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First published online 12 December 2007
doi: 10.1242/dev.006098
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,
,
1 Swiss Federal Institute of Technology, Department of Biology, and Brain
Research Institute of the University of Zurich, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland.
2 Institute of Zoology, University of Zurich, Winterthurerstrasse 190, CH-8057
Zurich, Switzerland.
3 Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076
Tübingen, Germany.
Author for correspondence (e-mail:
stephan.neuhauss{at}zool.uzh.ch)
Accepted 23 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Zebrafish development, Eye, Vision, Pigmentation, Liver, Vesicle trafficking, Lysosomes and lysosome-related organelles, Vam6p/Vps39
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (OMIM
#208085). Similarly, the Chediak-Higashi syndrome (CHS), Hermansky-Pudlak
syndrome (HPS) and Griscelli syndrome (GS) have been linked to a number of
proteins involved in the formation, trafficking and fusion of lysosome and
lysosome-related organelles [OMIM #214500 (CHS), #203300 (HPS), #214450 (GS1),
#607624 (GS2), #609227 (GS3)]. These inherited human diseases are associated
with hypopigmentation, prolonged bleeding and immunological defects. It has
also been reported that affected individuals display visual defects, including
photophobia, strabismus and nystagmus. Although mutations in several genes
have been linked to these diseases, it has not yet been possible to identify
the molecular defects in all cases.
Lysosome-related organelles are cell type-specific organelles that share
several physiological properties with lysosomes
(Raposo and Marks, 2002). They
include organelles, such as melanosomes in skin and RPE melanocytes, lamellar
bodies in type II lung epithelial cells, dense granules in platelets and MHC
class II compartments in antigen-presenting cells, and cytotoxic granules of
lymphocytes. Tethering and docking of lysosomes and lysosome-related
organelles depends on the HOPS complex (homotypic fusion and vacuole protein
sorting complex). This complex interacts with both SNAREs and Ypt7p/Rab7
(Collins et al., 2005;
Price et al., 2000a;
Price et al., 2000b;
Sato et al., 2000;
Wurmser et al., 2000) and is
required for SNARE complex assembly
(Stroupe et al., 2006).
Furthermore, the HOPS complex seems to be important for early-late endosome
transition in the Rab-conversion model in mammalian cells
(Rink et al., 2005). The HOPS
complex comprises the class C Vps protein Vam5p/Vps33p, the protein defective
in ARC syndrome; the class C proteins Vam1p/Vps11p, Vam9p/Vps16p and
Vam8p/Vps18p; and two class B Vps proteins, Vam6p/Vps39p and Vam2p/Vps41p
(Rieder and Emr, 1997;
Seals et al., 2000;
Wurmser et al., 2000)
(reviewed by Bowers and Stevens,
2005).
Vam6p/Vps39p was shown to be required for vacuolar protein sorting in
yeast. Yeast Vps39p-null mutants exhibit highly fragmented vacuolar
morphogenesis and mutant cells accumulate numerous vesicular structures
scattered throughout the cytoplasm
(Nakamura et al., 1997).
Similarly, overexpression studies in cultured HEK cells have implicated Vam6p
in the clustering and fusion of lysosomes and late endosomes
(Caplan et al., 2001) and
blocking Vam6p function with antibodies suggests a possible role in sperm
cells during their interactions with the zona pellucida in mice
(Brahmaraju et al., 2004).
We have identified a zebrafish mutant (leberknödel; lbk) that carries a mutation in the zebrafish homologue of Vam6p/Vps39p and displays phenotypes similar to those observed in humans suffering from ARC syndrome, CHS, HPS and GS. Our studies reveal multisystemic defects in lbk, including a hypopigmentation of skin melanocytes and the retinal pigment epithelium (RPE). Moreover, lbk displays defects in internal organs (liver, intestine) and the innate immune system. Affected cells display increased numbers and enlarged intracellular vesicles. Physiological and behavioural analyses of visual function in lbk uncovered a reduced visual ability. These analyses suggest that Vam6p/Vps39p has essential functions in a range of tissues during zebrafish development. Notably, no animal model to study the function of Vam6p/Vps39p in vivo has been reported to date.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), WIK
(Dahm et al., 2005) and
lbkt20290. Staging of embryos in hours (hpf) and days
post-fertilization (dpf) was carried out as previously described
(Kimmel et al., 1995).
Transplantations
Transplantations were performed as described
(Ho and Kane, 1990). Mosaic
animals were generated by transplanting 30-40 cells from 3-4 hpf wild-type
Tü embryos into the animal pole region, including prospective ectodermal
domains, such as the eye and neural crest domains
(Kimmel et al., 1995), of
age-matched embryos obtained by crossing two lbk+/-
carriers. Larvae displaying a clear lbk-/- phenotype were
scored for the presence of skin melanocytes and RPE cells displaying wild-type
levels of melanin.
