2B) Bioinformatic analyses of the zebrafish genome (Build 7, Ens

2B). Bioinformatic analyses of the zebrafish genome (Build 7, Ensembl) did not reveal a second copy of cdipt. To investigate whether knockdown of Cdipt can replicate the hi559 phenotype, we injected two different splice-blocking morpholinos against cdipt into wild-type embryos. To reduce nonspecific effects due to activation of tp53, we coinjected tp53 morpholino.26 Approximately 60% (53/90) of the injected embryos displayed hepatic abnormalities similar to hi559 (Supporting Fig. 2A-D). Injection of 100 pg of cdipt LBH589 ic50 mRNA

resulted in significant reduction of larvae with hepatic phenotype from a clutch, rescuing presumably mutant embryos (Supporting Fig. 2E). In a clutch (n = 147) of cdipt mRNA–injected embryos, only 5 (3.4%, expected 25%) Luminespib displayed the typical hi559 phenotype at 5 dpf; all remaining larvae appeared normal. In the control group (injected with gfp mRNA), ≈25% (19/70) showed the typical hi559 phenotype.

These results strongly suggest that the hi559 phenotype is due to loss of Cdipt function. At 24 hours postfertilization, cdipt mRNA expression was ubiquitous, but became restricted to the developing liver and intestine by 48 hours postfertilization and remained high in these tissues through 5 dpf (Fig. 2C-E). Cdipt was also expressed in the brain, retina, and branchial arches throughout development. As expected, hi559 embryos lacked cdipt expression (Fig. 2E [right] and Supporting Fig. 3). Because CDIPT plays a critical role in PtdIns synthesis, we wanted to confirm alteration of PtdIns levels in the absence of cdipt expression. Surprisingly, comparative phopholipid profile by way of thin layer chromatography revealed that levels of PtdIns and other phospholipids of deyolked wild-type and hi559 larvae were similar at 5 dpf (Supporting Fig. 4). However, we noticed that the embryonic yolk at 1-cell stage MCE contained abundant PtdIns, suggesting that it was maternally deposited. We reasoned that de novo PtdIns synthesis might be disrupted in hi559 embryos and tested PIS activity in 5-dpf wild-type and mutant larvae. PIS activity was

negligible in hi559, but robust in wild-type siblings (Fig. 2F). To further confirm that chemical inhibition of PIS replicates the hi559 phenotype, we treated wild-type larvae with δ-hexachlorocyclohexane, a drug with the same configuration as myo-inositol, known to inhibit myo-inositol incorporation into PtdIns.27 Treatment with δ-hexachlorocyclohexane resulted in hepatomegaly and a darkish liver similar to hi559 (Supporting Fig. 5). These results suggest that maternally deposited and de novo–synthesized PtdIns are not functionally equivalent, and that de novo synthesis of PtdIns is required for normal hepatic development. In sagittal sections, the hi559 liver appeared swollen and vacuolar, with enlarged hepatocytes and increased internuclear distance between adjacent hepatocytes compared with wild-type liver (Fig. 3A).

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