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/. Embryol. exp. Morph. Vol. 33, 1, pp. 243-257, 1975
Printed in Great Britain
243
Structural basis of the functional
development of the retina in the cichlid
Tilapia leucosticta (teleostei)
By GERD GRUN 1
From Spezielle Zoologie, Ruhr University, Bochum
SUMMARY
The differentiation of retinal cells has been studied with special reference to the formation
of functionally important structures. Three phases could be revealed: from day 3 to day 6
retinal cells in the mostly advanced central part show signs of general cell differentiation
(formation of ribosomes, endoplasmic reticulum, mitochondria). In the second phase from
day 6 to day 9 characteristic nerve cell structures appear (neurites, dendrites, synapses,
receptor outer and inner segments). In the last phase from day 9 to day 12 these special
structures attain their final, mature appearance, synapses seem ready for function, dendritic
invaginations and synaptic ribbons are formed, twin cones become arranged in mosaic
patterns. This developmental order conforms to a gradient running from the ganglion cells
to the receptors. Neurites and dendrites appear in the ganglion cells on day 3, in the intermediate neuron layer not before day 4. The horizontal cells are the last ones to differentiate
out of the intermediate neurons. The inner plexiform layer synapses are structurally mature
before those from the outer plexiform layer. The receptor inner and outer segments differentiate from the 5th day up to the time the youngfishis able to see (day 13). The last structures
to appear are dendritic invaginations and synaptic ribbons in the receptor terminals, and the
twin cone mosaic. It is assumed that the ability to see is achieved only when these structures
have been formed.
INTRODUCTION
Though there is a considerable amount of literature on developmental
processes in the nervous system, the number of studies concerning the correlation of developing structure and maturing function in the nerve cell is not
very great. If we only consider the easily accessible retina, those papers are
mainly concerned with structural (Hollyfield, 1972; Kahn, 1973; Yazulla, 1974),
biochemical (Nakano & Hasegawa, 1971; Lolley & Racz, 1972) or physiological
development (Beazley, Keating & Gaze, 1972; Carton & Appel, 1974), to cite
only some recent examples. Many of these and other less recent papers give a
correlation of different aspects, as do in particular the studies on neural specificity (Jacobson, 1968; Fisher & Jacobson, 1970; Straznicky & Gaze, 1971;
Dixon & Cronly-Dillon, 1972; Grillo & Rosenbluth, 1972), or those of
Coulombre (1955), Nilsson & Crescitelli (1969) and Moscona & Piddington
1
Author's address: Spezielle Zoologie, Ruhr University, Bochum, Federal Republic of
Germany.
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G. GRUN
(1966). However, for the retina an investigation combining structural, biochemical and physiological aspects at the cellular level is still lacking. Flexner
and his colleagues (e.g. 1950, 1951/2) have done this for the cerebral cortex. It
is the aim of the present author to use this approach with the retina and, by
means of different cytological research methods, to clarify the way cytological
differentiation leads to functional ability.
Since single neural cells cannot be regarded as functionally independent, one
always has to relate developmental steps in the neuron to the development of the
tissue as a whole. Therefore the present paper is intended to give the morphological basis of the maturation processes for different neuron types as well as
for the whole retina.
Observations have been made on a mouth-breeding cichlid species, because
the moment when function of the retina not only might be achieved, but is
actually needed, is easy to determine: it is the moment the young fish leaves the
mother's mouth. Moreover embryos are readily available in great number and
the time of embryonic development is rather short (about 2 weeks).
MATERIAL AND METHODS
Embryos were removed from the mother's mouth and kept in a simple
breeding apparatus. In other cases they were taken from the mouth at the time
they were to be fixed. The age of the embryos is given in hours or days from the
moment of fertilization and uptake into the mouth. This gives an uncertainty
of ± 30 min. Specimens were killed between the 3rd and 4th day every 2 h and
from the 4th day to the 15th day every 24 h. Total embryos or only heads or
eyes were fixed for electron microscopy in cold cacodylate buffered glutaraldehyde and postfixed in cold osmic acid, pH 7-2, for 90 min. After dehydration
through a series of acetones andpropylene oxide they were embedded in Maraglas
(Erlandson, 1964). Ultrathin sections were stained with uranium acetate and
lead citrate and viewed in a Zeiss EM 9 S-2. Semithin sections (1 jum) for light
microscopy were stained with methylene blue. Other specimens were fixed in
cold Carnoy for 24 h and embedded in paraplast; 5 fim. sections, intended for
other purposes too, were stained after the Feulgen method.
