First published online 20 April 2005
doi: 10.1242/dev.01832
Development 132, 2475-2487 (2005)
Published by The Company of Biologists 2005
T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis
Chen-Leng Cai1,2,
Wenlai Zhou3,
Lei Yang1,2,*,
Lei Bu1,2,*,
Yibing Qyang2,*,
Xiaoxue Zhang1,2,
Xiaodong Li2,
Michael G. Rosenfeld3,
Ju Chen2 and
Sylvia Evans1,2,
1 Skaggs School of Pharmacy, University of California, San Diego, 9500 Gilman
Drive, La Jolla, CA 92093, USA
2 Department of Medicine, University of California, San Diego, 9500 Gilman
Drive, La Jolla, CA 92093, USA
3 Department of Medicine and Howard Hughes Medical Institute, University of
California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
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Fig. 1. (A-D) Generation of Tbx20 targeted allele. (A) Two LoxP sites were induced
into Tbx20 exon 2 where the T-box domain starts. (B) Southern blot of ES cell
DNA digested with NcoI and hybridized with a genomic fragment
external to the targeting construct with wild-type band 5.5 kb and recombinant
band 4.3 kb. Recombinant ES cells were then transfected with a Cre plasmid in
order to remove PGKNeo, HSV-TK cassettes. Tbx20-null mice were
obtained by crossing mice with Tbx20 floxed allele to protamine-Cre
mice. (C) RT-PCR using whole heart RNA obtained from adult wild-type (lane 1)
and heterozygous mice (lane 2) with primers located in exon 1 (P-RT-5')
and exon 3 (P-RT-3') showed the wild-type band 677 bp and mutant band
427 bp. Excision of exon 2 of Tbx20 created a new immediate stop codon within
exon 3. (D) Genotypes determined by PCR of one littermate embryos from
heterozygous cross. The wild-type allele band was 310 bp and the mutant allele
band was 650 bp. (E-L) Whole-mount (E,F,I,J) and histological (G,H,K,L) views
of Tbx20-null mice and littermate controls at E8.5 and E9.5. The
left, middle and right columns give right, frontal and left views,
respectively. Hearts of Tbx20-null mice are severely hypoplastic
relative to control littermates at both stages. Arrows indicate the heart
region.
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Fig. 2. Whole-mount in situ analysis of Tbx20-null mutants and control
littermates. (A-H) Differentiation markers MLC2a, ß-MHC and MLC2v are
normally expressed in Tbx20 mutants;  -MHC, however, is
downregulated. (I,J) Tbx5 is expressed in left ventricular, atrioventricular
and atrial progenitors in an anteroposterior gradient. This expression profile
is maintained in Tbx20 mutants. (K,L) Wnt11 is expressed in outflow
tract and atrioventricular canal. In Tbx20 mutants, outflow tract
expression is present, but atrioventricular canal expression is lacking. (M,N)
GATA4 is expressed throughout the heart in an anteroposterior gradient that is
maintained in Tbx20 mutants. Arrows indicate the corresponding heart
regions in wild type and mutant embryos.
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Fig. 3. Tbx2 is upregulated in Tbx20 mutants and is a direct target for repression
by Tbx20. (A-F) Whole-mount RNA in situ and section analysis for Tbx2
expression in Tbx20-null embryos and wild-type littermates. Arrows
indicate the corresponding heart regions in wild type and mutant embryos. OFT,
outflow tract; A/V, atrioventricular canal. (G-L) Whole-mount RNA in situ
demonstrates downregulation of Nppa, chisel and cited 1 in Tbx20
mutants. (M-P) Expression of Tbx20 and Tbx3 is unaffected. Arrows indicate the
corresponding heart regions in wild type and mutant embryos. (Q) ChIP analysis
with embryonic heart extracts revealed binding of Tbx20 to region containing
conserved T-box sites within Tbx2 promoter (lane 1, primer P-813, P-573). ChIP
analysis with primers against an unrelated promoter region revealed no Tbx20
recruitment (lane 2) (see Materials and methods for primers). No recruitment
was found with beads, IgG or H2O. (R) Co-transfection of Tbx20
expression vector (500 ng) with Tbx2 promoter-luciferase constructs (100 ng)
into HEK293 cells demonstrates repression by Tbx20, which was abrograted by
mutation (Mu) of conserved T-box elements within the Tbx2 promoter.
*P<0.05, paired t-test.
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Fig. 4. Apoptosis and proliferation assays in Tbx20-null mutants and
control littermates. (A-D) TUNEL analysis revealed no increase in apoptosis in
Tbx20-null embryos relative to control littermates. Arrows indicate
cardiac crescent (A,B) and heart tube (C,D). (E-L) Antibody staining (whole
mount and sections) for phosphorylated histone H3 reveals decreased
proliferation in Tbx20-null mutants relative to control littermates.
Arrows indicate positive phosphorylated histone H3 staining in
cardiomyocytes.
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Fig. 5. Tbx2 directly binds Nmyc1 and represses its expression in regions
of relatively low proliferation within the heart. (A-H) Regions of relatively
low proliferation; outflow tract (OFT) and atrioventricular canal (A/V) are
indicated by arrows. Corresponding sections (C,D; G,H; K) are shown
progressively from anterior to posterior, respectively. Expression of Nmyc1
(A-D) and cyclin A2 (E-H) is downregulated in Tbx20-null mutants.
Section analysis of wild-type littermates reveals regional differences in
expression of Nmyc1 (C) and cyclin A2 (Ccna2) (G), with relatively low levels
in OFT and A/V. (I-K) Wild-type Tbx2 expression is complementary to that of
Nmyc1 and cyclinA2. (L) Expression of phosphorylated histone H3 in wild-type
embryos revealed regions of low proliferation within developing heart. The
left, middle and right panels show right, frontal and left side views,
respectively. (M) ChIP analysis with embryonic heart extracts demonstrated
recruitment of Tbx2 and Tbx20 to regions containing T-box consensus sites
within intron 1 of the Nmyc1 gene (lane 1, primer P-4030, P-4330;
lane 2, primer P-4630, P-4930). ChIP analysis with primers against an
unrelated promoter region revealed no Tbx2 recruitment (lane 3) (see Materials
and methods for primers). No recruitment was found with IgG. (N)
Co-transfections of Tbx2 or Tbx20 expression vectors with Nmyc1 intron
1-luciferase reporter into HEK293 cells demonstrated repression or activation,
respectively. **P<0.005, paired t-test;
*P<0.05, paired t-test. (O) Co-transfections
of Tbx2 expression vector with Nmyc1 intron 1-luciferase reporter into HEK293
cells demonstrated dose-dependent repression by Tbx2.
**P<0.005, paired t-test.
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Fig. 6. Tbx20 regulates expression of a subset of BMP genes. Expression of Bmp4
(C,D) and Bmp7 (G,H) are not downregulated in Tbx20 mutants, whereas
expression of Bmp2 (A,B) and Bmp5 (E,F) is severely downregulated specifically
in the heart, as indicated by arrows. The left and right images in A and B
show frontal and left views, respectively.
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Fig. 7. Tbx20 and Nkx2-5 independently regulate common downstream targets. (A-D)
Expression of Hand1 and Irx4 is downregulated in Tbx20 mutants. (E,F)
Hand2 and (G-J) Nkx2-5 expression are unaffected in Tbx20 mutants.
Images in A,B show left side views and images in C-J show frontal views.
Arrows indicate the corresponding heart regions in wild type and mutant
embryos.
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Fig. 8. Tbx20 directly binds and represses Isl1 in myocardium. (A-D) Isl1 is
expressed throughout myocardium in Tbx20 mutants. (E) ChIP analysis
revealed Tbx20 recruitment to region of Isl1 promoter with conserved T-box
site (lane 1, primer P-842, P-581). ChIP analysis with primers against an
unrelated promoter region revealed no Tbx20 recruitment (lane 2) (see
Materials an methods for primers). No recruitment was found with beads, IgG or
H2O. (F) Co-transfection of Tbx20 expression vector (500 ng) with
Isl1 promoter-luciferase reporter (100 ng) demonstrated repression of
Isl1 by Tbx20. Repression was abrogated by mutation of the consensus
T-box element within the Isl1 promoter.
*P<0.05, paired t-test. (G-J) The nebulette
promoter, expressed exclusively in myocardium during early embryogenesis
(J.C., unpublished) was used to drive expression of Isl1 in transient
transgenic mice. Whole-mount in situ hybridization (G,H) revealed expression
of Isl1 throughout myocardium in transgenic mice (H). Hearts of transgenics
appeared normal at E10.5. The left and right images in A-D,G,H show right and
left side views. Corresponding sections are shown progressively from anterior
to posterior (C,D; I,J). Arrows indicate the corresponding heart regions in
wild type and mutant embryos.
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© The Company of Biologists Ltd 2005
