Toxicology and Applied Pharmacology 235 (2009) 68–76

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Toxicology and Applied Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p

Cadmium affects retinogenesis during zebrafish embryonic development

Elly Suk Hen Chow 1, Michelle Nga Yu Hui, Chi Wa Cheng 2, Shuk Han Cheng ⁎
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China

a r t i c l e i n f o a b s t r a c t

Article history: Ocular malformations are commonly observed in embryos of aquatic species after exposure to toxicants.
Received 19 May 2008 Using zebrafish embryos as the model organism, we showed that cadmium exposure from sphere stage
Revised 10 November 2008 (4 hpf) to end of segmentation stage (24 hpf) induced microphthalmia in cadmium-treated embryos.
Accepted 11 November 2008
Embryos with eye defects were then assessed for visual abilities. Cadmium-exposed embryos were
Available online 27 November 2008
behaviorally blind, showing hyperpigmentation and loss of camouflage response to light. We investigated the
Keywords:
cellular basis of the formation of the small eyes phenotype and the induction of blindness by studying retina
Cadmium development and retinotectal projections. Retinal progenitors were found in cadmium-treated embryos
Zebrafish albeit in smaller numbers. The number of retinal ganglion cells (RGC), the first class of retinal cells to
Embryos differentiate during retinogenesis, was reduced, while photoreceptor cells, the last batch of retinal neurons to
Retina differentiate, were absent. Cadmium also affected the propagation of neurons in neurogenic waves. The
Neurogenesis neurons remained in the ventronasal area and failed to spread across the retina. Drastically reduced RGC
axons and disrupted optic stalk showed that the optic nerves did not extend from the retina beyond the
chiasm into the tectum. Our data suggested that impairment in neuronal differentiation of the retina,
disruption in RGC axon formation and absence of cone photoreceptors were the causes of microphthalmia
and visual impairment in cadmium-treated embryos.
© 2008 Elsevier Inc. All rights reserved.

Introduction Bannigan, 2001) and is linked with eye deformities. Cadmium

Toxic metals such as cadmium, mercury and lead are widely
disseminated in the environment because of the extensive use of
these metals in industry and consumer products. The retina, an
important component of the central nervous system (CNS), is known
to be a target for many toxicants. Lead exposure results in a loss of rods
and bipolar cells (Fox et al., 1997, 1998). Lead and cadmium have
depressive effects on rod potential response (Sillman et al., 1982).
However, little is known about cadmium toxicity in neurogenesis.
Upon cadmium exposure, zebrafish embryos exhibit loss of neural
tissues, which suggested that cadmium, like other toxic metals, exerts
adverse effects on neural development (Cheng et al., 2000). Induction
of neurogenesis in zebrafish embryos is susceptible to cadmium
exposure; consequently, formation of several types of neurons and
sensory ganglia are disrupted. In particular, formation of forebrain
commissures, hindbrain interneurons and cranial sensory ganglia are
severely perturbed by cadmium exposure (Chow et al., 2008).
Cadmium exposure gives rise to various developmental defects in
other vertebrate species (Barr, 1973; Chisolm, 1974; Thompson and
⁎ Corresponding author. Fax: +86 852 27887406.
E-mail address: bhcheng@cityu.edu.hk (S.H. Cheng).
1
Current address: Division of Biology, California Institute of Technology, 1200
California Boulevard, Pasadena, CA91125, USA.
2
Current address: Department of Biochemistry, The University of Hong Kong, 3/F
Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Hong Kong.
exposure leads to ocular malformations such as microphthalmia
during embryonic development in fresh water fish Ambassis commer-
soni Cuvier and induces ocular anomalies in Xenopus embryos and
damages the corneal endothelium of bullfrog (Pragatheeswaran et al.,
1989; Sunderman et al., 1991; Weidner and Sillman, 1997). Cadmium
exposure also causes apoptotic changes in photoreceptors and
ganglionic cells (Roozbehi et al., 2007). Previously, we report that
cadmium-exposed zebrafish embryos developed small eyes (Cheng et
al., 2000). Together, these cited studies concentrate on the occurrence
of ocular malformations that resulted from cadmium exposure.
However, there is no systematic study on visual behavior or
retinogenesis processes being affected by cadmium. This is partly
due to the fact that eye development is a highly orchestrated process
and presents multiple targets for cadmium toxicity.
Eye formation is conserved among vertebrate embryos (Land and
Fernald, 1992). In zebrafish, early eye morphogenesis is similar to
other vertebrates even though the optic primordium evaginate from
the forebrain as solid masses of cells (Schmitt and Dowling, 1994). The
optic primordium appears around 12 h post-fertilization (hpf), with
the first ganglion cells appearing in the ventronasal retina at about
28 hpf (Hu and Easter, 1999), while the first ganglion axons arrive at
the optic chiasm at about 32 hpf (Burrill and Easter, 1995). The cone
outer segments first appear at 60 hpf in a restricted area of ventral
retina, and then gradually elongate (Branchek and Bremiller, 1984).
Pax6, six3 and rx1 are eye-determination genes involved in early
processes of induction and regional specification (Gehring, 2005).

0041-008X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2008.11.013
 E.S. Hen Chow et al. / Toxicology and Applied Pharmacology 235 (2009) 68–76
Pax6 is the “master gene” of eye development and plays a critical role
in the early stages of eye formation and later in maintaining the multi-
potent state of retinal progenitor cells (Marquardt et al., 2001;
reviewed by Chow and Lang, 2001). Six3 is determinative for the
retina and is involved in specifying retinal identity (Hitchcock and
Raymond, 2004). Rx homeobox genes are important for regulating the
initial specification of retinal cells and subsequent proliferation
(Mathers et al., 1997; Zhang et al., 2000).
During gastrulation, the presumptive lens area is exposed to lens-
inducing signals. By 24 hpf, the lens vesicle develops as a solid cell
mass, acquires a spherical shape and buds off from the surface
ectoderm. The lens has an inductive role in addition to its optic role in
vision development. The retinotectal projections are essentially the
optic stalk, which connects the eye to the forebrain (Schmitt and
Dowling, 1994; reviewed by Easter and Malicki, 2002). They comprise
of ganglion cell axons, which project from the retina, navigate through
the midline at the chiasma and continue up the contralateral
diencephalon. Full vision depends on both the lens and the retina;
therefore, the completion of ocular cell differentiation events
coincides with development of vision-dependent behavior by 72 hpf
(Easter and Nicola, 1996).
Initiation of retinal neurogenesis is activated by expression of a
transcriptional factor, ath5, a vertebrate homolog to the Drosophila
proneural gene atonal (ato) (Jarman et al., 1993). Zebrafish retinal
neurogenesis takes on a characteristic neurogenic wave, whereby all
events begin in a tiny cluster of cells located in the ventronasal area
and spread to cover the whole retina (Hu and Easter, 1999; Masai et al.,
2000; Neumann and Nuesslein-Volhard, 2000). A key player mediat-
ing the progression of retinal cell differentiation is sonic hedgehog
(ssh), a member of the Hedgehog (Hh) family of secreted molecules
that are essential for many other developmental processes (reviewed
in Ingham and McMahon, 2001; Dakubo and Wallace, 2004). Hedge-
hog signaling controls the neurogenic wave propagation through the
retina but is not required for formation of the initial patch of post-
mitotic neurons (reviewed by Neumann, 2001). Similar to the wave in
Drosophila, shh is expressed in the RGC and drives a wave of
neurogenesis across the zebrafish retina (Neumann and Nuesslein-
Volhard, 2000). In zebrafish, shh is expressed in the ganglion cell layer
(GCL) and retinal pigment epithelium (RPE) and directs proliferation
and differentiation of several late-arising cell types, such as the
photoreceptors and the glia (Jensen and Wallace, 1997; Levine et al.,
1997; Stenkamp et al., 2000).
The assessment of pigmentation is one of the assays for blindness
in zebrafish (reviewed by Goldsmith and Harris, 2003). Zebrafish
produces several classes of pigment cells including black melano-
phores, yellow or orange xanthophores, red erythrophores and
iridescent iridophores (reviewed by Parichy, 2006). Teleosts, including
zebrafish larvae, adjust the distribution of skin melanin pigment to
ambient light levels. The retina projects sensory input (light intensity)
to the hypothalamus, which in turn induces the pituitary to secrete
two antagonistic hormones on melanophores, one for dispersal and
one for aggregation of melanin granules (reviewed by Balm and
Gröneveld, 1998). When placed on a light background, melanosomes
are aggregated and appear as star-shaped black spots on the skin.
When placed on a dark background, melanosomes become widely
distributed and expanded to form big clumps of black cells (Neuhauss
et al., 1999).
In this study, the possible causes of physiological visual defect in
zebrafish embryos upon cadmium exposure were examined. We first
investigated whether cadmium induces visual impairment by testing
the background adaptation ability. Specification and differentiation of
the presumptive eye, initiation of retinal neurogenesis and the
propagation of neurogenesis wave were examined in cadmium-
exposed embryos exhibiting the small eye phenotype. Our data
further confirmed that zebrafish embryonic eyes are susceptible to
cadmium exposure. For the first time in our knowledge, we
69
demonstrated that failure in neurogenesis wave propagation from
the initial ventral patch, which may lead to subsequent failure in
retinal ganglion cells and cone photoreceptors differentiation, might
be the major causes of cadmium-induced visual impairment in
zebrafish embryos.
Methods
Chemical and embryo culture medium. Cadmium chloride (CdCl2.
2H2O) was purchased from Sigma (St. Louis, MO). 1 mM stock solution
in culture medium (19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO4.
7H2O, 0.2 mM Ca(NO3)2, 1.67 mM Hepes [pH 7.2]) was prepared and
treatment doses were made from this stock solution in triple distilled
water.
Zebrafish maintenance and embryo treatment. Adult zebrafish were
maintained as previously described (Cheng et al., 2000). The median
lethal concentration (LC50), median combined adverse effect
concentration (EC50) and relevant cadmium exposure doses were
previously determined (Cheng et al., 2000). The LC50 and EC50 were
168 μM and 138 μM, respectively. In this study, embryos were
continuously exposed to 100 μM cadmium chloride in embryo
medium from sphere stage (4 hpf) to the end of segmentation stage
(24 hpf). Fifteen replicates (n = 15) containing 20 embryos in a 60-mm
diameter Petri dish were cultured in both control and cadmium
treatment group. The untreated control embryos were maintained in
culture medium only. For subsequent in situ hybridization and
immunohistochemistry experiments, embryos from 5 Petri dishes
containing 20 embryos in each dish (a total of 100 embryos) were
pooled into one eppendorf tube after treatment. At 24 hpf, embryos
were dechorionated for examination for eye abnormalities as
described previously (Cheng et al., 2000). The area of the eyes was
digitally measured for each embryo. Embryos with less than 70% of the
mean eye area compared with controls were classified as having small
eyes. The mean area of the eye was determined by measuring the area
of the eyes of control embryos and calculated by dividing the sum of
areas by the total number of embryos measured.
Behavioral study. Visual background adaptation was conducted as
previously reported (Neuhauss et al., 1999). Embryos were incubated
in culture medium containing cadmium until 24 hpf in dark, washed
three times with culture medium and transferred to new culture
medium for incubation until 7 days post-fertilization (dpf). Three
replicates (n = 3) containing 20 embryos in a Petri dish were cultured
in both control and cadmium treatment group and were incubated at
28.5 °C in light from 24 hpf and onward. As a positive control,
untreated embryos were kept in dark from 24 hpf until 7 dpf. Embryos
were mounted in dorsal view in 1% agarose. Pigmentation was
observed under Nikon DIC-camera and cadmium-treated zebrafish
larvae were scored for hyperpigmentation. Embryos exhibiting
merged melanophore granules were classified as hyperpigmentation.
TUNEL assay. Three replicates (n = 3) containing 20 embryos in a Petri
dish were cultured in both control and cadmium treatment group.
Control and cadmium-exposed embryos were examined for apoptosis
by TUNEL assay at 18 hpf, 20 hpf and 24 hpf using the In Situ Apoptosis
Detection Kit (Takara, Shiga, Japan). Embryos were fixed overnight in
phosphate-buffered saline (PBS) with 4% paraformaldehyde (PFA) at
4 °C. Embryos were dehydrated in methanol and stored at −20 °C. To
block endogenous peroxidase activity, embryos were treated with 3%
hydrogen peroxide (H2O2) at room temperature for 30 min. Embryos
were rehydrated and digested with 10 μg/ml proteinase K in PBS with
0.1% Tween 20 before incubation with Permeabilization Buffer on ice for
5 min. After several washes with PBT, embryos were incubated in
labeling reaction mixture at 37 °C for 2 h. Images were taken by confocal
microscopy (Leica TCS SPE).
70 E.S. Hen Chow et al. / Toxicology and Applied Pharmacology 235 (2009) 68–76

Whole-mount in situ hybridization. Whole-mount in situ hybridi-
zation was performed as described by Westerfield (1994), with
modifications (Chow and Cheng, 2003). Digoxigenin-labeled antisense
riboprobes were synthesized for Pax6 (Püschel et al., 1992), rx1
(Chuang and Raymond, 2001), six3 (Seo et al., 1998) and ath5 (Masai
et al., 2000) by linearizing the plasmid and transcribing with T7
polymerase and Digoxigenin-11-UTP (Roche, Basel, Switzerland).
Plasmids containing cDNA probes for Pax6, rx1, six3 were generously
provided by Dr. Uwe Strähle (IGBMC, Strasbourg, France). Plasmid
containing cDNA probe for ath5 was given as a gift from Dr. Masai
(Brain Science Institute, RIKEN, Saitama, Japan). In situ hybridization
was performed with 3 replicates (n = 3) containing 100 embryos in a
1.5 ml eppendorf tube for both control and cadmium treatment group.
At 24 hpf, embryos were dechorionated and fixed overnight with 4%
PFA/PBS at 4 °C. Embryos were dehydrated in methanol and stored at
− 20 °C. Embryos were rehydrated and digested with 10 μg/ml
proteinase K in PBS with 0.1% Tween 20 before incubation with
antisense probes (1: 200) overnight at 55 °C. Following hybridization,
probes were removed with high-stringency washes, 2 × SSC and
0.2 × SSC twice each for 30 min, at 62 °C. Embryos were subsequently
incubated with pre-absorbed sheep anti-digoxigenin-alkaline
phosphatase Fab fragments (Roche, Basel, Switzerland) for 2 h at
room temperature. The blue-purple color reaction was performed by
using 5-bromo-4-chloroindolyl phosphate (BCIP) as substrate and
nitro blue tetrazolium (NBT) as coupler (Roche, Basel, Switzerland).
Chicago, IL, USA). All data were analyzed by Mann–Whitney test to
determine significant difference between control and cadmium-
exposed embryos. The level accepted for statistical significance in all
cases was p b 0.05.
Results
Cadmium exposure during early embryonic stage caused eye
malformations
Zebrafish embryos exhibited eye malformations in response to
cadmium exposure during early embryonic development. Scoring for
eye malformations was conducted at 24 hpf. Control embryos
presented spherical eyes, consisting of the neural retina and the
spherical lens enclosed, distinguished by a clear boundary (Figs. 1A–C).
The outer layer of the optic cup gives rise to the retinal pigment
epithelium (RPE) surrounding the neural retina and pigmentation was
observed in control embryos (Fig. 1C). However, cadmium-exposed
embryos exhibited less than 70% of the mean area of the eye, which we
classified as the “small eyes” phenotype. In addition, loss of
pigmentation in the RPE (Figs. 1D–F) was observed in cadmium-
exposed embryos. Cadmium exposure during early embryonic stage
and before onset of retinal neurogenesis caused significant increase in
incidence of small eyes compared to the control embryos (Fig. 1G).

Immunohistochemistry. Embryos were collected at the end of

treatment (at 24 hpf), washed three times and transferred to fresh
culture medium without cadmium for further incubation. At 30 hpf or
48 hpf, embryos were dechorionated and fixed with 4% PFA in PBS at
4 °C overnight. Immunostaining with antibodies was performed as
described previously (Chow and Cheng, 2003), with 3 replicates (n = 3)
containing 100 embryos in a 1.5 ml eppendorf tube in both control and
cadmium treatment group. Embryos were frozen at −20 °C in acetone
to increase tissue permeability. To block endogenous peroxidase
activity, embryos were treated with 3% H2O2 for 30 min at room
temperature. Embryos were then treated with 2% goat serum in PBS
(0.8% NaCl, 0.02% KCl, 0.02 M PO4 [pH7.3])/1% BSA/1% dimethyl
sulfoxide (DMSO) for 30 min and incubated in primary antibodies
overnight at 4 °C. After thorough washing, embryos were incubated
for 30 min at room temperature in biotinylated goat anti-mouse
secondary antibody that was pre-blocked with zebrafish embryo
powder (DAKO, Carpinteria, CA, USA). Horseradish peroxidase–
Streptavidin was added before incubation with chromogenic substrate
(diaminobenzidine and hydrogen peroxide). Primary antibodies used
were: 39.4D5 for Isl-1 (1:200) from Developmental Studies
Hybridoma Bank, University of Iowa, IA, USA), zn-5 (1:100), zl-1
(1:20) and zpr-1 (1:20) from Oregon monoclonal bank. For zpr-1,
immunohistochemical signal was visualized with an FITC-conjugated
secondary antibody (Sigma, St. Louis, MO, USA) and optical images
were photographed with a confocal microscope (Zeiss, Germany).

Anterograde labeling of optic nerves. For both control and cadmium

treatment group, 3 replicates (n = 3) containing 20 embryos in a Petri
dish were dechorionated at 48 hpf and embedded in 1% agar in PBS on
a glass slide. The glass slide was mounted on a microscope stage and
by pressure injection, fluorescent lipophilic dyes DiI and DiO
(Molecular Probes, Eugene, OR) were injected into each of the eyes,
between the lens and the retina at the ventronasal position. The
injected embryos were incubated at room temperature in dark for 1 h
to allow the dyes to anterogradely label the optic nerve running out
from the eyes.
Statistical analysis. All data for morphology and gene expression
analysis were presented as percentages and the mean± SEM were
reported. Statistical analyses were performed using SPSS (SPSS Inc.,
Fig. 1. Small eyes phenotype developed in zebrafish embryos at 24 hpf after cadmium
exposure. Black arrows indicate the developing spherical eyes with clear boundary
between the lens and the neural retina and black arrowhead indicates pigmentation in
the eye showed by the retinal pigmented epithelium (RPE) in control embryos (A–C).
Red arrows indicate smaller eyes with unclear boundary between the lens and the
neural retina and red arrowheads indicate loss of pigmentation in the eye in cadmium-
exposed embryos (D–F). Panel G illustrates the frequency (%) of small eyes phenotype
developed after cadmium exposure (⁎⁎ denotes p b 0.01) in which the data are mean ±
standard error of the mean (vertical bars). Abbreviations: nr = neural retina; le = lens.
Scale bar = 500 μm in A, B, D and E and 200 μm in C and F.
 E.S. Hen Chow et al. / Toxicology and Applied Pharmacology 235 (2009) 68–76 71

Fig. 2. Phenotypes of ectopic melanophore granules in 7 days post-fertilization (dpf) control and cadmium (Cd)-treated zebrafish embryos. Cd-treated embryos developed
hyperpigmentation as a result of visual impairment. Black arrows indicate melanophore granules in control embryo incubated in light (A, B). White arrows indicate merged
melanophore granules and ectopic pigmentation in Cd-treated embryos kept in light (D, E) and in dark (F). White arrowhead indicates ectopic pigmentation in control embryo kept in
dark (C). Dorsal views are shown with anterior to the left. Scale bar = 200 μm.

Upon cadmium exposure, 11.2% ± 2.23% (p b 0.01) embryos developed
small eyes, while only 2% ± 2.73% embryos displayed eye malformation
in the control group.
Cadmium causes blindness in zebrafish embryos
The assessment of pigmentation is one of the assays for blindness
in zebrafish (reviewed by Goldsmith and Harris, 2003). Fish adapt
their skin melanin pigment levels in order to blend in with the
background as a camouflage response. This response is dependent to
visual function as blind fish are hyperpigmented in a well-lit
environment (reviewed by Goldsmith and Harris, 2003). Since
zebrafish embryos suffered from small eyes in response to cadmium
exposure, we asked whether cadmium also affected the physiological
function of embryonic eyes. Coloration of 7 days post-fertilization
(dpf) control and cadmium-exposed larvae were examined. In a dark
environment, both control and cadmium-exposed larvae showed dark
coloration and ectopic pigmentation (Figs. 2C, F). Hyperpigmentation
displayed in control larvae because of visual adaptation to the
background. Contrastingly, all control larvae showed normal pigmen-
tation pattern when they were grown in a bright environment (Figs.
2A, B). Hyperpigmentation throughout the body was observed in

Fig. 3. Confocal projected images of apoptotic cells in lens and retinal cells in control embryos and cadmium-treated embryos at 18 hpf, 20 hpf and 24 hpf. Lateral views are shown
with anterior to the left. Scale bar in A = 25 μm.
72 E.S. Hen Chow et al. / Toxicology and Applied Pharmacology 235 (2009) 68–76

detected in the prospective retina and lens (Figs. 4A–D) and rx1
transcripts were expressed in the neural retina (Figs. 4E, F) at 24 hpf. In
cadmium-exposed embryos, expression of these genes was unaltered
in the retina and lens albeit formation of the small eyes phenotype
(Figs. 4G–L). Hence, the expression of these transcription factors in
cadmium-exposed embryos accounted for the successful induction in
eye formation even though the eyes were smaller in area.

Cadmium affects initiation of retinal neurogenesis and axon formation

We investigated the initiation of retinal neurogenesis by ath5

expression by whole-mount in situ hybridization. Ath5 expression was
first detected in the ventronasal retina adjacent to the choroids fissure
at 25 hpf in both control and cadmium-treated embryos (Figs. 5A, B).
Ath5 was detected in cells throughout the retina at 30 hpf in control
embryos (Fig. 5C). Retinal neurogenesis inhibition was detected in
9.33% ± 2.49% (p b 0.01) of cadmium-exposed embryos, which showed
reduction in ath5 expression in the retina (Fig. 5F). Cadmium exposure
caused defects in the initiation of retinal cells differentiation. To
confirm the absence of retinal ganglion cells (RGCs), whole-mount

Fig. 4. Normal eye formation in developing zebrafish embryos revealed by localization

of Pax6, six3 and rx1 transcripts in control and cadmium (Cd)-treated embryos at
24 hpf. Cadmium exposure had no effect on eye formation in developing zebrafish
embryos. Expression of marker genes dictated normal eye formation: Pax6 and six3 for
commitment of retina and lens cell fates; rx1 for initial specification and proliferation of
retinal cells. Lateral views (A, C, E, G, I, K) and dorsal views (B, D, F, H, J, L) with anterior to
the left. Scale bar = 500 μm.

28.33% ± 6.01% (p b 0.01) cadmium-exposed zebrafish larvae incubated

in light (Figs. 2D, E). Zebrafish embryos became behaviorally blind in
response to cadmium exposure. The larvae developed hyperpigmen-
tation for camouflage as a result of visual impairment and this
suggested that cadmium may cause visual impairment. It should be
noted that hyperpigmentation was observed in the larvae with grossly
normal morphology, suggesting that visual impairment was not
simply caused by eye degeneration.

Cadmium induced ectopic apoptosis in zebrafish embryonic eyes

To investigate if apoptosis was induced in zebrafish embryonic eye

upon cadmium exposure, TUNEL assay was performed to detect
apoptotic cells in these embryos at 18 hpf, 20 hpf and 24 hpf. In
cadmium-exposed embryos, ectopic apoptotic cells were abundantly
detected in the retina and the lens (Figs. 3C, D, G, H, K, L), whereas very
few ectopic apoptotic cells were observed in the control embryos
(Figs. 3A, B, E, F, I, J). These results suggested that cadmium induced
ectopic apoptosis in zebrafish embryonic eyes, which could potentially
be one of the causes of vision loss in the larval stage.
Induction of eye development in cadmium-exposed zebrafish embryos
To gain insight into the molecular basis for the cadmium-induced
small eyes phenotype, expression patterns of transcriptional factors
involved in early stages of eye formation, namely Pax6, six3 and rx1,
were investigated. In control embryos, Pax6 and six3 transcripts were
Fig. 5. Retinal neurogenesis in embryonic zebrafish retina. Ath5 expression initiated in
the ventronasal retina adjacent to the choroids fissure in control (A) and cadmium-
treated embryos (D). Ath5 spreads throughout the retina in a wave-like manner (B, C) in
control embryos. Cadmium (Cd) — treated embryos have reduced neuronal differentia-
tion (E, F) at 28 hpf and 30 hpf by ath5 transcript detection. Black arrowheads indicate
retinal ganglion neurons at 30 hpf (G) and 48 hpf (H) in control embryos. White
arrowheads indicate reduction of retinal ganglion neurons in Cd — exposed siblings
(J, K). Black arrows indicate projection of retinal ganglion axons forming the optic
chiasm towards the dorsal midbrain in control embryo at 48 hpf (H). White arrows
indicate reduction of retinal ganglion axon in Cd-exposed siblings (K). Optic nerve in
one of the eyes failed to extend through the optic chiasm at the midline into the tectum
in Cd-exposed embryos (L) Ventral views are shown. Scale bar = 500 μm in A–H, J, K
and = 200 μm in I, L. Asterisks (⁎) indicate the position of the choroid fissure.
 E.S. Hen Chow et al. / Toxicology and Applied Pharmacology 235 (2009) 68–76
immunohistochemistry was performed using Isl-1 and zn-5 anti-
bodies. Nuclei of RGCs were detected in the ganglion cell layer at
30 hpf in control embryos (Fig. 5G). Strikingly, cadmium exposure
caused reduction of RGCs in 12.67% ± 3.09% (p b 0.01) cadmium-
exposed embryos as demonstrated by Isl-1 staining (Fig. 5J). Control
embryos developed retinal ganglion neurons and extended their
axons, forming the optic chiasm at the midline running through the
dorsal tectum at 48 hpf (Fig. 5H). In 13.33% ± 3.49% (p b 0.01) cadmium-
exposed embryos, cadmium exposure reduced RGCs in the ganglion
cell layer, thus their axons projecting to the brain as shown by zn-5
staining (Fig. 5K). Cadmium exposure at lower concentrations also
showed similar phenotype (Supplementary Figure and Table). We also
examined axonal growth of the optic nerve by anterograde labeling. In
control embryos, the optic nerve carrying the visual signals exits from
the eye and course through the optic chiasm at the midline, then run
to the dorsal midbrain where signals are received (Fig. 5I). In
cadmium-exposed embryos, failure in extension of the optic nerve
from the eye was observed in embryos with severe defect (Fig. 5L).
Taken together, it appears that exposure to cadmium caused defects in
retinal ganglion cell fate specification in zebrafish embryonic eyes.
Cadmium disrupts retinogenesis wave in zebrafish embryos and cone
cell formation
The absence of ath5 RNA expression suggested that the first wave
of retinal neurogenesis might also be affected in cadmium-exposed
Fig. 6. Confocal projected images of GFP-Shh expression in retinal ganglion cells (RGCs)
in the ganglion cell layer (GCL) in control embryos and cadmium-treated embryos at
31 hpf, 52 hpf and 72 hpf. White dotted circles indicate RGCs in the GCL (B, C) and cones
cells in the ONL (H, I) in control embryos. White arrows indicate GFP-Shh expressing
RGCs at the ventronasal retina which failed to spread out through the GCL in Cd-treated
embryos (E, F). Red arrows indicate loss of cone cells in the ONL (K, L). Lateral views are
shown with anterior to the left. Scale bar = 500 μm.
73
Fig. 7. Whole-mount immunohistochemistry of zpr-1 in cone cells of the outer nuclear
layer (ONL) at 72 hpf in control and cadmium (Cd)-treated embryos. Red arrows indicate
loss of cone cells in the ONL (E, F). Lateral views are shown with anterior to the left. Scale
bar = 500 μm.
embryos. To investigate the effect of cadmium on the wave of RGCs,
we exposed zebrafish embryos from a strain harboring green
fluorescent protein (GFP) transgene under the control of Shh promoter
from 4 hpf until 24 hpf. In zebrafish, ShhGFP and shh RNA expression
were activated from 28 hpf to 30 hpf in a patch of cells in the
ventronasal retina. At 31 hpf, ShhGFP was detected as a spot at the
ventronasal retina (Figs. 6A, D). Contrastingly, ShhGFP expression was
not detected in cadmium-treated embryos (Figs. 6G, J). At 52 hpf,
ShhGFP and shh RNA expression already spread from ventronasal
retina and filled the central retina (Figs. 6B, E). In cadmium-exposed
embryos, initiation of ShhGFP expression in RGCs was observed but
further spreading was retarded (Figs. 6H, K). At 72 hpf, ShhGFP
expression was detected in the RGCs and amacrine cells in control
embryos (Figs. 6C, F), while its expression was absent in cadmium-
treated embryos (Figs. 6I, L). We then asked whether cadmium
specifically affected the first retinal neurogenesis or whether it
affected subsequent retinogenesis wave propagation. The cone
photoreceptors formed as a result of the third retinogenesis wave
spreading and were reported to be sensitive to cadmium in bullfrog
(Sillman et al., 1982). Zpr-1 antibody was used to label cone
photoreceptors. At 72 hpf, the spread of zpr-1 immunoreactivity
appeared normal and filled the ONL in control embryos (Figs. 7A–C).
Contrastingly, no immunoreactivity of zpr-1 was detected in 32.49% ±
2.39% (p b 0.01) cadmium-exposed embryos (Figs. 7D–F). Cadmium
exposure at lower concentrations also revealed the same phenotype
(Supplementary Figure and Table). In correlation with the interrupted
retinal differentiation, it appeared that cadmium disrupted RGC
formation and hence spreading of retinogenesis wave through the
GCL. Failure in propagation of the wave of neurogenesis from the
initial ventral patch might account for the subsequent failure in
differentiation of retinal ganglion cells and cone photoreceptors.
Discussion
Cell commitment and differentiation in early eye development of
cadmium-exposed embryos
Eye development is complex which involves specification and
differentiation of different cell types. Pax6 and six3 are involved in
commitment of retina and lens cell fates and rx1 is specifically
expressed in the presumptive retina (reviewed by Mathers and
Jamrich, 2000). The present study demonstrates that cadmium-
74 E.S. Hen Chow et al. / Toxicology and Applied Pharmacology 235 (2009) 68–76
exposed embryos possessed normal expression of Pax6, six3 and rx1,
indicating that eye formation was successfully induced.
Initiation of retinal neurogenesis and determination of retinal
ganglion cells from multi-potent retinal progenitor cells are affected.
The adult retina consists of seven classes of retinal cells generated by
differentiation of multi-potent progenitor cells during retinogenesis.
In all vertebrates studied thus far, the RGCs are the first retina cells to
become post-mitotic and to differentiate (Livesey and Cepko, 2001).
They are the sole projection neurons of the retina and carry the
neural signals generated by light to the brain. In cadmium-exposed
embryos, expression of a RGCs determining gene, ath5, is markedly
reduced. Similar observation is found when cadmium-exposed
embryos were labeled with a retinal ganglion neuron specific marker,
Isl-1 antibody. This current data indicates that commitment of retinal
progenitor cells into RGC fate is disturbed by cadmium exposure.
Absence of cone photoreceptors is observed by immunostaining with
zpr-1. Cadmium-induced small eyes phenotype in zebrafish embryos
may be due to failure to initiate the process of retinogenesis early in
retinal development rather than to the commitment of their
progenitor cells.
Visual impairment and hyperpigmentation in cadmium-exposed
zebrafish embryos
Teleosts and amphibians adjust the distribution of skin melanin
pigment in order to blend in with changing ambient light levels. Blind
zebrafish larvae are unable to perform this visually controlled
background adaptation behavior and hence develop hyperpigmenta-
tion. Blindness of zebrafish mutant lakritz (lak) is resulted from
mutation of ath5 and is identified for pigmentation defects (Kelsh et
al., 1996). The fiddler crab, (Uca pugilator) adjusts to its environment
by dispersing black pigment granules and darkening its carapace
when placed on a black background or by concentrating the pigment
and lightening its carapace on a white background. Cadmium also
causes depletion of the black pigment-dispersing hormone in the eye
stalks and resulted in decreased ability to disperse black pigment in a
black background (Reddy and Fingerman, 1995). Behavioral analysis
shows that the pigmentation phenotype was a subsequent effect of a
visual system defect (Neuhauss et al., 1999). Physiological color change
is largely under hormonal control in lower vertebrates. Melanin-
concentrating hormone (MCH) is a neurohypophysial hormone which
induces aggregation of melanosomes (reviewed by Kawauchi and
Baker, 2004). It has also been proposed that melanosome transloca-
tion in teleosts requires microfilaments (Logan et al., 2006). In this
study, hyperpigmentation is observed in zebrafish embryos exposed
to cadmium albeit they were incubated in light. To further investigate
the cause of such defect, examination of retinal neuron axons that
course through to the midbrain where visual signals are received was
conducted. Cadmium-exposed embryos display reduction of retinal
ganglion cells and thinner axons. In agreement with the loss of retinal
neurogenesis, some embryos display severe phenotype and do not
develop retinal ganglion neurons and their extending axons to the
brain are completely interrupted. Loss of the optic nerve that extends
from the eye to the optic tectum is also observed. Although the
embryos are incubated in light, they are unable to detect light and
perceive that they are in dark ambient. Hyperpigmentation in
cadmium-exposed embryos may arise from a combination of retinal
defects such as the absence of cone photoreceptors and the loss of
retinal ganglion neurons as a consequence of retinal neurogenesis
interruption. However, we cannot exclude the possibility that
cadmium has a direct effect on hormonal control of pigmentation.
Wave of retinogenesis in cadmium-exposed zebrafish embryos
During early embryogenesis, many signaling pathways are
involved and are critical in regulation of normal patterning,
differentiation and proliferation. One of which is the hedgehog
signaling pathway. Mutations of sonic hedgehog (shh) gene and its
downstream components in the signaling pathway result in abnormal
somitogenesis and neurogenesis (Varga et al., 2001). The Hh signaling
pathway is of central importance in directing retinal differentiation in
zebrafish. At earlier stages, Hh signaling is required for the spread, but
not the induction of the first Hh-expressing neurons, by driving a
wave of shh expression and neurogenesis across the GCL (Neumann
and Nuesslein-Volhard, 2000). All differentiated cell types derive from
the neural retina depends on Hh signaling (reviewed by Stadler et al.,
2004). Cadmium-treated embryos resemble similar defects in spread-
ing of the first wave of retinogenesis as revealed by restricted
expressing of ShhGFP cells in the embryonic retina. Although cyclopia
is closely related to loss of Shh signaling in the midline, cadmium-
treated embryos did not have this developmental defect. From a
previous study, the production of cyclopia after ethanol exposure in
zebrafish is not due to suppression of Shh signaling (Blader and
Strähle, 1998). In addition, high dose of teratogen is required to
produce cyclopia (Loucks et al., 2007).
In addition to maintaining the spread of RGC genesis, Sonic
hedgehog (Shh) expressing in the GCL and RPE is suggested to play a
role in directing proliferation and differentiation of several late arising
cell types, such as the photoreceptors (reviewed by Neumann, 2001).
In mammalian cell cultures, Shh stimulates photoreceptor differentia-
tion (Levine et al., 1997). In zebrafish, hedgehog genes may be an
important player in generating propagation of photoreceptor differ-
entiation wave across the developing embryonic eye (reviewed by
Neumann, 2001). Mutations in Hh signaling pathway genes show
retardation of photoreceptor differentiation (Stenkamp et al., 2000).
Here, we report that there is a loss in cone photoreceptors in
cadmium-exposed embryos, suggesting a possible secondary conse-
quence to the loss of the first retinogenesis wave spreading. Taken
together, it seems that cadmium disrupts RGC formation by
interrupting the initiation of retinal neurogenesis, which in turn
affects the propagation wave of retinogenesis through the entire
retina, leading to loss of cone photoreceptors. These results also
suggest that the lack of retinogenesis wave spreading in cadmium-
exposed embryos may possibly be one of the causes to the failure of
continuous retinal neurogenesis.
Cell death
Apoptosis is a highly-regulated cell death process. Ectopic apoptotic
cells can be found in malformed tissues and mutants. Genetic
mutations or toxicant exposures lead to developmental abnormalities,
resulting in disruption of apoptosis (Zakeri and Ahuja, 1997; Mirkes,
2002). Previous studies showed that zebrafish mutant embryos with
neurodegeneration formed an abnormal central nervous system and
have ectopic apoptosis in their neural tissues (Furutani-Seiki et al.,
1996). Cadmium induces apoptosis or necrosis in rat cortical neuron
cultures in a dose-dependent manner (López et al., 2003). A high
number of apoptotic cells throughout the body is reported in the
anterior and posterior ends of zebrafish embryos having axial defect
after cadmium treatment (Chan and Cheng, 2003). In this study,
cadmium-exposed embryos develop small eyes and cell death is
observed in the eyes of cadmium-exposed embryos during early eye
development prior to differentiation of the committed lens primordial
cells and initiation of retina neuronal production. Here, we can only
postulate that cell death may be one of the causes of retinogenesis
reduction and hence formation of small eyes. How cadmium induces
ectopic apoptosis in the eye; however, remain to be investigated.
Mechanism of cadmium toxicity
The underlying mechanism of cadmium-induced eye defect is
poorly understood. Because shh expression was impaired in
 E.S. Hen Chow et al. / Toxicology and Applied Pharmacology 235 (2009) 68–76
cadmium-treated embryos, defective eye development may result
from the lack of inductive signals provided by shh during neurogen-
esis. Therefore, cadmium may act on the genetic pathway regulating
neurogenesis in the events either before or during shh expression. In
our previous work, we demonstrate reduction of midline shh
expression in cadmium-treated embryos (Yu et al., 2006).
The cell adhesion molecules such as cadherins and NCAM have
long been suspected as potential targets in metal toxicity (Prozialeck
et al., 2002). Cadherins are calcium-binding metalloproteins with
specific binding sites for calcium in the extracellular domain. It is this
critical interaction with calcium that provides one target for potential
perturbation through calcium mimicry by metals (reviewed in
Prozialeck et al., 2002). During early and late stages of neural
development, disruption of cadherin expression of function led to
abnormal development (Bronner-Fraser et al., 1992). Recent studies
suggest that cadmium, and possibly other toxic metals, may disrupt
cadherin function by displacing Ca2+ from its binding sites on the
cadherin molecules (Prozialeck et al., 2002). The observed effects in
our current study can be direct actions of cadmium interfering with
various cellular mechanisms, possibly by replacing other divalent ions.
Interestingly, studies have shown that after Ca2+ or Zn2+ supplemen-
tation, certain symptoms of cadmium toxicity may be eliminated
(Peraza et al., 1998). We can only speculate here that the presence of
cadmium may cause imbalances of the Ca2+ concentrations through
disruption of the cadherin complex.
Human exposure to cadmium can be occupational or environ-
mental. In human, the toxic level of cadmium varies among different
organs and tissues (ATSDR, 1999). Relatively low doses of cadmium
result in renal dysfunction, carcinogenicity, cardiovascular diseases
etc. despite various cadmium toxicities in animal studies (reviewed by
Satoh et al., 2002). A concentration of 100 μM cadmium chloride was
chosen in the current study because it resulted in a greater number of
affected embryos but was below the LC50 and EC50 (Cheng et al.,
2000). The exposure period of cadmium was examined previously in
Chow and Cheng, 2003. Cadmium exposure during the gastrulation
period is critical and exposure during both gastrulation and
segmentation periods induce the highest incidence of malformation.
Although the cadmium concentration used in this study is higher than
in the environment, chronic exposure to cadmium in zebrafish
embryos reveals potential risks of retinal defects to human. The eyes
have been reported as being susceptible to cadmium in humans.
Disturbance of lens optical performance result in cataract, the largest
single cause of blindness worldwide (Francis et al., 1999). Cadmium in
cigarettes has been evidenced as a major cause of cataract and age-
related macular degeneration which lead to blindness (reviewed by
DeBlack, 2003). Cadmium in rats has demonstrated to cause eye
defects upon acute prenatal exposure, indicating that cadmium is
teratogenic to fetus during pregnancy (Samarawickrama and Webb,
1981). Our current data provide further evidence that the retina is
susceptible to cadmium. Cadmium-exposed larvae are behaviorally
blind due to the loss of sole neurons, retinal ganglion neurons and
cone photoreceptors. This study shows that cadmium exposure to
embryos can have severe developmental consequences and terminate
in fatal ocular impairment.
Acknowledgment
The work described in this paper was fully supported by a grant
from the Research Grants Council of the Hong Kong Special
Administrative Region, China (Project No. CityU 1474/05M).
The authors declare that there are no conflicts of interest.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.taap.2008.11.013.
75
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