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
* Author for correspondence (e-mail: mpack{at}mail.med.upenn.edu)
Accepted 3 October 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Zebrafish, Arthrogryposis-renal dysfunction-cholestasis syndrome, Human, Disease, tcf2, onecut1
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutations in the vacuolar sorting protein VPS33B have been found in
individuals with the arthrogryposis-renal dysfunction-cholestasis syndrome
(ARC), a rare autosomal recessive disorder
(Gissen et al., 2004)
associated with joint contractures, altered kidney function and cholestasis.
Dysmorphic facial features, diarrhea and poor growth are also common in such
individuals (Eastham et al.,
2001
). Yeast vps33, and other class C yeast vacuolar
sorting proteins (Vps11, Vps16 and Vps18), play an essential role in
intracellular trafficking (Sato et al.,
2000
; Peterson and Emr,
2001
). Mammalian Class C VPS proteins have a similar subcellular
localization as their yeast orthologs and are thought to play related roles
(Huizing et al., 2001
;
Kim et al., 2003
). Consistent
with this idea, mis-sorted liver and kidney membrane proteins have been
reported in the pathological specimens from individuals with ARC
(Gissen et al., 2004
).
Here, we present data showing that knockdown of zebrafish vps33b
causes cholestasis, bile duct paucity and other defects compatible with a
partial ARC syndrome phenocopy. Ultrastructural analysis identifies
cytoplasmic inclusions in biliary and intestinal epithelial cells that
resemble those seen in yeast vps class C mutants. These findings suggest that
intracellular trafficking is altered in vps33b-deficient cells and
that the cholestasis, diarrhea and growth retardation seen in individuals with
ARC may arise autonomously. By contrast, reduced motor neuron density, which
probably accounts for the arthrogryposis in individuals with ARC
(Di Rocco et al., 1990), was
not evident in vps33b-deficient embryos.
Biliary defects in vps33b-deficient larvae closely resembled
defects associated with knockdown of the onecut family transcription factor
hnf6 (onecut1 - Zebrafish Information Network)
(Matthews et al., 2004).
Molecular analyses showed that vps33b expression is reduced by
knockdown of hnf6 and by mutation of vhnf1 (tcf2 -
Zebrafish Information Network) a gene that functions downstream of
hnf6 in zebrafish (Matthews et
al., 2004
) and mammals (Clotman
et al., 2002
). Forced expression of vhnf1 activated
vps33b expression. Further, vHnf1 protein could bind and activate the
vps33b promoter. These data show that hnf6 indirectly
regulates vps33b expression through its downstream target gene,
vhnf1, and thus identify a novel pathway active during zebrafish
biliary development.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Morpholino oligonucleotides
Morpholino oligonucleotides (MOs) were designed based on sequences
available from the zebrafish genome assembly. Morpholinos were designed to
target the 5th and 18th exons, as these are sites of VPS33B mutations
in individuals with ARC. A translational start site morpholino was also
designed (Table 1). For the IE5
and IE18 MOs, 1.5 pg was injected at the one-cell stage, whereas 0.2 pg of the
translation initiation MO was used. Higher doses were lethal at early
embryonic stages. The same dose of a 4 bp mismatch control IE18 MO was lethal
at early embryonic stages. Lower doses had no effect on biliary development.
For the complementation experiments, the IE18 MO and a previously described
hnf6 MO (Matthews et al.,
2004) were used at one-tenth the above amount (150 fg).
To demonstrate specific MO targeting, PCR primers were designed that flanked the 5th exon and the 18th exon (Table 1, vps33b-1, -2, -5 and -6). cDNA synthesized from RNA isolated from 24 hpf control embryos and embryos injected with increasing doses of morpholino was used as template for PCR reactions with these primers. The two distinct products from the IE18-injected embryos were sequenced. The single product from the IE5-injected embryos was also sequenced.
Morpholinos directed against the translation start site and the splice donor site of exon 4 (EI4) from zebrafish vps33a were also designed, based on sequence from the zebrafish genome assembly (Zv4). The sequences of the morpholinos are listed in Table 1. Injection with either vps33a morpholino (1.5 pg) produced an identical phenotype. PCR primers to document vps33a knockdown are also listed in Table 1 (vps33a-1, -2).
PED-6 treatment
Wild-type (vehicle-injected) and morpholino injected larvae at 5 dpf were
soaked overnight in 0.1 µg/ml PED-6; as a control, swallowing function was
confirmed using fluorescent beads (Farber
et al., 2001; Matthews et al.,
2004
).
Immunostaining and electron microscopy
Wild-type and morpholino-injected larvae at 3, 4 and 5 dpf were fixed and
prepared as described (Matthews et al.,
2004). For motor axon staining, 48 hpf larvae were fixed in 4%
paraformaldehyde. For electron microscopy, 5 dpf larvae were fixed in buffered
gluteraldehyde. Keratin immunostaining of wild-type and morpholino-injected
larvae were performed as previously described
(Matthews et al., 2004
;
Lorent et al., 2004
). Larvae
for motor axon immunostaining were treated with proteinase K and then
incubated with mouse anti-ZNP1 and goat anti-mouse Alexa 488-conjugated
secondary antibody as described (Lefebvre
et al., 2004
). Processing and analysis of electron microscopy
specimens was identical to previously described methods
(Matthews et al., 2004
).
For cell proliferation and apoptosis assays, control and IE18-injected larvae were fixed in 4% paraformaldehyde at 3 and 4 dpf. Following collagenase treatment or skin dissection, larvae were incubated in anti-PCNA antibody (Sigma), followed by incubation with HRP-conjugated anti-mouse secondary antibody (Vector Laboratories) and staining using diaminobenzidine. Larvae were then embedded in JB-4, sectioned and examined for nuclear staining in the liver. For the apoptosis assay, the larvae were assayed using the Apoptag system (Chemicon) sectioned, and assayed for staining in the liver. As a positive control, we pretreated fixed wild-type 4 dpf larvae with 0.1 N HCl, which caused widespread DNA fragmentation.
AM1-43 labeling
Larvae at 5 dpf were soaked in 5 µM AM1-43 (Biotium) overnight and then
30 µM for 1 hour. To remove membrane bound AM1-43, larvae were then washed
twice briefly in 10 µM ADVASEP-7, followed by incubation in the ADVASEP-7
for one hour (Kay et al.,
1999), at which point they were fixed in 4% paraformaldehyde. The
larvae were then embedded in glycol methacrylate and sectioned and examined as
described (Wallace and Pack,
2003
). For quantification, 200-300 cells per larva from three
wild-type and four morpholino-injected larvae were examined, and the ratio of
the number of red AM1-43 labeled vesicles to epithelial nuclei was
calculated.
Quantitative real-time PCR
cDNA was synthesized from RNA obtained from 3 dpf control and hnf6
morpholino-injected whole larvae derived from three independent experiments as
previously described (Matthews et al.,
2004). Additionally, RNA was collected from whole 3 dpf larvae
injected with 2 ng/µl vhnf1 or 20 ng/µl hnf6 mRNA, as
well as from sorted 3 dpf vhnf1 mutants
(Sun and Hopkins, 2001
). The
resulting cDNA was used as template for real-time quantitative PCR as
described previously (Matthews et al.,
2004
). Primers for tbp and vhnf1 are listed
elsewhere (Matthews et al.,
2004
); the vps33b primers are listed in
Table 1. Either 3' primer
(12 or 13) was used with the 5' primer (11). Analysis was performed as
described previously (Matthews et al.,
2004
). For quantification of the PCR bands documenting morpholino
efficacy, NIH Image 1.63f was used.
Promoter cloning, transient transfections and luciferase reporter gene assays
A 1.5 kb region of the vps33b promoter was cloned from wild-type
TLF and AB strain zebrafish embryos using the primers noted in
Table 1 (p-f2, p-r2), based on
sequence from the zebrafish genome assembly (Zv4). For the transient
transfections and gel-shift assays, BMEL cells were cultured as described
(Plumb-Rudewiez et al., 2004).
For luciferase reporter assays, BMEL cells were transfected using
lipofectamine 2000 (Life Technologies), 400 ng
pGL-basic-vps33b(-1560/+139)-luc, 10 ng of pCS2-hnf6 or
pCS2-vhnf1 expression vector
(Matthews et al., 2004
) and 15
ng of the Renilla luciferase coding pRL-SVK3 vector as internal control.
Luciferase activities were measured after 24 hours with the dual luciferase
assay system in a TD-20/20 luminometer (Promega). Luciferase activities were
expressed as the ratio of reporter activity (firefly luciferase) to internal
control activity (Renilla luciferase).
