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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. |
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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. |