Histology, immunohistochemistry and TUNEL assay
For histological analyses by light microscopy (LM), larvae were fixed in 4%
paraformaldehyde (PFA) in PBS (pH 7.2) at 4°C overnight and washed three
times in PBS. The embryos were dehydrated in a standard ethanol series,
infiltrated and embedded in Technovit 7100 (Heraeus Kulzer, Germany) for
sectioning. Sections (3 µm) of different developmental stages of mutant and
wild-type larvae were cut with a glass knife and mounted on Superfrost Plus
slides (Microm International, Switzerland). The sections were subsequently
stained with 0.5% Toluidine Blue in a 1% sodium tetraborate buffer (pH 9.2)
and analysed using an Axioscope 2Mot (Zeiss, Jena, Germany) connected to an
AxioCamHR colour camera using the AxioVision 3.0 software (Zeiss).
For ultrastructural analysis by transmission electron microscopy (TEM), larvae were fixed in 2% PFA and 2.5% glutaraldehyde in 0.1 M HEPES buffer (pH 7.2) at 4°C overnight and subsequently washed three times in HEPES buffer. Larvae were then postfixed with 1% OsO4 in 100 mM PO4 buffer (pH 7.2) for 1 hour on ice, washed with double distilled H20, treated with 1% aqueous uranyl acetate for 1 hour at 4°C, dehydrated through a graded series of ethanol, infiltrated with ethanol/resin mixtures and embedded in Epon (using glycidether 100 from Roth, Karlsruhe). Ultra-thin sections were collected on coated slot grids, stained with uranyl acetate and lead citrate and viewed in a Philips CM 10 electron microscope.
For immunohistochemistry, larvae were fixed in 4% PFA in PBS (pH 7.2) for 45 minutes at room temperature, cryoprotected in 30% sucrose for at least 2 hours, embedded in OCT TissueTek (Jung-Leica; Tissue Freezing Medium) and frozen in liquid nitrogen (N2). Sections (20 µm) were cut at -20°C, mounted on SuperFrost Plus slides, fixed in ice-cold acetone for 1 minute and stored at -20°C. For further use, slides were thawed, air-dried, washed three times in PBS (0.5x, pH 7.4) and blocked with a solution of 20% normal goat serum and 2% bovine serum albumin in PBS containing 0.3% Triton X-100 (PBST) for 1 hour. Sections were then incubated for 2 hours at 4°C in the primary antibody. Primary antibodies used in this study were anti-glutamine synthetase (1:700; Chemicon, Temecula, USA) and anti-rhodopsine (1:200; MilanAnalytica, LaRoche, Switzerland). Antibodies were diluted in PBST. After three washes in PBST, sections were incubated in Alexa546-coupled anti-mouse secondary antibody (1:500; Molecular Probes/Invitrogen, Eugene, OR) for 1 hour, washed three times in PBST, mounted in glycerol and analysed with an Axioscope fluorescence microscope as described above. Apoptosis was detected using the TUNEL (TdT-mediated dUTP nick-end labelling) method on 35 µm cryosections. TUNEL staining was performed using the Cell Death Kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. Oil red O in dextrin was used to visualise lipid-containing vesicles in cryosections (20 µm). Confocal laser scanning microscopy (CLSM) was performed with an LSM510 (Zeiss) and 25x PlanNeofluar and 63x PlanApochromat oil immersion objectives (Zeiss).
Genetic mapping of lbk and radiation hybrid mapping of vam6
Map crosses were set up between heterozygous lbk+/-
(Tü background) and wild-type WIK zebrafish. The offspring from these
crosses were inbred and homozygous lbk-/- and sibling F2
progeny were collected and their DNA extracted. Bulked segregant analysis on
48 lbk-/- larvae and 48 siblings, respectively, was
carried out using 192 simple sequence length polymorphisms (SSLP markers)
distributed over the entire genome
(Geisler, 2002). Further fine
mapping was performed using the total DNA of single homozygous mutant larvae.
DNA extraction and PCR were performed as described
(Geisler, 2002). The zebrafish
vam6 gene was radiation hybrid (RH) mapped on the Goodfellow T51 RH
panel as described (Geisler,
2002) using six independent primer pairs
(Table 1). PCRs for RH mapping
were carried out independently in triplicate.
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). Briefly, single larvae were placed in the centre of a
Petri dish (35 mm diameter) filled with 3% pre-warmed (28°C)
methylcellulose facing with their right eye a screen at an apparent distance
of 4.65 cm within their visual field. Moving sine-wave gratings were projected
by a HP vp6111 projector onto the screen. The projection size on the screen
was 8x6 cm, subtending a visual angle of 65.6° horizontally and
53.1° vertically. Eye movements were recorded by an infrared-sensitive CCD
camera triggered by the visual stimulation. A custom developed programme based
on the LabView IMAQ software (National Instruments, version 5.1) was used to
control the stimulation and the camera as well as to analyse the resulting
images. Contrast sensitivity functions for mutant and wild-type larvae were
measured by the eye velocity as a function of the contrast of the moving
grating. Statistics were derived as described previously
(Rinner et al., 2005a) by
bootstrap sampling of differences between randomly drawn samples
(n=10,000) from a pooled data set of wild-type and mutant larvae. The
significance level chosen was at 5% for all experiments.
For recordings of electroretinograms (ERGs)
(Makhankov et al., 2004), 5
dpf larvae were dark-adapted for at least 30 minutes prior to positioning them
in the recording chamber. The recording chamber was shielded from interference
from external sources of electromagnetic radiation by placing it inside a
tight Faraday cage. Each larva was placed on its side on the surface of a
moist sponge with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2
and 0.33 mM MgSO4) and paralyzed by directly adding a drop of the
muscle relaxant Esmeron (0.8 mg/ml; Organon Teknika, Eppelheim, Germany) onto
the larva. An Ag/AgCl electrode system was used to record the ERG responses.
The recording electrode was positioned on the centre of the cornea. The
reference Ag/AgCl pellet was placed under the body of the larva. All
pre-recording steps were carried out under dim red light illumination. An
additional 5-minute period in complete darkness was chosen to adapt the larva
to dark prior to measurements. White light stimuli (100 mseconds) were used to
elicit ERG responses with interstimulus intervals of 5 seconds. Light stimuli
were fixed at five relatively different light intensities ranging from 2 lux
(OD 5) to 20,000 lux (OD 0). Unattenuated light stimulus intensities were
measured over the head of the larvae using a light meter (Tektronix J17, Texas
Instruments, USA) and found to account for 3100 lux (optical density (OD)
equal to 0 log units). Different light intensities were adjusted using neutral
density filter wheels. A virtual instrument (VI) under NI LabVIEW 5.1 was
developed for use in all experiments. Sampling was carried out in buffered
acquisition mode with a sampling rate of 1000 Hz. The resulting ERGs, as the
corneal sum field potentials of the retina in response to light, are shaped
like in other vertebrates: a small negative deflection, termed the a-wave,
which reflects photoreceptor activation, is followed by a stronger positive
deflection, the b-wave, which reflects second order neuron activation. The
small a-wave is often masked by the much larger b-wave because of
interference. Therefore, in contrast to the b-wave, the a-wave is not a robust
measure in larval zebrafish. As a consequence, the b-wave was taken as an
indirect measure of outer retina activation. For rescue experiments, a light
intensity of 6000 lux was chosen to elicit ERG responses in sibling and
lbk-/- larvae. ERG responses obtained from one larva were
averaged three to seven times depending on signal-to-noise ratio. Statistical
analysis was performed using GraphPad Prism4 (GraphPad Software, San Diego,
CA) software and graphs were generated using Origin v.7 (OriginLab,
Northampton, MA).
Bacteria injections
Salmonella enterica serovar Typhimurium expressing DsRed
(van der Sar et al., 2003)
were injected with a FemtoJet injection apparatus (Eppendorf) at a constant
pressure of 80 hPa for 0.5 seconds into the common cardinal vein of 48 hpf
larvae embedded in low-melting point agarose as previously described
(van der Sar et al., 2003). To
ensure delivery of equal amounts of bacteria, mutant and sibling larvae were
injected with the same injection capillary and the injection of the bacteria
was monitored under a fluorescence stereo-microscope (Zeiss). For bacterial
counts, larvae were macerated in PBS and plated onto ampicillin-containing
bacterial plates as described (van der Sar
et al., 2003; van der Sar et
al., 2006). Bacterial growth was assessed after 24 hours of
incubation at 37°C with a fluorescence stereo-microscope.
Rescue experiments
The rescue was performed by injecting a pSGH2 vector containing the
vam6 cds under the control of a heat-shock promoter
(Bajoghli et al., 2004) into
one-cell stage embryos derived from a cross of two lbk+/-
individuals. At 3 dpf, injected larvae were heat shocked at 38°C for 2
hours and subsequently transferred back to 28°C. The extent of the rescue
was determined at 5 dpf by assessing the level of pigmentation in skin
melanocytes and RPE and in ERG measurements.
