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First published online November 10, 2005
doi: 10.1242/10.1242/dev.02140

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Zebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6

¹ Division of Gastroenterology and Nutrition, The Children's Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
² Hormone and Metabolic Research Unit, University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Brussels B1200, Belgium
³ Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
⁴ Section of Medical and Molecular Genetics, University of Birmingham, Birmingham B15 2TT, UK
⁵ Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

View larger version (59K): [in a new window]   Fig. 1. Sequence and mapping of zebrafish vps33b. (A) Deduced amino acid sequence of zebrafish and human vps33b. Identical amino acids are shaded black, similar residues in gray. VPS33B mutations from individuals with ARC are colored red (identical residue) and green (similar residue). Red lines refer to the location of ARC mutations at splice sites conserved in the zebrafish and human vps33b/VPS33B genes. (B) Map comparison of zebrafish supercontig NA54330 and human chromosome 15. Supercontig locations of vps33b and three other zebrafish genes (serf2,small EDRK-rich factor 2; mfap1, microfibrillar-associated protein 1; prc1, protein regulator of cytokinesis 1) are noted. Arrows indicate locations of the orthologous genes on human chromosome 15.

View larger version (94K): [in a new window]   Fig. 2. vps33b expression in zebrafish embryos and larvae. (A-I) Whole-mount RNA in situ hybridization. Sphere stage (A), 10 somite (B) and 24 hpf (C) embryos show diffuse vps33b expression. (D) vps33b expression at 48 hpf is evident in the developing liver (black arrow) and proximal intestine (blue arrow). (E) Lateral view of a 72 hpf larva showing vps33b expression in the liver (black arrow) and proximal intestine (blue arrow). Weak pancreas expression (white arrow) is also evident. (F,G) High-power lateral views of 4 dpf larvae processed for vps33b and ceruloplasmin whole-mount RNA in situ hybridization. Liver (black arrow) demonstrates a reticular pattern of vps33b expression (black arrow) (F) compared with a homogeneous pattern of ceruloplasmin expression (G). Intestinal vps33b expression is also evident (blue arrow, F). (H) Higher power view of liver depicted in F. (I) Enhanced view of H outlining putative ducts. (J,K) Histological cross-sections of a 4 dpf larva processed for vps33b (J) and ceruloplasmin (K) whole-mount RNA in situ hybridization. These panels show punctate regions of vps33b expression in presumptive bile ducts (green arrowheads) and a small number of hepatocytes (red arrowheads). Biliary epithelial cell size and cytoplasm in these panels are comparable with biliary epithelial cell ultrastructure (Fig. 5) (Lorent et al., 2004; Matthews et al., 2004).

View larger version (111K): [in a new window]   Fig. 3. vps33b knockdown disrupts zebrafish intrahepatic biliary development. (A,B) Left lateral views of 5 dpf wild-type (A) and vps33b (B) morpholino-injected larvae. Liver size (black arrowheads) is comparable in these larvae. (C,D) Right lateral fluorescent images of 6 dpf wild-type (C) and vps33b morpholino injected larvae (D) following ingestion of the PED-6 lipid reporter. Gallbladder fluorescence (white arrow) is decreased in morpholino-injected (D) larva relative to wild-type larva (C). i, intestinal fluorescence; y, endogenous yolk fluorescence. (E-G) Confocal projections through the liver of 5 dpf wild-type (E) and vps33b (F,G) larvae processed for keratin 18 immunohistochemistry. There are fewer bile ducts in F than in E; ducts are sparse, with fewer interconnecting ducts and terminal ductules. (H-J) Colorized schematics of bile ducts from E-G. Long ducts depicted in blue, interconnecting ducts in green and terminal ductules in red.

View larger version (20K): [in a new window]   Fig. 4. vps33b morpholino targeting and altered splice product from IE18. (A,B) Schematics showing exons 4 and 5 and intervening intron 4 (A), and exons 17 and 18 and intervening intron 17 (B) of the vps33b gene. The sequences targeted by the IE5 and IE18 morpholinos are depicted in green and red, respectively. The wild-type and morpholino-targeted mRNA transcripts are depicted below the genomic region. Targeting by the IE18 morpholino generates a novel cDNA that uses a cryptic splice acceptor site within exon 18. The site of splicing induced by the IE5 morpholino could not be determined. (C,D) RT-PCR shows altered vps33b expression induced by both the IE5 and IE18 morpholinos. For these experiments, cDNA derived from 24 hpf wild-type and morpholino-injected larvae was amplified using primers flanking exons 5 or 18. There is decreased amplification of the wild-type vps33b fragment relative to tbp, quantified below, in both sets of morpholino-injected larvae. Wild-type (blue) and truncated (red) fragments are amplified from IE18 morpholino-injected larvae. (E) The shorter cDNA fragment amplified from IE18 morpholino injected larvae has an in-frame deletion of 15 amino acids includes R438 (red), which is mutated in some individuals with ARC.

