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doi: 10.1242/10.1242/dev.00181

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BMP signaling is required for septation of the outflow tract of the mammalian heart

¹ Department of Orthopaedic Surgery, UCLA School of Medicine, Los Angeles, CA 90095, USA
² Department of Human Genetics, UCLA School of Medicine, Los Angeles, CA 90095, USA
³ Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA 90095, USA
⁴ Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095, USA
⁵ Department of Molecular Cell and Developmental Biology, UCLA, Los Angeles, CA 90095, USA

View larger version (27K): [in a new window]   Fig. 1. A functional truncated protein is encoded by the Bmpr2E2 allele. (A) Targeting strategy. Exon 2 of Bmpr2 was replaced by a neomycin resistance cassette. A, AatII; B, BamHI; Bg, BglII; E, EcoRI; Rv, EcoRV; K, KpnI; X, XbaI; Xh, XhoI. (B) Southern blot showing genotyping of pups with the internal probe. (C) Amino acid sequence comparison of the extracellular domains of mouse ActRII, TGFßRII and Bmpr2. The region encoded by exon 2 is boxed and conserved residues involved in 3D-structure formation are in red. This includes three out of seven cysteine residues, which are conserved among all type II receptors, that are required to maintain the conformation of the extracellular domain, and two out of five hydrophobic amino acids thought to participate in the hydrophobic interactions that stabilize the structure. (D) Structure of the Bmpr2 protein. Exon 1 encodes the initiator methionine. Exons 2 and 3 each encode half of the extracellular ligand-binding domain. Exon 4 encodes the transmembrane domain. The serine threonine kinase domain is encoded by exons 5-11. Exons 12-13 encode an intracytoplasmic tail of unknown function. Deletion of exon 2, which is in frame, should generate a truncated protein. (E) RT-PCR on RNA from wild type (+/+), heterozygous (+/-) and E2 mutants (-/-) with primers encompassing the deletion (exons 1-5) and downstream of the deletion (exons 3-5) shows that the Bmpr2E2 allele is transcribed. (F) Transfection of the mutant or wild-type Bmpr2 cDNA expression plasmid confers BMP responsiveness to Mv1Lu cells. BMP signaling activity was assayed with a reporter construct that contains luciferase under the control of a BMP-responsive element from the promoter of the Msx2 gene (Daluiski et al., 2001; Liu et al., 1994).

View larger version (75K): [in a new window]   Fig. 2. Skeletal phenotype of Bmpr2E2/E2 mutants. (A-C) Three siblings from a litter dissected at E15.5 showing that mutants die at various stages of gestation. (A) wild type (WT), (B) mutant dead at E14.5, (C) mutant dead at E12, as judged by the extent of limb development. (D-F) Cleared skeletal preparations of two siblings, recovered dead at birth. Dorsal views of the base of the skulls of WT (D) and mutant (E) illustrate the major ossification delay of the interparietal bone (Ipar), and of the ventral processes of the cervical vertebrae 1-4, in particular of the atlas (At). Socc, supraoccipital bone. (F) Ventral views of the half vertebral columns of wild type (left) and mutant (right) showing the absence of the thirteenth rib in the mutant (asterisk). Thoracic (T1-T13) and lumbar (L1) vertebrae are numbered. Note the ossification delay of the ventral and lateral ossification processes of the vertebrae in the mutant.

View larger version (44K): [in a new window]   Fig. 3. External aspect of the hearts of Bmpr2E2/E2 mutants. (A,B) Frontal views of wild-type (A) and Bmpr2E2/E2 mutant (B) hearts at E16 showing a normally septated ascending arch of the aorta (Ao) and pulmonary trunk (PT) distally, but not proximally to the heart. The black arrowhead in A indicates the semilunar valves of the PT. The pink arrowhead in B indicates a dimple at the apex of the ventricle, a common sign of ventricular septal defect. (C-G) Ink-injected E13.5 hearts. Left lateral views of whole-mount India ink-injected wild-type (C) and Bmpr2E2/E2 (D) hearts. The arrowhead in C indicates the semilunar valve level. Semilunar valves are absent in mutants. The white asterisk in D indicates the non-septated conotruncus of the mutants. DAo, descending aorta. (E-G) Interruption of the aortic arch (purple arrow) of varying severity in two mutants (F,G) is visible between the left common carotid artery (LCC) and the left subclavian artery (LSA) in the ink-injected outflow tract (upper panel) and derived cartoons (lower panels). The ductus arteriosus (Du) is enlarged. Pulmonary arteries (PAs) stem normally off of the pulmonary trunk in the mutants. RCC: right common carotid artery. (H,I) Cartoon of a WT (H) and Bmpr2E2/E2 mutant (I) heart showing the association of persistent truncus arteriosus (PTA), ventricular septal defect (VSD) and interrupted aortic arch type B (IAA) that constitute the type A4 persistent truncus arteriosus (Jacobs, 2000). Ao, aorta; DAo, descending aorta; Du, ductus arteriosus; IVS, interventricular septum; LA and RA, left and right atria, respectively; LCC and RCC, left and right common carotids, respectively; LPA and RPA, left and right pulmonary arteries, respectively; LSA, left subclavian artery; PT, pulmonary trunk.