Electrophoretic mobility shift assays (EMSAs)
For production of vHNF1, HNF6 or GFP, BMEL cells were transfected using
lipofectamine 2000 and 25 µg pCS2-hnf6 or pCS2-vhnf1
expression vector. Cells were washed with PBS and resuspended in hypertonic
buffer, and the nuclear extract was then incubated with 32P-labeled
oligonucleotide probe (see Table
1 for sequences). A 50-fold excess of unlabeled probe was added to
the binding reaction when testing for binding specificity. Antibodies against
HNF1ß (vhnf1) (polyclonal sc7411, Santa Cruz) or against HNF1
(polyclonal sc10791, Santa Cruz) were used in supershift experiments. After
incubation, the samples were separated by electrophoresis and the gels were
exposed.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Developmental expression of vps33b
Human VPS33B is expressed in a nearly ubiquitous pattern in adult
tissues (Huizing et al.,
2001). Staged in situ hybridization in embryonic and larval
zebrafish revealed widespread vps33b expression at sphere stage (4
hpf) through 24 hpf (Fig. 2).
At 48 hpf, vps33b expression localized to the brain, retina, ear,
liver and proximal intestine. This expression pattern was more pronounced at
72 hpf (Fig. 2) and persisted
through 5 dpf (not shown). Close examination of 3 dpf (not shown) and 4 dpf
(Fig. 2) larvae revealed a
reticular pattern of vps33b expression within the liver, suggesting
the possibility that vps33b is predominantly expressed in the
developing biliary epithelial cells. Histological analyses of these specimens,
and comparison with larvae that express the hepatocyte gene
ceruloplasmin, supported this interpretation
(Fig. 2). Neither kidney nor
spinal cord expression was evident in the whole-mount specimens at any stage
analyzed.
vps33b knockdown disrupts zebrafish biliary development
To assay the role of vps33b in zebrafish biliary development, gene
knockdown experiments using antisense morpholino oligonucleotides were
performed. Morpholinos were designed to target the 5' translational
start site and the splice acceptor sites for exons 5 ('IE5') and 18 ('IE18').
These latter sites correspond to regions that contain mutations found in
individuals with ARC syndrome (Gissen et
al., 2004).
Knockdown of vps33b did not affect the overall appearance or liver
size of larvae at 5 dpf (Fig.
3A,B). However, gallbladder fluorescence following ingestion of
the quenched fluorescent lipid, PED-6, was reduced in vps33b
morpholino larvae (n=12 of 14 larvae examined;
Fig. 3C,D). Transport of this
compound to the gallbladder can serve as an indicator of biliary secretion
(Farber et al., 2002), biliary morphology
(Matthews et al., 2004) and
intestinal lipid absorption. Thus, these data suggest the possibility that
vps33b deficiency might alter biliary development.
Keratin immunohistochemistry of 5 dpf morpholino-injected larvae strongly
suggest that altered biliary development contributed to the defects of PED-6
processing. In over 95% of larvae injected with 1.5 pg of the IE5 or IE18
morpholinos, the number of intrahepatic bile ducts was reduced compared with
control (n=50 larvae examined;
Fig. 3E-J), whereas the
extrahepatic biliary tree appeared normal in all morpholino-injected larvae
(data not shown). Within the intrahepatic biliary system, defects of the
interconnecting and terminal ducts were most pronounced
(Fig. 3H-J and
Table 2). Consistent with these
selective intrahepatic defects, biliary morphology appeared normal in 3 dpf
morpholino-injected larvae, whereas bile duct paucity was evident in 4 dpf
larvae and was more pronounced at 5 dpf (see Fig. S1 in the supplementary
material), the timepoints when the interconnecting and terminal ducts are
first evident (Lorent et al.,
2004; Matthews et al.,
2004
). Importantly, we did not identify alterations of cell
proliferation in the developing liver of morpholino-injected larvae, as
determined by the percentage of PCNA-positive cells, nor an increased number
of cells undergoing apoptosis (Table
3). Taken together, these data support the idea that bile duct
paucity in vps33b deficiency disrupts either growth of existing
biliary epithelial cells or the recruitment of new biliary cells from
undifferentiated progenitors.
|
|
|
|
|
One possible explanation for the lack of an effect in the spinal cord or
kidney would be the presence of another vps33 paralog in the
zebrafish genome. In mammals and insects, a second ortholog of yeast
vps33 has been identified. Mutation of this gene in the mouse
(buff) alters coat pigmentation and platelet function
(Suzuki et al., 2003), while
mutation in the fly (carnation) alters eye pigmentation
(Sevrioukov et al., 1999
). We
identified a zebrafish vps33a ortholog from the Ensembl database and
generated morpholinos to the translation initiation site and the exon 4 splice
acceptor site. An aberrant splice product of a 135 bp in-frame deletion was
identified from EI4-injected 24 hpf embryos (data not shown). Knockdown of
zebrafish vps33a did not disrupt motor neuron density (n=300
hemisegments), nor did it alter melanophore number or morphology
(n=80 larvae examined; data not shown). Thus, vps33a does
not perform the neuronal functions of vps33b, nor is it vital to
pigmentation development. Of note, vps33a knockdown had no effect on
biliary morphology (n=10 larvae examined; not shown). No other
potential orthologs of vps33b were identified in the zebrafish genome
assembly (Zv4).
|
We also examined intestinal ultrastructure in vps33b
morpholino-injected larvae. As depicted in
Fig. 6A-D, numerous vesicles
and enlarged Golgi cisternae were seen in intestinal cells from
vps33b morpholino larvae, but not in control larvae. This finding
suggested disruption of the secretory pathway. Supporting this idea,
intracellular vesicles accumulated in intestinal epithelial cells of
vps33b-deficient larvae following ingestion of AM1-43, a fixable form
of a styryl dye (FM1-43) used to track intracellular trafficking
(Cochilla et al., 1999)
(Table 4,
Fig. 6E-H). Disruption of the
secretory pathway could alter the uptake, intracellular transport or secretion
of dietary lipids by enterocytes, and thus might underlie the malabsorptive
diarrhea and poor growth frequently associated with ARC. Consistent with this
idea, vps33b-deficient larvae fail to process doses of the PED-6
lipid reporter (see Fig. 3)
that are readily processed by hnf6-deficient larvae with comparable
biliary defects (Matthews et al.,
2004
).
|
|
|
|
Regulation of vps33b by hnf6 and vhnf1 through its target gene vhnf1
To further define the mechanism whereby hnf6 and vhnf1
regulate vps33b expression, we searched sequences within the
vps33b promoter region for Hnf6 and vHnf1 binding sites.
TESS-assisted (transcription element search system) and visual scanning of
this sequence identified four putative Hnf1 sites and three Hnf6 sites
(Fig. 8). To determine whether
vHnf1 or Hnf6 regulated vps33b gene expression through binding at
these sites, 1.5 kb of sequence immediately 5' to the vps33b
translation initiation start site was cloned into a luciferase reporter gene
construct. This reporter construct was co-transfected with expression vectors
for hnf6 or vhnf1 into mouse embryonic liver cells.
Co-transfection of the vps33b reporter with the vhnf1
expression vector stimulated reporter gene expression 3.7 fold, while
co-transfection with the hnf6 expression vector had no significant
effect (Fig. 8).
Electrophoretic mobility shift assays demonstrated that the Hnf1-4 putative
binding site binds vHnf1 protein, whereas Hnf6 was not bound by any of the
putative binding sites within the vps33b promoter region
(Fig. 8; data not shown). These
data suggest that hnf6 regulates vps33b expression
indirectly, through vhnf1, and that vhnf1-mediated
regulation likely occurs through direct binding of vHnf1 to the
vps33b promoter. Interestingly, vps33b expression was
reduced in Hnf6-/- mice only postnatally (see Fig. S4 in
the supplementary material). These data suggest that the regulation of
vps33b expression in mammals may be more complex than in
zebrafish.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Knockdown of zebrafish vps33b suggests that bile duct paucity is
an important contributory factor to the pathogenesis of ARC liver disease. In
all affected vps33b morpholino-injected larvae, the number of bile
ducts was significantly reduced. Cytoplasmic inclusions reminiscent of the
vesicular-like defects seen with yeast class C vps gene mutations
were present in the biliary cells of vps33b morpholino-injected
larvae. This finding, plus the selective pattern of vps33b expression
within the larval zebrafish liver, suggests that biliary cells or committed
biliary progenitors are targeted by loss of vps33b function.
