1 Max Planck Institut fur Entwicklungsbiologie, Spemannstrasse 35, 72076
Tübingen, Germany
2 Brain Research Institute, University of Zurich and Department of Biology,
Swiss Federal Institute of Technology Zurich, Winterthurerstr. 190 8057
Zurich, Switzerland
3 Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science
University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA
* Author for correspondence (e-mail: nicolson{at}ohsu.edu)
Accepted 23 November 2004
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SUMMARY |
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Key words: Protocadherin 15, Hair cell, Photoreceptor, Zebrafish, Deafness, Blindness, Outer segment, Stereocilia, Gene duplication, orbiter
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Introduction |
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As in mammals, fish sense mechanical stimuli with hair cell receptors in
the inner ear and an additional sensory system unique to aquatic animals, the
lateral line (Manley and Koppl,
1998; Popper and Fay,
1997
). In recent screens for genes necessary for inner ear
function in zebrafish larvae, 24 loci linked to an
auditory/vestibular-specific phenotype were identified
(Nicolson et al., 1998
) (T.N.,
unpublished). Here we show that the zebrafish auditory/vestibular mutant
orbiter is affected in the gene pcdh15. Mutations in
PCDH15 have been recently shown to be the cause of human Usher
syndrome 1F (Ahmed et al.,
2001
; Alagramam et al.,
2001b
; Ben-Yosef et al.,
2003
) and deafness in Ames waltzer mice
(Alagramam et al., 2001a
).
Furthermore, we report the characterization of a second, duplicated
pcdh15 gene in zebrafish, which is expressed in the retina. Our
results indicate that one gene acquired an essential function in the eye and
the other one in the ear. Both genes are required for the structural integrity
of the respective receptor cells in these organs.
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Materials and methods |
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Mapping and cloning
Linkage analysis was performed using WIK wild-type fish crossed with
heterozygous orbiterth263b-Tü fish. The mutation was
mapped using SSLP (simple sequence length polymorphisms) and SSCP (single
strand conformation polymorphisms) (Cleangels SSCPplus gels were used
according to the manufacturer's instruction; ETC, Kirchentellinsfurt,
Germany). For fine-mapping, single mutant larvae were tested for recombination
events. To initiate a chromosomal walk, we used the marker z26404 to screen
PCR-able pools of genomic zebrafish BAC (BioCat, Heidelberg, Germany) or PAC
(RZPD, Berlin, Germany) libraries. All clones found were sized using
pulsefield gel electrophoresis. New SSLP or SSCP markers were created using
sequences from the ends of the identified BAC- or PAC-clones. After defining a
critical interval, six clones spanning the contig (PAC1, BAC1, PAC10, PAC13,
BAC6, BAC7) were sequenced at the Sanger Center (Welcome Trust Sanger
Institute, Cambridge, UK). The sequence within the critical interval was
analyzed using Genscan
(http://genes.mit.edu/GENSCAN.html)
and blast searches of the NCBI database
(http://www.ncbi.nlm.nih.gov/BLAST).
To clone the 3' and 5' ends of the genes several rounds of RACE
with either the Marathon or SMART RACE cDNA amplification kit (Clontech, Palo
Alto, CA) were performed according to the manufacturers' instruction. The
Goodfellow T51 panel was used to map pcdh15b
(Geisler et al., 1999).
Mapping prediction on the T51 panel map was done using the `Instant Mapping'
website from The Children's Hospital Zebrafish Genome Project Initiatives
(http://zfrhmaps.tch.harvard.edu/ZonRHmapper/instantMapping.html).
GenBank accession numbers and clone names
The official names of the PAC clones from RZPD are the following: PAC1:
BUSMP706C1835Q2; PAC5/7: BUSMP706H0192Q2; PAC9: BUSMP706M01204Q2; PAC12:
BUSMP706N24105Q2; PAC10: BUSMP706N24105Q2; PAC11: BUSMP706O192Q2; PAC13:
BUSMP706J031239. See the RZPD-Database
(https://www.rzpd.de)
for further information. GenBank accession number of the clones sequenced by
the Sanger center: BAC1: AL592289; BAC6: AL592062; BAC7: AL645689; PAC10:
AL592202; PAC13: AL592204; PAC1: AL592077. GenBank accession numbers for
zebrafish pcdh15a: AY772390; and pcdh15b: AY772391.
Mutation analyses
Total RNA was prepared from pools of 5-10 wild-type sibling or mutant
larvae using the NucleoSpin RNAII Kit (MACHEREYNAGEL, Easton, PA). Reverse
transcription reactions were performed using the Superscript reverse
transcriptase (Invitrogen, Carlsbad, CA) with an oligo (dT) primer.
In situ hybridization
Whole mount in situ hybridization was performed as described
(Schulte-Merker et al., 1994).
A fragment containing bp 180-1800 from the extracellular domain
(Fig. 2) or bp 4516-5908 from
the polymorphic intracellular part were used as probes for pcdh15a.
Both probes gave the same signal; the signal with the extracellular probe was
stronger and appeared more specific. bp 4075-5697 from the intracellular part
was used as probe for pcdh15b.
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Electron microscopy
For electron microscopy, larvae were processed as previously described
(Seiler and Nicolson, 1999).
Whole larvae were embedded in Epon and oriented such that the eye was
sectioned in a transverse dorsal to ventral direction. All sections were done
in the plane of the optic nerve. Images were taken from areas adjacent to the
optic nerve in the center of the retina.
Morpholino injections
Four different morpholinos were used in this study and injected at the one
cell stage as described (Nasevicius and
Ekker, 2000). One was directed against the translation start (ATG
Mo) of pcdh15b with the sequence
5'-CCACGACCTCTGGCGATCTTCATA-3', one against the 3' splice
site of coding exon number 4 (GT Mo) with the sequence
5'-AAAAGATAATCACATCTCGGTCCAG-3', and an unrelated control
morpholino (a 4 bp mismatched oligo against fkd6;
5'-CGACGAGCTCTGCCGATCTTGATA-3'). A second control morpholino was a
4 bp mismatched oligo against pcdh15b
(5'-TGCGCGCCAGACAGGGTGATGAC-3'). In the ATG Mo-injected larvae (40
ng), a slightly shorter body axis of the larvae was occasionally observed.
Differentiation of other organs like the heart, brain and ear appeared normal,
indicating that this effect was not due to a delay in development. To ensure
that the 40 ng dose had no effect on the onset of eye differentiation, we
stained 14 µm sections of eyes of these larvae with anti-tyrosine
hydroxylase antibody and counted the positive cells. No difference in number
could be detected between uninjected (1.4 cells/section n=13) and
larvae injected with 40 ng ATG Mo (1.5 cells/section n=14).
Fluorescent staining and microscopy
For confocal microscopy, an inversed Leica DM IRBE microscope equipped with
a 100x oil lens was used. For antibody staining, the larvae were fixed
with 4% formaldehyde in PBS for at least 24 hours prior to sectioning. The
sections were incubated with 1% bovine serum albumin. The following antibodies
were used in this study: zpr-1 (The zebrafish international resource
Center, Eugene, OR, USA), rhod-1 (Ret-P1, Milan, Analytica
AG/Switzerland), and Alexa488 goat anti-rabbit Ab (Molecular Probes, Eugene,
OR).
Optokinetic stimulation
Single larvae were placed dorsal side up in the center of a petridish (35
mm diameter) containing 3% prewarmed (28°C) methylcellulose. Moving
gratings were projected by a Proxima 4200 DLP projector onto a screen within
the visual field of the larva, at an apparent distance 4.65 cm from the
larva's right eye. Projection size on the screen was 8 cm x 6 cm,
subtending a visual angle of 65.6° horizontally and 53.1° vertically.
A custom-made graphics library running under Microsoft DOS, allowing full
control of timing and intensity of the projection was used to create the
stimuli. Mean intensity levels were adjusted by introducing neutral density
filters into the light beam. The contrast C of grating stimuli was
calculated from: C =(Imax -
Imin)/(Imax +
Imin).
