Using the Tg(nrd:egfp)/albino Zebrafish Line to
Characterize In Vivo Expression of
Jennifer L. Thomas1, Margaret J. Ochocinska2, Peter F. Hitchcock2, Ryan Thummel1*
1Department of Anatomy and Cell Biology and Department of Ophthalmology, Wayne State University School of Medicine, Detroit, Michigan, United States of America,
2Department of Ophthalmology and Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan, United States of America
In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of
neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration.
We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in
cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral
line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of
the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based
on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating
retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually
re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals
that neurod is not expressed in Mu ¨ller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as
they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration,
expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of
endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating
the fundamental processes that govern photoreceptor regeneration in adults.
Citation: Thomas JL, Ochocinska MJ, Hitchcock PF, Thummel R (2012) Using the Tg(nrd:egfp)/albino Zebrafish Line to Characterize In Vivo Expression of
eurod. PLoS ONE 7(1): e29128. doi:10.1371/journal.pone.0029128n
Editor: Mike O. Karl, Center for Regenerative Therapies Dresden, Germany
Received June 17, 2011; Accepted November 21, 2011; Published January 3, 2012
Copyright: ? 2012 Thomas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institutes of Health grants R21EY019401 (RT) R01EY018417 (RT), P30EY04068 (RT), R01EY07060 (PFH), P30EY07003
(PFH), the Research to Prevent Blindness (PFH), and start-up funds to RT, including an unrestricted grant from Research to Prevent Blindness to the Wayne State
University, Department of Ophthalmology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
NeuroD is a basic helix-loop-helix (bHLH) transcription factor
that plays a common role in persistently mitotic cells as an essential
link between cell cycle exit, cell fate determination, and cell
survival . In the vertebrates, neurod is expressed in areas of the
brain including the cortex, cerebellum, olfactory bulb, eye, and
midbrain [1,2,3,4]. Neurod is also expressed in the developing
endocrine pancreas , the auditory and vestibular neuroblasts of
the developing inner ear , and the lateral line of teleost fish .
In both mice and zebrafish, neurogenin is expressed in cells prior to
neurod, [2,4] and overexpression of the neurogenin homolog in
Xenopus (X-NGNR-1) induces ectopic expression of Xneurod
mRNA , suggesting that neurogenin is an upstream regulator of
neurod . During both zebrafish and mammalian retinogenesis,
neurod is first expressed in retinal neuroepithelial cells as they exit
the cell cycle. Once distinct cell types have formed, neurod is
expressed in a subset of cells in both the inner nuclear layer (INL)
and outer nuclear layer (ONL), but not in the ganglion cell layer
(GCL) [1,9]. By adulthood, neurod expression was previously
reported to persist in a subset of amacrine cells nascent cone
photoreceptors near the retinal margins [1,10].
NeuroD functions in both neuronal and non-neuronal tissues
and its specific role appears to be dependent of the mitotic state of
the cell. In mitotic cells, NeuroD specifically regulates proliferation
[11,12] and cell cycle exit . This was first demonstrated in
Xenopus embryos where ectopic expression of Xneurod results in
premature differentiation of neuronal precursors . In post-
mitotic cells, loss of NeuroD function can result in cell death
during after cell differentiation [12,14,15,16]. For example,
NeuroD-null mice are deaf due to apoptosis of the otic epithelium
and neurons that form the cochlear-vestibular ganglion . In
addition, loss of NeuroD in mice also causes age-related rod
photoreceptor degeneration .
During mouse retinogenesis, neurod expression in retinal
progenitors promotes the genesis of neurons versus glial cells,
and specifically promotes amacrine cell fates versus bipolar cell
fates [9,17]. In the developing chick retina, NeuroD is necessary
and sufficient for photoreceptor differentiation [18,19]. During
zebrafish retinogenesis, NeuroD regulates exit from the cell cycle
among late-stage photoreceptor progenitors .
The zebrafish is a unique model because of its ability fully
regenerate a variety of tissues, including the fin [21,22], heart ,
spinal cord  and retina . Numerous approaches have been
developed to induce retinal regeneration, including cytotoxins
, [27,28], laser ablation , stab wound  and constant
intense light treatment, which selectively kills rod and cone
photoreceptors [25,31]. Whereas each of these methods is unique
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in its severity of injury and selectivity of cellular damage, the
mechanisms of regeneration are conserved. Cell death elicits a
subset of Mu ¨ller glial cells to reenter the cell cycle and generate
retinal progenitors that differentiate into all the retinal cell types
lost to the original injury [25,32].
In this study, the Tg(nrd:egfp)/albino zebrafish line was used to
characterize neurod expression. In this line, the transgene is
expressed in the CNS, including the retina, olfactory bulbs,
midbrain, hindbrain, neural tube, lateral line, inner ear and
visceral organs, including the pancreas and gut. A detailed analysis
of neurod expression, as evidenced by EGFP localization, is shown
during retinal development in larvae and photoreceptor regener-
ation in adults. During regeneration we show that the neurod
transgene in not expressed in Mu ¨ller glial cells as they reenter the
cell cycle, nor is it expressed in their immediate progeny.
However, the transgene is expressed in progenitors of the
regenerating photoreceptors as they exit the cell cycle and begin
differentiating. We find that this neurod transgene is a useful tool to
visualize neurod expression during the development of multiple
organ systems and during the dynamic process of adult retinal
Materials and Methods
All protocols used in this study were approved by the animal use
committee at the University of Notre Dame and Wayne State
University School of Medicine (Protocol # A040310) and are in
compliance with the ARVO statement for the use of animals in
The Tg(nrd:egfp) line and zebrafish maintenance
The Tg(nrd:egfp) line was obtained as a gift from Alex
Nechiporuk, who generated the line . Briefly, a BAC clone
(dK33b12) was isolated that contained 67 kilobase pairs (kb) of
sequence upstream and 89 kb of sequence downstream of neurod.
Recombineering resulted in egfp positioned at the endogenous start
site. This construct (ZFIN ID: ZDB-TGCONSTRCT-080701-1)
was used to make transgenic animals. Adult fish positive for the
transgene were out-crossed to albino mutants. Fish were fed a
combination of brine shrimp and flake food three times daily and
maintainedundera daily light
(250 lux):10 hours dark at 28.5uC .
cycle of14 hours light
Constant intense-light treatment protocol
Photoreceptor degeneration was accomplished by constant
intense-light treatment as previously described . Adult
Tg(nrd:egfp)/albino zebrafish were subjected to dark adaptation for
10 days, and then transferred to a clear 1.8 liter tank positioned
between 4 halogen lamps (250 watts). The fish were continuously
exposed to the light (8000 lux) for up to four days, at which time
they were returned to standard light/dark conditions. During the
light treatment, water temperature remained between 30–33u C.
EdU labeling of retinal progenitors
59-ethynyl-29-deoxyuridine (EdU; Invitrogen, Carlsbad, CA)
was diluted in 1XPBS to 1 mg/mL and injected intraperitoneally
(50 microliters) into adult Tg(nrd:egfp)/albino zebrafish. Two
injection protocols were used. In order to label all of the
progenitors, daily injections were performed throughout the light
treatment . In order to label a subset of the progenitors, a
single injection was performed immediately prior to starting the
light treatment. Eyes were harvested 96 hours after light onset and
processed for immunohistochemistry as described below. For EdU
immunolocalization, Click-iT EdU AlexaFluor 594 Imaging Kit
was performed per the manufacturer’s instructions (Invitrogen),
followed by EGFP immunolocalization as described below.
Wholemount brightfield and fluorescent imaging
Live transgenic embryos and adult fish were anesthetized with
2-phenoxyethanol prior to microscopy. Images were captured on a
Spot digital camera (Diagnostic Instruments; Sterling Heights, MI,
USA) attached to a Leica M165 FC stereomicroscope.
Immunohistochemistry and microscopy
Tg(nrd:egfp)/albino zebrafish were collected at 24, 32, 42, 48, 72,
and 96 hour post-fertilization (hpf), dechorionated (if necessary),
and fixed in either 4% paraformaldehyde in 5% sucrose/16PBS
or 9:1 ethanolic formaldehyde (100% ethanol: 36% formaldehyde)
overnight at 4u C. Embryos and larva were cryoprotected in 5%
sucrose/16 PBS twice at room temperature, followed by a 30%
sucrose/16 PBS wash overnight at 4u C. Larvae were frozen in
Tissue Freezing Medium (TFM) (Triangle Biomedical Sciences,
Durham, NC) and cryosectioned at 18 mm. Sections were
transferred to glass slides, dried for up to 4 hours at 56u C, and
stored at 280uC.
For controls and those receiving photolytic lesions, fish were
euthanized and their eyes were harvested at various times after
light onset: 0, 42, 72, or 96 hours, or 7 or 11 days. Eye tissue was
fixed in either 4% paraformaldehyde in 5% sucrose/16 PBS or
9:1 ethanolic formaldehyde (100% ethanol: 36% formaldehyde)
overnight at 4u C, cryoprotected and embedded in TFM . Eyes
were cryosectioned at 18 mm and sections were transferred to glass
slides, dried at 56u C for 2 hours, and stored at 280u C.
Immunohistochemistry was performed as previously de-
scribed . The following primary antibodies and dilutions
were used: chicken anti-insulin polyclonal antisera (1:200,
Abcam, Cambridge, MA) mouse monoclonal anti-green fluo-
rescent protein (GFP) antibody (1:200, Sigma Chemical, St.
Louis, MO), mouse monoclonal anti-PCNA antibody (1:500,
Sigma Chemical, St. Louis, MO), rabbit polyclonal anti-PCNA
antisera (1:100, AnaSpec, Fremont, CA), mouse monoclonal
anti-glutamine synthetase antibody (1:500, Chemicon Interna-
tional, Temecula, CA), mouse monoclonal anti-HuC/D (1:30,
Invitrogen), mouse monoclonal anti-Zpr-3 antibody (1:200,
Zebrafish International Resource Center, Eugene, OR), and
mouse monoclonal anti-Zpr-1 antibody (1:200). Secondary
antibodies used for this study included goat anti-mouse 488
and 594, goat anti-rabbit 488 and 594, and goat anti-chicken
594 (Invitrogen, Carlsbad, CA). In addition, nuclei were labeled
using TO-PRO-3 (1:750, Invitrogen).
Confocal microscopy was performed using a Leica TCS SP2.
Approximately 12–15 retinal sections taken at or adjacent to the
optic nerve were examined for each time point.
