From the Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
Received for publication, November 20, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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To exploit zebrafish as a transgenic model,
tissue-specific promoters must be identified. We isolated a
20-kilobase (kbp) zebrafish rod opsin genomic clone, which
consists of 18 kbp of 5'-flanking region, the entire coding region, and
0.5 kbp of 3'-flanking sequence. Polymerase chain reaction, Southern
blotting, and DNA sequencing revealed the rod opsin gene lacks introns.
The transcription start site was localized 94 nucleotides upstream of
the translation initiation site. Sequence alignment with orthologous
promoters revealed conserved cis-elements including
glass, NRE,
OTX/Bat-1, Ret-1/PCE-1,
Ret-4, and TATA box. A 1.2-kbp
promoter fragment was cloned upstream of the enhanced green fluorescent
protein (EGFP) cDNA and microinjected into 1- to 2-cell stage
zebrafish embryos. EGFP expression was detected in the ventral-nasal
eye at 3 days postfertilization and spread throughout the eye. Progeny of the positive founder fish, which were identified by
polymerase chain reaction amplification of fin genomic DNA, exhibited
EGFP expression in the retina, confirming the germline transmission of
the transgene. Frozen eye sections demonstrated the EGFP expression was
rod-specific and exhibited a similar developmental expression profile
as the rod opsin protein. This stable transgenic line provides a novel
tool for identification of genes regulating development and maintenance
of rod photoreceptors.
The zebrafish, Danio rerio, has emerged as a novel
vertebrate model system that is amenable to mutagenesis and
transgenesis. High fecundity, rapid oviparous development, and a
translucent embryo make zebrafish a prolific experimental model (1).
Furthermore, the zebrafish eye possesses distinct advantages for
studying the development, function, and inherited diseases of the
retina. Eye ontogenesis proceeds rapidly, completing the laminae of the
adult retina by 3 days postfertilization (dpf1; Ref. 2).
The zebrafish eye is relatively large and
accessible, and the position and
morphology of the rod and cone classes are readily distinguishable (3).
Finally, the integrity of visual system structure and function can be
evaluated by morphological, behavioral, and electrophysiological
methods (4-6). Chemical mutagenesis screens previously identified over
65 zebrafish visual system mutants exhibiting either behavioral (4, 6,
7) or morphological abnormalities (5). Insertional mutagenesis screens
generated additional eye mutants (8, 9). Overall, Rhodopsin, comprising the rod opsin protein bound to
11-cis-retinaldehyde, constitutes the photosensitive visual
pigment of vertebrate rod photoreceptors (15). Rhodopsin is
concentrated in the outer segments of the rod photoreceptor cells,
which mediate vision in dim light. Mutations in the human rod opsin
gene (RHO) can result in retinitis pigmentosa, a slow
retinal degeneration resulting in blindness (16). Drosophila
melanogaster, Mus musculus, and Xenopus
laevis have served as genetic models for studying rhodopsin
function and generating models of inherited human visual disease
(17-19). However, none of these models combines the vertebrate eye
with features of amenability to mutagenesis screens and transgenesis, as found in zebrafish. The developmental expression of zebrafish rod
opsin initiates in two phases. It is expressed initially in a
ventral-nasal patch of cells, and subsequently rod opsin-expressing cells arise in a scattered pattern throughout the remainder of the
retina (20, 21). Furthermore, rod photoreceptors are added to the fish
retina throughout life from two retinal sources: stem cells in the
circumferential germinal zone and rod precursor cells, which are found
in the outer nuclear layer (22-24).
To develop tools for transgene expression in the zebrafish retina, we
cloned the rod opsin gene. Characterization of the gene revealed that
it lacks introns and contains conserved cis-regulatory sequences. We identified a 1.2-kbp promoter fragment that directs enhanced green fluorescent protein (EGFP) expression specifically to
rods. Furthermore, this transgene was integrated stably into the
germline and transmitted to the subsequent generation. The expression
of transgenes in zebrafish photoreceptors will enhance our ability to
investigate visual gene function, gene regulation, and gene therapy.
Materials--
Restriction enzymes, T4 polynucleotide
kinase, and Taq DNA polymerase were purchased from Fisher.
Hybond-N+ nylon membranes were purchased from Amersham Pharmacia
Biotech. The Ultraspec RNA II isolation system and Hybrisol II were
obtained from Cinna Biotecx and Intergen, respectively. The
zebrafish genomic library was purchased from Stratagene. The
Thermoscript reverse transcriptase system and ThermoSequenase
Radiolabeled Terminator cycle sequencing kit were obtained from Life
Technologies, Inc. and U. S. Biochemical Corp., respectively. The
pEGFP-1 plasmid and microcapillaries were purchased from
CLONTECH and World Precision Instruments, Inc., respectively.
Southern Blotting--
DNA was fractionated on 1% agarose gels
and transferred to Hybond-N+ positively charged nylon membranes by
downward alkaline capillary transfer (25). Rod opsin cDNA probes
were isolated by restriction digestion and labeled by random primers.
Membranes were hybridized with probe in Church's buffer (1% bovine
serum albumin, 1 mM EDTA, 0.5 M
NaHPO4 (pH 7.2), 7% SDS) at 65 °C for 16 hours and then
washed twice under high stringency conditions (1 mM EDTA/40
mM NaHPO4 (pH 7.2)/1% SDS) at 66 °C for 30 min prior to autoradiography.
Northern Blotting--
Total RNA was isolated using the
Ultraspec RNA II isolation system. RNA aliquots ( Genomic Library Screens--
Approximately 107
recombinant phage in a Polymerase Chain Reaction (PCR) Mapping--
Genomic, phage, and
plasmid DNA templates were PCR-amplified using primers spanning the
zebrafish rod opsin cDNA. PCR parameters were 30 cycles of
denaturation at 94 °C for 30 s, anneal primers at
65-67 °C for 30 s, and extension at 72 °C for 5 min using
Taq DNA polymerase.
Primer Extension--
A primer complementary to the 5' end of
the zebrafish rod opsin cDNA (RR1) was end-labeled with
32P using T4 polynucleotide kinase. 12 µg of zebrafish
eye or body total RNA were hybridized with the labeled primer for
1 h at either 60 or 42 °C (in 50% formamide). The annealed
primer was extended using the Thermoscript reverse transcriptase system
supplemented with 50 ng/µl actinomycin D. Products were extracted
with phenol/chloroform/isoamyl alcohol, ethanol-precipitated, and
electrophoresed on a 6% polyacrylamide-urea sequencing gel. The
ThermoSequenase Radiolabeled Terminator cycle sequencing kit was used
for DNA sequencing.
Embryo Microinjections--
A 1.2-kbp
(EcoRI/XbaI) promoter fragment of the zebrafish
rod opsin gene was subcloned upstream of the EGFP-coding sequence in
pEGFP-1, creating pZOP-EGFP. This vector was linearized and resuspended
in water plus India ink at 50 ng/µl. Zebrafish embryos were
microinjected with linearized vector at the 1- to 2-cell developmental
stage (28). Injection needles were pulled from 1-mm (inner diameter)
microcapillaries on a Sutter Instrument Co. model P-97 Flaming/Brown
micropipette puller. Embryos positioned on an agarose injection chamber
(28) were injected using air pressure on a Narishige U. S. A., Inc.
micromanipulator. Some fish were reared in water treated with 0.003%
1-phenyl-2-thiourea to inhibit melanin formation (28).
Screening for Transgenic Fish--
Fish were anesthetized and
placed on a depression slide for fluorescence microscopy using a Carl
Zeiss, Inc. Axiovert 100 microscope and cooled charge-couple
deviced camera under a ×10 objective. Fish were viewed using a
Carl Zeiss, Inc. narrow bandpass fluorescein isothiocyanate filter set
and compared with a rhodamine filter set to differentiate between a
true EGFP signal and autofluorescence. Fish exhibiting EGFP expression
were isolated and pooled for long term characterization. Caudal and
anal fins were clipped from adult founders, and genomic DNA was
isolated (28). These samples were screened for the presence of the EGFP
cassette using the EGFP-F (5'-GCCACAAGTTCAGCGTGTCC) and EGFP-R
(5'-GATGCCCTTCAGCTCGATGC) primers and the PCR parameters described above.
