Received: March 4, 2004
Accepted: June 23, 2004
Dev Neurosci 2004;26:308–317
DOI: 10.1159/000082272
The Lens Has a Specific Influence on
Optic Nerve and Tectum Development in
the Blind Cavefish Astyanax
Daphne Soares Yoshiyuki Yamamoto Allen G. Strickler William R. Jeffery
Department of Biology, University of Maryland, College Park, Md., USA
Key Words
Blind cavefish Lens Apoptosis Retinotectal
projections Optic nerve Optic tectum
Abstract
We used the teleost Astyanax mexicanus to examine the
role of the lens in optic nerve and tectum development.
This species is unusually suited for studies of nervous
system development and evolution because of its two
extant forms: an eyed surface dwelling (surface fish) and
several blind cave dwelling (cavefish) forms. Cavefish
embryos initially form eye primordia, but the lens eventually dies by apoptosis, then the retina ceases to grow,
and finally the degenerating eyes sink into the orbits.
Transplantation of an embryonic surface fish lens into a
cavefish optic cup restores eye development. We show
here that retinal nerve fibers are formed and project to
the optic tectum in cavefish embryos. In adult cavefish
that have completed lens degeneration, however, the
number of retinal axons in the optic nerve is substantially reduced compared to surface fish. The presumptive
brain domains of embryonic cavefish are not altered relative to surface fish based on expression of the regional
marker genes Pax6, Pax2.1, and engrailed2. In contrast,
the adult cavefish brain is elongated, the optic tectum is
diminished in volume, and the number of tectal neurons
is reduced relative to surface fish. Unilateral transplantation of an embryonic surface fish lens into a cavefish
optic cup increases the size of the optic nerve, the num-
© 2004 S. Karger AG, Basel
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
Accessible online at:
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ber of retinotectal projections from the restored eye, and
the volume and neuronal content of the contralateral optic tectum. The results suggest that the lens has a specific influence on optic nerve and tectum development
during eye growth in Astyanax.
Copyright © 2004 S. Karger AG, Basel
Introduction
For many years, studies of lens development were focused primarily on its induction in the early embryo. The
optic cup was once considered to be the exclusive lens
inducer, but recent studies have shown that it is the last
of a series of lens-inductive activities [Saha et al., 1992].
Early embryological studies also did not fully appreciate
the importance of the lens in regulating retinal development. For example, the appearance of a contorted, but
normally layered, retina after lens removal from a chick
embryo was interpreted to mean that retinal development
is independent of the lens [Coulombre and Coulombre,
1964; Coulombre, 1965]. The role of the lens as a major
regulator of eye growth and development has now been
recognized. Early studies showing that the lens organizes
the anterior eye sector, including the cornea, iris, ciliary
body, and anterior chamber, have been confirmed and
extended [Genis-Galvez, 1966; Beebe and Coats, 2000;
Thut et al., 2001]. Moreover, microsurgical [Yamamoto
and Jeffery, 2000], molecular [Breitman et al., 1987;
Landel et al., 1988; Kaur et al., 1989; Kurita et al., 2003],
William R. Jeffery
Department of Biology, University of Maryland
College Park, MD 20742 (USA)
Tel. +1 301 405 5202, Fax +1 301 314 9358
E-Mail jeffery@umd.edu
and mutational [Ashery-Padan et al., 2000] analyses have
established that the lens is required for normal organization of the retina.
We study visual system development in the teleost Astyanax mexicanus, a single species consisting of eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) forms [Jeffery, 2001]. Functional eyes are absent in
adult cavefish, although embryos form small optic primordia, which are delayed, arrested in growth, degenerate, and sink into the orbits. Many of the regressive
changes in the cavefish eye appear to be related to abnormalities of the lens. Cavefish lens fiber cells do not terminally differentiate, instead they activate the hsp90 gene
and initiate apoptosis [Jeffery and Martasian, 1998;
Yamamoto and Jeffery, 2000; Hooven et al., 2004]. Subsequently, the Pax6 gene is downregulated in the corneal
epithelium, the anterior eye sector does not differentiate,
and retinal growth and photoreceptor cell differentiation
are arrested [Yamamoto and Jeffery, 2000], although new
cells are still produced in the ciliary marginal zone [Strickler et al., 2002].
Lens extirpation and transplantation experiments
have established the central role of the lens in cavefish eye
degeneration [Yamamoto and Jeffery, 2000; Jeffery et al.,
2003]. When a cavefish embryonic lens is transplanted
into a surface fish optic cup after its own lens is removed,
the surface fish eye reduces its growth rate and sinks into
the orbit. Similar results are obtained when the lens is
extirpated from a surface fish optic cup. In contrast, when
a surface fish lens vesicle is transplanted into a cavefish
optic cup, eye development is restored, indicating that the
surface fish lens induces the eye to resist degeneration and
that the inductive capacity of the cavefish lens has been
lost. Thus, the Astyanax system provides an unusual opportunity to study the role of the lens in visual system
development.
Although retinal organization appears to be dependent
on the lens, nothing is known about how the lens mediates this process, which retinal layers may be responsive
to lens signaling, and the consequences on visual center
development in the brain. In cavefish, lens degeneration
results in reduced rhodopsin expression and regression of
the retinal photoreceptor layer [Yamamoto and Jeffery,
2000]. However, the inner nuclear and ganglion cell
(GCL) layers develop normally based on Prox1 and Pax6
gene expression [Jeffery et al., 2000; Yamamoto and Jeffery, 2000; Strickler et al., 2001]. Here we use the Astyanax system to explore the role of the lens in retinal axon
and optic tectum development, critical components of
the developing visual system.
Lens and Visual System Development
Materials and Methods
Biological Materials and Procedures
Astyanax mexicanus surface fish were collected at Balmorhea
Springs State Park, Texas and Pachón cavefish [Dowling et al.,
2002] at Cueva de El Pachón in Tamaulipas, Mexico. Surface fish
and cavefish were maintained in a flow-through circulating aquarium system at 25 ° C. Procedures for maintenance of Astyanax colonies, spawning, and raising embryos to adults were carried out as
described previously [Jeffery et al., 2000; Strickler et al., 2001,
2002]. Lens transplantation was done at 24 h postfertilization (hpf)
by microsurgery [Yamamoto and Jeffery, 2002]. All methods were
approved by the University of Maryland Animal Care and Use
Committee and conformed to NIH guidelines.
Histology and TUNEL Assay
Routine histology was carried out by overnight fixation in 4%
paraformaldehyde (PFA) at 4 ° C. Fixed specimens were dehydrated
in a graded ethanol series, embedded in Paraplast, sectioned at
8 m, and stained with hematoxylin-eosin.
For TUNEL analysis, PFA-fixed specimens were washed twice
in 100% methanol and twice in PBST (1! PBS, 0.1% Triton X100) for 5 min at room temperature. The TUNEL assay was performed using the In Situ Cell Death Kit (Molecular Biochemicals,
Indianapolis, Ind., USA) with the detection of fragmented DNA by
peroxidase and Sigma fast, 3,3-diaminobenzidine (Sigma Chemicals, St. Louis, Mo., USA).
Molecular Biology and in situ Hybridization
The isolation and characterization of the Astyanax Pax6 cDNA
clone was described previously [Strickler et al., 2001]. Astyanax
Pax2.1 and engrailed (eng2) DNA fragments were obtained by RTPCR using the following primers: Pax2.1: 5-GTTATTGGBGGVTCYAARCCHAARGTKGC-3 (forward) and Pax2.1: 5-TGYTCWGRYTTGATRTGYTC-3 (reverse) and eng2: 5-GYARCGGAGGAAWGGVGGC-3 (forward) and eng2: 5-GGCSAMCAARACYTTGGTC-3 (reverse). RNA isolation, RT-PCR, DNA
sequencing, Blast analysis, construction of trees to verify paralogous genes, and in situ hybridization with RNA probes were carried
out as described previously [Jeffery et al., 2000; Strickler et al.,
2001, 2002].
DiI-DiO Labeling
Embryos and juveniles were anesthetized in 0.02% methane
tricaine sulfonate (Sigma), fixed for 1 h in phosphate-buffered, 4%
PFA (pH 7.4), and washed in 0.1 M phosphate buffer (PB; pH 7.4).
