Warkman AS , Krieg PA .

P01 HL063926-01A10004 NHLBI NIH HHS , R01 HL074184 NHLBI NIH HHS , R01 HL074184-01 NHLBI NIH HHS

Xla Wt + myocd MO (Fig 1. D)

	Fig. 1. MO inhibition of myocardin expression results in reduced cardiac gene expression and defective cardiac morphogenesis. (A) Ventral view of embryo injected with control MO and then assayed by in situ hybridization for MHC expression. The paired heart patches are clearly visible. Injected side is indicated with an arrow. (B) Section through heart of embryo injected with control MO. Heart tissue has been stained for Nkx2–5 transcripts. At this stage the myocardium is a simple linear tube surrounding the endocardial precursor cells. (C) Ventral view of embryo injected with MO directed against myocardin and assayed for MHC expression. Note that no MHC expression is detected on the injected side of the embryo (arrow). (D) Section through heart of embryo injected with myocardin MO. Heart tissue has been stained for Nkx2–5 transcripts. Note that the morphology of the developing heart is disrupted on the injected side (arrow). Adapted from [27] with permission from Elsevier.
	Fig. 2. The Xenopus animal cap explant procedure. SyntheticmRNAencoding a potential regulator of cardiac development is injected into the fertilized Xenopus egg at the one cell stage. When the embryo has reached the blastula stage, the animal cap (ectodermal tissue overlying the blastocoel cavity) is carefully removed using fine forceps and it placed into explant culture. After an appropriate period in culture, the cap tissue and is subjected to appropriate assays for gene expression. Reprinted from [68] with permission from Elsevier.
	Fig. 3. Mutational analysis of the ANF promoter using Xenopus transgenesis. (A) Alignment of the proximal promoter region of the human (top) and Xenopus (bottom) promoter sequences, showing conservation of sequence, spacing and orientation of transcription regulatory sequences. Two conserved sequences that do not correspond to binding sites for known transcription factors are indicated (Unknown 1 and 2). (B) Fluorescent image of the heart of Xenopus embryo expressing GFP reporter under control of the wild type ANF promoter. Note that GFP expression is restricted to the atrial chambers (a). (C) Fluorescent image of the heart of a Xenopus embryo expressing GFP under control of an ANF promoter in which the NKE (Nkx2–5 binding site) has been mutated. Note that mutation of the NKE prevents restriction of expression to the atria, with prominent expression persisting in the ventricular myocardium. Part A is adapted from [68] with permission from Elsevier, and Parts B and C are reprinted from [55] with permission from Elsevier.

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