Opposing Roles of Bmal1 and Period2 in Regulation of Developmental Angiogenesis(A) Zebrafish embryos received injections of specific morpholinos targeting Bmal1 (B1MO), Bmal2 (B2MO), Clock3 (C3MO), or Period2 (P2MO) (0.2 pmol/embryo) and were subsequently raised under LL or LD conditions. Scrambled morpholino (scrMO), five base-pair mismatch bmal1 (B1misMO), or five base-pair mismatch period2 (P2misMO) were used as controls. Dashed lines indicate the maximal length of ISVs. Bar = 50 μm.(B) Quantification of the average length ± SEM of ISVs in 24 hpf embryos under the LD condition (n = 8–48 embryos/group).(C) Quantification of the average length ± SEM of ISVs in 24 hpf embryos under the LL condition (n = 6–37 embryos/group).(D) Morphology of zebrafish embryos was examined at 24 hpf and 48 hpf under LD and LL conditions. Bars = 500 μm. S, somite; NC, notochord; H, hindbrain; M, mesencephalon; E, eye.(E) Quantification of average ± SEM somite lengths and head angles under LD and LL conditions (n = 7–17/group).(F) Zebrafish embryos received injections of specific morpholinos targeting Bmal1 (B1MO), Period2 (P2MO), scrambled morpholino (scrMO), bmal1 mRNA (B1mRNA), period2 mRNA (P2mRNA), B1mRNA plus B1MO, or P2mRNA plus P2MO and were subsequently raised under LD or LL conditions. Dashed lines indicate the maximal length of ISVs. Bar = 50 μm.(G) Quantification of the average length ± SEM of ISVs in 24 hpf embryos under the LD condition (n = 8–27 embryos/group).(H) Quantification of the average length ± SEM of ISVs in 24 hpf embryos under the LL condition (n = 6–28 embryos/group).(I) qPCR of average ± SEM period2 mRNA expression levels after 48 or 72 hpf LD or LL exposure (n = 3–4 × 20–50 embryos/group). p < 0.05; p < 0.01; and p < 0.001. NS, not significant.See also Figures S1 and S2.
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