DiI (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) or DiO (3,3-dilinoleyloxacarbocyanine perchlorate; Molecular Probes, Eugene, Oreg., USA) were dissolved in 1% chloroform/100% ethanol and pressure-injected directly into the eyes of
fixed animals. In the case of embryos, optic tecta were visualized
intact, while juveniles had their tecta removed and flat-mounted
on a glass slide. All animals were visualized using a BioRad confocal microscope.
Optic Nerve Analysis
Two adult surface fish and two adult cavefish were anesthetized
with methane tricaine sulfonate (Sigma) and perfused transcardially with normal saline followed by glutaraldehyde/PFA buffer mixture (2.5% glutaraldehyde, 1% PFA, 3% sucrose in 0.06 M PB, pH
Dev Neurosci 2004;26:308–317
309
Fig. 1. Lens apoptosis and eye development in surface fish (A, C, E) and cavefish (B, D, F). A–D TUNEL labeling
shows the dying lens (L) in cavefish (B, D) but not in surface fish (A, C) at 5 (A, B) and 10 (C, D) dpf. E, F Eye
morphology in adult surface fish (E) and cavefish (F) showing differences in size of the neural retina (NR), and the
absence of a lens and a cornea in cavefish. C Cornea in surface fish (E). The degenerate cavefish eye is covered by
an epidermal plug (EP). A Scale bar = 30 m (A–D are the same magnification). E Scale bar = 100 m (F is 2! E).
7.4). The heads were excised from the specimens and fixed at 4 ° C
for 1–2 days, and the eyes and attached optic nerves were dissected
from the brain on both sides. Specimens were postfixed in 1% OsO4
in PB for 1–2 h at room temperature, washed in PB, dehydrated in
a graded ethanol series, cleared in propylene oxide, and embedded
in epon. Semithin sections were cut and stained with 0.2% toluidine
blue in 0.2% borax solution. For each specimen, outlines of the entire nerve and counts of axonal bundles and individual axons were
done with a Neurolucida system (Microbrightfield; Williston, Vt.,
USA) using a 100! oil immersion objective.
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Dev Neurosci 2004;26:308–317
Optic Tectum Analysis
Adult surface fish, cavefish, and cavefish with a transplanted
lens were anesthetized with methane tricaine sulfonate (Sigma) and
perfused transcardially with normal saline followed by fixation in
4% PFA, 0.9% NaCl in 0.1 M PB (pH 7.2). Brains were removed
as described above, postfixed overnight, dehydrated, cleared in xylene, embedded in paraffin, and cut into 10-m transverse sections.
The sections were stained with cresyl violet (Kodak, Rochester,
N.Y., USA). The sections were outlined, the optic tectum areas
were measured, and neurons were counted in every third section
using the Neurolucida system with standard stereological techniques.
Soares/Yamamoto/Strickler/Jeffery
Results
Table 1. Comparison of areas and retinal axon numbers in adult
surface fish and cavefish optic nerves
Lens Apoptosis
Previous studies have shown that lens fiber cells undergo programmed cell death during early cavefish eye
development [Jeffery and Martasian, 1998; Yamamoto
and Jeffery, 2000]. In spite of the presence of dying cells
in the lens core, cell division continues in the lens epithelium [Strickler et al., 2002], indicating that lens development is not completely suppressed. To determine the status of the lens during later development, we compared
apoptosis in surface fish and cavefish larvae by TUNEL
analysis (fig. 1). TUNEL-labeled cells were seen throughout the cavefish lens but not in surface fish lens at 5 and
10 days postfertilization (dpf) (fig. 1A–D). Probably as a
result of apoptosis and removal of dying cells, the lens
could not be identified in most adult cavefish (fig. 1E, F),
whereas in others it degenerated into a small empty vesicle (data not shown). The results suggest that lens apoptosis continues during late cavefish development, canceling the effects of cell division in the epithelial layer and
ultimately resulting in complete lens degeneration. We
conclude that the cavefish retina is unlikely to receive late
developmental input from the lens.