View larger version (133K): [in a new window]   Fig. 4. Histology sections through the hearts at E12.5, E14.5 and E16.5. (A-D) Transverse sections through E14.5 wild-type (A) and Bmpr2E2/E2 (B-D) hearts show a common origin to the aorta (Ao) and pulmonary trunk (PT) and total absence of semilunar (SL) valves (B), while the valves of the atrioventricular canal develop normally (arrowhead in C). A ventricular septal defect is associated (arrowhead in D). The left subclavian artery was occasionally retro-esophageal (arrowhead in B). Oe, Esophagus. T, Trachea. (E-H) Sagittal sections through E16.5 wild-type (E,G) and mutant (F,H) hearts show the aorta (E,F) and the pulmonary trunk (G,H) exiting the same ventricle in the mutants. The pulmonary trunk is the main vessel connecting to the descending aorta (DAo), as a result of the hypoplasia of the aortic isthmus. Note that the thymus (Th) is of normal size in the mutant. (I-N) Series of frontal sections (dorsal to ventral) of an E12.5 wild-type (top panels) and a mutant (lower panels) heart show that, posteriorly, ventricular septation is normal (I), but at the base of the pulmonary trunk, a large ventricular septal defect is visible (black arrow in J). One of the conal cushions appears to form correctly (arrowhead in mutant, K) but most conal tissue (asterisk in wild type, J) is already missing (K). The outflow tract starts septating into two arteries at about the same level that the semilunar valves form in the wild type (L), and aorta and pulmonary trunk are fully septated and in normal relative position beyond that point (M). The aorta branches into the right subclavian artery, while the pulmonary arteries (PAs) arise normally from the pulmonary trunk (N).

View larger version (108K): [in a new window]   Fig. 5. Impaired OFT cushion growth in Bmpr2E2/E2 mutants. (A-D) Sagittal sections at E11.5 show that the conotruncal ridges (arrow) form in the mutant (B), but not to the extent that they do in the wild type (A). (C,D) Higher magnifications of the boxed areas in A,B, respectively. (E-G) Immunostaining for PCNA in OFT cushions of E11.5 wild type (E) and a mutant (F,G) littermate. The arrowheads indicate the myocardium. (H) Tbx1 expression at E10.5 in a Bmpr2E2/E2 mutant is found in the posterior part of the otic vesicle (ov), head mesenchyme (hm) and the mesenchyme surrounding the paired dorsal aortae (pm), as well as the dorsal wall of the aortic sac, with higher intensity in the posterior-most region (arrowheads) as described in wild type (Garg et al., 2001; Merscher et al., 2001; Vitelli et al., 2002). Anterior is towards the left and ventral towards the top. paI, pharyngeal arch I; Ve, ventricle; lb, left anterior limb bud. (I,J) Pax3 expression in neural crest cells (arrowhead) migrating in the neck region at E10.5 was normal in mutants (I, wild type; J, mutant). Pharyngeal arches are numbered in Roman numerals. Ve, ventricle. (K,L) Smooth muscle actin-positive neural crest cells (arrows) reach the outflow tract endocardial ridges in both the wild type (K) and mutant (L) E11.5 embryos. (M,N) At E13.5, in wild type embryos, Ctgf expression was detected in the pulmonary trunk and aorta, predominantly in the cell layers closer to the lumen, as well as in a punctate pattern in the ventricles (M). Myocardial expression is predominantly in the trabecular zone of the ventricles. Expression was not modified in mutants (N). (O,P) The expression of periostin, a BMP-regulated gene, was downregulated in the endocardial ridges of E11.5 mutants (P) compared with wild-type littermates (O). To help visualize the tissues in absence of background expression, P is an overlay of two different light exposures of the same section.