Whole-mount and Retina Section Antibody Labeling--
Larval and
juvenile transgenic G1 fish were fixed in 4%
paraformaldehyde, 5% sucrose, PBS pH 7.4. Antibody labeling of
whole-mount and frozen sections (10 µm) was performed as described
using polyclonal anti-rhodopsin diluted 1:5,000 (29).
The Zebrafish Genome Contains a Single Rod Opsin Gene--
We
examined the potential for duplicate rod opsin genes in the zebrafish
genome by Southern blots. The zebrafish rod opsin cDNA typically
detects single genomic fragments, although digestion with either
ApaI or EcoRI yielded two hybridizing fragments
(Fig. 1). The presence of two
ApaI fragments is consistent with an internal ApaI site in the cDNA probe. However, because no
internal EcoRI sites are known, the two EcoRI
fragments are likely due to a sequence polymorphism. Overall, the
pattern indicates the zebrafish rod opsin gene is likely single
copy.
Isolation of a Zebrafish Rod Opsin Genomic Clone--
A 20-kbp rod
opsin genomic clone was isolated from a zebrafish library. The phage
clone was mapped by Southern blot analysis using regions of the rod
opsin cDNA as probes (data not shown). The data suggested that the
zebrafish rod opsin gene contained minimal, if any, intron sequence. A
3-kbp (EcoRI-NotI) fragment detected by the
cDNA probe was subcloned into the pCR2.1 plasmid. We examined this
genomic subclone for the presence of introns by comparing the PCR
amplification products from genomic DNA, the genomic clone, and the
cDNA clone using primers spanning the zebrafish rod opsin cDNA
(Fig. 2). The genomic and cDNA
templates consistently produced PCR products of the same size (Fig. 2), which further demonstrates that the zebrafish rod opsin gene lacks introns.
Localization of the Transcription Start Site for Zebrafish Rod
Opsin--
We examined the size of the rod opsin mRNA to
approximate the length of the 5'-untranslated sequence. The rod opsin
cDNA clone that we previously characterized was 1,576 nucleotides
with 92 nucleotides of 5'-untranslated sequence (26). We probed
zebrafish eye and body total RNA with the rod opsin cDNA clone on
Northern blots. A 1.9-kb transcript was detected in the eye RNA but not in the body RNA (Fig. 3A). The
size and tissue specificity are consistent with other rod opsin
orthologues (15). The transcription start site of the zebrafish rod
opsin gene was determined by primer extension. Consistent with the
Northern results, primer extension products were observed with eye RNA
templates but not with body RNA (Fig. 3B). DNA sequencing of
the 3-kbp genomic subclone with the identical primer used for primer
extension localized the transcriptional start sites to the cytosine and
guanine nucleotides 94 and 91 base pairs (bp) upstream of the
translation start site, respectively. The presence of more than one
primer extension product may be because of differential methylation at
the 5' end of the transcript (30).
Sequence of the Zebrafish Rod Opsin Gene--
We characterized the
3-kbp rod opsin genomic subclone (pzfrh3CR). This clone contains A Zebrafish Rod Opsin Promoter Directs EGFP Expression to
Photoreceptors--
A 1.2-kbp fragment containing the putative
zebrafish rod opsin promoter, the transcriptional start site, and 52 bp
of 5'-untranslated sequence were cloned upstream of the EGFP cassette
to generate pZOP-EGFP. Zebrafish embryos at the 1- to 2-cell stage were
microinjected with linearized pZOP-EGFP. Approximately 50% of injected
embryos continued developing, with 90% of them hatching and 65%
surviving beyond 15 dpf. An average of 6% of the injected embryos
expressed the EGFP transgene at variable levels, although expression
was always restricted to the eye (Fig. 6,
A and B). EGFP expression was detected first at 3 dpf in the ventral-nasal eye and retinal sections localized the EGFP
expression to the photoreceptor layer (data not shown). The
localization and morphology of the EGFP-expressing cells was consistent
with rod photoreceptor-specific expression.
Germline Transmission of the ZOP-EGFP Transgene--
24 fish that
were EGFP-positive by fluorescence microscopy survived to adulthood.
The EGFP cassette was PCR-amplified from fin genomic DNA in seven of
these fish (data not shown). Based on fluorescence microscopy, one of
these fish exhibited germline transmission of the transgene to The advent of zebrafish as a prolific model system for vertebrate
genetics directed us to isolate and characterize the zebrafish rod
opsin gene. As previously reported for other teleosts, the zebrafish
gene lacks introns, contrasting with the typical gene structure of five
exons and four introns found in nearly all other vertebrate opsin genes
(42). Our localization of the transcription start site predicts a
1,660-bp nonpolyadenylated mRNA, which is consistent with the
1.9-kilobase polyadenylated message identified by Northern blot. In
zebrafish, genome duplication has resulted in multiple alleles of some
zebrafish genes (43). Occasionally, this creates distinct regulatory
profiles for the alleles that additively account for the expression
pattern of the gene (43). Also, gene duplication can mask recessive and
dominant mutations generated in mutagenesis screens. For zebrafish rod
opsin, our Southern data are consistent with a single-copy gene or
copies that are highly conserved.
We identified evolutionary conserved sequences in the 5'-flanking
region of the rod opsin gene that may control rod opsin expression in
zebrafish. The conserved cis-elements (glass,
NRE, Ret-1/PCE-1, and
Ret-4) include sites for transcription factors (Crx, Erx, Glass,
Mash-1, Nrl, and Rx) that
regulate retina/photoreceptor/rod-specific expression and that
segregate with visual defects (32, 34, 37-41, 44-47). Rod opsin
expression is regulated in a combinatorial fashion that depends on the
synergistic activities of several transcription factors (34, 39). The
presence of these regulatory elements in the ZOP-EGFP transgene likely
helped to ensure its robust level of rod-specific expression. Although
rod opsin gene expression may be regulated in a complex fashion, its
promoter is excellent for expressing transgenes in the rod
photoreceptors. Rod opsin promoters have successfully directed gene
expression to rod photoreceptors in both mouse and Xenopus
(48-51). Also, the promoter in its native state drives a high level of
expression during the terminal differentiation of rod cells and
maintains that expression level through the life of the cell.
In this study, we identified a 1.2-kbp zebrafish promoter that directs
EGFP expression to rod photoreceptors. The expression pattern of the
transgene appears to recapitulate the tissue-specific and developmental
progression of the zebrafish rod opsin gene (20, 21, 52). At 3 dpf,
EGFP-positive cells are clustered in the ventral-nasal retina and begin
to appear singly in other retinal regions at 6-7 dpf (data not shown).
However, a more critical analysis at the cellular level reveals two
subtle divergences from endogenous rod opsin expression. First, the
temporal expression of ZOP-EGFP is delayed slightly relative to rod
opsin. Rhodopsin expression is observed first in the ventral retina,
nasal to the choroid fissure. Although differentiating rods accumulate
rapidly and to a high density in this ventral patch, rods are added at a slower rate and in a random, isolated fashion in other retinal regions (20). In contrast, the EGFP expression pattern at 4 dpf appears
like the pattern of rhodopsin expression seen in developing fish at
55-60 h postfertilization (20). Thus, rods appear to differentiate and express rhodopsin prior to expressing EGFP. This
result may suggest that an additional factor necessary for the
initiation of rod opsin expression during early differentiation acts
beyond our 1.2-kbp cloned promoter fragment. Second, in adults, not all
rod photoreceptors express EGFP, and the expression may occur in a
dorsal-ventral gradient. A gradient of transgene expression was
reported previously with a bovine rod opsin promoter directing transgene expression in mice (51). These temporal and spatial divergences from rhodopsin expression may be because of restricted expression of the transgene to a subpopulation of rods such as the rod
precursor cells, position effects related to the integration site of
the transgene, or perhaps cytotoxicity by the EGFP (53). Further
examination of the transgenic retinas may shed light on native
regulation of rod opsin gene expression. Although the expression of
EGFP is delayed, the persistent rod-specific expression makes this a
useful promoter for expressing transgenes in rod cells during both
development and adulthood.