Voneida and Sligar [1976], who originally demonstrated
the presence of retinotectal projections in a related cavefish. Our DiI-DiO tracing results and quantitative morphological data suggest that retinotectal projections initially develop during cavefish embryogenesis but then either do not increase or possibly even decrease in number
following lens degeneration, thus resulting in a diminished optic nerve in adults.
Optic Nerve Development
Despite lens death, the GCL appears to form normally in the early cavefish retina [Langecker et al., 1995; Jeffery et al., 2000; Strickler et al., 2001]. However, later in
development the GCL begins to show disorganization,
and in adult cavefish the retina is disorganized and much
smaller than in surface fish (fig. 1E, F). Retinal axon development in the cavefish optic nerve was determined in
two ways. First, DiI or DiO was injected into the eye of
early cavefish or surface fish embryos and the extent of
dye movement was traced through the optic tract into the
tectum. The results were the same in cavefish and surface
fish embryos. The dye spread into the optic tract and advanced past the embryonic midline after injection at
36 hpf but was not observed in the contralateral optic
tectum until approximately 42 hpf (fig. 2A–D; data not
shown). Second, retinal axons were quantified in EM cross
sections of cavefish and surface optic nerves. The results
showed that an optic tract containing retinal axons is still
present in adult cavefish, although the number of fiber
bundles and therefore the total number of axons was substantially reduced compared to surface fish (fig. 2E, F;
table 1). It is interesting to note that each fiber bundle
showed approximately the same number of axons in both
surface fish and cavefish. These results extend those of
Brain and Optic Tectum Development
The expression of Pax6, Pax2.1, and eng2 was used to
compare the specification of presumptive brain regions
in cavefish and surface fish. In teleost embryos Pax6 expression is restricted to the presumptive forebrain and
hindbrain [Amirthalingam et al., 1995], Pax2.1 expression to the midbrain-hindbrain boundary and posterior
hindbrain [Kelly and Moon, 1995], and eng2 expression
to a wide stripe overlapping the midbrain and including
the future optic tectum [Fjose et al., 1992]. According to
the expression patterns of these genes, there are no significant differences in presumptive brain regions in cavefish and surface fish embryos at the 18-somite or 24-hpf
stages (fig. 3A–J). For example, the position of the Pax2.1
stripe at the midbrain-hindbrain boundary, the Pax6-expressing domain in the presumptive forebrain (exclusive
of the optic primordia, which are larger in surface fish
embryos; [Strickler et al., 2001]), and the eng2 expression
domain in the midbrain primordium were similar in both
forms (fig. 3A–J). The results suggest that early brain specification is similar in cavefish and surface fish embryos.
To compare the morphology of adult brains, whole
brains were dissected from cavefish and surface fish of the
same approximate size and age. As described previously
[Reidel, 1997], the cavefish brain is more elongate and
Lens and Visual System Development
Dev Neurosci 2004;26:308–317
Fish type
Surface fish
1
2
Cavefish
1
2
ON area
(m2 ! 103)
Number of
axon bundles
Number
of axons
388.1
378.1
265
171
6,162
3,728
8.6
7.3
4
1
99
30
ON = Optic nerve.
311
Fig. 2. Optic nerve development in surface
fish (A, C, E), cavefish (B, D, F), and cavefish
with a transplanted surface fish lens (G, H).
A–C DiI or DiO tracing of optic nerve (ON)
fibers at 36 (A, B) and 72 (C, D) hpf showing
growth toward the midline (A, B) and targeting (C, D) of the optic tectum (OT). DiI
or DiO was injected into the developing eye
(E). E, F Cross sections through optic nerves
of adult surface fish (E) and cavefish (F)
showing bundles of stained fibers (arrowheads). BV = Blood vessel; M = muscle.
G–H Transverse sections through the optic
nerves of an adult cavefish with a transplanted lens on one side (asterisk in G) showing increased optic nerve thickness on the
transplant (G) compared to the control (H)
side. A Scale bar = 100 m. E Scale
bar = 20 m. G Scale bar = 150 m. Magnification is the same in A–D, E, F, and G, H.
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Dev Neurosci 2004;26:308–317
Soares/Yamamoto/Strickler/Jeffery
slender than its surface fish counterpart (fig. 3K). However, the size of the various brain regions is similar in both
forms with the notable exception of the optic lobes, which
are much larger in surface fish than in cavefish (fig. 3K,
4A, B). To quantify the difference in optic lobes, the volumes and neuron content of surface fish and cavefish optic tecta were determined by analysis of serial cross sections. In these analyses, volumes were calculated by measuring areas, the number of neurons was counted in every
third optic section, and the data were compiled and compared for the left and right optic tecta of single individuals (fig. 4A, B; table 2). Several conclusions can be made
from these results. First, the difference in volume and
neuron number between optic tecta in the same animal
is very small (4% or less), a point that will be important
below in considering the results of unilateral lens transplantation. Second, the volume of cavefish optic tectum
is only 40–50% of surface fish. Third, the number of neurons in the cavefish optic tectum is reduced to less than
20% of surface fish.
Effects of Lens Transplantation on Optic Tectum
Development
To determine the role of lens on optic tectum development, lens transplantation was carried out between surface fish donors and cavefish hosts, and the adult eye phenotypes of the hosts were determined. Because lens transplantation was unilateral in these experiments, the
degenerate eye on the unoperated side of the head served
as a control for the restored eye. As shown previously
[Yamamoto and Jeffery, 2000], a complete anterior sector, including a cornea, iris, an anterior chamber, and a
Fig. 3. Surface fish (A–C, G, H, K/left) and cavefish (D–F, I, J, K/
right) embryonic (A–J) and adult (K) brains and the adult brain of
a cavefish with a transplanted surface fish lens (L). A–J In situ hybridization showing Pax6 (A, D, G, I), Pax2.1 (B, E), and eng2 (C,
F, H, J) expression patterns at the 18-somite (A–F) and 24-hpf (G–J)
stages. K, L Dissected brains of adult surface fish (K/left), cavefish
(K/right), and cavefish in which a surface fish lens was transplanted
into the optic cup on one side (asterisk) during embryogenesis (L).
OT = Optic tectum. A Scale bar = 200 m. G Scale bar = 300 m.
K Scale bar = 200 m. Magnification is the same in A–F, G–J, and
K, L.
Lens and Visual System Development
Dev Neurosci 2004;26:308–317
313
Fig. 4. Adult surface fish (A) and cavefish (B) optic tecta (OT) and
optic tecta of an adult cavefish with a transplanted surface fish lens
(C). Each optic tectum is shown in transverse section. Asterisk in
C indicates the side with the restored eye. Scale bar = 150 m; magnification is the same in each frame.
Table 2. Optic tectum development in
surface fish, cavefish, and cavefish with a
restored eye
Fish type
larger and more organized retina was formed in the restored eye (data not shown). Transverse sections showed
an increase in the size of the optic tract and number of
retinal nerve fibers on the lens transplant side (fig. 2G, H;
table 1), suggesting that additional axons were formed by
the GCL. To determine the extent of tectal innervation,
the restored eye of cavefish with a transplanted lens were
injected with DiI and dye movement into the brain was
determined. After confocal visualization of flat-mounted
optic tecta, many more fibers were seen extending from
the restored eye and innervating the contralateral optic
tectum than were observed to innervate the ipsilateral
optic tectum from the degenerate eye (fig. 5).
To quantify the effects of lens transplantation on the
optic tectum, the brain was removed from an adult cavefish (fig. 3L) with a transplanted lens and the volume and
neuron number of the contralateral and ipsilateral optic
tecta were compared by analysis of serial cross sections.
As described above, the volumes and number of neurons
in surface fish and cavefish left and right optic tecta differ
by less than 4% in the same animal (table 2). However,
in cavefish with a restored eye, the contralateral optic tectum showed a 13% increase in volume and an 8% increase
in neuron number relative to its ipsilateral counterpart.
Thus, the results show that lens transplantation and accompanying eye restoration can enhance optic tectum development in cavefish. We conclude that the lens has an
important role in development of these visual system
components.
