36 The in vivo functions SAR245409 datasheet of a few SNX genes have been investigated. For example, SNX1 and 2 have been knocked out in the mouse, and mice lacking either one of them are viable and fertile. However, the double-knockout mice die at midgestation, which complicates the detailed analysis of the in vivo functions of SNX1 and 2.37 SNX13 knockout mice are also embryonic lethal,38

whereas SNX27 plays essential roles during postnatal growth and survival.39 We started to investigate the in vivo functions of SNXs in the zebrafish model. We identified six SNX genes expressed in the embryonic liver and found that one of them (SNX7) was indispensable for hepatogenesis. The specification and proliferation of hepatoblasts were normal when SNX7 was blocked. However, these cells underwent extensive apoptosis during the budding stage of hepatogenesis. We concluded that an antiapoptotic activity of SNX7 was crucial for the survival

of hepatoblasts during liver budding. BMP, bone morphogenetic protein; c-FLIP, cellular FLICE-like inhibitory protein; c-FLIPL, the long form of c-FLIP; c-FLIPS, the short form of c-FLIP; CHX, cycloheximide; cp, ceruloplasmin; DAPI, 4′,6-diamidino-2-phenylindole; Wnt inhibitor dnmt, DNA methyltransferase; EGFR, epidermal growth factor receptor; FACS, fluorescence-activated cell sorting; FGFs, fibroblast growth factors; foxA3, forkhead box protein A3; gata6, GATA-binding factor 6; hdac, histone deacetylase; Hhex, hematopoietically expressed homeobox; hpf, hours postfertilization; HNF, hepatocyte nuclear factor; ifabp, intestinal fatty acid binding protein; ins, insulin; LDL, low-density lipoprotein; leg1, liver-enriched gene 1; lfabp, liver fatty acid binding protein; Mib1, mindbomb 1; MO, morpholino; mRNA, messenger RNA; mypt1, myosin phosphatase target subunit 1; PARP, poly(ADP-ribose) polymerase; P-H3, phosphorylated histone

3; Prox1, prospero homeobox protein 1; RA, selleck inhibitor retinoic acid; RT-PCR, reverse-transcription polymerase chain reaction; siRNA, short interfering RNA; SNX, sorting nexin; TGF-β, transforming growth factor beta; TNFα, tumor necrosis factor alpha; tomm22, translocase of outer mitochondrial membrane 22; try; trypsin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; uhrf1, ubiquitin-like protein containing PHD and Ring finger domains-1; vps18, vacuolar protein sorting protein 18; WT, wild type. Detailed protocols, including zebrafish manipulation, cell culture and short interfering RNA (siRNA) treatment, immunostaining, fluorescence-activated cell sorting (FACS) analysis, real-time reverse-transcription polymerase chain reaction (RT-PCR), and western blotting, can be found in the Supporting Materials and Methods. We performed a BLAST search against the zebrafish genome and EST databases, using human SNX sequences as references, and identified 38 zebrafish SNX family genes.