Nuclear localization of Pk1b is required during FBMN migration. (A) The rescue construct. (B-J′′) Maximum projection dorsal views of FBMNs (red) in 48 hpf Tg(zCREST1:membRFP) zebrafish embryos. (B,C) FBMNs migrate to r6 in uninjected embryos (B), whereas migration is completely blocked in Pk1b morphants (C). (D-J) Representative embryos injected with Pk1bMO and/or Venus-tagged rescue constructs (green). CBMN-specific expression of full-length Venus-Pk1b can partially rescue FBMN migration to varying extents; embryos with higher transgenesis efficiency (D) generally display greater rescue capacity than those with lower efficiency (E). Expression of Venus-Pk1bΔCaaX (F), Venus-Pk1bΔNLS (G) or myr/palmVenus-Pk1bΔNLS (H) significantly reduces rescue capacity. Neither Venus-Pk1b (I) nor myr/palmVenus-Pk1bΔNLS (J) expression significantly disrupts migration in the absence of Pk1bMO. (F′-J′′) Magnified, single-slice views of boxed regions of F-J, showing nuclear exclusion of Venus in a subset of neurons (arrowheads). (K-L′) Dorsal views of CBMNs (red) in representative 48 hpf Tg(zCREST1:membRFP) embryos uninjected (B) and injected with Venus-Pk1b (green). Rhombomeres are indicated. (L′) Magnified view of boxed region in L. Arrowheads highlight TgBMNs migrating out of r2 into r3 and r4. (M,N) Venus-positive neurons were scored for their anteroposterior position. The percentage of neurons in r4-6 is shown. The number of neurons scored is indicated in parentheses. ***, P<0.001; **, P<0.01; χ2 test (relative to MO + Venus-Pk1b). (O) Embryos that displayed at least one Venus-positive TgBMN were scored. The percentage of embryos with at least one Venus-positive migratory TgBMN is displayed. The number of embryos scored is indicated in parentheses. Scale bars: 20 μm.
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