RNA in situ hybridization and subsequent
For in situ hybridizations, eyes were dissected and preserved (as
described above), cryosectioned at 10 mm and processed as
described previously (Ochocinska and Hitchcock, 2009). Briefly,
sections were rehydrated in decreasing concentrations of ethanol,
permeabilized with Proteinase K, and treated with acetic
anhydride to reduce non-specific binding of the probe. The
2,158 basepair Digoxygenin-labeled probe was synthesized from a
full-length cDNA of neurod (kindly provided by Zhiyuan Gong,
National University of Singapore) . The probe was applied to
the sections and incubated overnight at 55u C. Sections were then
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washed at 55u C to remove unbound probe, and processed for
immunocytochemistry with antibodies against DIG that were
conjugated to alkaline phosphatase. NBT/BCIP (Roche) was used
as a substrate for the alkaline phosphatase. The reaction was
stopped (generally after 1 hr) with Tris-HCl buffer at pH 8.0. The
reaction product was preserved by briefly fixing the sections with
4% paraformaldehyde prior to the GFP immunohistochemistry
Tg(nrd:egfp) expression is observed in multiple tissues
from embryonic development through adulthood
The expression of the nrd:egfp transgene was first examined by
wholemount fluorescence microscopy. Consistent with previously
submitted gene expression data of endogenous neurod , the
transgene is not maternally expressed (data not shown), and was
not observed during gastrulation at 6 hours post-fertilization
(Fig. 1A). EGFP expression was first observed at 24 hours post-
fertilization (hpf) in the olfactory bulbs, pineal gland, inner ear,
midbrain, hindbrain, pancreas and neural tube (Fig. 1B, C), but
was not observed in the developing eye (Fig. 1C). This expression
pattern was identical to the previously reported expression pattern
of endogenous neurod . In the developing zebrafish retina,
endogenous neurod was first observed in the ventral nasal patch at
31 hpf  (Fig. 1E), which coincides with the initiation of a
ventral-to-dorsal wave of neurogenesis. At 32 hpf, very weak
EGFP expression (note the over-saturation of the surrounding
tissues) was observed in the retina immediately dorsal to the
ventral nasal patch (Fig. 1F). At 48 hpf, EGFP-positive cells were
observed throughout the inner retina (Fig. 1G, arrows) and outer
retina (Fig. 1G, arrowhead), indicating that the wave of
neurogenesis had completed. Persistent EGFP expression was also
observed in areas of the central nervous system, lateral line, and
the pancreas (Fig. 1H–L).
In the adult zebrafish, we observed persistent and intense EGFP
expression in the eye, pineal gland, and cerebellum (Fig. 2B, D and
F). This is consistent with previous reports indicating expression of
endogenous neurod in the adult pineal gland [2,37] and
cerebellum [38,39]. Expression was also observed surrounding
the anus (Fig. 2H and I). Closer examination of the zebrafish body
revealed weak EGFP expression in an extension of the lateral line,
which was especially visible near the tail fin girdle (Fig. 2J and K).
This expression revealed intricate nerve arborization and synaptic
boutons (Fig. 2K and L). In addition, EGFP expression was
observed in ganglia associated with the nerve that extends through
each bony hemiray of the caudal fin, which are anchored in the fin
girdle and give support for fin structure (Fig. 3B, C9, D9). The
transgene is not upregulated in the wound epithelium or
proliferative blastema during fin regeneration, but is re-expressed
in ganglia associated with the regenerating nerve (data not shown).
In addition, EGFP was observed in the adult endocrine pancreas
and in presumptive enteroendocrine cells in the gut epithelium.
Specifically, EGFP co-labeled with Insulin in the endocrine
pancreas, but was not expressed in the surrounding exocrine
pancreas (Fig. 4A90). Finally, EGFP was observed in a small
number of cells within the intestinal epithelium (Fig. 4B and C).
Neurod has previously been shown to be expressed in enteroendo-
crine cells and be required for proper enteroendocrine cell
differentiation. Based on these data and the location, distribution,
and morphology of the EGFP-positive cells observed in the gut,
the transgene appears to label both endocrine cells of the pancreas
and enteroendocrine cells in the adult gut.
The nrd:egfp transgene is expressed in cells as they exit
the cell cycle and in a subset of differentiated retinal
During retinal development in zebrafish, neurod is required for
photoreceptor progenitors to exit the cell cycle . We examined
expression of the nrd:egfp transgene in relationship to retinal
progenitors immunolabled with Proliferating Cell Nuclear Antigen
(PCNA), a marker for proliferating cells [25,40]. At 42 hpf, we
observed PCNA-positive cells restricted to the circumferential
marginal zone (CMZ) and EGFP expression in the central retina
with colocalization of cells in the overlapping regions of EGFP and
PCNA expression (Fig.5A). Following retinal lamination, at 72 and
96 hpf, PCNA-positive cells were restricted to the CMZ and no
longer colocalized with the transgene, and EGFP expression was
seen in a subset of amacrine and bipolar cells (Fig. 5B and C).
Closer examination of the nrd:egfp transgene expression during
retinal development and in adulthood revealed similarities and
differences between EGFP expression and the previous report of
endogenous neurod expression. Similar to the previous observation
, EGFP expression was not observed in undifferentiated
neuroepithelium 24 hpf (Fig. 6A) and at no age was EGFP
observed in the retinal progenitors located in the circumferential
marginal zone (CMZ) (Figs. 5 and 6). EGFP was first observed in
the retina immediately adjacent to the ventral nasal patch at 32
hpf (Fig. 6B). EGFP expression expanded throughout the inner
and outer retina at 48 hpf (Fig. 6C). At 72 hpf, endogenous neurod
expression was reported to be expressed only in amacrine cells and
in the ONL . In contrast, EGFP was present in a subset of
ganglion cells, amacrine cells, and bipolar cells, but was not
detected in the ONL (Fig. 6D). In addition, the EGFP signal grew
slowly in the population of rod photoreceptors, starting at 2 weeks
post fertilization (wkpf) (Fig. 6F), and was present in all rod
photoreceptors in adults (Fig. 6H). Although expression in the
Figure 1. Wholemount brightfield and flourescent images showing nrd:egfp transgene expression in the developing Tg(nrd:egfp)/
albino zebrafish. (A) Brightfield image with fluorescent inset showing the absence of transgene expression at 6 hpf. Arrowhead notes the
gastrulation site (B) Fluorescent image with a brightfield inset at 24 hpf showing EGFP expression in the developing pancreas (arrow), olfactory bulbs
(single arrowhead), and regions of the midbrain and hindbrain (double arrowheads). (C) High magnification overlay of brightfield and fluorescent
images at 24 hpf. EGFP is detected in the olfactory bulbs (arrowheads), pineal gland (arrow), and inner ear (top right of panel). At this time it is not
observed in the developing eye. (D) High magnification overlay of brightfield and fluorescent images at 24 hpf showing EGFP expression in the neural
tube. (E) RNA in situ hybridization at 31 hpf, showing endogenous neurod expression in the ventral nasal patch (arrow), immediately adjacent to the
choroid fissure (arrowhead). (F) Fluorescent image showing EGFP-positive cells in the retina at 32 hpf that are within a region (white arrows)
immediately adjacent to the ventral nasal patch (black arrow). The choroid fissure is marked with a white arrowhead). (G) High magnification overlay
of brightfield and fluorescent images at 48 hpf showing EGFP expression in throughout the inner retina (arrows) and in the outer retina (arrowhead).
