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

First published online 18 October 2006
doi: 10.1242/dev.02625

This Article Summary Full Text Full Text (PDF) Supplementary Material 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 Risebro, C. A. Articles by Riley, P. R. Search for Related Content PubMed PubMed Citation Articles by Risebro, C. A. Articles by Riley, P. R. Social Bookmarking                         What's this?

Hand1 regulates cardiomyocyte proliferation versus differentiation in the developing heart

¹ Molecular Medicine Unit, UCL Institute of Child Health, London WC1N 1EH, UK.
² Developmental Biology Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

View larger version (28K): [in a new window]   Fig. 1. Incorporating the Tet system into the Hand1 locus to generate an overexpression model. (A) Wild-type Hand1 consists of two exons (boxes: non-coding regions indicated in white, coding region is shaded, bHLH domain is shown in black) separated by a 1.5 kb intron. (B) The targeted Hand1 locus contains the transactivator tTA (red) under the control of the endogenous Hand1 promoter. (C) The tet-responsive Tre2 (blue)-Hand1 cDNA (black) upstream of a ß-globin poly(A) (green) randomly integrated as a transgene. The primers PrA, PrB, PrC and PrD are as shown in A,B. (D) Southern blot analysis to confirm the targeting of the endogenous Hand1 locus for the driver and varying copy number founder lines for the responder. (i) BglII digest with the 5' probe illustrated in A. (ii) HindIII digest with the probe illustrated in C. (E) PCR genotyping of F1 following driver x responder matings (asterisk denotes compound driver/responder heterozygote). (F) Quantitative real-time PCR on RNA from compound heterozygote embryos at E9.5 reveals twice as much expression of Hand1 compared with control littermates containing only driver or responder. Bars represent mean±s.e.m. of three real-time qRT-PCR experiments on single founder overexpression and control embryos. co, control; ns, non-specific; oe, overexpression.

View larger version (70K): [in a new window]   Fig. 2. The targeted tTA transactivator is expressed in an equivalent spatial pattern to endogenous Hand1. Whole-mount RNA in situ hybridisation analysis of Hand1 (A) and tTA (B) in control embryos at E9.5. (C,D) Frontal sections through the distal OFT region. (E-H) Sagittal sections through the first branchial arch (E,F) and heart (G.H). as, aortic sac; ba, branchial arch; oft, outflow tract; lv, left ventricle. Scale bars: in B, 200 µm for A,B; in H, 50 µm for C-H.

View larger version (114K): [in a new window]   Fig. 3. Primary heart tube expansion in Hand1-overexpressing embryos at E8.0-8.5. Bright-field whole-mount left lateral views of hearts from somite-matched Hand1-overexpressing (oe) embryos and control (co) littermates dissected pre-embryonic turning (nine somites; A,B) and during turning (12 somites; C,D). Haematoxylin and Eosin stained frontal sections through the heart tube of a control (E-H) and overexpression embryo (I-L) at the 12-somite stage. Whole-mount in situ hybridisation for Hand1 on control (M,O) and overexpression embryonic hearts (N,P). (A,B) The primary heart tube pre-turning is expanded (white arrowheads) and early stages of looping are accentuated in overexpressing embryos compared with control. Extension and exaggerated ventral and left lateral displacement of the heart tube can be seen as looping progresses during turning (depicted by white arrows; C,D; compare E-H with I-L). (M-P) Confirmation of appropriate spatial overexpression of Hand1 in the mutant embryos at the 12-somite stage. co, control; oe, overexpression; s, somites. Anterior (A) - posterior (P) and left (L) - right (R) axes are indicated for E-L. Scale bar: in D,P, 50 µm for A-P.

View larger version (85K): [in a new window]   Fig. 4. Hand1 overexpression promotes extension of the distal OFT, extraneous cardiac looping and impaired LV development. Rendered optical projection tomography of embryos at E9.5 (pericardium removed), viewed from left, ventral and right aspects, in control embryos (containing just the responder transgene; A-C), in Hand1-overexpressing embryos with a moderate phenotype (D-F) or in severe phenotype embryos (G-I). Frontal sections through the hearts of control (J) and moderate Hand1-overexpressing embryos (K) in the planes depicted in B and F, respectively. Sagittal sections through the hearts of control (L) and severe phenotype Hand1-overexpressing embryos (M) in the planes depicted in C and H, respectively. co, control; lv, left ventricle; oe, overexpression; oft, outflow tract. Scale bar: 100 µm in A.