Spatial frequency of the grating stimulus was varied between 0.025 and 0.14 cycles/°. All measurements were made under a mean luminance of 120 cd/m2, temporal frequency of 0.4 Hz, pattern contrast of 62.5% and a grating velocity of 7.6°/second.
We measured the CSF in 4-day-old larvae injected with either an ATG Mo (40 ng n=7 or 30 ng n=7) or the control Morpholino (40 ng n=7). The experiment has been repeated with similar results in an independently injected clutch.
ERG recordings
Electroretinograms (ERGs) were performed on larvae at 4.5 dpf as previously
described (Makhankov et al.,
2004). All pre-recording steps were done under red illumination to
minimize bleaching of the visual pigment. Preparation and recordings were
performed in a tight Faraday cage. All specimens were dark-adapted for 30
minutes prior to positioning them in the recording chamber. Each larva was
placed on its side on the surface of a moist sponge with E3 medium (mM: 5
NaCl, 0.17 KCl, 0.33 CaCl2 and 0.33 MgSO4) and paralyzed
by directly adding a droplet of the muscle relaxant Esmeron (0.8 mg/ml in
larval medium; Organon Teknika, Eppelheim, Germany). The Ag/AgCl electrode
system was used to record the ERG response. The recording electrode was
positioned in the center of the cornea. The reference Ag/AgCl pellet was
placed under the body of the larva. A 3-minute period was chosen to adapt the
larvae to both dark- and light-adapted states prior to measurement. Relative
background light intensitiy was 175 lux for the light-adapted state. The
duration of light stimulus was 100 mseconds with an interstimulus interval of
5 seconds. Stimulus illumination was increased in 0.5 log unit steps over the
range from -5 log unit to -1 log unit. Unattenuated light intensity over the
subject's head was 20,000 lux (optical density, OD was equal 0 log unit), as
measured by a light meter (Tekronix J17, Texas Instruments, USA). A virtual
instrument (VI) under NI LabVIEW 5.1 was developed to use in all experiments.
Amplified analog signals were sampled by means of NI PCI 6035E DAQ board
connected to a NI BNC-2090 BNC terminal block. Sampling was done in buffered
acquisition mode with a sampling rate of 250 Hz. Recording was triggered by
the shutter signal. To analyze the ERG response with respect to the actual
onset of the light stimulus, mechanical shutter delay was measured by means of
a photodiode. Traces were normalized to the baseline by subtracting the
average potential before the stimulus onset. Responses were averaged between
three and five, times depending on the signal-to-noise ratio.
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Results |
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Sequencing of pcdh15a cDNA from the mutants revealed that it is the gene affected in orbiter mutants. For the alleles of the strong class, we found that the mutation in t25708 leads to a valine to glutamine exchange (V142Q) in close proximity to the conserved cadherin calcium-binding motif (with the consensus DXNDN) of the first cadherin domain. The mutation in th263b causes a truncation of the protein in the third cadherin repeat (R360X), presumably resulting in a nonfunctional protein. t288 causes a leucine to proline (L386P) exchange, adjacent to the calcium binding domain of the third cadherin domain (here DENNQ instead of DXNDN) (Fig. 1B).
Sequencing of the alleles of the hypomorphic class revealed that tc256e causes an isoleucine to asparagine (I419N) exchange in the linker between the third and fourth cadherin domain. t23281 causes an asparagine to lysine (N1143K) exchange in the tenth cadherin domain, changing the calcium-binding motive from DENDH to DEKDH (Fig. 1B). The weaker phenotype seen in homozygous t23281 animals suggests that this cadherin domain is less critical for Pcdh15 function.
A second zebrafish pcdh15 gene
In addition to the pcdh15 gene mutated in orbiter, we
identified a second ortholog of pcdh15. We designated the gene
mutated in orbiter as pcdh15a, and the other gene
pcdh15b. pcdh15b is located on chromosome 12 between the markers
dkk1 and z1473 (T51 RH panel). The predicted Pcdh15b protein has the
same domain structure and shares high amino acid similarity (70%) with the
extracellular domain of zebrafish Pcdh15a, and mouse and human PCDH15
(Fig. 1C). The intracellular
domain is less similar to the intracellular domains of Pcdh15a and mouse and
human protocadherin 15 (17-33%, Fig.
1C), but the intracellular tail is divergent among all species
(Fig. 1C). Nevertheless, all
Pcdh15 intracellular domains share the feature of being proline- and
serine-rich, and there are short stretches of high similarity. Pcdh15b has a
putative PDZ class I binding motif at its C-terminus [consensus X-S/T-X-L
(Sheng and Sala, 2001)]. This
motif is also conserved in human and mouse PCDH15, but is not present in
zebrafish Pcdh15a, which is shorter at the C-terminus.
Phylogenetic analysis based on a ClustalW alignment revealed that the two
zebrafish proteins are more related to each other than to the mammalian
orthologs (Fig. 1D). This
suggests that they were duplicated after the split of the mammalian and fish
lineage, probably during the suggested genome duplication event in ray-finned
fish (Prince and Pickett,
2002; Taylor et al.,
2003
).
Expression of pcdh15a and pcdh15b in the ear and the eye
To determine the spatial and temporal expression of pcdh15a and
pcdh15b, we performed mRNA in situ hybridization. At the
four-somite stage (11 hours post fertilization, hpf), pcdh15a is
expressed in the anterior neuroectoderm and in the first forming somites
(Fig. 2A). At 24 hpf,
pcdh15a is expressed in the eye
(Fig. 2B) and hatching gland
(out of focus). Expression is also detectable in the newly developed
mechanosensory hair cells (Fig.
2B,C). Expression of pcdh15a in hair cells was visible in
all later stages investigated (Fig.
2D-F'). At day 3, additional expression in regions of the
brain is present, together with expression at the lateral borders of the
epiphysis (Fig. 2D). At day 4,
expression in the brain persists and is detectable in the hair cells of
neuromasts (Fig. 2E,F').
Within the neuroepithelia of the ear and lateral line, levels of
pcdh15a mRNA are highest in the hair cells
(Fig. 2F,F').
pcdh15b is weakly expressed in the whole embryo at 24 hpf (data
not shown). At 48 hpf, strong expression in the epiphysis is visible but not
in the eye (data not shown). At day 2.5, the gene is expressed at the ventral
margin of the eye (Fig. 3A),
where the first photoreceptors differentiate. Following the wave of
differentiation of photoreceptors (Larison
and Bremiller, 1990; Raymond
et al., 1995
), pcdh15b is expressed in the proximal
regions of the eye around day 3 and restricted to the lateral margin at day 5
(Fig. 3B,C). In thick sections,
expression is evident in photoreceptors
(Fig. 3E,F). In addition,
pcdh15b is expressed in the brain and center of the epiphysis at day
3 (Fig. 3D) and 5. Weak
expression was detected in the ear and neuromasts (data not shown). No signal
was detected with the corresponding sense probes.
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pcdh15b morphants have reduced contrast sensitivity and retinal function
To determine the function of Pcdh15b, we designed two morpholinos: one
against the ATG translational start site (ATG Mo), and one against the GT
splice site of the fourth coding exon (GT Mo). The ATG Mo is predicted to
inhibit the initiation of translation of pcdh15b message, whereas the
GT Mo should prevent the correct splicing of exon 4. Because the ATG Mo
inhibits protein synthesis of Pcdh15b, its efficiency cannot be evaluated at
the mRNA expression level. However, we could test the efficiency of the GT Mo
by amplifying cDNA with primers flanking the targeted exon. RT-PCR analysis
confirmed that the GT Mo indeed caused aberrant splicing of the targeted exon.
Wild-type transcript levels are reduced by approximately 60% on day 3, and
reduced by 40% on day 4 (Fig.
5D). The results of the histological analysis and
electroretinograms (see below) suggest that the ATG Mo was more effective than
the GT Mo. Morpholino-injected animals appeared to develop normally and
overall morphology was normal (see Fig. S2 in the supplementary material). No
obvious defects in balance or the acoustic startle reflex or hair bundle
morphology were observed in pcdh15b morpholino-injected (morphant)
larvae (data not shown). In addition, we examined uptake of the vital dye,
FM1-43, an indicator of mechanotransduction in zebrafish hair cells (Seiler et
al., 1999). Uptake was unaffected in pcdh15b morphants (see Fig. S3
in the supplementary material).
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To determine whether injection of pcdh15b morpholinos results in physiological defects that are specific for retinal function, the electroretinogram (ERG) b-wave was measured in 4.5-day-old larvae. Larvae injected with 40 ng of the ATG Mo (n=6) or 16 ng of the GT Mo (n=6) were compared with uninjected larvae (n=5) and larvae injected with the control Mo (n=5). As shown in Fig. 5B and C, the b-wave increases in amplitude with increasing stimulus intensity in all experimental groups. Injection of both morpholinos resulted in a significant reduction in b-wave amplitude. (3-way ANOVA d.f.=3, F=11.7, a<0.001). Although both morpholinos are targeted against different sequences, they cause a comparable reduction in b-wave amplitude, indicating that this effect is specific for the knockdown. Comparison of the pcdh15b morphants with control-injected and uninjected larvae is significant for 40 ng ATG MO (a<0.001) and 16 ng GT MO (a<0.001; Scheffe test). The weaker b-wave amplitude reduction between the GT Mo compared to the ATG Mo injected larvae was not significant, though. Representative traces from individual specimens are shown in Fig. 5E,F. Due to a very low amplitude and differing width, we were not able to evaluate the a-wave data conclusively. Nevertheless, our behavioral and physiological analyses indicate a clear defect in retinal responses in pcdh15b morphants.
Reduction of pcdh15b activity affects photoreceptor morphology
To determine if the visual defect is due to a morphological defect in the
retina, we examined cryosections of morpholino-injected larvae using
immunohistochemistry. In larvae injected with 30 ng (n=5) or 40 ng
(n=7, 2 independent experiments) of the ATG Mo, we observed a
dose-dependent phenotype in the retinal photoreceptor cell layer
(Fig. 6). The zpr-1
antibody labels double cone photoreceptor cell bodies and outer segments
(Larison and Bremiller, 1990).
In 4-day old morphants, the general morphology of the photoreceptors labeled
with zpr-1 antibody appeared disorganized. Labeled cells appeared
wider, shorter, and deformed (Fig.
6A-F). Effects were also evident using an anti-rhodopsin
(rhod-1) antibody that labels outer segments. Outer segments of the
rod photoreceptors appeared shorter and less organized than wild-type outer
segments (Fig. 6G-I). The
effects on both rods and cones seen with the antibodies were stronger in
larvae injected with 40 ng (Fig.
6F,I) than in larvae injected with 30 ng ATG Mo
(Fig. 6E,H).
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Discussion |
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In a recent study, PCDH15 protein was shown to be localized at the
stereociliary bundle of hair cells in mammals
(Ahmed et al., 2003a). This
result is consistent with the bundle phenotype we detected in orbiter
mutants, suggesting that orbiter/pcdh15a encodes a component of the
stereociliary bundle. The large extracellular domain of Pcdh15a, along with
the localization data from mammals, suggests that Pcdh15a may be part of the
outer membrane calyx or a component of the extracellular links spanning
neighboring stereocilia. Although the bundle phenotype is mild in
orbiter mutants, microphonic potentials are not detectable in larvae
carrying the strong allele. This phenotype raises the possibility that Pcdh15a
plays a functional role in hair bundles.
In the pcdh15b morphants, the tilted outer segment phenotype
suggests at least two possible functions of pcdh15b during
differentiation. First, Pcdh15b may be necessary for adhesive contact between
the outer segment of photoreceptors and the retinal pigment epithelium.