Morpholino knock-down
A 25 nucleotide morpholino antisense oligonucleotide was designed against
the ATG region of the vam6 mRNA: GAACTGGTTCGTATGCGTCGTGCAT. The
morpholino was injected using a pressure of 30 hPa for 4 mseconds into
one-cell stage wild-type embryos at different oligonucleotide concentrations:
100 µM, 500 µM and 700 µM, corresponding to approximately 1.5 ng, 7.5
ng and 10.5 ng of morpholino. As a control, the generic control morpholino
provided by GeneTools (Philomath, OR) was used.
Detection of vam6 expression
For PCR analysis, total RNA was isolated from 100 wild-type zygotes and 24
hpf embryos, respectively, using the RNAeasy kit and cDNA synthesised using
the SuperScriptIII Reverse Transcriptase kit. PCR was performed at an
annealing temperature of 60°C for 35 cycles using primers listed in
Table 2.
Whole-mount in situ hybridisation
Sense and antisense probes for in situ hybridisation were in vitro
transcribed from a linearized pCRII-Vector containing the full-length
vam6 cds using SP6 and T7 polymerases, respectively, in the presence
of digoxigenin-coupled nucleotides (DigRNA labelling kit; Roche Molecular
Biochemicals, Switzerland). Transcripts were hydrolysed to obtain fragments of
300-500 nucleotides in length. To prevent melanisation of skin
melanocytes and the RPE, embryos used for in situ hybridisation were treated
with 0.2 mM PTU (1-phenyl-2-thiourea; Sigma). PTU-treated embryos were staged,
fixed in 4% PFA in PBS (pH 7.25) at 4°C overnight, dehydrated in a
standard methanol series and stored in 100% methanol at -20°C. In situ
hybridisation was performed using an automated in situ hybridisation apparatus
(Hölle&Hüttner, Tübingen, Germany). Hybridisation was
carried out overnight at 58°C. Probe detection was performed with an
alkaline phosphatase-coupled anti-Dig Fab-fragment (Roche) and NBT/BCIP
staining buffer. Stained embryos were cleared with 50% methanol/50% glycerol
and transferred into 100% glycerol for imaging.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). External analyses revealed that, starting at 3 dpf,
lbk-/- larvae show a pronounced hypopigmentation of skin
melanocytes and the RPE. Instead of the evenly black melanocytes
characteristic of wild-type larvae, lbk melanocytes show only scarce
and patchy pigmentation (Fig.
1, see Fig. S1 in the supplementary material). It is interesting
that in addition to melanosome-containing cells the iridophores are affected
in lbk. Iridophores derive their reflective properties from vesicular
organelles containing guanine crystals
(Morrison and Frost-Mason,
1991). These light reflections are absent in lbk, both in
the eye and body, at all stages of development
(Fig. 1E-H), indicating that
the biogenesis of these organelles is disrupted. We cannot, however, exclude
the possibility that iridophores are missing altogether, although, in view of
the overall lbk phenotype, this possibility appears unlikely. Further
to the pigment cell phenotypes, starting at 4 dpf, the intestinal tract of
lbk develops a brownish discolouration
(Fig. 1A-F,I,J) and the liver
becomes enlarged (Fig. 1I,J).
lbk-/- larvae fail to develop swim bladders and die
between 7 and 8 dpf.
Mapping and candidate gene approach identify a mutation in the vam6 gene as underlying the lbk phenotype
To identify the mutation underlying the lbk phenotype, we mapped
the mutation on the zebrafish genome. Pooled DNA of 48
lbk-/- and 48 sibling 5 dpf larvae, respectively, was
tested with 192 SSLP markers (Knapik et
al., 1998) resulting in a linkage of the mutation to chromosome 17
between markers z22083 and z4053 (Fig.
1K). To narrow down the critical interval of the mutated locus, we
tested additional SSLP markers located in this genomic region. Analysis of 282
meioses identified z9692 and z4053 as the closest SSLP markers
(Fig. 1K).
As the phenotype observed in lbk suggests a defect in endosomal
vesicle trafficking (see below), we performed RH mapping of several genes
involved in this process. This approach resulted in a linkage of the zebrafish
vam6 gene to the EST fc27c07.x1 on chromosome 17. This EST is located
in close proximity to z9692 on the T51 RH map
(Fig. 1K)
(Geisler et al., 1999). Given
this close linkage, we cloned and sequenced the zebrafish vam6-coding
sequence (cds), which has a length of 2628 bp corresponding to a protein of
875 amino acids (see Fig. S2 in the supplementary material).
Sequencing of pooled vam6 cDNA from lbk-/-
larvae and phenotypically wild-type siblings, respectively, revealed a
nonsense mutation at position 1066 of the vam6 cds
(Fig. 1L), which segregated
with the mutant phenotype. This point mutation was confirmed by sequencing
exon 11 from genomic DNA of the larvae identified as recombinant in the fine
mapping approach. All 15 individuals displaying the lbk phenotype
were genotypically homozygous for the point mutation, whereas all
phenotypically wild-type individuals were either heterozygous or homozygous
for the wild-type allele (not shown). The putative truncated protein product
lacks the C-terminal 520 amino acids, including the central clathrin homology
(CLH) repeat domain, which has been demonstrated to play an essential role in
the clustering and fusion of lysosomes
(Caplan et al., 2001), and
Ypt7p-interacting sequences (Wurmser et
al., 2000) (Fig.
1M,N).
To identify additional lbk alleles and thus confirm that a
mutation in vam6 underlies the lbk phenotype, we performed
complementation analyses with described mutants displaying similar external
phenotypes (Glass and Dahm,
2004; Kelsh et al.,
1996). These analyses identified no additional lbk
alleles. We therefore performed an allele screen by crossing heterozygous
lbk+/- individuals with ENU-mutated Tü zebrafish.
Over 1000 genomes were screened for external phenotypes equivalent to that
observed in lbk, indicating non-complementation of the novel allele
with the lbk mutation. This approach identified a compound
heterozygous larva (lbk*; see Fig. S3 in the supplementary
material) harbouring in one allele of the vam6 gene the original
C
T exchange found in lbk and in the second allele a TA
exchange at position 374 of the vam6 cds
(Fig. 1O). The latter mutation
results in the exchange of a conserved methionine residue for a lysine at
amino acid position 125 in the N-terminal citron homology (CNH) domain of the
Vam6p protein (see Figs S2, S3 in the supplementary material). This domain has
been shown to be required for lysosome clustering and fusion
(Caplan et al., 2001).
Importantly, siblings from the same cross carrying the lbk nonsense
allele, did not carry the TA exchange identified in
lbk* compound heterozygotes
(Fig. 1O), confirming that this
mutation disrupts Vam6p in the lbk* larva. Sequencing of
142 vam6 alleles confirmed that this TA exchange does not occur
in the genetic pool from which the founder fish for the ENU-mutagenesis were
derived (see Fig. S3 in the supplementary material). The compound heterozygous
mutant was not viable and the allele was not recovered to generate a permanent
line. Therefore, the information that can be derived from the analysis of the
lbk* mutant is limited to the one experiment reported
here.
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lbk larvae have a compromised RPE and are severely visually impaired
Hypopigmented retinas often have additional defects and affected zebrafish
were shown to display decreased visual ability
(Schonthaler et al., 2005).
Similarly, in humans several pathological conditions with RPE hypopigmentation
are associated with more generalised ocular symptoms. This prompted us to
examine the eye phenotype in lbk more closely. Histological analyses
revealed that the overall patterning of the neural retina is normal
(Fig. 2A-F). However, sections
taken at different developmental stages revealed not only a severe
hypopigmentation of the RPE, but also a progressive thickening
(Fig. 2A-H) up to three times
the thickness of the wild-type RPE (Fig.
2R). This thickening results from an increase of RPE cell size,
rather than increased numbers of RPE cells
(Fig. 2S). TEM analyses of
lbk revealed substantially fewer and mostly immature and aberrantly
shaped melanosomes (Fig.
2I-N,Q). As melanin is, however, present in the
lbk-/- RPE and skin melanocytes (see Fig. S5 in the
supplementary material), the mutation affects the biogenesis of melanosomes
rather than melanin biosynthesis. This is supported by the observation that
the number of mature melanosomes is substantially reduced in lbk
(Fig. 2V).
In addition to optically shielding photoreceptor cells (PRCs), the RPE is essential for PRC function by phagocytosing shed PRC outer segments. Outer segments harbour the photopigments that allow the detection of light and the shedding of parts of these segments is an essential process in the maintenance of normal PRC function. The RPE cytoplasm in lbk is filled with numerous vesicles whose number increases between 5 and 7 dpf (Fig. 2I-L). Some of these vesicles contain thick membrane stacks reminiscent of the membrane discs in PRC outer segments (Fig. 2M,N), suggesting a defect in the fusion of endocytic vesicles with lysosomes.
To test this hypothesis, we used rhodopsin as a molecular marker for rod outer segments (Fig. 2O,P). lbk larvae (5 dpf) show an increase in rhodopsin-positive vesicles inside the RPE, while in the wild-type, rhodopsin antibodies only label continuous rod outer segments. Quantification confirmed that the lbk RPE contains increased numbers of outer segment-containing vesicles (Fig. 2T). Staining of eye cryosections with Oil Red O confirmed the presence of numerous lipid-containing vesicles in the lbk RPE (Fig. 4J). These data further suggest a failure in the mutant RPE to metabolise phagocytosed PRC membrane stacks.