OT volume
m3
% OT
difference
Neurons
% Neuron
difference
Surface fish
Left OT
Right OT
87.0
89.0
2.2
24,903
24,109
3.2
Cavefish
Left OT
Right OT
41.8
41.5
2.4
4,074
3,908
4.0
One-eyed cavefish
Left OT
Right OT
12.5
15.4
13.3
5,743
6,256
8.2
OT = Optic tectum. Percent differences are the dividend of the OT with the greater
volume and the lesser volume ! 100. One-eyed cavefish has a restored eye on the left side
of the head.
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Soares/Yamamoto/Strickler/Jeffery
Fig. 5. DiI tracing of retinotectal projections in flat-mounted optic tecta (OT) from a cavefish with a restored eye.
A Abundant retinal neurons projecting from the restored eye (left out of view) into the contralateral optic tectum.
B Sparse retinal neurons projecting from the degenerate eye (left out of view) into the ipsilateral optic tectum.
Contralateral and ipsilateral are designated with respect to the position of the restored eye. A Scale bar = 100 m;
magnification is the same in A and B.
The purpose of the present investigation was to determine the effects of the lens on visual system development
in Astyanax. We used two approaches to determine
whether the lens exerts an influence over optic nerve and
tectum development. First, we compared normal optic
nerve and tectum development in surface fish, which
have an actively growing lens, and in cavefish, in which
the lens degenerates during early development. Second,
we examined the effects of lens transplantation and subsequent eye restoration on optic nerve and tectum development in cavefish. The results of both approaches suggest that the lens has a specific influence on optic nerve
and tectum development.
The first step in our investigation was to assess the capacity of the lens to influence late eye development in
cavefish. We demonstrated previously that primary lens
fiber cells do not terminally differentiate but instead undergo apoptotic cell death beginning about 12 h after lens
vesicle formation [Jeffery and Martasian, 1998; Yamamoto and Jeffery, 2000]. Based on PCNA and BrdU labeling studies, however, it was subsequently shown that
lens epithelial cells continue to divide following the initial
round of apoptosis in fiber cell precursors [Strickler et al.,
2002], potentially allowing the lens to be revived during
later cavefish development. Here we document extensive
and virtually complete lens apoptosis at 5 and 10 dpf and
show that adult cavefish lack a detectable lens or have a
structureless lens vestige. We therefore conclude that continuous apoptosis acts to cancel the effects of late cell division in the embryonic lens, which is structurally obliterated by the early larval stage, when the surface fish lens
and eye are growing rapidly. Consequently, the degenerated lens is unlikely to be a factor during the later stages
of cavefish visual system development.
There are two phases of optic development in teleosts:
an embryonic phase and a larval/adult growth phase [Hu
and Easter, 1999]. During the embryonic phase, surface
ectoderm and neural plate cells proliferate and are specified to become the lens and neuroretina, respectively. The
presence of an embryonic lens and normal early differentiation of the retina [Langecker et al., 1995; Yamamoto
and Jeffery, 2000; Strickler et al., 2001] suggest that (aside
from the smaller size of the optic primordia) the initial
stages of eye development are normal in cavefish. Accordingly, we show that the embryonic cavefish GCL forms
axons, which extend through the optic tract to the contralateral optic tectum. During the growth phase of optic
development, both the lens and retina add new cells primarily at their margins, and these cells subsequently differentiate. As in other teleosts [Kroger and Fernald, 1994],
the surface fish eye is characterized by growth in accordance with increasing body size. We have shown here and
in previous studies [Jeffery and Martasian, 1998; Yamamoto and Jeffery, 2000] that the retinal growth phase is
Lens and Visual System Development
Dev Neurosci 2004;26:308–317
Discussion
315
radically changed and uncoupled from body growth in
cavefish due to programmed cell death and degeneration
of the lens. The cavefish retina continues to produce new
cells at the ciliary marginal zone, although its net growth
is very small [Strickler et al., 2002], suggesting that most
newly born cells are removed by cell death [Langecker et
al., 1995]. We further demonstrate that retinal axons persist in adult cavefish, probably stemming from the original retinal ganglion cells produced during the embryonic
phase, although they are decreased in number. Our results
are consistent with an arrest of postembryonic GCL differentiation and axon extension to the optic tecta during
the growth phase of retinal development in cavefish.
