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Fig. 5 Expression of npas4l-target genes requires npas4l, arnt1 and arnt2. Zebrafish embryos, derived from the indicated parental genotypes, were injected at the one-cell stage with 50 pg of npas4l mRNA or with 50 pg each of arnt1 and arnt2 mRNA (100 pg total RNA). Embryos were fixed at shield stage and probed for tal1 or lmo2 and npas4l-target genes, using whole-mount in situ hybridization. Embryos were then genotyped. (A-C) In wild-type embryos, tal1 is expressed after injection of npas4l RNA. tal1 mRNA was not detected in uninjected embryos or after injection of arnt1 and arnt2 RNA. (D-F) Heterozygous npas4l mutant fish were crossed to each other and the resulting embryos were injected with the indicated RNA. npas4l RNA, but not arnt1 and arnt2 RNA, upregulated expression of tal1. Genotype of embryos in E: n=11 npas4l+/+, n=15 npas4ls5/+ and n=10 npas4ls5/s5. Genotype of embryos in F: n=12 npas4l+/+, n=20 npas4ls5/+ and n=8 npas4ls5/s5. (G-I) arnt1bcm2/+;arnt2bcm3/+ adults were crossed to each other and the resulting embryos were injected with npas4l RNA. Double homozygous embryos failed to upregulate lmo2 (I), whereas sibling embryos upregulated lmo2 (H). Genotype of embryos in H: n=1 arnt1+/+;arnt2+/+, n=7 arnt1bcm2/+;arnt2bcm3/+, n=1 arnt1+/+;arnt2bcm3/+, n=8 arnt1bcm2/+;arnt2+/+, n=4 arnt1+/+;arnt2bcm3/bcm3, n=7 arnt1bcm2/bcm2;arnt2+/+, n=7 arnt1bcm2/+;arnt2bcm3/bcm3 and n=4 arnt1bcm2/bcm2;arnt2bcm3/+. Genotype of embryos in I: n=3 arnt1bcm2/bcm2;arnt2bcm3/bcm3. Embryos are oriented in a lateral view with the animal pole towards the top. Fractions in the bottom right corner indicate the number of embryos with the represented phenotype over the total number of embryos assayed. Scale bar: 100 μm.