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Fig. 3.
Knockdown of P-TEFb components leads to loss of expression of neural crest specifying genes. (A) All embryos were injected with 100 ng standard control morpholino, 100 ng CDK9.S morpholino and 60 ng CYCLINT1 morpholino in both cells of a two cell stage embryo and fixed at stage 38. Loss of melanocytes in the tail head and lateral stripe are indicated by black arrow heads. (B) All embryos were injected in one cell of a two cell stage embryo with either a control morpholino CoMo, 100 ng CDK9.S morpholino or 60 ng CYCLINT1 morpholino. 300 pg 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. c-Myc (stage 13) shows tissue specific loss of expression (black arrows). Sox10 (stage 15) shows loss of expression (black arrows). Slug/Snail2 expression (stage 15) and FoxD3 expression (stage 15) appear partially lost or altered. Zic1 expression (stage 12), Zic3 expression (stage 12), Pax3 expression (stage 12) and Sox2 expression (stage 15) appear unchanged. Quantification of these results can be found in Fig. S5D. Scale bar=200 µm. (C) Knockdown of P-TEFb components leads to a loss of trigeminal placode markers. All embryos were injected in one cell of a two cell stage embryo with either 100 ng control morpholino (CoMo), 100 ng CDK9.S morpholino or 60 ng CYCLINT1 morpholino. 300 pg 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 trigeminal placode markers. Tbx2 expression (stage 18) shows loss of expression specifically in the trigeminal placode region (black arrow). Expression in the otic placode and dorsal root ganglia in not altered. NeuroD expression (stage 18) shows specific loss of expression in the trigeminal placode region (black arrow). ElrD expression (stage 18) is lost specifically in the trigeminal placode region (black arrow). Expression in the dorsal root ganglia remains unaltered. |
