XB-ART-41564Development. June 1, 2010; 137 (11): 1919-29.
TBX20 has been shown to be essential for vertebrate heart development. Mutations within the TBX20 coding region are associated with human congenital heart disease, and the loss of Tbx20 in a wide variety of model systems leads to cardiac defects and eventually heart failure. Despite the crucial role of TBX20 in a range of cardiac cellular processes, the signal transduction pathways that act upstream of Tbx20 remain unknown. Here, we have identified and characterized a conserved 334 bp Tbx20 cardiac regulatory element that is directly activated by the BMP/SMAD1 signaling pathway. We demonstrate that this element is both necessary and sufficient to drive cardiac-specific expression of Tbx20 in Xenopus, and that blocking SMAD1 signaling in vivo specifically abolishes transcription of Tbx20, but not that of other cardiac factors, such as Tbx5 and MHC, in the developing heart. We further demonstrate that activation of Tbx20 by SMAD1 is mediated by a set of novel, non-canonical, high-affinity SMAD-binding sites located within this regulatory element and that phospho-SMAD1 directly binds a non-canonical SMAD1 site in vivo. Finally, we show that these non-canonical sites are necessary and sufficient for Tbx20 expression in Xenopus, and that reporter constructs containing these sites are expressed in a cardiac-specific manner in zebrafish and mouse. Collectively, our findings define Tbx20 as a direct transcriptional target of the BMP/SMAD1 signaling pathway during cardiac maturation.
PubMed ID: 20460370
PMC ID: PMC2867324
Article link: Development.
Grant support: R01 DE018825 NIDCR NIH HHS, R01 HL089641 NHLBI NIH HHS
Genes referenced: acvr1 myh4 myh6 smad1 tbx20 tbx5 smad9 tpm1
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|Fig. 4. XTbx20 is expressed throughout the myocardium and endocardium of the X. laevis heart. (A,B) Tbx20 is expressed in both the anterior and posterior regions of the X. laevis stage 46 heart. (C-F) Immunohistochemistry of serial sections shows that Tbx20 expression overlaps with that of the myocardial marker tropomyosin (C,D) and with phospho-SMAD1/5/8 expression in the endocardium (E,F); anti-tropomyosin (Tm) staining is labeled green, anti-pSMAD1/5/8 is labeled red, and nuclei are labeled blue with DAPI. LA, left atrium; OFT, outflow tract; TA, truncus arteriosus; V, ventricle.|
|Fig 1. A regulatory element 5′ to the Tbx20 genomic locus is sufficient to drive gene expression in the Xenopus cement gland and heart. (A) Schematic of the X. tropicalis Tbx20 genomic locus. X. tropicalis Tbx20 consists of eight exons spanning approximately 20 kB. The Tbx20 transcriptional start site is located 287 bp upstream of the translation start site in exon 1. A putative cardiac regulatory element is located at the 5′ end of the Tbx20 locus (dashed box). (B) Schematic of the 2464 bp region of the 5′ end of Tbx20 cloned in frame to the EGFP reporter to examine its regulatory capacity in X. laevis transgenics. (C-F) As with endogenous Xenopus Tbx20 expression, the Tbx20 EGFP reporter is expressed in the cement gland and heart of living X. laevis transgenic embryos. (C) Ventral views of the anterior ends of stage 46 sibling non-transgenic (left) and transgenic (right) embryos. (D) Fluorescence views of siblings in C. (E,F) Magnified view of the EGFP expression driven by the Tbx20 regulatory element in the cement gland (E) and heart (F) of the transgenic embryo in D.|
|Fig 2. A 334 bp regulatory element recapitulates the endogenous expression of Tbx20 throughout the X. laevis heart. A deletion series of the 5′ regulatory element was created to determine a reduced element sufficient to drive EGFP transgene expression. (A) Schematic of the deletion series of Tbx20 elements fused to EGFP for X. laevis transgenesis. (B,E,H) Ventral view of the anterior regions of living stage 46 (late tadpole) X. laevis embryos (left) and siblings transgenic for constructs shown in A (right) under white light. (C,F,I) Embryos in B, E and H as viewed under fluorescent light. Green autofluorescence in the gut can be noted in both control and transgenic embryos. (D,G,J) Magnified views of the EGFP-expressing hearts of embryos in C, F and I demonstrating that EGFP expression in the heart is maintained under the control of a Tbx20(34) element. (K-P) Transverse sections were cut through the embryos expressing Tbx20-EGFP shown in B-J, and expression of the Tbx20(464)-EGFP (K,L), Tbx20(483)-EGFP (M,N) and Tbx20(34)-EGFP (O,P) transgenes was demonstrated by antibody staining for EGFP. Anterior (K,M,O) and posterior (L,N,P) sections show EGFP transgene expression throughout the heart. CA, carotid arch; EC, endocardial cushion; LA, left atrium; OFT, outflow tract; PA, pulmocutaneous arch; RA, right atrium; SA, systemic arch; T, trabeculae; TA, truncus arteriosis; V, ventricle.|
|Fig 5. SMAD1 activation is required for cardiac-specific expression of Tbx20 in X. laevis. (A-F′) Immunohistochemistry of transverse sections through the heart of stage 40 anterior explants shows loss of nuclear phospho-SMAD1/5/8 (arrows) in the myocardium of dorsomorphin-treated explants (D-F,D′-F′) compared with DMSO-treated controls (A-C,A′-C′). In the merged images, anti-phospho-SMAD1/5/8 (pSMAD1/5/8) staining is labeled red, anti-myosin heavy chain (MHC) is labeled green, and nuclei are labeled blue with DAPI. (G-L) In situ hybridization for Tbx20 performed on stage 40 anterior and cardiac explants shows complete loss of Tbx20 expression in the heart (H,L) but not the hindbrain (J) of dorsomorphin-treated anterior and cardiac explants compared with DMSO-treated controls (G,I,K). (M-P) Whole-mount antibody staining of stage 40 anterior explants shows normal expression of the myocardial marker MHC in dorsomorphin-treated explants (N,P) compared to DMSO-treated controls (M,O). Dorso, dorsomorphin. Scale bars: 20 μm in A-F′; 1 mm in G-P.|
|Fig. 8. The Xenopus Tbx20 334 bp cardiac regulatory element drives EGFP expression in a cardiac-specific manner in zebrafish. (A,B) In situ hybridization depicts expression of Tbx20 in wild-type zebrafish embryos and alk8sk42 (Marques and Yelon, 2009) mutant siblings at the 10-somite stage; dorsal views, anterior to the top. Tbx20 expression is reduced in both the anterior lateral plate mesoderm, including the bilateral cardiac primordia, and the midline mesenchyme of zygotic alk8 mutants. (C-E) Lateral views of a live zebrafish embryo at 48 hpf, following injection with the XTbx20(34)-EGFP transgene. Injected embryos express EGFP in the myocardium (arrows).|