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Figure 3. Xic1 is expressed in the differentiating heart. In situ hybridization to detect Xic1 in ventral or lateral view (A–E) of stage 27–41 embryos. Xic1 is weakly expressed in the hearts of embryos from stage-27 onwards (arrows). Picture B was taken following clearing embryo with Benzyl-benzoate:Benzyl-alcohol. (G) RT–PCR confirming expression of Xic1 transcript in the dissected hearts (F) of stages-33/4 to -42. |
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Figure 5. The requirement for Xic1 in cardiomyocyte differentiation is distinct from its ability to arrest the cell cycle. Stage-29/30 embryos either uninjected (A and H) or injected with CTRMo (B and I), Xic1Mo (C, J, and P), or Xic1Mo plus FL-Xic1 (30 pg) (D, K, and Q), NT-Xic1 (15 pg) (E and L), CT-Xic1 (30 pg) (F and M), Xic1(35–96) (30 pg) (G and N), Xic1CK- (R), or CTRMo plus Xic1CK- (S) detecting either Nkx2.10 (A–G) or TIc (H–S). (T) Percentage of stage-29/30 embryos with heart sizes 1–3 (cf. Figure 4) following injection with Mo and mRNA combinations as described. n = 26 – 53 embryos/treatment group. |
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Supplementary Figure 1: (A) The antisense Xic1Mo morpholino oligonucleotide Xic1Mo (red) binds to the first 25-nucleotide sequences of p27Xic1 RNA (black) while the Control Morpholino ConMo (blue) with 5-nucleotide substitutions cannot, as detected by Western blotting for Xic1 protein (B). The Xic1 mutant RNA (green) used for rescue experiments was designed to contain 8 transcriptionally silent nucleotide substitutions at the 5’ end. (B) Xic1Mo, but not CTRMo, inhibits the in- vitro translation of wild-type Xic1 but cannot target the nucleotide-substituted Xic1 rescue construct. (C) Western blot demonstrating that injection of Xic1Mo effectively inhibits the expression of Xic1 protein in stage 17 embryos. (D) A ventral and lateral view of stage 33/4 Xic1Mo injected embryo following TIc ISH and clearing with Benzyl Benzoate: Benzyl alcohol, compared to staged matched uninjected embryo.
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