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

First published online 8 April 2004
doi: 10.1242/dev.01086

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 Stottmann, R. W. Articles by Klingensmith, J. Search for Related Content PubMed PubMed Citation Articles by Stottmann, R. W. Articles by Klingensmith, J.

BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium

¹ Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
² Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
³ Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA

View larger version (83K): [in a new window]   Fig. 1. Wnt1-Cre recombination in the neural crest cells is marked by the expression of ß-galactosidase in Wnt1-Cre; R26R embryos. (A,B) At 4 somites, Wnt1-Cre mediates loxP recombination in the forming NCCs of the hindbrain, but not the neural tube. (C,D) Multiple neural crest cells are positive for ß-galactosidase activity in the neural tube at 6 somites. (E,F) NCCs are more plentiful at the eight-somite stage and are present at more caudal levels of the neural tube. Black arrowheads indicate the NCCs. B,D,F are dorsal, higher magnification views of embryos shown in A,C,E, respectively. Anterior is towards the top in B, towards the left in D,F. (G) A transverse section through the first pharyngeal arch (pa) of an embryo at similar stages to E,F. (H) E9.0 (15 somites) and (I) E10.5 embryos show caudal progression of Wnt1-Cre activity and increased NCC induction and migration.

View larger version (54K): [in a new window]   Fig. 2. Ablation of Bmpr1a in neural crest cells. We have used the Wnt1-Cre transgene to ablate Bmpr1a activity in mouse NCCs. In the mating scheme outlined here (A), one quarter of the resulting embryos cannot transduce signals through BMPRIA in the neural crest. (B) PCR primers designed to detect recombined Bmpr1a alleles from genomic DNA show recombination from 15 s embryos only in the presence of Cre. (C-F) At E11.0, wild-type and Wnt1-Cre; Bmpr1aflox/null (mutant) embryos were morphologically indistinguishable. (G-J) Dissections at E11.5 showed an apparent lack of blood flow in mutant yolk sacs despite the presence of yolk sac blood vessels (I, arrows). Mutant embryos at E11.5 (J) show pooling of blood in peripheral vessels, heart, and liver. (K) Genotyping of dissected embryos reveals that Wnt1-Cre; Bmpr1aflox/null embryos are fully represented (Mendelian expectation is 25%) until E12.5. At E12.5, half of embryos recovered are in the process of resorption (red) and half are clearly necrotic. No mutants have been recovered after E12.5.

View larger version (85K): [in a new window]   Fig. 3. NCCs develop in BMPRIA NCC mutant embryos. (A,C,E) Wild-type, stage-matched control embryos for Wnt1-Cre; Bmpr1aflox/null mutants (B,D,F). (A,B) Expression of Crabp1 and other neural crest markers showed normal NCC specification in mutants. (C,D) Immunostaining for neurofilaments with the 2H3 antibody showed proper differentiation of NCCs in mutants. (E,F) Mutant embryos expressing the R26R reporter, to label NCCs, show a normal distribution of NCCs and proper formation of many target tissues, including pharyngeal arches (arrows) and dorsal root ganglia (arrowheads).

View larger version (71K): [in a new window]   Fig. 4. Normal vasculature in BMPRIA NCC mutants. (A) No Wnt1-Cre expressing cells were observed in yolk sacs (corresponding embryo in A'). Expression of a Tie2-lacz transgene marking endothelial cells showed no structural defect in mutant yolk sacs (B) or in mutant embryos (D) compared with controls (C). (E-J) Immunostaining for the endothelial marker, PECAM1 (E,F; sagittal sections through descending aorta) and for smooth muscle actin (G,H; coronal sections through pharyngeal arch arteries, highlighted by arrowheads; and I,J, coronal sections through outflow tract tissue) further confirm the lack of structural defects in peripheral vessels of mutants. p, pharynx. All paired images are shown at identical magnification.

View larger version (94K): [in a new window]   Fig. 5. BMPRIA mutant embryos show cardiac outflow tract defects. (A-D) Dissections at E9.5 show mutant embryos (B,D) to have shortened outflow tract and future ventricular tissue (rv and lv indicate future right and left ventricle, respectively; broken lines indicate limit of the future right ventricle). These defects continue through E10.5 (E-H) and E11.5 (I-L) with reduced contributions of NCCs to outflow tract tissue clearly visible by E10.5 (H; black arrows highlight extent of NCC migration into outflow tract). (M-P) Frontal sections of E11.0 Wnt1-Cre;Bmpr1aflox/null; R26R embryos (NCCs stained blue) show no occlusion of the outflow tract lumen (N) or aortic arch arteries (P, black arrowheads) but do reveal a reduction in endocardial cushion (ec) size compared with wild type (M). (Q,R) Tie2-lacZ transgene expression marks endothelial cells, highlighting the lack of outflow tract septation in mutants at E12.0: red arrowhead indicates aorta, blue arrowhead indicates pulmonary artery, purple arrowhead indicates persistent (unseptated) truncus arteriosus. (S,T) Injection of ink into E11.5 ventricles shows dispersion through aortic sac to peripheral vasculature in both control and mutant embryos. Note the hypoplastic arch arteries evident in mutants (T; iii, third aortic arch artery; iv, fourth aortic arch artery). (U,V) Frontal section through a necrotic Wnt1-Cre;Bmpr1aflox/null embryo at E12 still reveals a blood filled, but unoccluded outflow tract. White arrows indicate lumenal edge. All paired images are shown at identical magnification.

