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Drosophila tinman is an NK-class homeobox gene required for formation of the dorsal vessel, the insect equivalent of the vertebrate heart. Vertebrate sequences related to tinman, such as mouse Nkx-2.5, chicken cNkx-2.5, Xenopus XNkx-2.5 and XNkx-2.3 are expressed in cardiac precursors and in tissues involved in induction of cardiac mesoderm. Mice which lack a functional Nkx-2.5 gene die due to cardiac defects. To determine the role of tinman-related sequences in heart development, we have overexpressed both XNkx-2.3 and XNkx-2.5 in Xenopus laevis embryos. The resulting embryos are morphologically normal except that they have enlarged hearts. The enlarged heart phenotype is due to a thickening of the myocardium caused by an increase in the overall number of myocardial cells (hyperplasia). Neither ectopic nor precocious expression of cardiac differentiation markers is detectable in overexpressing embryos. These results suggest that both XNkx-2.3 and XNkx-2.5 are functional homologues of tinman, responsible for maintenance of the heart field.
Fig. 1. XNkx-2.3 and XNkx-2.5 are expressed in similar regions of the developing embryo. In situ hybridization with antisense XNkx-2.3 and XNkx-2.5 probes demonstrates that both of these sequences are expressed in early heart progenitors (h) and are maintained in cardiac tissue throughout development. XNkx-2.3 and XNkx-2.5 are also expressed in similar, but distinct regions of anteriorendoderm (e) and pharynx (p). (A) Stage 18 XNkx-2.5 stained embryo. (B) Stage 18 XNkx-2.3 stained embryo. (C) Stage 28 embryos. (D) Stage 37 embryos. In C and D the XNkx-2.3 stained embryo is on top.
Fig. 2. Overexpression of XNkx-2.3 and XNkx-2.5 in heart progenitors results in enlarged cardiac structures. Embryos injected with lineage tracer plus water, control Mix.2 RNA, XNkx-2.3 RNA or XNkx-2.5 RNA are shown. (A) Fluorescence micrograph of an embryo injected with fluoresceinated dextran in water into the dorsal vegetal blastomere at the 8-cell stage. The heart region is indicated by an arrowhead. (B) Embryo injected as in A into the ventral vegetal blastomere. These embryos are labeled primarily in the posteriorgut and ventralmesoderm. (C-K) In situ hybridization depicting cardiac troponin I expression (blue stain) in stage 37 embryos that were injected into the dorsal vegetal blastomere as in A. (C,K) Water injected (D) Mix.2 injected (E,G,I) XNkx-2.3 injected (F,H,J) XNkx-2.5 injected (L) XNkx-2.5 injected embryo stained by in situ hybridization with XMLC2 probe. The heart is enlarged in XNkx-2.3 and XNkx-2.5 injected embryos (E,F,G,H,I,J,L), but not in water or Mix.2 injected embryos (C,D,K). This is not a non-specific effect of overexpressing homeobox sequences since Mix.2 injected embryos have normal size or small hearts (D). No ectopic cardiac differentiation is detected by in situ hybridization with either XTnIc or XMLC2 probe (I,J,L). Embryos in C-H were photographed at 0 magnification and embryos in I-L were photographed at 0 magnification. The brown color in all embryos is natural pigmentation.
Fig. 3. Increased regions of marker gene expression at early stages of cardiac differentiation. In situ hybridization of stage 26 embryos to detect cardiac troponin I expression indicates that cardiac differentiation begins in two symmetrical regions within the heart field (A) and that these regions are enlarged in XNkx-2.5 and XNkx- 2.3 injected embryos (B,C). (A) Water injected embryo. (B) XNkx- 2.3 injected embryo with symmetrically large troponin expressing regions. (C) XNkx-2.3 injected embryo with asymmetrically large troponin expressing region. The left side which received the injection is larger. Fluorescence microscopy of stage 25 embryos injected into the left dorsal vegetal blastomere at the 8-cell stage demonstrates extensive cell mixing between the left and right sides in the heart region. This cell mixing may result in significant overexpression of injected RNAs on both the left and right sides leading to expansion of the heart field on both sides. (D) Anterior view of stage 25 embryo injected with tetramethylrhodamine-conjugated dextran showing mixing on the ventral side. (E) Anterior view of stage 25 embryo injected as in D but with much less cell mixing. In each case, the plane of the dorsal/ventral axis is indicated by opposing arrowheads.
Fig. 4. Paraffin sections demonstrate an increase in myocardial volume and in the number of myocardial cells in XNkx-2.3 and XNkx-2.5 injected embryos. In situ hybridization to detect cardiac troponin I transcripts (the purple color in A,B,E,F) was completed before sectioning. Nuclei were visualized in C,D,G, and H with the fluorescent nuclear stain SYTOX Green (Molecular Probes). (A) Cross section through the heart region of a water injected embryo at stage 33. (B) Section equivalent to that in A from a XNkx- 2.5 injected embryo. (C) Section adjacent to that in A. (D) Section adjacent to that in B. (E) Cross section through the heart region of a water injected embryo at stage 31. (F) Section equivalent to E from a XNkx-2.3-injected embryo. (G) Same section as in E. (H) Same section as in F. A-B were photographed at 00. C-H were photographed at 00.
nkx-2.3 e expression in bisected Xenopus laevisembryo, mid-sagittal section, assayed via in situ hybridization, NF stage 18, anterior left, dorsal up.
