hi459 is a zygotic null allele of arl13b (sco). (A) arl13b transcripts in embryos at 5 dpf shown by RT-PCR of cDNA from hi459 mutant embryos (lanes 2, 4, 6) and wild-type siblings (lanes 1, 3, 5). m, size marker; lanes 1-2, RT-PCR with a pair of primers spanning the proviral insertion site in arl13b; lanes 3-4, RT-PCR with a pair of primers for beta-actin as loading controls; lanes 5-6, RT-PCR with a pair of primers 3′ to the proviral insertion site in arl13b. (B) Absence of Arl13b in hi459 mutant embryos at 5 dpf. A band of the predicted size can be seen in samples immunoprecipitated with anti-Sco from lysate of wild-type siblings (w), but not in the sample from hi459 mutant embryos (m), nor in the lysate precipitated with protein A beads alone. (C) RT-PCR time-course for arl13b expression. 4-32, four- to 32-cell stage; 256-1k, 256- to 1000-cell stage; 30%, 30% epiboly. (D) Presence of Arl13b in embryos at the 64-cell stage. Lysates from embryos at the 64-cell stage were subjected to immunoprecipitation using anti-Sco (Sco) or pre-immune IgG (Pre) and precipitated proteins detected by western analysis using anti-Sco. (E-H) Wild type (wt) and arl13b morphant (mph) embryos in dorsal view (E,F) and side view (G,H) at around the 8-somite stage. Arrows in E and F point to somites. In G and H, body gap angle was labeled using ImageJ. (I) Gap angle in degrees in wild type (wt, n=13) embryos and arl13b morphants (mo, n=14; *P<0.001). (J) Gap angle in degrees in arl13b morphants co-injected with eGFP (eGFP, n=13) or sco mRNA (sco, n=15; *P<0.001).
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