View larger version (65K):
[in this window]
[in a new window]
|
Fig. 2. lbk larvae display a severely compromised RPE and shorter PRC
outer segments. (A-H) Sections of lbk (B,D,F,H) and
sibling (A,C,E,G) eyes show that overall eye development proceeds normally in
lbk, including the formation of a multi-layered neural retina.
Beginning at 4 dpf (A,D), however, the retinal pigment epithelium (RPE) is
severely hypopigmented and becomes progressively inflated. At 7 dpf (E-H), the
RPE in lbk is threefold thicker than in age-matched siblings (G,H;
brackets). The inner retina in lbk is morphologically
indistinguishable from wild-type siblings. INL, inner nuclear layer; PRC,
photoreceptor cells; RGC, retinal ganglion cells. (I-N) TEM sections
reveal that the lbk RPE contains very few mature melanosomes. The
majority of melanosomes are aberrantly shaped and show regions that lack
pigment. Moreover, the mutant RPE is filled with numerous vesicles. The number
of vesicles increases from 5-7 dpf (compare J with L). The microvilli of the
RPE (I, arrowheads) that normally interdigitate with the outer segments of
PRCs are significantly reduced in lbk (J; arrow). Furthermore, the
outer segment length is reduced. Some lbk PRCs virtually lack outer
segments (L, arrowheads). (M,N) Higher magnification of vesicles containing
undigested PRC outer segments (arrows) in the lbk RPE (N shows a
higher magnification of the boxed area in M). M, PRC mitochondria; N, PRC
nucleus; OS, PRC outer segment. (O,P) Merged phase contrast and
fluorescence images of eye sections stained for rhodopsin (red) show an
increase of discrete rhodopsin-positive vesicles in the lbk RPE (P,
arrows) versus continuous PRC OS in the wild type (O, arrows). Insets show
higher magnifications of parts of the RPE from the sibling and mutant retinas
shown in O and P. (Q) Images showing melanosomes at different stages of
maturation (I-IV) in the sibling (top) and lbk (bottom) RPE at 5 dpf.
Arrowheads indicate melanosomes of the respective stages. (R-U)
Statistical analyses of RPE thickness (R), average RPE cell area per nucleus
(S), average number of vesicles containing PRC outer segments (T) and PRC
outer segment length (U) in sibling and lbk larvae at 5 dpf. Error
bars indicate standard deviations; P values were calculated using
Student's t-test. (V) Quantification of melanosomes at
different stages of maturation in the lbk and sibling RPE at 5 dpf.
Counts represent the numbers of melanosomes in areas of 10 µm2.
Although there are no substantial differences between the numbers of stage
I-III melanosomes, mutant larvae contain significantly fewer mature (stage IV)
melanosomes. (W) TUNEL labelling of cryosections shows increased levels
of apoptosis in lbk (bottom right). The relatively dark appearance of
the mutant RPE results from the thickness of these sections (40 µm). All
images were derived from mid-transversal sections of the eye. Scale bars: 50
µm in A-F; 10 µm in G,H; 5 µm in I-M; 2.5 µm in N; 25 µm in
O,P.
|
,
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
T (arrowheads), resulting in
a premature STOP codon. (M) Domain structures of the wild-type Vam6p
(bottom) and the protein truncated in lbk lacking the C-terminal 520
amino acids (top), including the clathrin homology (CLH) repeat domain and
Ypt7p-interacting sequences. (N) Sequence alignment of human, mouse,
chicken and zebrafish Vam6p proteins shows the high level of conservation in
the region affected by the mutation (red box). (O) Sequencing of the
complete vam6 cds from the lbk* mutant revealed
that this larva was heterozygous at two positions: the C
) with a
significance level of 5%. (B) Electroretinograms (ERGs) of 5 dpf
lbk and sibling larvae measured over five log intensities of light.
Mutant larvae (right) show a dramatically reduced b-wave compared with
age-matched siblings (left). Red brackets indicate 100 ms light stimuli.
20% (C). Insets show head regions
of vam6 vector-injected sibling (A) and lbk larvae (B,C) and
the extent of rescue in RPE and skin melanocyte pigmentation. Arrowheads
indicate skin melanocytes with near wild-type levels of melanin in rescued
larvae. (D) Quantification of the observed ERG rescue: sibling larvae
display an average b-wave amplitude of 286 µV, vam6
vector-injected lbk larvae show an average b-wave amplitude of 83
µV (n=7 for both sets of larvae; error bars show standard
deviations). (E) Phenotype of the vam6 knock-down at 36 hpf
showing the hypopigmentation of the RPE and skin melanocytes (insets;
arrowheads) characteristic of lbk. However, the knock-down results in
additional phenotypes not observed in lbk, including a small head and
eyes and a shortened body axis. Larvae displaying wild-type levels of melanin
are age-matched control morpholino-injected individuals. (F) PCR
analysis for the presence of vam6 transcripts in RNA isolated from
wild-type zebrafish zygotes (upper half of gel) and 24 hpf embryos (lower
half). β-Actin (expressed at all developmental stages) and
pmel17 [onset of expression: