Fig. 3

Inpp5b morpholino affects multiple organ development in zebrafish.

(A) Immunoblot analysis of 40 microgram of total lysates of zebrafish embryo injected with control MO (4 ng) or inpp5b MO (4 ng) at 48 hpf with anti-INPP5B and anti-beta-actin antibodies. (B) Zebrafish embryos were injected with p53 MO (2 ng) or p53 MO (2 ng) and inpp5b MO (4 ng). Representative phenotypes of microphthalmia (black arrowhead), pericardial edema (small arrow), body axis asymmetry, kinked tail (white arrow), pronephric cyst formation (red arrow), and hypopigmentation were observed at 48 hpf. Scale bar 250 micron. (C) Dose-dependent effect of morpholinos in zebrafish. Control or inpp5b MO at indicated doses was injected into zebrafish embryos, and phenotypes of microphthalmia, kinked tail, and body asymmetry were quantified at 48 hpf (ANOVA, F = 92, p = 3.6E-10), kinked tail (ANOVA, F = 3.6, p = 0.08), and body asymmetry (ANOVA, F = 5.2, p = 0.04). (N = the number of injected embryos N >50). (D) Quantification of eye size of morphants at 24 hpf, 48 hpf and 72 hpf. The eye size was determined by the longest diameters in dorsal view. (N >40 embryos, three independent experiments, unpaired t-test, * p = 4.98E-07 ** p = 2.1E-10, ns, not statistically significant). (E) Cresyl violet staining of ocular sections of zebrafish larvae (5 dpf) injected with control MO (4 ng) or inpp5b MO (4 ng). Scale bar 30 micron. (F) Zebrafish embryos were injected with control MO (4 ng), inpp5b MO (4 ng) or inpp5b MO (4 ng) and Inpp5b WT mRNA (Inpp5b mRNA, 500 pg). Representative phenotypes of microphthalmia (arrowhead), pericardial edema (arrow), body axis asymmetry, kinked tail (white arrow), pronephric cyst formation (red arrow), and hypopigmentation were observed at 48 hpf. Scale bar 250 micron. (G) Quantification of eye size of zebrafish morphants at 24 hpf and 48 hpf. (N >40 embryos, three independent experiments, unpaired t-test, * p = 4.5E-05). (H) The ventral sides of embryos were injected with control MO (4 ng), inpp5b MO (4 ng) or inpp5b MO (4 ng) and Inpp5b WT mRNA (500 pg). Scale bar 100 micron.