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Dev Biol
1998 Dec 01;2041:187-96. doi: 10.1006/dbio.1998.9080.
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Tinman function is essential for vertebrate heart development: elimination of cardiac differentiation by dominant inhibitory mutants of the tinman-related genes, XNkx2-3 and XNkx2-5.
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In Drosophila, the tinman gene is absolutely required for development of the dorsal vessel, the insect equivalent of the heart. In vertebrates, the tinman gene is represented by a small family of tinman-related sequences, some of which are expressed during embryonic heart development. At present however, the precise importance of this gene family for vertebrate heart development is unclear. Using the Xenopus embryo, we have employed a dominant inhibitory strategy to interfere with the function of the endogenous tinman-related genes. In these experiments, suppression of tinman gene function can result in the complete elimination of myocardial gene expression and the absence of cell movements associated with embryonic heart development. This inhibition can be rescued by expression of wild-type tinman sequences. These experiments indicate that function of tinman family genes is essential for development of the vertebrate heart.
FIG. 1. Single-sided expression of dominant inhibitory XNkx2-3 and XNkx2-5 inhibits myocardial gene expression. In all cases the arrowhead indicates the injected side of the embryo. All embryos were assayed for expression of the myocardial marker, TnIc, using whole-mount in situ hybridization. (A) Stage 28, water-injected control embryo, showing normal expression of TnIc. At this early stage, TnIc expression marks bilaterally symmetrical precardiac tissues, located on each side of the ventral midline. (B) Stage 28 embryo injected with 250 pg of wild-type Nkx2-5 mRNA showing increased expression of TnIc on the injected side. (C) Stage 28 embryo, after single-sided injection of 250 pg of Nkx2-3LP mRNA. Expression of TnIc is dramatically reduced on the injected side (indicated by the arrowhead). (D) Stage 29 embryo, after single-sided injection of 250 pg of Nkx2-5LP mRNA. Expression of TnIc is dramatically reduced on the injected side (arrowhead). (E) Lateral view of control and experimental stage 30 embryos. The water-injected control embryo is shown on top. The bottom embryo was injected with 250 pg of Nkx2-3LP mRNA and shows severe inhibition of TnIc expression in the cardiac region. Note that the overall morphology of the experimental embryo is indistinguishable from the water-injected control. (F) Histological section through the heart region of a stage 30, water-injected control embryo assayed for TnIc expression. Two regions of TnIc expression are visible on either side of the ventral midline. (G) Section through the heart region of a stage 30 embryo injected with 250 pg of Nkx2-3LP. Note the absence of TnIc expression on the injected side (arrowhead).
Double-sided injection of a dominant inhibitory tinman construction can completely eliminate myocardial differentiation. Lateral view of control and experimental stage 30 embryos. The top embryo was injected with water and shows normal expression of TnIc in the developing heart tube. The bottom embryo was injected on each side with 125 pg of Nkx2-5LP mRNA. No expression of the TnIc myocardial marker is visible. Note that the overall morphol- ogy of both the control and experimental embryos is normal
FIG 3. Double-sided injection of a dominant inhibitory tinman construction eliminates the cellular movements associated with heart formation. Histological sections through the heart region of control and experimental stage 30 embryos assayed for TnIc expression. (A) Control embryo showing expression of TnIc in the developing heart tube. The myocardial layer has completely separated from the overlying endoderm and the underlying somatic mesoderm layers. The myocardial tube surrounds endothelial cells that will later form the endocardium. (B) Experimental embryo injected with 125 pg of Nkx2-5 LP mRNA on each side. No detectable expression of the myocardial marker TnIc is visible. In addition, there is no visible delamination of the tissue layers and no evidence of heart tube formation.
