| |
Supplementary figure 4:Knockdown of P-TEFb components CDK9b and a combination of CDK9a and b leads to loss of expression of neural crest genes. All embryos were injected in one cell of a two cell stage embryo with either a control morpholino CoMo, 100ng CDK9b morpholino or 40ng CDK9.S morpholino with 60ng CDK9.L morpholino. 300pg of Lac-Z cRNA was co-injected with morpholinos and β-gal activity was detected using a red gal stain to monitor lineage tracing. in situ hybridisation was then carried out on these embryos for neural crest specifying, neural plate border and neural plate genes. (A) c-Myc expression (stage 13) shows tissue specific loss of expression in the anterior neural crest region (black arrows) after injection of CDK9b morpholino and CDK9.S+L morpholinos. Sox10 (stage 15) shows loss of expression after injection of CDK9.L morpholino and CDK9.S+L morpholinos. Slug expression (stage 15) appears partially lost or altered after injection of CDK9.L morpholino and CDK9.S+L morpholinos. FoxD3 expression (stage 15) appears partially lost or altered after injection of CDK9.L morpholino and CDK9.S+L morpholinos. Sox9 expression (stage 15) appears partially lost or altered after injection of CDK9.L morpholino and CDK9.S+L morpholinos. Zic1 expression (stage 12) appears unchanged after injection of CDK9.L morpholino and CDK9.S+L morpholinos. Zic3 expression (stage 12) appears unchanged after injection of CDK9.L morpholino and CDK9.S+L morpholinos. Pax3 expression (stage 12) appears unchanged after injection of CDK9.L morpholino and CDK9.S+L morpholinos. Sox2 expression (stage 15) appears unchanged after injection of CDK9.L morpholino and CDK9.S+L morpholinos. (B) Graphs showing the percentage of embryos displaying an expansion, wild type, partial loss or loss of expression pattern by in situ hybridisation after 100ng CDK9.L morpholino (top panel) and CDK9.S+L morpholinos (bottom panel) injection. Cdk9.L mo injected c-myc n=45, sox10 n=35, slug n=33, foxd3 n=43, sox9 n=33, zic1 n=27, zic3 n=30, pax3 n=25, sox2 n=34. Cdk9.S and L injected c-myc n=50, sox10 n=30, slug n=53, foxd3 n=58, sox9 n=57, zic1 n=42, zic3 n=63, pax3 n=49, sox2 n=43 (C) qRT-PCR showing level of mRNA expression after CDK9.S or CYCLINT1 knockdown compared to standard control morpholino injected whole embryos. Top panel shows the level of expression of Zic1, Zic3, Pax3, Sox2 and a negative reverse transcription (RT) control. No significant change in expression was seen for any of these genes ns = not significant.
Right panel shows the level of expression of c-Myc, Sox10, Slug, FoxD3 and a negative RT control. Some of these show a significant decrease in expression level. *=p<0.05, **=p<0.0000001 and ***=p<0.0000000001. (D) The effect of CDK9.S and CyclinT1 morpholino knockdown on trigeminal placode markers. Graph showing the percentage of embryos displaying a wild type, partial loss or loss of expression pattern by in situ hybridisation after 100ng control morpholino, 100ng CDK9.S morpholino and 60ng CYCLINT1 morpholino injection. Wholemount in situ hybridisation was carried out for Tbx2, NeuroD and ElrD. Como injected Tbx2 n=36, NeuroD n=33, Elrd n=33. Cdk9a mo injected Tbx2 n=53, NeuroD n=51, Elrd n=33. CyclinT1 mo injected Tbx2 n=34, NeuroD n=44, Elrd n=40 (E) The effect of CDK9 and CyclinT1 morpholino knockdown on trigeminal placode markers. Como injected Runx1 n=37, Islet1 n=31, Ngnr1 n=38. Cdk9.S mo injected Runx1 n=46, Islet1 n=32, Ngnr1 n=39. CyclinT1 mo injected Runx1 n=36, Islet1 n=35, Ngnr1 n=33. |
