx2 null embryos. (I,J) 12.5 dpc wild-type (I) and Pitx2 abccreneo;abcnull (J) embryos hybridized with cre probe showing expression in outflow tract and right ventricular myocardium (arrows).

View larger version (80K): [in a new window]   Fig. 6. Fate mapping with Pitx2 abccreneo and Rosa26 reporter allele. (A,B) lacZ staining of 10.5 dpc Pitx2 abccreneo+/- (A) and Pitx2 abccreneo;abcnull (B), and Rosa26 reporter trans-heterozygous embryo. Arrow indicates the lacZ-positive cells in the OFT that have crossed the midline (broken line). (C,D) lacZ staining (C) and Pitx2c whole-mount in situ (D) of 12.5 dpc Pitx2 abccreneo+/- and Rosa26 reporter trans heterozygous embryos. Signal is indicated by the arrows. (E-H) lacZ staining (E,G) and Pitx2c whole-mount in situ (F,H) of 14.5 dpc (E,F) and 16.5 dpc (G,H) Pitx2 abccreneo+/- and Rosa26 reporter trans heterozygotes. lacZ-positive cells are indicated by arrows (E,G). Pitx2c expression has been extinguished in Pitx2 daughter cells that would be lacZ positive and are marked with and asterisk (F,H). (I,J) Coronal sections through a 16.0 dpc Pitx2 abccreneo+/- and Rosa26 reporter trans-heterozygotes at slightly different dorsoventral planes. Arrows indicate lacZ-positive cells in myocardium (I) and in interatrial and interventricular septum (J). (K,L) Whole-mount lacZ staining of 14.5 dpc wild-type (K) and Pitx2 abccreneo;c Pitx2 mutant (L), and Rosa26 reporter trans-heterozygous embryos. Arrows indicate lacZ-positive cells. Circled area in L indicates region with fewer lacZ-positive cells. (M,N)Whole-mount lacZ staining of 16.5 dpc wild-type (M) and Pitx2 abccrenoe;c Pitx2 mutant (N), and Rosa26 reporter trans-heterozygotes. Arrows indicate lacZ-positive cells. Circled area in N indicates region with fewer lacZ-positive cells. (O-R) Transverse sections through a 16.5 dpc Pitx2 abccreneo+/- (O,Q) and Pitx2 abccreneo;c (P,R) and Rosa26 reporter trans-heterozygous embryo. More rostral sections show lacZ-positive Pitx2 daughter cells (arrow) in myocardium of both Pitx2 abccreneo+/- (O) and abccreneo;c mutants (P), while more caudal sections near cardiac apex show lacZ-positive cells in abccreneo+/- (Q) but not in abccreneo;c mutants (R) as denoted by the asterisk. (S,T) Transverse sections through a 12.5 dpc Pitx2 abccreneo+/- (S) and Pitx2 abccreneo;abcnull (T) and Rosa26 reporter trans-heterozygote showing lacZ expression in AV cushion in heterozygote (arrow) but absent in the mutant (asterisk). (U) Coronal section through a 14.5 dpc Pitx2 abccreneo;abcnull and Rosa26 reporter trans-heterozygote showing exclusion of lacZ-positive cells from AV cushion (asterisk). (V,W) Coronal section through a 16.5 dpc Pitx2 abccreneo+/- (V) and abccreneo;c (W) and Rosa26 reporter trans-heterozygote showing lacZ-positive cells within valve leaflet in the Pitx2 abccreneo+/- embryo (arrows) but exclusion of lacZ-positive cells from the AV valve leaflets of Pitx2 mutants(asterisk). rv, right ventricle; lv, left ventricle; ivs, interventricular septum; scv, superior caval vein.

View larger version (82K): [in a new window]   Fig. 7. Analysis of pulmonary vein morphology in Pitx2 c-/- embryos and fate mapping with Pitx2 abccreneo allele. (A-D) Scanning electron microscopy of corrosion casts of wild type (A,C) and the Pitx2 c-/- (B,D) embryos. In wild-type embryos, the LSCV drains into the coronary sinus, guarded by the valve of the coronary sinus. The RSCV and ICV are connected to right atrium by thin strips at the valves. The left and right pulmonary veins join to a common pulmonary vein that drains into left atrium. White stars indicate left atrium, just superior to entry of common pulmonary vein (A,C). By contrast, in Pitx2 c-/- embryos, all these veins converge into a common, medial venous sinus (B,D). Stars indicate inferior caval vein, just inferior to entry of pulmonary vein and the left superior caval vein. This embryo also has bilateral inferior caval veins. (E-H) Fate mapping with Pitx2 abccreneo allele. Transverse sections through lungs of 16.5 wild-type (E) and Pitx2 mutant (F). lacZ positive cells marking Pitx2 daughter cells are present in wild type but are severely reduced in mutant (arrows). (G,H) Whole mounts of 16.5 dpc lungs from wild type (G) and Pitx2 mutants (H), showing lacZ-positive cells in pulmonary veins of wild type and reduced staining in mutant (arrows).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter