Mandel EM , Kaltenbrun E , Callis TE , Zeng XX , Marques SR , Yelon D , Wang DZ , Conlon FL .

R01 DE018825 NIDCR NIH HHS, R01 HL089641 NHLBI NIH HHS

             BMP signaling pathway

	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).
	Fig. S1. The Tbx20-EGFP reporter directs EGFP expression reproducibly in the heart and cement gland of transgenic siblings. (A,B) Ventral view of the anterior end of stage 46 non-transgenic (left) and transgenic siblings (right) generated from one batch of injections of the Tbx20(-2464)-EGFP reporter demonstrate consistent and reproducible EGFP expression within the heart and cement gland of transgenic embryos. Brightfield views of living embryos (A) and corresponding EGFP fluorescence (B) are shown.
	Fig. S2. Further deletion of the Tbx20(-334)-EGFP reporter leads to a decrease in activity in response to SMAD4 and an increase in non-specific Tbx20 expression. (A,B) Tbx20(−251)-luciferase reporter (A) and Tbx20(−81)-luciferase reporter (B) and corresponding SMAD4 transcriptional assays. Fold induction reflects changes in induction relative to induction of the reporter alone; error bars represent standard deviation of three replicates. (C-F) Tbx20(−251)-EGFP (C,D) or Tbx20(−81)-EGFP (E,F) reporter constructs were introduced into X. laevis transgenic embryos. Transgenic embryos are located at the right of each image, while non-transgenic siblings are at the left. Brightfield views of living stage 46 embryos (C,E) and EGFP expression of corresponding embryos (D,F) are shown. non-Tg, non-transgenic; Tg, transgenic.
	Fig. S3. SMAD1 inhibition during cardiac chamber differentiation does not affect expression of the cardiac markers tropomyosin and Tbx5. (A,B) In situ hybridization for Tbx5 on stage 40 DMSO (A) and dorsomorphin-treated (B) anterior explants shows normal expression of Tbx5 in the heart and cardinal vein after SMAD1 inhibition. (C-F) Whole-mount antibody staining for tropomyosin of a stage 40 DMSO (C,E) or dorsomorphin-treated (D,F) anterior explants. Ventral view reveals no change in expression of tropomyosin in the heart after treatment with dorsomorphin. Dorso, dorsomorphin. Scale bars: 1 mm.
	tbx20 (t-box 20) is expressed throughout the myocardium and endocardium, in both the anterior and posterior regions of the X. laevis heart, at NF stage 46. Key: OFT, outflow tract; TA, truncus arteriosus; V, ventricle.

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