EGFP was transiently expressed in The identification of a functional zebrafish rod opsin promoter will
provide several significant applications. The promoter will be used to
drive the expression of putative dominantly active transgenes to
generate models of human retinal diseases. Also, as candidate genes are
identified for zebrafish retinal mutants, the expression of wild-type
genes in the mutant will be a powerful method to confirm gene
identification. This type of "gene rescue" has been widely
exploited in Drosophila. In addition, transgenic lines that
express markers such as EGFP will be useful tools in mutagenic screens,
in which subtle phenotypes may be identified more easily by the loss of
a readily observable marker like the EGFP. Because the
cis-elements in rod opsin promoters are conserved between
zebrafish and mammals, transgenic zebrafish may prove useful in
characterizing the developmental regulation of rod opsin and other
photoreceptor genes. Transgenic zebrafish also may prove useful in
elucidating the current controversy over the nature of circadian
photoreception. The controversy stems from whether the circadian
photoresponse is initiated in the eye, pineal, or other tissues and
whether the photoreceptor is opsin-based, a cryptochrome, or an unknown
photoreceptor (55, 56). Apparent system redundancy and species
differences complicate the issue (57, 58). In zebrafish, six opsin
genes expressed in four retinal photoreceptor classes and six
cryptochrome genes expressed in the body, brain, and eye have been
characterized (26, 57). Mutagenesis screens to identify circadian
mutants and the ablation of specific retinal cell populations by
targeted expression of toxic genes may prove useful to reveal genes
that function in circadian photoreception.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
35 mutants possess
visual system-specific defects (4-7). Transposition, random insertion,
and retroviral integration provide the means for introducing transgenes
into the zebrafish genome (10-12). Accurate tissue-specific and
developmental expression has been recapitulated in nonretinal tissue
using endogenous zebrafish promoters (13, 14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
18 µg) were
electrophoresed in 1% agarose gels containing 6.6% formaldehyde,
blotted to Hybond-N nylon membranes in 20× SSC, and UV cross-linked.
Membranes were hybridized with a probe in Hybrisol II containing 35%
(v/v) formamide at 55 °C. Final high stringency washing conditions
were 0.08× SSC, 0.1% SDS at 65 °C for 30 min twice.
Fix II zebrafish genomic library were
screened with a radiolabeled zebrafish rod opsin cDNA (26).
Escherichia coli strain XL1-MRAP2 was infected with titrated
phage and plated on 150-mm NZY Petri dishes. Plaques were lifted and
UV-crosslinked onto Hybond-N+ nylon membranes (27). Hybridization
conditions were as described under "Southern Blotting."
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Genomic Southern blot of the zebrafish rod
opsin gene. Aliquots of zebrafish genomic DNA (18 µg) were
digested with EcoRI, EcoRV, SpeI,
XhoI, or ApaI, electrophoresed through agarose
gels, and hybridized to the zebrafish rod opsin cDNA. The positions
of the 1-kbp DNA ladder markers are shown to the left.
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Fig. 2.
Mapping the zebrafish rod opsin genomic
clone. Primers spanning the rod opsin cDNA were used to PCR
amplify the following DNA samples: rod opsin genomic phage DNA
(a), zebrafish genomic DNA (b), rod opsin
cDNA (c), and the pzfrh3CR plasmid (d). A
control reaction that lacked template was also used ( ). The primer
pairs used in each PCR reaction (RF1-RR1, RF1-RR2, RF2-RR2, RF2-RR3,
and RF3-RR4) are shown at the top of the gels. The
positions of the primers relative to the cDNA are shown at the
bottom of the figure.
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Fig. 3.
