Fig. 4

Defects of cilia formation in Inpp5b zebrafish morphants.

(A) INPP5B WT mRNA rescue the loss of inpp5b. KV cilia of zebrafish embryos injected with control MO (4 ng), inpp5b MO (4 ng) or inpp5b MO (4 ng) and INPP5B WT mRNA (500 ng) at 6-somite stage were immunostained with acetylated α-tubulin (red), representative images are shown (dash line indicates border of KV). Scale bar 10 micron. (B–C) Quantification of number (B) and length (C) of KV cilia in zebrafish embryos injected with control MO (4 ng), inpp5b MO (4 ng) or inpp5b MO (4 ng) and INPP5B WT mRNA (500 ng). (N >20 embryos, three independent experiments, unpaired t-test, * p = 3.4E-03 in B and * p = 1.9E-23 in C). (D) INPP5B WT mRNA rescue of inpp5b pronephric cilia formation. Representative image of pronephric cilia of zebrafish embryos at 24 hpf stage, injected with control MO (4 ng), inpp5b MO (4 ng) or inpp5b MO (4 ng) and INPP5B WT mRNA (500 ng), immunostaining with acetylated α-tubulin (red). Scale bar 10 micron. (E) Pronephric cilia length of control and inpp5b MO. Pronephric cilia of zebrafish embryos injected with control MO (4 ng), inpp5b MO (4 ng) or inpp5b MO (4 ng) and INPP5B WT mRNA (500 ng) at 24 hpf stage were analyzed by immunostaining with acetylated alpha-tubulin and cilia length was measured. (N >200 cilia; three independent experiments, unpaired t-test, * p = 5.45E-18). (F) Ocrl and Inpp5b morphants showed slowed retrograde melanosome transport. Representative photos are shown for the melanosomes in Ocrl and Inpp5b morphants before and after treatment with epinephrine in 5 dpf embryos (box, region of pigment evaluation). (G) Quantification of the response time for epinephrine treatments in the control MO (2 ng), ocrl MO (2 ng), ocrl MO (2 ng) and OCRL WT mRNA (500 pg), inpp5b MO (2 ng), and inpp5b MO (2 ng) and Inpp5b WT mRNA (500 pg) embryos (N >30 embryos, three independent experiments, unpaired t-test, * p = 4.6E-50, ** p = 2.3E-43).