(H) Overlay of brightfield and fluorescent at 48 hpf (with brightfield image inset), showing EGFP in the developing pancreas. (I–J) Fluorescent (I) and
brightfield overlay (J) of image shown in H. EGFP expression is observed in the neural tube and lateral line (arrow). (K) Fluorescent image of the dorsal
head at 48 hpf. EGFP expression is observed in the pancreas (arrow), inner ear (arrowhead, with bracket to indicate location of ear), and regions of the
CNS. (L) Corresponding overlay of brightfield and fluorescent images. Abbreviations: L (Lens), Re (Retina), AYE (Anal Yolk Extension), Nc (Notochord),
Nt (Neural tube). Scale bar: 250 (A); 250 microns (B, H); 100 microns (C, D, I, J); 50 microns (E); 50 microns (K, L).
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ONL and bipolar cells was not reported previously, we find that
endogenous neurod is expressed in each of these cell types in adults
(Fig. 6I–I0). Specifically, weak expression of neurod was observed in
Figure 2. Wholemount brightfield and fluorescent images
showing nrd:egfp trangene expression in adult Tg(nrd:egfp)/
albino zebrafish. (A) A multiple brightfield image overlay showing the
entire adult fish. Arrowhead indicates the location of the pineal gland
and cerebelum (shown in Panels E and F). The boxes and corresponding
panel letter indicate the location of the higher magnification images
shown in Panels C–K. (B) Corresponding fluorescent image to Panel A,
showing EGFP expression in the eye (white arrow) and the pineal (white
arrowhead). (C–D) Brightfield and corresponding fluorescent image
showing EGFP expression in the eye. (E) Brightfield image of the dorsal
side of the head showing the pineal gland (arrow), telecephalon (Te),
and cerebellum (Ce, arrowhead). (F) Corresponding fluorescent image
showing EGFP expression in the pineal gland (arrow) and cerebellum
(arrowhead). (G–I) Brightfield, overlay, and fluorescent images of the
anus and its expression of the transgene. (J–K) Brightfield and
corresponding fluorescent image showing EGFP expression in nerves
located near the girdle of the tail fin. The box indicates the location of
image shown in Panel L. (L) A high magnification fluorescent image of a
branch of the nerve shown in Panel K, most likely of the posterior lateral
line, showing EGFP expression in each of the terminating synaptic
buttons. Scale bar: 2 mm (A).
Figure 3. Wholemount brightfield and flourescent images
showing nrd:egfp transgene expression in the adult caudal tail
fin. (A) Brightfield image of the adult caudal fin. (B) Corresponding
fluorescent image to panel A. EGFP expression is visualized in the nerve
coursing through each bony hemiray of the caudal fin, however at this
level of magnification, it is difficult to visualize. (C–C9) High
magnification brightfield and corresponding fluorescent overlay
showing multiple bony lepidotrichia. The arrows point to the nerve
running within each bony hemiray and arrowheads point to EGFP-
positive ganglia associated with the nerve. (D and D9) A section of a
single bony ray immunolabeled with EGFP to show the transgene and
co-labeled with TO-PRO-3 to show all nuclei (magenta). The white line
in Panel D shows the location of the nerve. Ep=Epithelium, Bn=bony
ray, CT=connective tissue.
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the ONL, with strong expression in the rod photoreceptor inner
segments. In the INL, every EGFP-positive cell exhibited at least
some neurod expression. However, many cells that were strongly
expressing neurod, showed only weak EGFP, and vice versa,
perhaps reflecting the dynamic regulation of neurod transcription in
Adult retinas were characterized further using morphological
analysis and antibody markers to identify cell types that express the
nrd:egfp transgene. EGFP was observed in all rod photoreceptor cell
bodies and in rod inner and outer segments (Fig. 7A and B9), but
not in double cones (Fig. 7C and D9). Further, EGFP was observed
in a subset of the amacrine cells, and very weak expression was
detected in a small population of ganglion cells (Fig. 7E and F9),
but not observed in Mu ¨ller glia (Fig. 7G and H9). Since adult
zebrafish contain at least 17 subtypes of bipolar cells, EGFP-
positive bipolar cells were identified by the location, size and shape
of the somata, shape of the dendritric tree, and the sublaminal
innervation level in the inner plexiform layer (IPL). Based on the
previously described characteristics of each subtype, we observed
seven subtypes of EGFP-positive OFF bipolar cells (Boff-s1, Boff-
s2w, Boff-s3, Boff-s1/s2, Boff-s1/s3, Boff-s2/s3, and Boff-s1/s4) in
adult nrd:egfp retinas, including many cases where the projections
could be traced from the photoreceptors to the IPL (Fig. 7F9).
