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Fig. 2. HDAC activity is essential for proper gene expression in pluripotent blastula cells. (A,B) In situ hybridization examining tfap2, id3, oct25, vent2 and sox3 expression in pluripotent blastula cells following treatment with vehicle or inhibitor [500 nM TSA (A) or 20 mM VPA (B)]. Embryos were treated at the two-cell stage and collected at the late blastula stage (stage 9). (C) qRT-PCR of animal pole explants examining the expression of pluripotency genes after treatment with vehicle or inhibitor (500 nM TSA) (***P<0.005). Explants were cultured alongside sibling embryos grown until late blastula stages (stage 9). (D) In situ hybridization examining epk and sox3 expression in animal pole explants treated with vehicle or inhibitor (500 nM TSA). Explants were cultured alongside sibling embryos until late gastrula stages (stage 13). Scale bars: 250 μm. |
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Supplementary Figure 2. (A) In situ hybridization examining HDAC1 expression in early embryos from
2-cell to late neurula stages. (B) In situ hybridization examining neural plate border markers Msx2, Ap2,
and Zic1 expression after treatment with vehicle or inhibitor (TSA- 200nM or VPA- 20mM). Embryos
were treated at mid-gastrula stages (stage 11) and collected at mid-neurula stages (stage 15). (C) Explant
assay examining Snail2 and FoxD3 expression in Wnt/Chordin induced explants that were treated with
vehicle or inhibitor (TSA-200nM or VPA-10mM). Explants were cultured alongside sibling embryos
grown until late neurula stages (stage 18). (D) Explant assay examining Snail2 and FoxD3 expression in
Wnt/Chordin induced explants that were treated with vehicle (DMSO) or inhibitor (RMD – 15uM).
Explants were cultured alongside sibling embryos cultured until late neurula stages (stage 18). |