Localization of the transcription start site
for zebrafish rod opsin. A, zebrafish eye or body total
RNA were probed with the zebrafish rod opsin cDNA. The positions of
the RNA size markers are indicated. B, primer extension
reactions were performed on zebrafish RNA samples annealed to rod opsin
primer RR1. RNA was annealed with 1-, 0.4-, or 0.2-pmol labeled primer.
Shown to the left is the sequencing reaction of pzfrh3CR
using the RR1 primer.
1.2
kbp upstream of the transcriptional start site, which is the entire rod
opsin coding sequence and 468 bp of 3'-flanking sequence (Fig.
4). Consensus polyadenylation signals
(AATAAA) were found at positions 1535 and 1553, located 18-51 bp
upstream of two transcription terminator signals (YGTGTTYY) (31). The
first polyadenylation signal is in agreement with our cDNA
sequence, which deviates from the genomic sequence at position 1559 by
the presence of 19 adenine residues. Alignment of proximal promoter
sequences of zebrafish, Xenopus, chicken, mouse, and human
rod opsin genes identifies conserved cis-elements (Fig.
5) that are implicated in regulating
retina-specific gene expression: glass (32, 33);
NRE (34, 35); Otx/Bat-1
(36, 37); Ret-1/PCE-1
(38-40), and Ret-4 (41).
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Fig. 4.
Sequence of the zebrafish rod opsin
gene. The determined 2,222-bp sequence of the zebrafish rod opsin
gene is shown. The 5'- and 3'-flanking sequences are shown in
lowercase, the coding sequence is shown in
uppercase, and the translated sequence is shown in
bold type. The transcriptional start site, as determined by
primer extension, is numbered +1 and underlined; the ATG
translation initiation and TAA translation termination codons are
underlined also. Conserved regulatory sequences and the
XbaI site used in generating the 1.2-kbp promoter fragment
are boxed and labeled. The GenBankTM EBI
accession number for the reported sequence is AF331797.
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Fig. 5.
Identification of
cis-elements conserved in rod opsin promoters.
Alignment of proximal promoter sequences from human, mouse, chicken,
Xenopus, and zebrafish rod opsin genes is shown. The
sequence alignment was generated using ClustalW alignment in Mac Vector
6.5.1 software (Oxford Molecular, Inc.) with a gap penalty of 5 and an
extend gap penalty of 1.4. Conserved nucleotides are boxed,
conserved cis-elements are labeled, and dashes
were inserted by the computer program to maximize the alignment. The
nucleotide positions are numbered. GenBank accession numbers
for the sequences are: human, U49742; mouse, M55171; chicken, M98497;
and Xenopus, U23808.
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Fig. 6.
The zebrafish rod opsin promoter directs EGFP
expression to rod photoreceptors. A and
B, the eye of a G0 embryo (8 dpf) that was
microinjected with pZOP-EGFP and viewed under epifluorescent
illumination (A) and reflected light (B). The
arrowheads demarcate the dorsal aspect of the pigmented eye,
and the arrow points to the fluorescence emerging through
the pupil. C and D, anti-rhodopsin-labeled
whole-mount images from a 4-dpf G1 transgenic fish. The
anti-rhodopsin in the rod outer segments was detected with a
Cy3-conjugated secondary antibody. The, EGFP-expressing cells are
located predominately in the ventral-nasal aspect of the retina
(double arrows in upper eye; arrowhead points to
choroid fissure). The single arrow in C points to an
isolated double-labeled cell that is shown at higher magnification in
D (arrow points to EGFP-containing cell body;
arrowhead points to rhodopsin-positive rod outer segment).
E, a 10-µm frozen section of a 17-dpf G1 fish
reveals EGFP-expressing cells in the dorsal and ventral aspects of both
retinas (arrows point to dorsal retinas of each eye). The
arrowhead on the left eye indicates the optic nerve exit
point. F, frozen section from a 54-dpf G1 fish
reveals EGFP-containing nuclei in the outer nuclear layer
(ONL; arrow) and many long rod outer segments
that also contain the EGFP (ROS; arrow).
