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Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption

¹ Department of Neuroscience, University of Pennsylvania School of Medicine, 1115 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104, USA
² Department of Medicine, Cardiovascular Division, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
³ The Rockefeller University, New York, New York 10021, USA
^* Author for correspondence (e-mail:raperj{at}mail.med.upen.edu )

View larger version (36K): [in a new window]   Fig. 1. Generation of Sema3C mutant mice and mRNA analysis. (A) A diagram of the targeting vector used to disrupt Sema3C is shown. A majority of the targeted exon was replaced by a cassette that included an internal ribosomal entry site followed by a tau-lacZ gene and a neomycin resistance cassette flanked by loxP sites (see text). (B) Southern blot analysis shows wild-type (lane 1) and three targeted ES cell lines (lanes 2, 3 and 4). (C) PCR genotyping of wild-type (WT), heterozygous (HET) and Sema3C mutant (KO) mice. PhiX, X174 DNA-HaeIII digest (D) Schematic of Sema3C transcript showing the locations of primers used for RT-PCR analysis. White inset represents the coding region replaced by the targeting vector. Primer sets 1, 2 and 3 (see text) used in RT-PCR reactions are color coded in blue, red, and green, respectively. (E) Analysis of RT-PCR from mRNA extracted from whole E12.5 wild-type and knockout embryos reveals that Sema3C mRNA is produced in mutants but that the mutant message does not contain the exon replaced by the targeting construct (primer set #3; `green' product).

View larger version (118K): [in a new window]   Fig. 2. Cardiovascular defects in Sema3C mutant mice. Gross dissections of P0 wild-type (A) and Sema3C mutant (B-D) mice with atria removed facilitates visualization of the great arteries. The arch of the aorta is indicated by an arrowhead. The ductus arteriosus is labeled with an asterisk in each panel. The great vessels are labeled as follows: a, ascending aorta; b, brachiocephalic artery; c, left common carotid artery; d, left subclavian artery; e, descending aorta; f, pulmonary artery and g, persistent truncus arteriosus (PTA). (B) Fusion of aortic and pulmonary trunks (PTA) and type B interruption of the aortic arch in a Sema3C mutant mouse. The left common carotid artery originates from the PTA. (C) PTA and type C interruption of the aortic arch in a Sema3C mutant mouse. The left common carotid artery originates from the descending aorta. (D) Duplication of the left common carotid artery in a Sema3C mutant mouse. Insets in each frame present a schematic of the aortic arch with the aorta and tributaries in red, the ductus arteriosus in yellow, and the pulmonary artery in blue.

View larger version (102K): [in a new window]   Fig. 3. Cardiovascular phenotype characterized by corrosion casting. Methyl methacrylate was injected into the left ventricle of anesthetized P0 wild-type and mutant mice. (A) Cast of wild-type showing normal vascular architecture and a nearly closed ductus arteriosus (arrowhead). (B) Cast of a Sema3C mutant mouse reveals interruption of the aortic arch with normal septation of aorta and pulmonary trunks. The pulmonary vasculature is not filled as the ductus arteriosus has closed. Note the absence of filling of the descending aorta. (C) Another cast of a Sema3C mutant P0 mouse shows persistent truncus arteriosus, interruption of the aortic arch, and a single four-leaflet valve (inset-cusps are labeled 1-4). The descending aorta has filled because the ductus arteriosus (arrowhead) has not yet closed completely. (D) Hematoxylin and Eosin-stained paraffin wax section through the heart of a P0 Sema3C mutant heart demonstrates a ventricular septal defect in the membranous portion of the ventricular septum. (E) A more posterior section reveals an intact muscular ventricular septum. (F) Higher magnification of the ventricular wall demonstrates normal myocardium and trabeculations in Sema3C mutant mice. RV, right ventricle; LV, left ventricle; a, ascending aorta; b, brachiocephalic artery; c, left common carotid artery; d, left subclavian artery; e, descending aorta; f, pulmonary artery.

View larger version (88K): [in a new window]   Fig. 4. Sema3C expression in the cardiac outflow tract. In situ hybridization using digoxigenin-labeled antisense Sema3C riboprobe at (A,D) E10.5, (B) E11.5 and (C,E,F) E12.5. Sema3C is expressed in the conotruncus throughout the period of outflow tract septation and remodeling of branchial arch arteries. (A-C) Whole-mount images reveal strong Sema3C expression restricted to the outflow tract within the heart. (D) Sema3C is expressed by cells surrounding the branchial arch arteries (long arrow) and in the left lateral wall of the conus (short arrow). (E) At E12.5, Sema3C is expressed by mesenchymal tissue surrounding the great vessels and myocardium in the right ventricular outflow tract. (F) In a more caudal section closer to the left ventricular outflow tract, Sema3C mRNA is expressed in the condensed mesenchyme of the aorticopulmonary septation complex. aAorta, ascending aorta.

View larger version (147K): [in a new window]   Fig. 5. Altered cardiac neural crest population of outflow tract endocardial cushions in Sema3C mutant embryos. E12.5 (A,C,E) wild-type or (B,D,F) Sema3C mutant littermates were sectioned and probed for markers of cardiac neural crest cells. mRNAs for (A,B) Foxc1, (C,D) endothelin receptor A (EdnrA), or (E,F) plexinA2 were visualized with 35S-labeled riboprobes. In wild-type embryos cardiac crest cells are detected populating the endocardial cushions of the cardiac outflow tract (arrows) and surrounding the aortic arch arteries, while in Sema3C mutant embryos few if any cardiac crest cells are detected in comparable locations (arrows). Ao indicates the aorta and PA the pulmonary artery.

View larger version (162K): [in a new window]   Fig. 6. Normal formation of branchial arch arteries in Sema3C mutant embryos. Hematoxylin and Eosin-stained sections of (A,C,E,G,I) wild-type and (B,D,F,H,J,K) Sema3C mutant littermate embryos. (A,B) The left fourth branchial arch (arrows, 4) is apparent in both wild-type and mutant E10.5 embryos. (C,D) The right fourth branchial arch (arrows, 4) is present in wild-type and mutant E10.5 embryos. (E,F) Branchial arches 2, 3 and 4 (arrows) appear normal in wild-type and mutant E10.5 embryos cut in sagittal section. (G,H) The left fourth aortic arch is present in wild-type and mutant E11.5 embryos (arrows, 4). (I) An intact ventricular septum and the proximal aorta (Ao) arising from the left ventricle (LV) in a wild-type E13.5 embryo. J and K show a ventricular septal defect (VSD, in J) and the proximal aorta arising from the right ventricle (RV, in K) in a Sema3C mutant littermate. This Sema3C mutant embryo had a double outlet right ventricle. Ht, heart.