Interestingly, when the retinal pigment epithelium is absent in the zebrafish
retina, the outer segments are tilted sideways in a similar way (C. Seiler and
A. Rojas-Munoz, unpublished). This phenotype suggests that the outer segments
are actively intercalated into the pigmented epithelium. This paradigm
requires trans interactions of at least two cadherins expressed in
photoreceptors and the retinal pigment epithelium, but we could not detect
pcdh15b in the pigment epithelium. Another possibility is that
Pcdh15b is required for stabilization of photoreceptor outer segments. Such
contacts may help to align the outer segments, making interdigitation with the
retinal pigment epithelium possible. The localization pattern of PCDH15 in
photoreceptor outer segments in the human and monkey retina
(Ahmed et al., 2003a) appears
to be consistent with these notions. PCDH15 protein was also detected in other
layers of the fetal and adult human retina, especially the synaptic layers,
and may play a role in synaptogenesis as well
(Alagramam et al., 2001b
).
To date, the only mouse mutant in an Usher gene reported to have a
morphological defect in the eye is the shaker1/Myo7a mutant. In
shaker1 mutants, the melanosomes of the retinal pigment epithelium do
not migrate between photoreceptors, suggesting that myosin VIIA is needed for
transport of these pigmented structures into the apical processes of retinal
pigment cells (Liu et al.,
1998). Slight reductions in ERG responses have also been detected
in myosin VIIA mutant mice (Libby and
Steel, 2001
). Moreover, myosin VIIA has been shown to be expressed
and required in the retinal pigment epithelium for uptake of outer segment
disks (Gibbs et al., 2003
).
These results suggest that myosin VIIA also participates in photoreceptor and
retinal pigment epithelium interactions.
In human Usher syndrome, retinitis pigmentosa develops in the second to
third decade of life. This late defect in humans contrasts with the very early
onset of photoreceptor malformation in zebrafish pcdh15b morphants.
It is worth noting that in zebrafish, the eye develops much faster than in
mammals (O'Brien et al., 2003;
Sharma et al., 2003
). Within
2.5 days, the zebrafish eye develops from a undifferentiated neuroephithelium
to a layered and differentiated functional organ
(Easter and Malicki, 2002
).
Furthermore, a fully populated photoreceptor cell layer develops within 30
hours (Raymond et al.,
1995
).
Thus, the contact and migration of outer segments into the retinal pigment
epithelium happens much faster in zebrafish, and proper function of Pcdh15
appears critical at this timepoint. In various mouse models for retinal
degeneration, the photoreceptor cell layer is initially disorganized and shows
severe degeneration later in life [for example rd8/Crb1
(Mehalow et al., 2003)
rd6/Mfrp (Hawes et al.,
2000
; Kameya et al.,
2002
)]. Due to the limited efficiency of antisense morpholinos, we
were not able to investigate juvenile stages to look for degeneration.
However, photoreceptor interactions with the retinal pigment epithelium have
been shown to be important for the outer segment disc phagocytosis, visual
pigment renewal, and nutrient supply
(Boulton and Dayhaw-Barker,
2001
; Pacione et al.,
2003
). Moreover, progressive death of photoreceptors was observed
in the vitiligo/Mitf mouse mutant in which retinal pigment epithelium
initially failed to interdigitate with photoreceptors
(Sidman et al., 1996
), and in
mutant vestigial outer segments zebrafish, which exhibit a similar
phenotype to pcdh15b morphants
(Manzoor-Ali et al., 2003
).
Thus, impaired contact between the retina pigment epithelium and the outer
segments may cause degeneration at later stages.
The phenotypes observed in either mutant orbiter/pcdh15a zebrafish or pcdh15b morphants indicate a clear requirement for Pcdh15 proteins in hearing and vision in zebrafish. The sensory receptors in either the ear or the eye rely upon these protocadherins for structural integrity. Our study establishes a role for Pcdh15 in maintaining the structural integrity of the photoreceptor outer segment, and highlights the usefulness of an alternative animal model in studying the function of Usher genes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/3/615/DC1
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ACKNOWLEDGMENTS |
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