Tg(nrd:egfp) expression in the light-damaged adult
We examined the spatial and temporal expression of the nrd:egfp
transgene following photolytic lesions and during photoreceptor
regeneration. Specifically, we examined expression of the nrd:egfp
transgene in relationship to retinal progenitors immunolabled with
PCNA and Mu ¨ller glia immunolabeled with Glutamine Synthe-
tase. In the INL, 48 hours after light onset, Mu ¨ller glial reenter the
cell cycle and express PCNA (Fig. 8A; see Vihtelic and Hyde
2000). At this time, EGFP was not detected in the Glutamine
Synthetase-positive Mu ¨ller glial or their immediate progeny
(Fig. 8A and B). At 72 and 96 hours after light onset, large
numbers of progenitor cells were observed (Fig. 8C and D). Very
weak EGFP expression was also observed in clusters of cells in the
INL (Fig. 8E and F). Further characterization of these EGFP-
positive clusters revealed a down-regulation of Glutamine
Synthetase (Fig. 8G and H) and PCNA co-immunolocalization
(Fig. 8I–L9). This is consistent with a previous report that showed
that Mu ¨ller glia down-regulate cell-specific markers after the re-
enter the cell cycle to produce large clusters of PCNA-positive
Figure 5. Retinal sections from embryonic Tg(nrd:egfp)/albino
zebrafish immunolabeled with PCNA (red) and EGFP (green).
(A) At 42 hpf, EGFP is detected throughout the retinal neuroepithelium
in the central retina. PCNA immunolocalization, showing proliferating
cells, is primarily restricted to the CMZ (arrows). In the overlapping
region of PCNA and EGFP co-labeling can be visualized (arrowhead). (B)
At 72 hpf, EGFP is detected in a subset of ganglion, amacrine and
bipolar cells, and is not present in the outer nuclear layer. Proliferating
cells are restricted to the CMZ (arrows), and there is no evidence of
PCNA and EGFP co-immunolabeling. (C) At 96 hpf, there is persistent
expression of EGFP detected in a subset of ganglion, amacrine and
bipolar cells. Proliferating cells are restricted to the CMZ (arrows) and do
not co-label with the transgene. Scale bars: 25 microns (A) and 50
microns (B, C).
Figure 4. Section from Tg(nrd:egfp)/albino zebrafish showing
nrd:egfp trangene expression in the endocrine pancreas (A–A90)
and gut (B–C). (A–A90). Immunolocalization of EGFP (A, green) co-
labels with Insulin (A9, red) in the endocrine pancreas (A90, En). Co-
labeling with TO-PRO-3 shows all nuclei (A90, blue) and indicates the
surrounding exocrine pancreas (A90, Ex) and adjacent lumen of the gut
(A90, Lu). (B) EGFP expression can be visualized in enteroendocrine cells
within each villus and in the surrounding smooth muscle. The adjacent
pancreas is also visible (arrow). (C) High magnification image of the box
in panel B.
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progenitors . At 96 hours after light onset, weak, somewhat
disorganized, EGFP-positive cells were present in the ONL
(Fig. 8F), and co-labeled with PCNA (Fig. 8K). 7 days after light
onset, proliferating cells in the INL were not observed, however,
EGFP co-labeled with a large number of PCNA-positive
progenitors in the ONL (Fig. 8G). 11 days after light onset, the
transgene was weakly expressed in the newly formed rod
photoreceptors in the ONL (Fig. 8H).
A closer examination of the outer retina was performed using
Zpr-3, which labels rod photoreceptor outer segments. 48 hours
after light onset, the number of EGFP-positive rod photoreceptors
was greatly reduced, along with their Zpr-3-positive outer
segments (cf. Figs. 9A and 9B). By 72 hours after light onset,
newly-formed rod progenitors were observed in the ONL (Fig. 9C).
These could be readily discerned from existing rod photoreceptors
due to their comparatively weak expression of the transgene
(Fig. 9C, inset). 96 hours after light onset, the number of EGFP-
positive rod progenitors was greatly increased, although they were
still somewhat disorganized (Fig. 9D). 7 days after light onset, the
newly formed rod photoreceptors had become more organized
(Fig. 9E) and 11 days after light onset regenerated rod inner
segments and Zpr-3-positive outer segments were observed
(Fig. 9F). Full regeneration of rod outer segments was not
achieved until 28 days after light onset (data not shown).
In order to determine whether the weakly-EGFP positive cells in
the ONL (Fig. 9C, inset) were derived from progenitors or were
undamaged photoreceptors that simply down-regulated EGFP, we
performed an EdU labeling experiment. As was previously
reported , daily injections of EdU following light onset results
in labeling of many, if not all, of the neuronal progenitors. We
repeated this method (Fig. 10A) and found that at 96 hours after
light onset all the weakly-EGFP-positive cells in both the INL and
ONL were also EdU-positive (Fig. 10B, B9). For a better resolution
of individual cells in the ONL, we performed a single injection of
EdU immediately prior to the light treatment, which only labeled
a subset of the progenitors. At 96 hours after light onset, we found
that the EdU-positive cells in the ONL were weakly stained with
EGFP (Fig. 10F, F9), indicating that they were derived from
progenitors. Importantly, with either injection method, we found
that none of the strongly-EGFP-positive rod nuclei in the ONL
Figure 6. Retinal sections from embryonic Tg(nrd:egfp)/albino zebrafish showing EGFP in green and a nuclear stain, TO-PRO-3, in
blue (A–H). All retinas are oriented with dorsal toward the top and ventral toward the bottom. RNA in situ hybridization in adult Tg(nrd:egfp)/albino
retinas comparing endogenous expression of neurod to the transgene (I–I0). (A) At 24 hpf, EGFP is not detected in the retinal neuroepithelium. (B) At
32 hpf, EGFP is detected in a few cells immediately adjacent to the ventral nasal patch (arrow). (C) At 48 hpf, distinct retinal layers can be visualized.
EGFP is detected in the ganglion cell layer (G), inner nuclear layer (I) at the level of the amacrine cells, and in the outer nuclear layer (O), but from this
timepoint onward, is not detected in the CMZ (arrowheads). (D) At 72 hpf, EGFP expression is restricted to very few ganglion cells (G), a subset of
amacrine (A) and bipolar cells (arrow), as well as the inner plexiform layer (arrowhead). (E) At 96 hpf, persistent EGFP expression is visible in a subset
of amacrine and bipolar cells, in the inner plexiform layer, and very weakly expressed in a few ganglion cells (G). (F) At 2 wkpf, EGFP begins to
reappear in a subset of rod photoreceptors (arrowhead). (G) At 6 wkpf, a majority of rod photoreceptors express EGFP as well as a subset of amacrine
and bipolar cells, and the inner plexiform layer. (H) In the adult eye, all rod photoreceptors express EGFP, as well as a subset of amacrine and bipolar
cells. (I–I90) Comparing endogenous neurod (I) to transgenic neurod (I0) in the adult retina. Endogenous neurod is weakly expressed in rod
photoreceptor soma in the outer nuclear layer (ONL, double arrowheads) and their corresponding rod inner segments (asterisk). Expression is also
observed in individual neurons in the inner nuclear layer (INL). Strong EGFP was observed in the ONL (double arrowheads) and rod inner segments
(above), and in individual neurons in the INL. Every EGFP-positive cell contains at least some endogenous neurod, but the levels vary greatly. Some
cells have strong EGFP expression but weak endogenous neurod (single arrowhead), while other show the opposite expression profile (arrow). Scale
bars: 50 microns (A–G), 300 microns (H), 50 microns (I–I90).
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were EdU positive (Fig. 10B, B9, F, F9), indicating that this line can
be used to distinguish between undamaged and newly-formed rod
Tg(nrd:egfp) expression in comparison to endogenous
neurod expression during photoreceptor regeneration
In situ hybridization was used to compare endogenous and
transgenic expression of neurod during photoreceptor regeneration.
Prior to light treatment, dark-adapted adult Tg(nrd:egfp)/albino
retinas showed endogenous neurod in a subset of amacrine and
bipolar cells in the INL, weak expression in rod photoreceptor
soma, and strong expression in rod inner segments (Figs. 6I, 11A).
The expression of endogenous neurod in the rod inner segments was
not observed in non-dark treated animals (data not shown),
indicating dynamic expression changes of neurod in photoreceptors
during dark adaptation. Similarly, EGFP was strongly expressed in
Figure 7. Retinal sections from adult Tg(nrd:egfp)/albino zebrafish. (A) Immunolocalization of GFP (green) to visualize the nrd:egfp transgene
and Zpr-3 to visualize rod photoreceptors. (B) Higher magnification inset of (A) showing Zpr-3 immunolocalization in rods. (B9) Overlay image
showing that the transgene is present in rod photoreceptors and co-labels with rod inner and out segments. (C) Immunolocalization of GFP (green)
to visualize the nrd:egfp transgene and Zpr-1 (red) to visualize double cones. (D) Higher magnification inset of (A) showing Zpr-1 immunolocalization
in double cones. (D9) Overlay image showing that EGFP is restricted to rod photoreceptor soma and outer segments and does not co-label with
double cones. (E) Immunolocalization of GFP (green) to visualize the nrd:egfp transgene and HuC/D (red) to visualize all amacrine and ganglion cells.
(F) Higher magnification inset of (C) showing HuC/D expression in amacrine and ganglion cells only. (F9) Overlay image showing co-labeling of EGFP
with a subset of HuC/D-positive amacrine cells (arrows) and a HuC/D-negative bipolar cell extending its processes from the photoreceptors to the IPL
(arrowhead). (G) Immunolocalization of GFP (green) to visualize the nrd:egfp transgene and Glutamine Synthetase (G.S.; red) to visualize all Mu ¨ller glial
cells. (H) Higher magnification inset of (E) showing G.S.-positive Mu ¨ller glial cells. (H9) Overlay image showing that EGFP does not co-label with G.S.
-positive Mu ¨ller glial cells. ROS, rod outer segments; CC, cone cells; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL,
ganglion cell layer. Scale bar: 50 microns (A, C, E, G).
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all rod photoreceptors, and a subset of amacrine and bipolar cells
(Fig. 11B). 72 hours after light onset, nearly all rod and cone
photoreceptors are destroyed (Fig. 10D and E, asterisk). Endog-
enous neurod was observed in isolated INL progenitors as they
migrated to the ONL (Fig. 11F9). Weak EGFP expression was
observed in these cells using GFP immunohistochemistry alone
(Fig. 8E and F), but not when GFP immunohistochemistry was
combined with in situ hybridizations. At 7 days after light onset,
two distinct bands of endogenous and transgenic neurod were
observed in the ONL (Fig. 11G and H). EGFP was observed in a
band of the cell bodies of newly regenerated rods immediately
adjacent to the outer plexiform layer (i.e. toward the inner retina)
(Fig. 11I9). Endogenous neurod was strongly expressed in a band of
rod cell bodies immediately distal to the EGFP band (Fig. 11I9),
with only an occasional co-labeling among the cells residing in
these two bands (Fig. 11I9).
Figure 8. Retinal sections from adult Tg(nrd:egfp)/albino zebrafish over a time course of light treatment and immunolabeled with
EGFP (green) to visualize the nrd:egfp transgene and co-labeled with either PCNA (A, C, D, I, J, J9, K, L, L9, M, N) or Glutamine
Synthetase (B, G, H). (A) At 48 hours after light onset, almost all rod photoreceptors have been ablated and proliferating cells can be seen in the in
the INL. Nuclei are labeled in blue with TO-PRO-3 (TP3). (B) At this time point, Mu ¨ller glial cells express Glutamine Synthetase (G.S., red, arrow), and do
not co-label with EGFP (arrowhead). (C) At 72 hours after light onset, clusters of proliferating progenitors begin to migrate towards the ONL
(arrowheads). (D) At 96 hours post light onset, PCNA-positive progenitors (arrowheads) are present in both in INL and ONL, with occasional aberrant
migration to the GCL. (E–F) At 72 and 96 hours after light onset, respectively, clusters of progenitors weakly express EGFP (arrowheads). (F) At
96 hours after light onset, EGFP is observed in a newly-formed and disorganized ONL. (G–H) At 72 and 96 hours after light onset, respectively, weakly-
EGFP-positive clusters in the INL (arrowheads) down-regulated Glutamine Synthetase. Mu ¨ller glial cells that did not re-enter the cell cycle strongly
express G.S. (arrows), but are EGFP-negative. (I–J9) At 72 hours after light onset, weakly-EGFP-positive cells in both the INL and ONL co-label with
PCNA. The box in I represents the PCNA and EGFP labeling shown in J and J9, respectively. (K–L9) At 96 hours after light onset, weakly-EGFP-positive
cells in both the INL (arrowheads) and ONL continue to co-label with PCNA. The box in K represents the PCNA and EGFP labeling shown in L and L9,
respectively. (M) At 7 days after light onset, a subset of PCNA-positive progenitors in the ONL co-label with EGFP (N) At 11 days after light onset, only
a few PCNA-positive progenitors remain in the ONL. EGFP can be visualized in rod photoreceptors and newly-formed rod inner segments
Neurod Transgene Expression in Zebrafish
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Figure 9. High magnification images of retinal sections from adult Tg(nrd:egfp)/albino zebrafish over a time course of light
treatment. Sections were immunolabeled with EGFP (green) to visualize the nrd:egfp transgene and Zpr-3 (red) to visualize rod photoreceptors. (A)
Prior to light treatment (0 hr), EGFP co-labels with Zpr-3 and is observed in rod photoreceptor soma, rod inner segments (RIS) and rod outer
segments (ROS). (B) At 48 hours after light onset, the ROS and RIS are almost completely destroyed and only a few EGFP-positive cells remain in the
ONL. (C) At 72 hours after light onset, newly-formed rod progenitor cells are present in the ONL. These could be readily discerned from existing rod
photoreceptors due to their comparably weak expression of the transgene (inset shows new rod progenitor on the left). (D) At 96 hours after light
onset, a greater number of new regenerated cells are present in the ONL, although it still somewhat disorganized. (E) At 7 days after light onset,
newly differentiated rod photoreceptors appear more organized and greater in abundance. (F) At 11 days after light onset, EGFP is expressed in the
newly formed rod photoreceptors and co-labels with Zpr-3-positive and newly-formed RIS and R0S. Scale bar: 50 microns (A–F).
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To evaluate the utility of the nrd:egfp transgenic line, we
compared the expression of the transgene to that of endogenous
neurod during retinal development, in the adult retina and during
photoreceptor regeneration. Previously, RNA in situ hybridization
showed that during early retinogenesis neurod is first expressed in
the ventral nasal patch and then throughout the neuroepithelium.
Subsequently, neurod is transiently expressed in the nascent
photoreceptors in the outer nuclear layer and persistently
expressed in a subset of amacrine cells in the inner nuclear layer
. Similarly, we show that the nrd:egfp transgene is initially
expressed adjacent to the ventral nasal patch (Figs. 1F and 6B),
and then throughout the neuroepithelium and nascent photore-
ceptor layer (Fig. 6C). In contrast to the in situ data, however,
EGFP is also present in bipolar cells, in a small fraction of rod
photoreceptors at 2 wkpf, and in all rod photoreceptor cell bodies
There are potential explanations for the subtle temporal and
cellular disparities in the expression of neurod, as detected by in situ
hybridizations, and the expression of the nrd:egfp transgene. One
possibility is that the neurod transgene lacks a required silencer or is
influenced by neighboring enhancers near the site of integration.
However, it would have to lie far outside the coding region, as the
transgene contains 67 kb of sequence upstream and 89 kb of
sequence downstream of neurod open reading frame . Another
possibility is that mature bipolar cells and rod photoreceptors, not
observed following in situ hybridizations, produce very low levels of
endogenous neurod, and the stability of EGFP more readily allows
for the detection of these cells. In support of this interpretation,
prior to light treatment we observed weak expression of
endogenous neurod in all rod photoreceptor cells by in situ
hybridization, and strong expression of EGFP in the same cells
(Figs. 6I–I09, 11A and B).
We observed both overlapping and distinct expression profiles
for endogenous and transgenic neurod expression during retinal
regeneration. In both cases, neurod was not observed in dividing
Mu ¨ller glia or in the early stages of neuronal progenitor
amplification. Both endogenous and transgenic neurod were first
observed in INL progenitors in later stages of proliferation as these
progenitors were migrating to the ONL (Fig. 11F9 and 8E). At this
point endogenous neurod expression is very strong in these
progenitors, whereas EGFP is very weak (cf. Figs. 11F9and 8E).
By 3 days post light treatment, two distinct bands of expression
were observed. At this point, endogenous neurod is downregulated
in the first wave of newly regenerated rod photoreceptors that are
closest to the INL, whereas EGFP was strongly expressed in these
cells. In contrast, endogenous neurod is highly expressed in the next
wave of rod photoreceptors located distal to the first band of cells,
but EGFP is not yet present. These differences in endogenous and
transgene expression may be explained by dynamic changes in
endogenous neurod expression compared to the relatively long
(,24 hour) half-life of EGFP. In each case, endogenous neurod
expression proceeded EGFP expression and EGFP was visualized
after the downregulation of endogenous neurod.
