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First published online 2 February 2005
doi: 10.1242/dev.01640

Development 132, 1137-1146 (2005)
Published by The Company of Biologists 2005
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Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart

Bin Zhou1,*, Bingruo Wu1, Kevin L. Tompkins1, Kathleen L. Boyer1, Justin C. Grindley1 and H. Scott Baldwin1,2

1 Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
2 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA



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  		Fig. 2. (A) Schematic diagram of the Nfatc1 promoter/enhancer reporter constructs. (B) Transient transgenic embryos following X-gal staining documents that the 6.2-kb NheI-XhoI P1 promoter-reporter (NX-lacZ) does not produce endocardial gene expression at E11.5. However, the BB-HSP-lacZ enhancer-reporter, containing 4.1 kb BssHII-BssHII fragment of the P2 (intron 1) regulatory region, linked to the HSP68 minimal promoter, is able to drive expression specifically in the endocardial lumen of the atrioventricular canal (AVC) and in the conal (c) and truncal (t) regions of the developing outflow tract. No expression is detected in the atrium (a) or ventricle (v) or in the extracardiac vasculature. (C) Table summaries this group of transgenic experiments. TG, transgenic embryos; ECS, endocardial-specific expression; ET, ectopic expression.

 


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  		Fig. 4. Analysis of ß-galactosidase activity in embryos from four independent stable transgenic lines shows consistent pro-valve endocardial enhancer activity of the 4.1 kb BssHII-BssHII P2 fragment in whole-mount-stained E8.5 to E12.5 embryos (A-E). X-gal staining is restricted to the endocardial cells in AVC (arrow) and OFT (arrowhead), especially intensified at later stages in the regions of forming valves and septa. (F-J) Sectioning of stained E8.5, E9.5, E12.5 and E14.5 embryos highlights this enhancer activity for the pro-valve endocardial cells of the forming valves. ao, aorta; av, aortic valve; pt, pulmonary trunk; pv, pulmonary valve; la, left atrium; ra, right atrium; lv, left ventricle; rv, right ventricle; mv, mitral valve; tv, tricuspid valve.

 


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  		Fig. 1. (A) Structure of the 5' regulatory region of mouse Nfatc1, which contains two independent promoters, P1 and P2, for transcription of Nfatc1.{alpha} and Nfatc1.ß isoforms, respectively. (B) RT-PCR analysis using exon (isoform)-specific primers showing that the Nfatc1.{alpha} isoform is abundant in the cultured primary E11.5 endocardial cells (ECC) and its transcripts is detected by a 35-cycle amplification, whereas the Nfatc1.ß isoform is detected only after a 40-cycle amplification. The structures of the isoforms are shown on the left.

 


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  		Fig. 3. X-gal staining in transient transgenic embryos shows endocardial-specific enhancer activity of the 4.1 kb BssHII-BssHII P2 fragment in whole-mount E9.5 embryos (A) and sections of whole-mount-stained E10.5 (B) and E11.5 (C) embryos. ß-Galactosidase activity is restricted to the pro-valve endocardial cells in AVC (arrowhead) and OFT (arrow). The enhancer is not activated in the mesenchymal cells derived from transformed endocardial cells in the AVC and OFT endocardial cushions. Top row, OFT; bottom row, AVC. a, atrium; c, conus; t, truncus; v, ventricle.

 


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  		Fig. 5. Deletional analysis of the endocardial enhancer. (A) Schematic depicting the unique features of the 4.1 kb BssHII-BssHII intron 1 region and the deletional reporter constructs (d1-d7). (B) Representative whole-mount staining of E11.5 embryos demonstrates the presence of the pro-valve endocardial enhancer activity in d1-d5 but not d6 and d7. (C) Cross-sectional analysis of d5 transgenic embryos (E11.5) shows that the enhancer activity is exclusively restricted to the pro-valve endocardial cells in AVC (arrow) and OFT (arrowhead). The enhancer is not activated in the transformed cells of the endocardial cushions and endothelial cells outside of the heart.