Alternative explanations for paucity, such as degeneration of biliary cells or
failure of multipotent progenitors to adopt a biliary fate seem unlikely, as
we saw no evidence of dying or degenerating biliary cells, nor did we see
immature hybrid cells such as those seen in larvae injected with
jagged morpholinos (Lorent et
al., 2004).
Proteins encoded by class C vps genes form a complex in both mammals and
yeast that is essential for intracellular protein trafficking
(Rieder and Emr, 1997;
Peterson and Emr, 2001
;
Kim et al., 2003
). Mutations
in yeast Class C vps genes are reported to interfere with
Golgi-to-endosome and endosome-to-vacuole protein transport. Mammalian class C
vps genes have been identified in both late and early endosomes, and
bind to Golgi proteins (Kim et al.,
2003
; Richardson et al.,
2004
). Although we cannot precisely localize which component of
the intracellular protein sorting pathway is disrupted in the
vps33b-deficient biliary cells, we hypothesize it leads to the
mislocalization or degradation of cellular proteins required for biliary
epithelial cell development.
Intestinal epithelial defects in vps33b-deficient larvae
Although growth retardation is common in many cholestasis syndromes, severe
fat malabsorption, such as occurs in ARC syndrome, is unusual. Here, we
present several lines of evidence that knockdown of zebrafish vps33b
phenocopies this feature of the ARC syndrome. First, Golgi abnormalities are
present in the cytoplasm of vps33b morpholino-injected but not
control intestinal epithelial cells. Second, vps33b
morpholino-injected larvae fail to process conventional doses of PED-6, a
quenched fluorescent lipid that is absorbed in the intestine, that are
normally processed by hnf6-deficient larvae with a comparable degree
of bile duct paucity (Matthews et al.,
2004). Third, labeling experiments using a fluorescent lipid
(AM1-43) frequently used to track exocytosis
(Cochilla et al., 1999
) showed
evidence of altered intestinal vesicle transport. These data suggest a model
in which fat malabsorption arises from altered intracellular trafficking of
dietary lipids within enterocytes. Although ultrastructural analysis of
intestine from individuals with ARC has not been reported, we predict that
defects related to those of vps33b-deficient zebrafish will be
identified.
Disparity of the zebrafish vps33b knockdown and human ARC syndrome phenotypes
We found no evidence that vps33b knockdown generated the
motoneuron defects responsible for congenital joint contractures
(arthrogryposis) associated with ARC syndrome. This could be due to the
existence of an unidentified vps33b paralogue, incomplete gene
knockdown or a non-conserved role for vps33b in nervous system
development. We believe the latter explanation is most likely. With respect to
a possible additional paralogue, we have found only two vps33 genes
in zebrafish in silico, corresponding to vps33a and vps33b.
Second, although the phenotype discrepancy could be due to incomplete gene
knockdown (see below), we did not observe specific spinal neural expression of
vps33b, suggesting that the function of vps33b in zebrafish
spinal cord may be assumed by another gene. We did examine whether knockdown
of zebrafish vps33a disrupted motoneuron development, but did not
observe defects using either of two non-overlapping morpholinos. Cutaneous
pigmentation was also normal in vps33a-deficient embryos, in contrast
to coat hypopigmentation associated with mutation of mouse vps33a
(Suzuki et al., 2003).
Together, these findings suggest that the roles of Class C vps genes have
evolved during vertebrate evolution. Interestingly, mutation of another
zebrafish Class C vps gene, vps18
(Golling et al., 2002
), causes
cutaneous hypopigmentation, hepatocyte defects and bile duct paucity, but
neither motoneuron nor kidney defects (K. Sadler and N. Hopkins, personal
communication). Overlapping biliary phenotypes of vps33b- and
vps18-deficient larvae support the idea that zebrafish Class C
proteins function in a complex, as described in yeast and mammals, while the
difference in phenotypes suggests that there are cell-specific roles for the
individual genes as well.
Kidney defects are another cardinal feature of the ARC syndrome. Several
types of abnormalities have been described, including renal tubular epithelial
degeneration, proximal renal tubular dysfunction in (renal contrast Fanconi
syndrome), nephrocalcinosis and mis-sorting of apical proteins in kidney
tubules (Nezelof et al., 1979;
di Rocco et al., 1990
;
Horslen et al., 1994
;
Eastham et al., 2001
;
Gissen et al., 2004
). Other
than the absence of renal cell degeneration, we could not determine whether
comparable kidney defects were present in vps33b-deficient larvae
because we lack suitable markers and assays. A role for vps33b in
zebrafish kidney seems unlikely, though, given the apparent lack of expression
of vps33b expression in developing zebrafish glomerulus or pronephric
duct of late embryos or larvae.
Partial loss-of-function or gene dose effects may be an alternative
explanation for why motoneuron and renal defects were not identified in any of
our vps33b morpholino-injected larvae. Although the amino acids
deleted by the IE18 morpholino are contiguous with a binding motif essential
for VPS33B function (Gissen et al.,
2005), a significant percentage of vps33b transcripts
were not efficiently targeted by either the IE18 or the IE5 morpholinos.
Although there is currently no way of knowing whether the seemingly normal
transcripts that persist in larvae injected with either of these morpholinos
are translated in vivo, the data suggest that there is at least some residual
vps33b activity in these fish. Consistent with this idea, a
morpholino targeting the vps33b translation initiation codon was
significantly more effective than either of the intron-exon morpholinos.
Regulation of vps33b by hnf6 and vhnf1
Knockdown of zebrafish vps33b causes a paucity phenotype nearly
identical to that associated with knockdown of hnf6. This, as well as
several other pieces of evidence, supports the idea that hnf6,
through its downstream target vhnf1, regulates vps33b
expression in zebrafish. First, knockdown of hnf6, as well as
mutation of vhnf1, reduces vps33b expression. Second,
ectopic vhnf1 expression, and to a lesser extent hnf6
expression, increases endogenous vps33b expression in zebrafish
embryos. Third, expression of zebrafish vhnf1, but not hnf6,
in mammalian embryonic liver cells activates a zebrafish vps33b
reporter construct, and vHnf1 binds a putative binding site within the
vps33b promoter. Fourth, partial knockdowns of hnf6 and
vps33b act synergistically to disrupt biliary development. These data
strongly support the idea that vps33b is a target of Hnf6/vHnf1
signaling.
Our data suggest that vps33b is not the only essential target of
Hnf6/Hnf1 signaling required for biliary development. The cytoplasmic vesicles
seen in the vps33b deficient biliary cells were not present in the
biliary epithelial cells of hnf6 morpholino-injected larvae. In
addition, vps33b mRNA injections did not rescue
hnf6-deficient larvae (R.P.M. and M.P., unpublished). We speculate
that altered expression of other zebrafish hnf6 target genes accounts
for these findings. What role hnf6-directed expression of
vps33b or other class C VPS genes plays during mammalian biliary
development is uncertain. Our data show that vps33b expression was
reduced in hnf6 mutants on post-natal day 3, but not during embryonic
stages. To account for these findings, we speculate that another class C VPS
gene may function downstream of hnf6 during bile duct development.
Consistent with this idea, bile duct paucity has recently been reported in
zebrafish vps18 mutant larvae, although vesicle accumulation in
hepatocytes, not bile duct cells, is reported in these mutants
(Sadler et al., 2005).
The role of vps33b in biliary development
Knockdown of vps33b affects late stages of biliary development in
zebrafish larvae, as reflected in the reduction of interconnecting and
terminal ducts. Currently, the mechanism underlying expansion of the larval
zebrafish biliary system is not known. We speculate that vps33b and
possibly other Class C vps genes may play a role in either the differentiation
of biliary progenitors or the growth of established biliary cells. Low-level
apoptosis of biliary progenitors beyond the level we could detect with our
assays, or non-apoptotic cell death of biliary cells, are alternative
explanations. The finding that vps33b knockdown leads to
ultrastructural defects in existing biliary cells could be interpreted as
supporting any of these mechanisms. Further experimentation will be required
to distinguish between these possible mechanisms, each of which could arise
from altered trafficking of membrane or secreted proteins.
The importance of vps33b in biliary development lies in the
identification of proteins whose trafficking is dependent upon the function of
class C vps genes. Proteins not properly localized to the cell membrane may
include signaling receptors or ligands, as well as transporters that may
function in biliary progenitors or other cell types. There may also be
non-membrane cytoplasmic proteins that require the C complex for proper
intracellular localization. Numerous human cholestatic conditions exist in
which there are defects in members of these classes of proteins.
