Fig. 6

The Insulin Receptor Is a Substrate for Bace2 and Regulates Melanophore Dendricity

(A and B) Inhibition of the insulin receptor in bace2^−/− embryos using the insulin receptor inhibitors BMS-754807 (7.5 μM) and NVP-AEW541 (60 μM) rescue the hyperdendritic melanophores in the mutant, quantified in (B). bace2^−/− embryos are treated from 24 to 72 hpf, and the data are from three independent experiments. One-way ANOVA followed by Holm-Sidak's multiple comparisons test, ^∗p < 0.1, ^∗∗∗∗p < 0.0001.

(C and D) Morpholino knockdown of insulin receptor (Insr) in bace2^−/− embryos rescues the hyperdendritic melanophores, quantified in (D). Insra and insrb morpholinos were coinjected into bace2^−/− embryos to deplete all insulin receptors. The data are from three independent experiments, two-tailed t test, ^∗∗∗∗p < 0.0001.

(E and F) Bace2 cleaves the insulin receptor. Expression of zebrafish Insra-Myc (E) and Insrb-Myc (F) fusion protein in HEK293T cells give rise to full-length insulin receptor and the β chain. Inhibition of γ secretase using DAPT (2 μM, 24-hr treatment) prevents the CTF from being cleaved, leading to CTF accumulation. Expression of zebrafish Bace2 (zBace2) on top of the Insra-Myc fusion protein leads to production of single CTF band (E). zBace2 addition leads to the production of two fragments from Insrb-Myc, one that migrates similarly to the CTF and additional larger fragment designated as CTF^∗ (F). This is not seen with the enzymatic dead version of Bace2 (zBace2 dead) or after Bace2 inhibition with PF-06663195 (24-hr treatment). zBace2 dead was constructed by site-mutagenesis of two conserved catalytic sites aspartic acids into alanines (D98A and D292A).

(G and H) Bace2 inhibition with PF-06663195 (25 μM, 24-hr treatment) in ZMEL1 cells, which normally express both Bace2 and the insulin receptor, leads to accumulation of endogenous insulin receptor β chain, quantified in (H). The data are from five independent experiments, two-tailed t test, ^∗∗p < 0.01.

(I and J) Melanophore-specific inhibition of insulin signaling in bace2^−/− mutants rescues melanophores hyperdendricity in the tailfin. A mosaic F0 transgenic line was created in which the fugu tyrp1 promoter drives dominant-negative insulin receptor substrate 2 (dnIRS2) fused to EGFP in the melanophores (along with a cmlc2: mCherry transgene marker). At 72 hpf, tailfin melanophore cell area was quantified in uninjected control (Control) and transgene-positive injected embryos (J). The data are from three independent experiments, two-tailed t test, ^∗∗∗∗p < 0.0001. All bar graphs are presented as means ± SEM. Scale bar, 100 μm.

See also Figures S5 and S6.