At 5 dpf, the PRC outer segments are fully developed in the zebrafish. In lbk mutants, however, they are reduced in length and their regular palisade arrangement is disrupted (Fig. 2I-L,U). Moreover, the microvilli from the RPE that normally interdigitate with the outer segments, cannot be detected. As development proceeds, these phenotypes get stronger resulting in PRCs with very short or even no outer segments (Fig. 2L).
Although we found the outer retina to be severely affected, the inner retina showed no evidence of morphological alterations in lbk (Fig. 2A-F). Similarly, staining of retinal Müller glia cells, which span the entire inner retina and are thus a good marker for inner retinal architecture, revealed no differences in their number, arrangement and morphology between wild-type and lbk-/- larvae (see Fig. S6 in the supplementary material). Starting at 7dpf, however, apoptotic cells can be detected in the neural retina (Fig. 2W).
To analyse the physiological effect of the lbk mutation on visual performance, we tested larvae by optokinetic response (OKR) measurements. In this test, eye tracking movements of immobilised larvae exposed to moving visual stimuli are used to assess visual ability. In contrast to wild-type larvae, moving gratings consistently failed to evoke eye movements in all lbk-/- larvae tested, indicating that mutants are behaviourally blind (Fig. 3A). However, this effect is not due to the inability of the mutants to move their eyes, as spontaneous eye movements were regularly observed. We further performed electroretinogram (ERG) analyses of 5 dpf wild-type and lbk larvae (Fig. 3B). In wild-type larvae the b-wave amplitude increased with increasing light intensities. In lbk, however, it remained small even at high light intensities. At 7dpf, the ERG of lbk-/- is flat, indicating that at this stage the mutant retina no longer responds to light stimuli (not shown).
lbk larvae display vesicle phenotypes in the liver and intestinal tract
Subsequent analyses revealed additional phenotypes in internal organs,
including the liver and intestine. Starting at 6dpf, the liver of lbk
larvae becomes progressively enlarged and darkly discoloured
(Fig. 1I,J). Histological
analyses confirmed this enlargement and showed a significant swelling of liver
cells (Fig. 4A,B). This
phenotype was confirmed when lbk-/- mutants were crossed
to a transgenic line, Tg (ef1:GFP), that expresses GFP under the control of
the intestinal promoter ef1 (Field et al.,
2003) and sections imaged by confocal microscopy
(Fig. 4C). To quantify the
increase in liver size observed in lbk, we related the total liver
area to the number of hepatocyte nuclei in a given section. By this measure, 5
dpf lbk larvae show a 30% increase of liver cell area compared to
their siblings (Fig. 4D).
TEM analyses revealed that the cytoplasm of hepatocytes in lbk is filled with numerous, sometimes very large vesicles (up to 30 µm in diameter; Fig. 4E,F), indicative of a defect in vesicle trafficking in this cell type. At 7 dpf, the hepatocytes of lbk larvae show substantial necrotic changes (Fig. 4G,H). These changes might also explain the discoloration of the liver observable in external views (Fig. 1J). Oil Red O staining further showed an accumulation of vesicles containing large amounts of lipids in the lbk liver (Fig. 4I).
We further examined the intestines of lbk and age-matched sibling larvae. TEM analyses of 7 dpf larvae revealed a vesicle phenotype also in the cells of the mutant intestinal bulb. Although wild-type cells contain few large vesicles, the number of vesicles is significantly increased in lbk with a concomitant reduction in vesicle size (Fig. 4K,L; see Fig. S7 in the supplementary material). This further indicates a possible defect in the fusion of intracellular vesicles in lbk.
The innate immune system is compromised in lbk mutants
Similar to RPE cells, macrophages are highly phagocytotically active and
contain numerous lysosomes and lysosome-related organelles, such as major
histocompatibility complex class II compartments
(Raposo and Marks, 2002). This
prompted us to examine the morphology of vesicles in this cell type by
incubating 7 dpf lbk and sibling larvae in Neutral Red, which is
selectively retained in macrophage lysosomes
(Herbomel et al., 2001). This
staining suggests that intracellular vesicles in lbk macrophages may
be enlarged, seem to display an amorphous shape and are more heterogeneous in
size than in wild-type larvae (Fig.
5A,B; inserts).
To obtain functional information on the innate immune system in lbk, we injected DsRed-expressing bacteria into mutant and sibling larvae at 48 hpf (Fig. 5C). Phagocytosis of the injected bacteria was complete within 10 minutes of the injection. Clearance of the fluorescent bacteria was monitored by in vivo fluorescence microscopy as well as by plating dissociated larvae onto selective media plates and counting of the resulting bacterial colonies. We found that while siblings efficiently cleared injected bacteria within 32 hours, mutants still contained numerous bacteria (Fig. 5D-M). Importantly, although phagocytosis appeared unaffected in lbk, the subsequent failure of the mutant phagocytes to degrade the bacteria indicates a defect in the fusion of phagosomes with lysosomes.