Haploinsufficiency of the Notch ligand JAGGED1 causes Alagille syndrome, a
developmental disorder of which bile duct paucity is a cardinal feature
(Kamath and Piccoli, 2003).
Mutations of genes required for bile secretion, such as the ABC superfamily
members FIC1, BSEP, MDR3 and MRP2 cause chronic cholestasis
in various PFIC syndromes (Tomer and
Shneider, 2003
). Finally, cystic fibrosis and
1-antitrypsin
deficiency, both of which can be associated with cholestasis, result from
mislocalization of their respective gene products. Whether mislocalization of
these or other proteins in either hepatocyte or biliary cells leads to bile
duct paucity or contributes to cholestasis in individuals with ARC is not
known. Further knockdown experiments in zebrafish may help address this
question.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5295/DC1
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clotman, F., Lannoy, V. J., Reber, M., Cereghini, S., Cassiman, D., Jacquemin, P., Roskams, T., Rousseau, G. G. and Lemaigre, F. P. (2002). The onecut transcription factor HNF6 is required for normal development of the biliary tract. Development 129,1819 -1828.[Medline]
Cochilla, A. J., Angleson, J. K. and Betz, W. J. (1999). Monitoring secretory membrane with FM1-43 fluorescence. Annu. Rev. Neurosci. 22,1 -10.[CrossRef][Medline]
Di Rocco, M., Reboa, E., Barabino, A., Larnaout, A., Canepa, M., Savioli, C. and Cremonte, M. (1990). Arthrogryposis, cholestatic pigmentary liver disease and renal dysfunction: report of a second family. Am. J. Med. Genet. 37,237 -240.[Medline]
Eastham, K. M., McKiernan, P. J., Milford, D. V., Ramani, P.,
Wyllie, J., van't Hoff, W., Lynch, S. A. and Morris, A. A. M.
(2001). ARC syndrome: an expanding range of phenotypes.
Arch. Dis. Child 85,415
-420.
Farber, S. A., Pack, M., Ho, S. Y., Johnson, I. D., Wagner, D.
S., Dosch, R., Mullins, M. C., Hendrickson, H. S., Hendrickson, E. K. and
Halpern, M. E. (2001). Genetic analysis of digestive
physiology using fluorescent phospholipid reporters.
Science 292,1385
-1388.
Gissen, P., Johnson, C. A., Morgan, N. V., Stapelbroek, J. M., Forshew, T., Cooper, W. N., McKiernan, P. J., Klomp, L. W., Morris, A. A., Wraith, J. E. et al. (2004). Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome. Nat. Genet. 36,400 -404.[CrossRef][Medline]
Gissen, P., Johnson, C. A., Gentle, D., Hurst, L. D., Doherty,
A. J., O'Kane, C. J., Kelly, D. A. and Maher, E. R. (2005).
Comparitive evolutionary analysis of VPS33 homologues: genetic and functional
insights. Hum. Mol. Genet.
14,1261
-1270.
Golling, G., Amsterdam, A., Sun, Z., Antonelli, M., Maldonado, E., Chen, W., Burgess, S., Haldi, M., Artzt, K., Farrington, S. et al. (2002). Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat. Genet. 31,135 -140.[CrossRef][Medline]
Horslen, S. P., Quarrell, O. W. J. and Tanner, M. S. (1994). Liver histology in the arthrogryposis multiplex congenital, renal dysfunction, and cholestasis (ARC) syndrome: report of three new cases and review. J. Med. Genet. 31, 62-64.[Abstract]
Huizing, M., Didier, A., Walenta, J., Anikster, Y., Gahl, W. A. and Krämer, H. (2001). Molecular cloning and characterization of human VPS18, VPS11, VPS16 and VPS33. Gene 264,241 -247.[CrossRef][Medline]
Johnson, C. A., Gissen, P. and Sergi, C.
(2003). Molecular pathology and genetics of congenital
hepatorenal fibrocystic syndromes. J. Med. Genet.
40,311
-319.