View larger version (112K): [in a new window]   Fig. 5. vps33b knockdown disrupts biliary ultrastructure. (A,B) Electron micrographs of biliary epithelial cells from 5 dpf wild-type (A) and vps33b morpholino injected (B) larvae. The wild-type biliary cell cytoplasm has a homogeneous appearance. (B) A small bile duct comprising two bile duct cells from a vps33b morpholino-injected larva. Cytoplasm appears heterogeneously with multiple vesicles (black arrows). hep, hepatoctye; bd, bile duct lumen; bdc, bile duct cell.

View larger version (137K): [in a new window]   Fig. 6. vps33b knockdown disrupts intestinal vesicle transport. (A-D) Electron micrographs of enterocytes from 5 dpf wild-type (A) and vps33b morpholino injected (B-D) larvae. The apical surface is to the right of each panel. Insets are at twice the magnification of the indicated regions of cytoplasm. No vesicles can be identified within the wild-type cell cytoplasm. (C) Multiple vesicles (arrows) are seen in the cytoplasm of vps33b-deficient enterocyte. Dilated stacks of Golgi cisternae are also evident within these cells (D). g, golgi; m, mitochondria; n, nucleus. (E-H) Histological cross-sections from the anterior intestine of 5 dpf wild-type (E,G) and vps33b morpholino-injected (F,H) larvae that have ingested the styryl dye AM1-43. Nuclei stained with DAPI. (G,H) Magnified views from E and F, respectively. There was a 1.8-fold increase in the number of fluorescent vesicles in the vps33b-deficient larvae (see Table 4).

View larger version (44K): [in a new window]   Fig. 7. vps33b expression is regulated by hnf6/vhnf1. (A,B) vps33b expression in 3 dpf wild-type (cont) hnf6 morpholino-injected (A) and vhnf1mutant larvae (B) as determined by quantitative PCR. vps33b expression is reduced by 75% and 60%, respectively, in the hnf6 morpholino-injected larvae and vhnf1 mutants. Amplification levels normalized to wild type. (C-F) Whole-mount RNA in situ hybridization of 3 dpf wild-type (C,E) and hnf6 (D,F) morpholino-injected larvae using a ceruloplasmin (C,D) or a vps33b (E-F) riboprobe. Liver (black arrows) ceruloplasmin expression is unchanged in the hnf6 morpholino-injected larva, whereas decreased liver vps33b expression is evident. (G) Comparison of vps33b expression in 3 dpf wild-type larvae (cont) and in 3 dpf larvae injected with hnf6 morpholino (hnf6 MO), hnf6 morpholino and vhnf1mRNA, vhnf1 mRNA, or hnf6 mRNA. These experiments are normalized to control expression and show decreased vps33b expression (by 75%, as above) in hnf6-deficient larvae that is restored with co-injection of vhnf1 RNA. Microinjection of vhnf1 on its own increases vps33b expression (2.2x), as does hnf6 RNA to a lesser degree (1.3x). Error bars represent s.e.m. from six separate experiments.

View larger version (60K): [in a new window]   Fig. 8. Zebrafish vps33b promoter is regulated and bound by Hnf1. (A) Sequence of the putative zebrafish promoter. Predicted Hnf6 binding sites are highlighted in yellow, predicted vHnf1 binding sites are underlined and in blue, and the predicted sequence of the first untranslated exon is noted in green. (B) Expression of a luciferase vps33b reporter gene [vps33b(-1560/+139)-luciferase] is activated in BMEL cells by vHnf1, but not Hnf6 or GFP proteins. *P<0.05. (C) Electrophoretic mobility shift assays performed using nuclear extracts from BMEL cells that were transfected with GFP (lane 1) or vHnf1 (lanes 2-5) expression constructs. Lanes in which the extracts are incubated with anti-vHnf1 antibody (vHnf1; lane 3), anti-Hnf-1 antibody (Hnf-1; lane 4), or 50x excess cold probe directed against the vHnf1-4 site (lane 5) are noted. Addition of cold probe eliminates supershift seen in lane 3. Arrows indicates the gel shift resulting from vHnf1 binding (bound vHnf1) or vHnf1/anti-vHnf-1 antibody binding (supershifted vHnf1) to the labeled probe.