Expression of neurod is often found in tissues with persistent
mitotic activity. Although the zebrafish retina continues to grow
throughout its life, we did not observe the neurod transgene in
known locations of persistent neurogenesis in the retina. For
example, consistent with previously published in situ hybridiza-
tions, neurod transgene expression was not observed during
retinogenesis in the progenitors located in the circumferential
marginal zone (CMZ), but did overlap with PCNA-positive cells as
they exit the CMZ and begin differentiating (Fig. 5A). Similarly,
during retinal regeneration, endogenous and transgenic neurod was
not observed in Mu ¨ller glial or their immediate progeny, but in
later stage progenitors prior to photoreceptor differentiation
(Figs. 8, 10, 11). This is consistent with anti-sense morpholino
Figure 10. Retinal sections from adult Tg(nrd:egfp)/albino zebrafish at 96 hours after light onset showing transgene expression
(green) and EdU labeling (red). (A). Schematic representation of EdU injections during the light time course with corresponding
immunolocalization shown in Panels B–D9. (B–B9) EGFP and EGFP/EdU co-labeling, respectively, showing weakly-EGFP-positive cells in the INL
(arrowheads) and ONL co-label with EdU. The boxes in B9 represent the panels shown in C–D9. (C) Higher magnification image of the box shown in
the top right of Panel B9. Note that the weakly-EGFP-positive progenitors co-label with EdU (arrowheads), but strongly-EGFP-positive rod nuclei
(arrow) are EdU-negative. (D–D9) Higher magnification image of the box shown in the left of Panel B9, showing EGFP and EdU immunolocalization,
respectively, in a cluster of INL progenitors. (E) Schematic representation of a single EdU injection prior starting the light treatment in order to label a
subset of the progenitors. (F–F9). High magnification confocal microscopy showing EGFP and EGFP/EdU co-labeling in the ONL at 96 hours after light
onset. An individual EdU-positive cell in the ONL (arrowhead) co-labels with weak EGFP expression. The strongly-EGFP-positive cell, in contrast, is
Neurod Transgene Expression in Zebrafish
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studies in early zebrafish development which show that in the
absence of NeuroD, rod and cone progenitors fail to exit the cell
cycle . In addition, the developing chick retina requires neurod
for photoreceptor differentiation [18,19]. Together, these data
suggest that the major function of NeuroD in the developing retina
is in regulating mechanisms that promote cell cycle exit. It has yet
to be determined whether NeuroD plays a similar role during
retinal regeneration in the adult.
One potential use would be to utilize the line to visualize the
reestablishment of the synapses connecting rod photoreceptor and
bipolar cells. During intense light damage, rod photoreceptors are
lost, but the underlying bipolar cells remain (Fig. 9B). Once
disconnected from the photoreceptor, the bipolar cell processes
hypertrophy and bud out, presumably in an attempt to re-establish
the lost connection (data not shown). Once the new photoreceptor
is regenerated, this connection is re-established. Since a subset of
bipolar cells and newly formed rod photoreceptors are both
EGFP-positive, this line could be used for in vivo imaging and
genetic manipulation of this dynamic and poorly understood
This line also has potential uses for studies on the endocrine
pancreas. NeuroD has been shown to be expressed in the
endocrine pancreas in a variety of vertebrates [5,41]. Loss of
NeuroD in mice results in abnormal pancreatic b-cell maturation
and function , severe hyperglycemia and neonatal death .
We show the neurod transgene is expressed the endocrine pancreas
and could be used as a visual marker for b-cell function,
particularly in the growing field using zebrafish as a vertebrate
model for diabetes [44,45,46].
In summary, given the diverse areas of neurod expression in the
developing and adult zebrafish, we anticipate that the Tg(nrd:egfp)/
alb line will be a useful tool in multiple disciplines, including future
studies on photoreceptor differentiation and retinal progenitor
Figure 11. RNA in situ hybridization on retinal sections from adult Tg(nrd:egfp)/albino zebrafish comparing endogenous neurod
expression (purple) to transgene expression (green) during light-induced retinal regeneration. (A) Before light treatment (0 hr),
endogenous neurod is expressed in a subset of amacrine and bipolar cells, weakly expressed in rod photoreceptors in the ONL (arrowhead), and
expressed in rod inner segments (RIS). (B) The nrd:egfp transgene is expressed in a subset of amacrine and bipolar cells, in rod photoreceptors in the
ONL (arrowhead), and weakly in RIS. (C) Overlay of panels (A) and (B). (C9) Higher magnification inset of (C) showing co-labeling of endogenous and
transgenic neurod expression in a subset of cells in the INL (arrowheads). (D) 72 hours after light onset (72 hr), all rod photoreceptors have been
ablated (indicated by the asterisk), and endogenous neurod is persistently expressed in a subset of amacrine and bipolar cells. (E) The nrd:egfp
transgene is persistently expressed in a subset of amacrine and bipolar cells. (F) Overlay of panels (D) and (E). (F9) Higher magnification inset of (F)
showing co-labeling of endogenous and transgenic neurod expression in a column of progenitor cells (indicated by the arrowhead), and a subset of
cells of the INL. (G) 7 days after light onset (7 d), endogenous neurod is strongly expressed in newly formed rods in the ONL (black arrowhead), and
persistently expressed in a subset of amacrine and bipolar cells. The inset shows expression of neurod in newly-formed rod inner segments (white
arrowhead). (H) The transgene is more weakly expressed in newly formed rods, and persistently expressed in a subset of amacrine and bipolar cells. (I)
Overlay of panels (G) and (H). (I9) Higher magnification inset of (I) showing co-labeling of endogenous and transgenic neurod expression in a subset of
INL cells (arrowhead), and in newly formed rod progenitors. Scale bar: 50 microns (A–C, D–F, G–I).
Neurod Transgene Expression in Zebrafish
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The authors would like to thank Alex Nechiporuk at Oregon Health
Sciences University for the Tg(nrd:egfp) line and Xixia Luo at Wayne State
University School of Medicine for zebrafish husbandry and technical
Conceived and designed the experiments: RT. Performed the experiments:
JLT MJO PFH RT. Analyzed the data: JLT PFH RT. Contributed
reagents/materials/analysis tools: PFH RT. Wrote the paper: JLT PFH
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