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). This vector was injected at the one-cell stage and
expression induced by heat shock at 3 dpf. In total, three independent
experiments were performed and evaluated. To assess the rescue of the mutant
phenotype, the larvae were screened at 5 dpf for their external morphology.
vam6-injected lbk larvae developed darker skin melanocytes
and RPE (Fig. 6A-C; insets)
than uninjected and control vector-injected (no vam6 cds)
lbk larvae. The larvae were further tested by ERG analysis to assess
the rescue of the visual ability. vam6-injected lbk larvae
showed an increase in the b-wave amplitude to 20-60% of wild-type levels
(Fig. 6A-D). By contrast,
uninjected control lbk larvae did not show a b-wave
(Fig. 3B). To provide further evidence that a mutation in vam6 underlies the lbk phenotype, we designed an antisense morpholino against a region containing the start codon (ATG) of the vam6 mRNA. Injection of the ATG-morpholino into wild-type embryos at the one-cell stage resulted in a similar, but stronger phenotype than that observed in lbk (Fig. 6E). Injection of a control morpholino did not result in larvae displaying any phenotypes (Fig. 6E). The weaker phenotype of lbk larvae compared with the vam6 knock-down could be explained by the presence of maternally supplied wild-type vam6 mRNA, which could serve as a template for the synthesis of functional Vam6p during early embryonic development. To test this hypothesis, we isolated mRNA from wild-type zygotes, reverse transcribed it into cDNA and performed a PCR analysis. This showed that vam6 mRNA is maternally supplied in zebrafish (Fig. 6F) and its expression persists through later stages of development (Fig. 6F,G; see Fig. S8 in the supplementary material). The Vam6p protein is part of the HOPS complex, a large multi-subunit protein complex. In this context it is interesting that the mRNA for Vps18, another member of the HOPS complex, has been found to be maternally supplied in zebrafish zygotes in a microarray experiment (http://zf-espresso.tuebingen.mpg.de/).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
including the ARC syndrome, CHS, GS and HPS. For example, CHS, GS and HPS are
characterised by a hypopigmentation of the skin and RPE, as well as a
significant visual impairment (Barak and
Nir, 1987; Clark and Griffiths,
2003; Griscelli et al.,
1978; Hermansky et al.,
1975). These symptoms are also observed in lbk
mutants.
Melanosomes are part of the large group of lysosome-related organelles and
serve to synthesize and store melanin. The melanocytes in lbk mutants
display significantly fewer and lighter melanosomes than are observed in
wild-type animals. The presence of melanosomes with wild-type levels of
melanin pigmentation in the lbk skin and RPE shows that the mutation
does not cause a block in melanin synthesis or melanosome biogenesis per se.
The increase in immature melanosomes and the substantially decreased number of
mature (stage IV) melanosomes in lbk rather suggests a significantly
reduced efficiency of melanosome maturation, a process critically dependent on
the fusion of endosomal vesicles with premelanosomes
(Dell'Angelica, 2003).
Alternatively, the stop codon mutation we identified in lbk, UAG, has
been suggested to have intermediate read-through fidelity. Thus, the mutant
embryos might retain a low level of functional protein, as has been observed
in the human syndromes described in this manuscript and other lysosomal
storage disorders (Brooks et al.,
2006).
|
|
).
Similarly, the intestinal phenotype observed in lbk is reminiscent of
the granulomatous colitis described for individuals with HPS (OMIM
#203300).
Defects in lysosome-related organelles might also be responsible for the
immunological deficiencies observed in individuals with CHS and GS
(Faigle et al., 1998;
Griscelli et al., 1978).
Macrophages, for instance, are crucial for the body's innate immune response.
One of their key functions is the phagocytosis of pathogens.
Pathogen-containing endosomes subsequently fuse with lysosomes and major
histocompatibility complex class II (MHCII) compartments, a lysosome-related
organelle, in which the pathogens are degraded and foreign peptides loaded
onto MHCII complexes. Loaded MHCII complexes are subsequently delivered to the
surface of macrophages for presentation to cells of the adaptive immune
response. We found that lbk macrophages display an increase in
intracellular vesicles, which might be indicative of a defect in the endosomal
pathway and/or a defect in MHCII vesicle delivery to the plasma membrane.
Importantly, our analyses show that the innate immune response to bacterial
infection is significantly compromised in lbk.
