T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis

Abstract

Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. We provide evidence that Tbx2 is a direct target for repression by Tbx20 in developing heart. We have also found that Tbx2 directly binds to the Nmyc1 promoter in developing heart, and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. Our data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. We present a model whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, we have found that Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development.

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.

Fig. 2

Whole-mount in situ analysis of Tbx20-null mutants and control littermates. (A-H) Differentiation markers MLC2a, β-MHC and MLC2v are normally expressed in Tbx20 mutants; α-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.

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.

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.

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.

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.

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.

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.