 


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  		Fig. 6. (A) The activation of P1 promoter by the 781 bp sequence of the P2 regulatory region. Two short conserved elements (mouse/human) are located in this sequence with a cluster of binding sites for known transcription factors. (B) A schematic shows deletional analysis of the conserved putative endocardial enhancers (ECEs). (C-F) Whole-mount staining of E11.5 embryos (C,D) or isolated hearts (E,F) demonstrates that the ECE-HSP-lacZ, but not the ECE{Delta}-HSP-lacZ reporter (data not shown), is sufficient to direct gene expression specifically in the pro-valve endocardial cells of forming valves and septa. ao, aorta; pt, pulmonary trunk; la, left atrium; ra, right atrium; lv, left ventricle; rv, right ventricle; avc, atrioventricular canal; arrow, AVC; arrowhead, OFT.

 


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  		Fig. 7. Autoregulation of Nfatc1 enhancer activity. The BB-HSP-lacZ reporter transgenic line was crossed into the existing Nfatc1-null mutant line. Compared to the heterozygous littermates (A-C), inactivation of Nfatc1 greatly reduced the pro-valve endocardial enhancer activity of the BB-HSP-lacZ reporter construct (D-F). A consistent reduction of endocardial lacZ expression is shown in the OFT (arrow) and AVC (arrowhead) of the E11.5 heart when crossed into Nfatc1-null background

 


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  		Fig. 8. (A) Primary culture of embryonic (E11.5) endocardial cells (ECCs). ECCs form a colonized monolayer surrounded by fibroblastic-like OP9 feeder cells at passage 1 (p1). At p3, ECCs exhibit uniform, endothelial-like morphology with a typical `cobblestone' appearance. Approximately 80% of the ECCs expresses nuclear localized Nfatc1 (green; negative cells are indicated by arrowheads in DAPI staining). Negative control (no primary antibody) is shown in ECC(-Ab). (B) The 781 bp enhancer region. Two short stretch conserved sequences, ECE1 and ECE2, are shown with Nfat-binding sites highlighted in red and deleted GG or CC bi-nucleotides of core binding site are underlined. The arrows indicate the location of primers for the ChIP assays. (C) EMSA demonstrating that mutation of the Nfat-binding sites in either the N1 or N2 regions results in attenuation of Nfatc1 binding (arrows). (D) Results of the ChIP assay document PCR amplification of the chromatin region encompassing ECE1 and ECE2 from wild-type ECCs following immunoprecipitation with an Nfatc1 antibody (7A6) (arrow) and absence of chromatin amplification without antibody or using cultured Nfatc1-null ECCs.

 


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  		Fig. 9. Whole-mount (A-D) and section (E-H) analysis reveals that mutation of the Hox site results in activation of the enhancer outside the pro-valve endocardial cells. Activity of the mutated enhancer is observed in umbilical cord (uc), intersomitic artery (isa) (A) and the head vasculature (C). Sectional analysis shows lacZ expression in the endothelial cells of head vasculature (inset in C), ductus venous of E12.5 (F, arrow) and E11.5 embryos (H, arrowhead in inset). Within the heart, aberrant enhancer activity was observed in the endocardial cells of the trabeculated ventricular outlet in E12.5 (E, arrowhead in the inset) and E11.5 hearts (G, marked by an asterisk), and sinus venous valves (G, arrowhead). The dysregulated enhancer activity was also observed in the cushion mesenchymal cells (E, inset). Activity of the mutated enhancer appeared to be increased in the pro-valve endocardial cells (B and D, arrowheads). (I) A table summarizes transient transgenic experiments with the mutated constructs. TG, transgenic embryos; EC, endocardial cell expression; ET, ectopic expression.

 

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© The Company of Biologists Ltd 2005