View larger version (109K): [in a new window]   Fig. 5. Hand1 overexpression promotes extension of the distal OFT, extraneous cardiac looping and impaired LV development. (A-D) Hand1 overexpression in somite-stage matched embryos at E9.5 leads to moderate (B) and significant (C) extension of the distal OFT compared with control littermates (A); quantified in relative units (D), measurements are mean±s.e.m., values of n=9 (A), n=11 (B) and n=5 (C), based on distance depicted by black lines in A-C, *P<0.05, **P<0.01. The heart tube is extended (E) and displaced ventrally (F; red arrowheads) in overexpression embryos compared with controls. Looping is accentuated and highly convoluted (G) and, in severe cases, the presumptive LV (red asterisk) is small and necrotic (H). co, control; L, left; L/V, left-ventral; oe, overexpression; R, right. Scale bars: 100 µm in C,H; 200 µm in F.

View larger version (157K): [in a new window]   Fig. 6. Contribution of extracardiac cells from the AHF is unaffected in Hand1-overexpressing embryos. RNA in situ hybridisation analysis of Isl1 and Mef2c in control embryos (A,C,E,G,I,K) and moderate phenotype Hand1-overexpressing embryos (B,D,F,H,J,L) at E9.5. Right lateral views of whole-mount embryos (A,B,G,H). Sagittal sections of the control (C,E,I,K) and Hand1-overexpressing hearts (D,F,J,L). There are no apparent differences in expression of the AHF marker Isl-1 between control and overexpressing embryos (compare A,C,E with B,D,F) or in expression of markers of AHF derivatives, as indicated by Mef2c (compare G,I,K with H,J,L). aa, aortic arch(es); at, atrium; ba, branchial arch; co, control; oe, overexpression; oft, outflow tract; rv, right ventricle. Scale bars: 200 µm in A for A,B,G,H; 50 µm in E for C-F,I-L.

View larger version (81K): [in a new window]   Fig. 7. Markers of cardiomyocyte differentiation are down regulated in Hand1-overexpressing embryos. RNA in situ hybridisation analysis of Anf and Wnt11 in control embryos (A,C,E,G,I) and moderate phenotype Hand1-overexpressing embryos (B,D,F,H,J) at E9.5. Sagittal sections of the control (C,I) and Hand1-overexpressing hearts (D,J). Boundary of Anf expression in proximal outflow tract of overexpressing embryos is depicted by a broken red line in F. Wnt11 in distal outflow tract (black and white arrowheads) is downregulated in overexpressing embryos (H,J) compared with control embryos (G,I). at, atrium; co, control; do, distal outflow tract; lv, left ventricle; oe, overexpression; oft, outflow tract; po, proximal outflow tract. Scale bar: 100 µm in A.

View larger version (90K): [in a new window]   Fig. 8. Primary heart field markers are downregulated in Hand1-overexpressing embryos. RNA in situ hybridisation analysis of Nkx2.5 and Gata4 in control embryos (A,C,E,G,I,K,M,O) and moderate phenotype Hand1-overexpressing embryos (B,D,F,H,J,L,N,P) at E9.5. (I-P) Sagittal sections of the control (I,K,M,O) and Hand1-overexpressing (J,L,N,P) hearts. (Q) Quantitative real-time PCR analysis on RNA extracted from control or severe phenotype overexpression embryos for an array of cardiac markers. Means and standard deviation bars with 95% confidence limits are shown. *P<0.05, **P<0.01. at, atrium; co, control; lv, left ventricle; oe, overexpression; oft, outflow tract; rv, right ventricle. Scale bar: 100 µm in A.

View larger version (83K): [in a new window]   Fig. 9. Over-proliferation of cardiac precursors in the distal OFT of Hand1-overexpressing embryos. Sagittal (A-D) and frontal (E-H) sections through the distal OFT region of control or Hand1-overexpressing embryos at E9.5. Immunofluorescence with antibody specific to phospho-histone H3 (-PH3) to mark proliferating cells (A,C,E,G). (B,D,F,H) Bis-benzamide counterstaining (DNA) of nuclei. White arrowheads in C and G indicate areas of proliferating cells. co, control; oe, over-expression.

View larger version (46K): [in a new window]   Fig. 10. Hand1 regulates cardiomyocyte proliferation versus differentiation in vitro via G1 cell-cycle progression or exit. (A) Myocardial beating in embryoid bodies (differentiated for up to 14 days) derived from wild-type (+/+), Hand1-null (-/-), control (responder transgene alone, co) and Hand1-overexpressing (oe) ES-cell lines. (B) Quantitative real-time PCR analysis on RNA extracted from embryoid bodies following 14 days of differentiation, for an array of cardiac markers. (C) Western blots of day-14 EBs using antibodies for cyclin D2, Cdk4 and Gapdh. (D) Quantification of protein levels from C normalised to Gapdh using scanning densitometry.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter