Fig. 3

Knockdown of lfng produces a neurogenic phenotype. (A-H) Confocal images showing dorsal views of control MO (A-D) and lfng^E1I1 MO (E-H) injected embryos at 30 hpf, immunostained for HuC/D (red) and EphA4 (green) to mark postmitotic neurons and r3/r5, respectively. B-D and F-H are higher magnifications of the areas indicated in A and E, respectively. Arrowheads and arrows indicate rhombomere boundaries and centres, respectively. (I,J) Confocal images of the expression of RFP (red) and H2B-Citrine (green) in r3 and r5 in Tg(r3/r5-Gal4::UAS-RFP) embryos at 18 hpf, either noninjected (I) or injected with UAS-H2B-Citrine plasmid (J). Tg(r3/r5-Gal4::UAS-RFP) is an enhancer trap that drives Gal4 and mCherry expression in r3/r5; the expression of UAS-citrine in r3/r5 illustrates the targeting of transgene expression to these rhombomeres. (K) Quantification of the number of differentiating neurons (HuC/D expression) in Tg(r3/r5-Gal4::UAS-RFP) 18 hpf embryos injected with control MO or lfng^E1I1 MO together with either a control vector (UAS-H2B-Citrine) or a vector to overexpress Lfng (UAS-Lfng::UAS-H2B-Citrine). Transgenic expression of Lfng in r3/r5 leads to a decreased number of differentiated neurons and partly rescues the effect of lfng knockdown, whereas neurogenesis in r4 is not affected. Values are mean ± standard error, neurons in four embryos were counted for each experimental group; ^*, P<0.05, ^**, P<0.01. Scale bars: 50 μm in A for A-H; 50 μm in I for I,J.