G, high magnification image of a single EGFP-positive cell
from the section shown in F. The rod outer segment,
ellipsoid (E), myoid (M), and nucleus
(N) are identified. H, sections were labeled with
anti-rhodopsin and detected with Cy3-conjugated secondary antibody. The
EGFP signal is present in both the ONL and the layer of ROS, whereas
rhodopsin expression was detected in only the ROS. EGFP was not
detected elsewhere in the retina. D, dorsal; V,
ventral; L, lens; INL, inner nuclear layer;
GCL, ganglion cell layer. (Scale bars: C and
G, 50 µm; D, 10 µm; G, 5 µm;
E and F, 100 µm.)
6%
of its progeny. These EGFP-positive G1 progeny were
isolated, and their EGFP expression patterns were examined. At 4 dpf,
the EGFP expression was limited to the ventral-nasal portion of the
retina, although rhodopsin protein expression was detected throughout
the retina (Fig. 6C). Careful inspection of the
EGFP-expressing cells in the whole-mount-labeled retinas confirmed rhodopsin expression in the outer segments of the EGFP-positive cells
(Fig. 6D). At 17 and 54 dpf, EGFP expression in
G1 fish spanned the photoreceptor layer of the retina, but
with an apparent dorsal-ventral gradient (Fig. 6, E and
F, respectively). Close examination of the EGFP-positive
cell morphology in the 54-dpf retina revealed the characteristic rod
photoreceptor structure (Fig. 6G). Furthermore, labeling
frozen sections with rhodopsin antiserum demonstrated that the retinal
distribution of EGFP was restricted to rod outer segments and rod
nuclei (Fig. 6H).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6% of injected embryos,
consistent with percentages previously described (13, 14). The
frequency of injected embryos exhibiting germline transmission was
0.25-1.75%. However, the frequency of transmission may have been
underestimated because of gene silencing and technical difficulties in
detection of EGFP in the eye. In addition to screening for transgene
expression by fluorescence microscopy, adult fish were screened
secondarily for stable transgene integration by PCR amplification of
fin genomic DNA. Adults that typed positive were outcrossed, and their
progeny were screened by fluorescence microscopy to verify transmission
of the transgene in the germline. As the fish age, microscopic
observations of the retina become obscured by the envelope of
melanophores and iridiphores around the eyes. Treatment of fish with
1-phenyl-2-thiourea inhibits the synthesis of melanin, thus permitting
observation of EGFP through the translucent eye at 3 dpf (the onset of
rhodopsin expression). However, the 1-phenyl-2-thiourea does not
inhibit the production of the gold- or silver-reflecting components of
the iridiphores so that at 6-8 dpf, EGFP fluorescence is often
observed only as it exits through the lens. Thus, proper orientation of
the fish, as previously reported for Xenopus (54), is
critical to ensure the accurate detection of EGFP expression.
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ACKNOWLEDGEMENTS |
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We thank T. David Ponder for helpful discussions, Bill Archer for assistance with microphotography, and Zebrafish Management, Ltd. for providing Tetra AZ baby powder. We also thank Emily Cassidy, Rachel Hartzell, and Debbie Bang in the University of Notre Dame's Zebrafish Facility.
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FOOTNOTES |
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* This work was supported by a grant from the Foundation for Fighting Blindness (to D. R. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF331797.
Recipient of a postdoctoral fellowship from the Keck Center for
Transgene Research, University of Notre Dame. Present address: Howard
Hughes Medical Inst./Dept. of Biochemistry, P.O. Box 357370, University
of Washington, Seattle, WA 98195.
§ Supported by a National Science Foundation-Research Experience for Undergraduates award.
¶ To whom correspondence should be addressed. Tel.: 219-631-8054; Fax: 219-631-7413; E-mail: hyde.1@nd.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M010490200
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ABBREVIATIONS |
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The abbreviations used are: dpf, days postfertilization; kbp, kilobase pairs; EGFP, enhanced green fluorescent protein; PCR, polymerase chain reaction; bp, base pair(s)..
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