Kamath, B. M. and Piccoli, D. A. (2003). Heritable disorders of the bile ducts. Gastroenterol. Clin. N. Am. 32,857 -875.[CrossRef][Medline]
Kay, A. R., Alfonso, A., Alford, S., Cline, H. T., Holgado, A. M., Sakmann, B., Snitsarev, V. A., Stricker, T. P., Takahashi, M. and Wu, L. G. (1999). Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat. Neuron 24,809 -817.[CrossRef][Medline]
Kim, B. Y., Ueda, M., Kominami, E., Akagawa, K., Kohsaka, S. and Akazawa, C. (2003). Identification of mouse Vps16 and biochemical characterization of mammalian Class C Vps complex. Biochem. Biophys. Res. Commun. 311,577 -582.[CrossRef][Medline]
Lefebvre, J. L., Ono, F., Puglielli, C., Seidner, G.,
Franzini-Armstrong, C., Brehm, P. and Granato, M. (2004).
Increased neuromuscular activity causes axonal defects and muscular
degeneration. Development
131,2605
-2618.
Lorent, K., Yeo, S.-Y., Oda, T., Chandrasekharappa, S., Chitnis,
A., Matthews, R. P. and Pack, M. (2004). Inhibition of
Jagged-mediated Notch signaling disrupts zebrafish biliary development and
generates multi-organ defects compatible with an Alagille syndrome phenocopy.
Development 131,5753
-5766.
Matthews, R. P., Lorent, K., Russo, P. and Pack, M. (2004). The zebrafish onecut gene hnf-6 functions in an evolutionarily conserved genetic pathway that regulates vertebrate biliary development. Dev. Biol. 274,254 -259.
Nezelof, C., Dupart, M. C., Jaubert, F. and Eliachar, E. (1979). A lethal familial syndrome associating arthrogryposis multiplex congenita, renal dysfunction, and a cholestatic and pigmentary liver disease. J. Pediatr. 94,258 -260.[Medline]
Peterson, M. R. and Emr, S. D. (2001). The class C vps complex functions at multiple stages of the vacuolar transport pathway. Traffic 2,476 -486.[CrossRef][Medline]
Plumb-Rudewiez, N., Clotman, F., Strick-Marchand, H., Pierreux, C. E., Weiss, M. C., Rousseau, G. G. and Lemaigre, F. P. (2004). Transcription factor HNF-6/OC-1 inhibits the stimulation of the HNF-3alpha/Foxa1 gene by TGF-beta in mouse liver. Hepatology 40,1266 -1274.[CrossRef][Medline]
Richardson, S. C. W., Winistorfer, S. C., Poupon, V., Luzio, J.
P. and Piper, R. C. (2004). Mammalian late vacuole protein
sorting orthologues participate in early endosome fusion and interact with the
cytoskeleton. Mol. Biol. Cell
15,1197
-1210.
Rieder, S. E. and Emr, S. D. (1997). A novel
RING finger protein complex essential for a late step in protein transport to
the yeast vacuole. Mol. Biol. Cell
8,2307
-2327.
Sadler, K. C., Amsterdam, A., Soroka, C., Boyer, J. and Hopkins,
N. (2005). A genetic screen in zebrafish identifies the
mutants vps18, nf2, and foie gras as models of liver
disease. Development
132,3561
-3572.
Sato, T. K., Rehling, P., Peterson, M. R. and Emr, S. D. (2000). Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol. Cell. 6,661 -671.[CrossRef][Medline]
Sevrioukov, E. A., He, J. P., Moghrabi, N., Sunio, A. and Kramer, H. (1999). A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila. Mol. Cell. 4,479 -486.[CrossRef][Medline]
Sun, Z. and Hopkins, N. (2001). vhnf1, the
MODY5 and familial GCKD-associated gene, regulates regional specification of
the zebrafish gut, pronephros, and hiindbrain. Genes
Dev. 15,3217
-3229.
Suzuki, T., Oiso, N., Gautam, R., Novak, E. K., Panthier, J. J.,
Suprabha, P. G., Vida, T., Swank, R. T. and Spritz, R. A.
(2003). The mouse organellar biogenesis mutant buff results from
a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation.
Proc. Natl. Acad. Sci. USA
100,1146
-1150.
Tomer, G. and Shneider, B. L. (2003). Disorders of bile formation and biliary transport. Gastroenterol. Clin. N. Am. 32,839 -855.[CrossRef][Medline]
Wallace, K. N. and Pack, M. (2003). Unique and conserved aspects of gut development in zebrafish. Dev. Biol. 255,12 -29.[CrossRef][Medline]
Related articles in Development: