Fig. 1

Fig. 1 Dnmt3 knockdown in zebrafish embryos results in neurogenesis defects. A, splice blocking by dnmt3 splice-blocking morpholino (dnmt3 Mo1) was monitored by RT-PCR in mRNAs from control and dnmt3 morphants at 80 hpf. PCR was performed using a forward primer in exon 6 and a reverse primer in exon 7. Note that the dnmt3 Mo1 stabilizes the unspliced transcript containing introns 6 and 7 (400 bp), whereas the spliced version (300 bp) is detected in control injected embryos. Percent knockdown (% KD) shown is the ratio of intensity of unspliced product to combined intensity of unspliced and spliced products normalized over β-actin intensity. B, morphology defects in dnmt3 morphants at 80 hpf. Note the smaller head, a pericardial edema, and a curled tail in dnmt3 morphants compared with control morphants. Bar equals 0.5 mm. C, whole mount in situ analysis of ngn-1, ascl1a, and ascl1b expression in dnmt3 morpholino alone or with p53 morpholino-, dnmt1 morpholino-, or control morpholino-injected embryos at 30 hpf. Whereas ngn-1 expression was normal in dnmt3 and dnmt1 morphants, ascl1a and ascl1b were selectively is absent. This defect can be complemented by co-injection of the wild-type (Dnmt3^WT) but not by a catalytically inactive derivative (Dnmt3^C1240S). D, protein expression levels of Dnmt3 wild-type (Dnmt3^WT) and catalytically inactive (Dnmt3^C1240S) derivatives. HEK293 cells were transfected with Dnmt3^WT and Dnmt3^C1240S, and Western blots were performed using antibodies against His tag. Both of these show equal expression of these derivatives. β-Actin was used as a loading control.