The embryonic expression pattern of zebrafish zfyve9a. Embryos were hybridized to an antisense probe to zfyve9a (A-C, G-J) or a sense control probe (D-F). (A) Maternal zfyve9a mRNA was detected at 2-cell stage. (B-C) Low level expression of zfyve9a at the shield and 10-somite stages. (G-I) zfyve9a was enriched in the liver at day 2 and 3. It was also highly expressed in the brain and neural tube at day 3. (H) is the cross-section of (G) at the position indicated by the red line. (J) is the cross-section of (I) at the indicated position. NT, neural tube; L, liver; G, gut.

Zfyve9a is required for liver development. (A) A cartoon of the zfyve9a gene structure shown the positions of MO-AUG, MO-SP and primers used in RT-PCR reaction in (C). (B) MO-AUG (4 ng) effectively inhibited the translation of Zfyve9a-GFP mRNA (100 pg) as determined at about 70% epiboly stage. (C) RT-PCR analysis for the effect of MO-SP. PCR products from wild type and zfyve9a morphants were cloned and sequenced. The lower band in MO-SP treated sample corresponds to a deletion of the C-end of exon 2. (D) Representative live images of wild type embryo and zfyve9a morphants at day 3. (E-F) Expression of fabp10a in WT, MO-AUG or MO-SP injected embryos at day 3 (E) and day 4 (F). (G) Expression of another liver marker, cp, in WT embryos and zfyve9a morphants at day 4. (H-J) Knockdown of zfyve9a did not affect the development of pancreas or gut at day 4. insulin labels the pancreatic beta-cells, trypsin indicates the exocrine pancreas and fabp2 marks the intestine.

The specification of hepatoblasts is normal in zfyve9a morphants. (A) The expression of pan-endoderm marker foxa3 in WT embryos and zfyve9a morphants at 30 hpf. (B,C) The expression of prox1a in liver bud was clearly detected in zfyve9a morphants at 30 and 48 hpf. The neuronal expression of prox1a was severely reduced in morphants at these stages. (D) Expression of gata6 at day 2 in the endoderm of WT embryos and zfyve9a morphants. (E,F) Hepatic expression of myca and cp were initiated normally in zfyve9a morphants at day 2. Arrows in all panels indicate the liver bud.

Zfyve9a regulates the proliferation but not survival of hepatic cells. (A) Representative images of P-H3 staining of embryos at day 2. Green indicates the GFP-labeled endoderm cells. The outlined area is the liver. (B) Statistical results of the percentage of P-H3 positive hepatic cells in MO-CTL or MO-AUG injected embryos at 48 and 60 hpf. Stars indicates P<0.05 as analyzed by the t-test method. (C) Representative images of TUNEL assay at day 2. (D) The percentage of apoptotic hepatic cells in MO-CTL or MO-AUG treated embryos at 48 and 60 hpf. Error bars in (B) and (D) present the SD.