View larger version (61K): [in a new window]   Fig. 6. Embryos lacking BMPRIA in NCCs show defective ventricular myocardium by the 45-50 somite stage. Transverse sections of ventricles from embryos at E11.0 [36-40 somites (A-D)] and E11.5 [45-50 somites (E-H)]. The lower row shows higher magnification images from the sections immediately above. (B,D,F,H) Mutant Wnt1-Cre;Bmpr1aflox/null embryos; (A,C,E,G) wild-type (control) littermates. (A-D) Histological analysis of ventricular tissue revealed no mutant phenotype at E11.0, with mutant hearts (B,D) having similar degrees of compact myocardium (cm) as control hearts (A,C). (E-H) Control hearts at E11.5 (E,G) have robust compact myocardium as well as trabeculated myocardium occupying much of the ventricular chamber. Mutant hearts (F,H) have reduced compact and trabeculated myocardium, observable prior to global necrosis (inset in F shows the embryo from which heart was taken; embryo appears healthy). (I) Proliferation rates were measured by counting cells stained for anti-phosphohistone H3 (pHH3+) as a percentage of the total at E10.5. A significant reduction (asterisks; P<0.015) was seen in mutant embryos. Scale bars: 250 µm for A,B,E,F; 32 µm for C,D,G,H.

View larger version (74K): [in a new window]   Fig. 7. NCCs populate epicardium and ventricular myocardium. (A-C) Wnt1-Cre; R26R embryos showed blue ß-galactosidase-positive cells (arrowheads) immediately caudal to the heart (abutting the septum transversum) and on the cardiac ventral surface, in the epicardium at E9.5 (A), E10.5 (B) and E11.5 (C). A transverse section of the heart (C,F) reveals stained cells in the epicardium (epi) and myocardium (m). ivs, interventricular septum; pp, parietal pericardium. (D,E) Neonatal Wnt1-Cre; R26R hearts show ß-galactosidase-positive cells (arrowheads) contributing to the coronary vasculature, consistent with an epicardial lineage (inset shows entire heart shown in detail in D). (E) Sections through E17 hearts (relative plane of section indicated in D inset) indicate ß-galactosidase-positive cells surrounding an artery containing red blood cells. (G,H) Similar ß-galactosidase-positive cells are seen in the epicardium of PO-Cre; R26R embryos. (I) RT-PCR analysis to assay for expression of Wnt1-Cre in ventral tissues. From each of two Wnt1-Cre; R26R embryos (Cre+) and one control R26R embryo (Cre–), RNA was isolated from tissues ventral to the dorsal aorta (ventral), including the heart and septum transversum, and the neural tube (neural). RNA was reverse-transcribed and subjected to PCR amplification of Cre or Hprt (positive control). HPRT was amplified from all samples except water (H2O) or those prepared without reverse transcriptase (RT–). Cre was amplified only in the neural tube samples from Cre+ embryos. (J-L) Bmp4-lacz expression. Whole-mount embryos at E9.5 (J) and transverse sections at E9.5 (K) and E11.5 (L), showing high levels of Bmp4 expression in the septum transversum (s.t.) and parietal pericardium (pp), but not epicardium. cm, compact ventricular myocardium.

View larger version (74K): [in a new window]   Fig. 8. Epicardium appears normal in BMPRIA mutants. (A,B) NCCs appear in the same limited quantities in mutants as in control embryos at E9.5 (A) and E11.5 (B: compare to Fig. 7A,B). (C-F) Histological analysis (transverse sections) of the epicardium (e) at E11.5 shows no significant difference in the appearance of the epicardium of mutant tissue (D,F) which shows a clear ventricular myocardial phenotype (D, ivs: interventricular septum, pp: parietal pericardium). Dotted lines in E,F delineate the boundary between epicardium and compact ventricular myocardium. (G,H) In situ hybridization for epicardin also shows no defect in mutant epicardium (insets show that hybridization is specific to epicardial layer, pericardium was removed prior to in situ hybridization). (I,J) Immunohistochemistry for the WT-1 protein shows no decrease in expression in the mutant in either the epicardium or the coelomic epithelial cells of the parietal pericardium (cm, compact ventricular myocardium). PA1, first pharyngeal arch; a, atrium; e, epicardium. All paired images are shown at identical magnification.

View larger version (40K): [in a new window]   Fig. 9. A model for the promotion of ventricular myocardium proliferation by BMP signaling in neural crest derivatives. (A) A limited population of cells in a Wnt1-Cre lineage (blue) migrates to the epicardium, located between the pericardial wall and ventricular myocardium. (B) Within the epicardium, these cells transduce signals through BMPRIA, resulting in the production of a proliferative signal, both Bmpr1a dependent (solid arrows) and Bmpr1a independent (broken arrows), for the underlying ventricular myocardium. BMP ligands for BMPRIA are expressed in both the pericardium and ventricular myocardium. In mutant embryos (C), although BMPs and cells of the Wnt1-Cre lineage are both still present, BMPRIA is not present in Wnt1-Cre positive cells. This results in decreased production of the unidentified trophic factor downstream of BMPRIA, leading to decreased myocardial proliferation. (D) An alternative explanation for the myocardial proliferation defects involves the cardiac neural crest of the outflow tract (OFT). These cells may produce a long range signal (red arrows) stimulating proliferation throughout the myocardium. (E) In mutant embryos, decreased migration of NCCs into the OFT results in decreased production of myocardial proliferation signals.