Figure 5

Activation of kinesin-1 or Inhibition of cytoplasmic dynein-1 rescues the phenotype of high melanosome aggregation in adcy3a^-/-;adcy5^-/- double mutants. (A?C) WT embryos treated with either kinesore or ciliobrevin D from 24 hpf to 96 hpf show high melanosome dispersion when compared with DMSO controls. (D,E) dync1h1-MO-injected WT embryos display significant melanosome dispersion compared with control-MO-injected embryos. (F?H) adcy3a^-/-;adcy5^-/- double mutants show significant dispersion of melanosomes when treated with either kinesore or ciliobrevin D from 24 hpf to 96 hpf compared with DMSO-treated groups. (I,J) adcy3a^-/-;adcy5^-/- double mutants injected with dync1h1-MO exhibit significant dispersion of melanosomes in comparison with controls. (K,L) Statistical analysis of pigmentation coverage in WT embryos (K) and adcy3a^-/-;adcy5^-/- double mutants (L) treated with DMSO, kinesore, and ciliobrevin D, respectively, as well as either injected with dync1h1-MO or control-MO (Student?s t-test, ** p < 0.01. **** p < 0.0001). (M?O) The dispersion of melanosomes throughout the body is detected in kinesore (N,N?) or ciliobrevin D-treated adcy3a^-/-;adcy5^-/- double mutants (O,O?), compared with DMSO-treated groups (M,M?). (M??O?) are the partially magnified images of boxes in (M?O). (P) dync1h1-MO injected adcy3a^-/-;adcy5^-/- double mutants display melanosome dispersion throughout the body. (P?) is the partially magnified images of boxes in (P). (Q) A working model for Adcy3a and Adcy5 signaling cascades involving melanosome dispersion and melanin synthesis: ?-MSH binds to Mc1r and activates Adcy3a and Adcy5, leading to intracellular cAMP increase; the increased cAMP stimulates Pka to guide melanosome dispersion by activating kinesin-1 and suppressing cytoplasmic dynein-1, and promotes melanin synthesis through increasing the expression of mitf and its downstream target genes tyr, trp-1, and trp-2. Scale bar = 100 ?m in (A?J). Scale bar = 250 ?m in (M?P).