Voneida and Fish [1984] showed that in a related cavefish retinal axons do not respond to a light stimulus. Thus,
the function of cavefish retinotectal projections is not
presently understood; they could target a portion of the
tectum not associated with vision or they could be vestigial.
Aside from the size of the optic vesicles, no marked
changes were detected in the presumptive brain of cavefish embryos, according to expression of the Pax6, Pax2.1,
and eng2 regional gene markers. The eng2 transcription
factor controls early specification of the midbrain and the
posterior optic tectum [Itasaki et al., 1991]. Identical eng2
expression patterns in cavefish and surface fish are noteworthy since this gene has been implicated in the control
of axon targeting to the optic tectum by the retina [Itasaki and Nakamura, 1996]. In contrast to the embryonic
brain, the adult cavefish brain shows a substantial reduction in the volume and neuronal content of the optic tectum. These results suggest that changes in the cavefish
optic tectum may occur in concert with arrest of the retina during the growth phase of optic development. There
are two possible explanations for smaller optic tectum
development in cavefish, which are not mutually exclusive: First, there may be an intrinsic change in the ability
of the tectal cells to divide and differentiate, or alternatively they may undergo an enhancement in programmed
cell death. Second, reduced extrinsic input from fewer
retinotectal fibers may be responsible for the diminished
size of the optic tectum. Further studies will be necessary
to distinguish between these possibilities, although the
lens transplantation results described below suggest that
extrinsic factors are at least in part responsible for comparatively smaller optic tecta in adult cavefish.
Thus far, we have described the results of studies implicating the degenerate lens in modifying the visual system in cavefish. Additional evidence supporting a role for
the lens in this process was obtained from lens transplan-
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Dev Neurosci 2004;26:308–317
tation studies. Unilateral transplantation of a surface fish
embryonic lens into a cavefish optic cup can restore a
complete eye in adult cavefish [Yamamoto and Jeffery,
2000; Jeffery et al., 2003]. Prior to the present investigation, however, it was not known whether the restored eye
is connected to the optic tectum or whether the transplanted lens affects tectal development. Here we have
shown that lens transplantation enhances the number of
retinotectal projections and increases the volume and
neuronal number of the contralateral optic tectum. Although the increase in the optic tectum is relatively modest, it is higher than expected from natural deviation of
the right and left cavefish optic tecta. Thus, the cavefish
phenotype is similar to the medaka mutant eyeless, which
is characterized by small, misplaced retinae, a low number of optic nerve fibers, and extreme optic tectum diminution [Ishikawa et al., 2001]. We conclude that the lens
has an indirect positive effect on optic tectum differentiation through mediating the production of additional
retinal axons during the optic growth phase.
The increase in optic tectum development impels us
to consider whether cavefish with a restored eye are able
to respond to light. Recently, this question was addressed
by behavioral studies in which cavefish with a restored
eye were scored for their location in the illuminated or
dark side of an aquarium [Romero et al., 2003]. The results showed that these cavefish were indifferent to such
illumination, behaving similarly in this regard to cavefish
with two degenerate eyes. However, this experimental design could only have detected a large recovery in phototactic behavior, and coupled with the modest extent of
visual system restoration in cavefish with a transplanted
lens, we believe that this important issue is still open. We
are currently conducting detailed physiological and
behavioral experiments to determine if light elicits responses in the optic tectum and whether cavefish with a
restored eye can regain optomotor responses.
In conclusion, the results of this investigation suggest
that the lens promotes retinal ganglion cell differentiation
during the growth phase of optic development, and thus
has an indirect effect on projection of retinotectal fibers
and optic tectum development.
Acknowledgments
This research was supported by an NSF Postdoctoral Fellowship (DBI-0208257) to D.S. and NSF (IBN-0110275) and NIH
(EY014619) grants to W.R.J. We also thank Tim Maugel of the
Laboratory for Biological Ultrastructure at the University of Maryland for his technical assistance.
Soares/Yamamoto/Strickler/Jeffery
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