ARC syndrome, CHS, GS and HPS are caused by defects in vesicle trafficking,
particularly of lysosomes, lysosome-related organelles and late endosomes, and
mutations in genes involved in vesicle transport, sorting, docking and fusion
were shown to underlie these diseases. Moreover, ARC syndrome was demonstrated
to be caused by mutations in the vps33b gene, a member of the HOPS
complex, in humans (Gissen et al.,
2004). Similarly, morpholino knock-down experiments targeting the
zebrafish vps33b gene result in a phenotype resembling ARC syndrome
(Matthews et al., 2005).
Interestingly, the buff (bf) mouse, which harbours a mutation in the
Vps33a gene, has been proposed as a model for HPS
(Suzuki et al., 2003),
suggesting a link between ARC syndrome and the CHS, GS and HPS group of
diseases. The phenotypes observed in lbk encompass both symptoms
observed in individuals with CHS, GS and HPS (liver, macrophage,
hypopigmentation and visual phenotypes) and ARC syndrome (liver and intestinal
phenotypes), providing further evidence for a link between these
disorders.
Moreover, we have identified the gene mutated in lbk as
vam6. Like Vps33p, the Vam6p protein is a component of the HOPS
complex, which is required for SNARE complex assembly during vesicle docking
and fusion. Mutations in the two alleles identified in this study lead to a
premature STOP upstream of the CLH repeat domain and Ypt7p-interaction
sequences, and the exchange of a highly conserved amino acid in the N-terminal
CNH domain, respectively. The latter has been shown to be essential for
lysosome clustering and fusion (Caplan et
al., 2001). The Ypt7p-interaction domain mediates protein-protein
interactions between Vam6p/Vps39p and Ypt7p/Rab7, a Rab-GTPase with essential
functions in early to late endosome transport and late endosome-lysosome
fusion, as well as in axonal retrograde transport
(Deinhardt et al., 2006;
Stein et al., 2003). It has
been shown in yeast that Vam6p acts as a guanine nucleotide exchange factor
for Ypt7p (Wurmser et al.,
2000). When this exchange fails, Ypt7p is locked in its GDP-bound
state and vesicle docking is blocked. This probably contributes to the vesicle
phenotypes observed in lbk. In mammalian cells, both Rab7 and the
HOPS complex are essential for the conversion of early into late endosomes
(Rink et al., 2005).
Interestingly, RNAi-mediated knock-down of Rab7 in HeLa cells leads to a
cellular vesicle phenotype reminiscent of that observed in fibroblasts from
individuals with CHS (Davies et al.,
1997). Similarly, knock-down of RAB-7 in C. elegans leads
to enlarged early and late endosomes and knock-down of HOPS complex members
yield defects in gut lysosome formation
(Poteryaev et al., 2007).
|
;
Price et al., 2000a;
Price et al., 2000b;
Sato et al., 2000). Mutations
in Vam6p that abolish the functions of the CLH or CNH domains, respectively,
might hamper these interactions and additionally contribute to the
accumulation of vesicles in lbk.
The mutation in the vam6 gene in lbk acts in a
cell-autonomous fashion and affects cells derived from all three germ layers
(skin melanocytes, RPE, PRCs in ectoderm; liver and intestine in endoderm; and
macrophages in mesoderm). The most severe phenotypes are detected in cells
that display high levels of phagocytic (RPE cells, macrophages) and secretory
activity (hepatocytes, intestinal cells). Owing to the importance of vesicle
trafficking in these cells, they are probably the first and most severely
affected. The occurrence of only partially overlapping symptoms in ARC
syndrome, CHS, GS and HPS, as well as the zebrafish lbk mutant could
be due to the fact that lysosome-related organelles can be cell type-specific
(Raposo and Marks, 2002) and
thus defects in proteins required for these organelles might only affect
certain cell types. The comparatively widespread organ dysfunction observed in
lbk suggests that Vam6p is a core component with non-redundant
functions in vesicle trafficking in a range of cell types.
It has recently been shown that a viral insertion disrupting the
vps18p gene, another member of the HOPS complex, in zebrafish results
in similar phenotypes as those observed in lbk, including a
hypopigmentation of skin and RPE melanocytes, a lack of iridophore
reflections, visual defects, hepatomegaly and an accumulation of vesicles in
hepatocytes (Maldonado et al.,
2006; Sadler et al.,
2005). This provides further evidence for the importance of the
HOPS complex in various cell types and organs.
In conclusion, the overlap in symptoms in human syndromes caused by defective lysosome trafficking and phenotypes observed in lbk suggest this mutant as a model for these inherited human diseases and suggests a novel disease gene. Importantly, there is currently no mouse model for loss of function of Vam6p. Studies on lbk may offer important insights into the mechanisms underlying the observed symptoms and make these pathologies amenable to experimental manipulation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/387/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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These authors contributed equally to this work
Present address: Center for Brain Research, Medical University of Vienna,
Spitalgasse 4, A-1090 Vienna, Austria
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