RESULTS
Since differentiation starts in a certain region in the centre of the optic cup,
one always finds various stages of differentiation in a given section. Here only
those cells which are most advanced in cellular and histological development
will be looked at, i.e. the part of the eye-cup lying in a central region and differing
markedly from the peripheral parts (Fig. 1 A).
Development of retina in Tilapia
245
Fig. 1. Histogenesis of eye and and retina. (A) Three days, (B) 5 days, (C) 9 days, (D)
13 days. The formation of the neuronal layers is clearly seen (abbreviations, see
text). Note mitosis in (A), arrow.
Light microscopy
3-5 days. In the 3-day-old embryo (Fig. 1 A) the central part of the eye-cup
shows densely packed layers of cells, while in the large peripheral regions no
layers are to be seen and mitoses still occur. At this age the layers in the most
advanced part of the retina are as follows. A pigment layer is hardly distinguishable on the first days, pigment being only just visible. By the 5th day it
has become a broad dark layer surrounding the whole eye-cup. Subjacent to it
are the future receptor nuclei (m), which separate more and more from the rest
of the retina, and attain an ordered array.
The main bulk of the retina consists of cells forming two clearly distinguishable
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G. GRUN
layers after 4 and 5 days (Fig. IB, be, ac). They are the future bipolar and
amacrine cells and differ in the appearance of their nuclei. The bipolar nuclei
are dark with rather concentrated chromatin, the amacrine cells make up a
slightly looser layer. Their nuclei are lighter. They display chromatin masses
but no uniform concentration. Some dark nuclei in the amacrine cell layer
might belong to glial cells (Rasmussen, 1973). After 4 days the first nuclei of
horizontal cells appear in the outermost part of the bipolar cell layer (he). They
probably arise from the bulk of the intermediate neuron layer.
The future ganglion cell layer (gc) is about four cells thick and their nuclei
are very similar to the amacrine nuclei. The neurites of the optic tract are clearly
visible after 4 days.
The outer plexiform layer (opl) is a small zone connecting the receptor cells
with the bipolar cell layer. The inner plexiform layer (ipl) develops from a small
gap between the amacrine cells and the ganglion cells to a rather broad zone
containing displaced ganglion cells.
6-8 days. At this stage the layers of the retina appear more distinct, mainly
due to the plexiform layers separating them (Fig. 1C). The pigment layer is
becoming very broad and surrounds all the eye, nearly touching the lens. The
most conspicuous change is the formation and elongation of the receptor outer
and inner segments. In this period both segments grow from ca. 6 to 17 /im (rs).
The perinuclear part of the receptor cells at first remains unchanged. After 8
days two layers of nuclei have clearly separated, a scleral one consisting of
oblong light nuclei and a vitreal one of round, dark nuclei.
The two layers of the inner bulk by now are well characterized. The nuclei
of the bipolar cells are becoming still darker and smaller, with the chromatin
concentrated. The amacrine nuclei are larger and light. The cytoplasmic volume
however seems to be rather large in the bipolar cells. At the scleral border there
is a zone of horizontal cells increasing steadily in number. The ganglion cells
are the largest of all, due to their great cytoplasmic masses. Their nuclei are
slightly larger than the amacrine cell nuclei.
The outer as well as the inner plexiform layer have become clearly bordered
zones. These borders are formed by the cell membranes of the different nuclear
layers. While the outer plexiform layer has broadened only very slightly, the
inner one by the end of day 8 has become about 20 jum thick. It is essentially
free of cells, no displaced ganglion or amacrine cells lying in it. By now there
are numerous neural processes running through the layer of the intermediate
neurons.
9-13 days. This is the final part of the retinal cell differentiation, at the end
of which the young fish hatch from their mother's mouth. It is about this time
that the ability for visual perception has been achieved, though this is inferred
only from behavioural response.
Light microscopic observation does not reveal great changes in this period,
however (Fig. ID). The pigment layer broadens all the time and processes of
Development of retina in Tilapia
[mil
fitn
4A
Fig. 2. Future receptor cells, 3 days 8 h. Scleral to the large ovoid nuclei (rn) the
future inner segments (is), containing mitochondria (mi), vesicles (vs) and ribosomes.
Note difference between light and dark inner segments. The pigment epithelium
(pe) has formed only scattered granules.
Fig. 3. Ganglion cell, 2>\ days. At this stage ganglion cells are the only retina cells to
contain a well developed endoplasmic reticulum (er). Note the axon hillock with a
lipid granule (Ig).
Fig. 4. Outer retina, 5 days. (A) Inner segments (is) have elongated and contain
more mitochondria now. Pigment granules are well developed (pg). (B) Outgrowing
ciliary structure (ci) demonstrating the formation of the outer segment. (C) The
dendrites (de) of the OPL have no synaptic contact with the slim receptor terminal (rt).
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G. GRUN
the pigment epithelium reach far between the outer segments of the receptor
layer. The two layers of nuclei in the receptor layer make up two ordered arrays
of nuclei. The horizontal cells appear smaller than in the preceding stages.
Perhaps they ceased, growing some time before. The number of nerve fibres
crossing the intermediate nuclear layer has increased very much, as is seen by
the formation of columns in the amacrine cell layer. The other layers show no
light microscopic changes, except for the further widening of the inner plexiform
layer and the extension of the receptor outer segments.
Electron microscopy
3-5 days. From the 3rd day on, there are numerous free ribosomes and mitochondria in the future receptor cell cytoplasm (Fig. 2). By day 4 their amount
seems to have increased and there are lots of vesicles of the endoplasmic reticulum, which appears after 90 h and increases from then on. The first Golgi
apparatuses appear at this time too. The cytoplasmic part of the cells which
is to form the outer and inner segment enlarges and its special differentiation is
indicated by the formation of mitochondria (Fig. 4 A). There appears a ciliary
structure at the lateral part of the receptor cell (Fig. 4 B), indicating the beginning
of the formation of an outer segment. At this stage the receptor cells are very
similar to ependymal cells, which fact is underlined by the occurrence of desmosome-like cell junctions and rudimentary cilia, both in the region of the inner
segments.
The outer membrane of the receptor nucleus is densely covered with ribosomes
and there seems to be extrusion of substances out of the nucleus. Regularly
near the nucleus there is a Golgi apparatus or Golgi vesicles. The ribosomes,
which are very abundant, seem to be arrayed in a spiral or similar order.
The receptor terminals and their vitreal border are immediately subjacent to
the nuclear region (Fig. 4C). In no case are the receptor terminals separated by
glial processes. On the 5th day the terminal contains some vesicles and numerous
ribosomes. It touches dendrites of the outer plexiform layer, though synaptic
boutons are not yet to be found. The processes in the outer plexiform layer,
which are mostly, if not all, dendritic, seem to contain neurotubules.
In both bipolar and amacrine cells on the 3rd day the ribosomes are still more
abundant than in the receptor cells. Endoplasmic reticulum develops poorly
after 3 days, most of the ribosomes lying free in the cytoplasm. Mitochondria
are present. The difference now arising between the two types of cell is mainly
due to the distribution of the ribosomes and the broader cytoplasmic rim in the
amacrine cells, which gives them a clearer appearance. By the end of day 5 the
ribosomes in the bipolar cells form nuclear caps or lie in regions similar to axon
hillocks. Those axon hillocks sometimes contain filamentous structures. There
are only single endoplasmic and Golgi lamellae, as is the case also in the amacrine
cells. The bipolar nuclei contain dark chromatin masses (Fig. 4 A). In the whole
layer longitudinal sections of neurites are to be found.
Development of retina in Tilapia
249
Fig. 5. Seven days. (A) The outer segment of a rod contains membrane stacks with
narrow interlaminar space. In the inner segment there are densely packed mitochondria. (B) Inner segment. In the scleral part there are many mitochondria (note
dark and light types), while in the vitreal part the endoplasmic cisternae (er) are in
continuity with the nuclear envelope. (C) Receptor terminal containing synaptic
vesicles of different size. The terminals are in immediate contact with dendrites (de)
of the OPL, but there are no true synaptic structures. (D) Bipolar (bn) and amacrine
cell (an) nuclei.
The cells of the future ganglionic layer (Fig. 3) are the only ones which already
contain endoplasmic reticulum on the 3rd day. This increases on the following
days, as do mitochondria and Golgi apparatuses. Ribosomes are not so abundant
as in the other cells. They form a dense cap around one half of the nucleus 12 h
before similar caps appear in the bipolar cells. By day 5 there are the first
synaptic structures on to the perikaryon of the ganglion cells. As the intermediate
neuron layers do not yet develop neurites and synapses (see below) it must be
assumed that these synaptic structures are formed by other ganglion cells.
The outer plexiform layer consists of a number of short neuronal processes
on the 3rd day. As they are relatively large and seem to contain nothing but an
occasional mitochondrion, they must be looked on as dendrites. There is no
change up to day 5. The inner plexiform layer on day 5 attains a considerable
thickness by the formation of numerous neurites of the intermediate neurons.
They are smaller than the dendrites of the ganglion cells, the number of which
does not increase proportionally. The very large dendrites occupy the vitreal
one third of the whole inner plexiform layer, lying adjacent of the ganglionic
layer, while the rest of the inner plexiform layer now consists of the bipolar and
amacrine axons. True synaptic structures could not be detected nor are there
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G. GRUN
Fig. 6. Cone outer segment, 9 days. Lamellae are now separated by wide spaces. In
the vitreal part large cisternae are visible. A filamentous process (pr) passes from the
inner segment along the outer one. Note the possible discontinuous outer segment
formation.
Fig. 7. Double cone inner segment, 9 days. For further explanation see text.
Fig. 8. Nine days. Multiple membranes (arrow) between two partners of a double
cone.
Fig. 9. Thirteen days. Receptor mosaic pattern of four double cones surrounding
one central pair. Cross-section through scleral part of inner segment, pp, Pigment
cell process.
Development of retina in Tilapia
251
any tubules or filaments in the neuronal processes. It must be noted, however,
that the fibres of the optic tract contain neurotubules.
6-8 days. In the receptor layer membrane discs develop now - a seemingly
rapid process (Griin, 1974)-and it is possible to distinguish rods (Fig. 5A)
and cones (Fig. 6), according to the description given by Cohen (1972). The
inner segments enlarge enormously during this period and the mitochondria
increase in number (Fig. 5B). In the scleral part of the inner segment they are
densely packed. In the vitreal part there are swollen cisternae of rough endoplasmic reticulum. They are not numerous but very conspicuous, forming a sort
of network between each other and the nuclear membrane. Besides the aggregated
ribosomes there are many Golgi lamellae, vitreo-scleral directed filaments and
sometimes a ciliary structure in the inner segment. The ciliary structure arising
from the lateral part of the inner segment now continues into the process
accompanying the outer segment. Often the inner segments seem to be separated
by glial processes.
The terminals of the receptor cells (Fig. 5C) have elongated. Their scleral
parts contain more ribosomes, the vitreal parts mostly synaptic vesicles, some
of which are filled by an electron-dense substance. Junctions of the zonulae
adhaerentes type occur at different parts of the terminal, but synaptic junctions
have not attained their typical aspect.
In the bipolar cells (Fig. 5D) the numbers of ribosomes and mitochondria
decrease markedly. There are less vesicles too. The amacrine cells however still
contain abundant ribosomes, but only single lamellae and vesicles. The ganglion
layer remains unchanged.
The number of axons in the inner plexiform layer still increases, thus widening
this layer (compare light microscopic observations). Some of the longitudinally
or transversely sectioned neural processes show tubules or filaments. Mostly
however they appear empty or contain only mitochondria. There are synaptic
regions running like tapes through the layer. They lie in different parts of the
plexiform layer and presumably form the contact zone between the bipolar/
amacrine neurites and ganglion cell dendrites. This is corroborated by the fact
that vitreal to these regions the neural processes are mostly of the dendritic
type. The first synaptic vesicles appear on the 6th day.
9-13 days. The receptor outer segments become very elongated during this
period, the cone membrane discs displaying a very regular appearance (Fig. 6),
tapering towards the scleral end. The inner segment (Fig. 7) contains still more
mitochondria, more endoplasmic reticulum and perhaps more ribosomes than
before. Already from day 7 on, 'twin cones' (Cohen, 1972) have been appearing,
paired cones touching each other very intimately (Figs. 7, 8). There are multiple
membranes formed at the inner side of each of the participating cell membranes,
thus giving the appearance of subsurface membranes (Rosenbluth, 1962). In
the inner segment, the mitochondria always lie near to these membranes, while
the other, mitochondria-free part contains vesicles, Golgi elements and ribosomes.
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G. G R U N
10
Fig. 10. Receptor terminals, 13 days. By now dendritic infoldings (di) and synaptic
ribbons (sr) have been formed, de, Dendrites; sv, synaptic vesicles. Note immediate
contact between adjacent terminals; they are not separated by glial processes.
Fig. 11. Thirteen days. Bundle of neurites (ri) cross the intermediate neuron layer
and fill the intercellular space between amacrine cells (ac).
Later on (day 11) four or five twin cones come together to surround one
central pair, thus forming a mosaic pattern (Fig. 9), a rather common feature
of the fish retina (Lyall, 1957; Meyer-Rochow, 1972).
In the receptor perikarya, the cytoplasmic rim where the endoplasmic reticulum seems to have increased a little, has become very slim. The nuclei of both
rods and cones contain dark material contacting the inner membrane. From
the perikaryon thin processes which contain neurotubules can be traced in some
instances. They probably lead to the terminals. These terminals are enlarged
and filled with synaptic vesicles. On day 10 there are dendritic infoldings and
synaptic ribbons (Fig. 10). The terminals always touch other terminals laterally,
glial processes have never been found to separate them, though there are glial
processes in the receptor layer. Zonulae adhaerentes between these processes
and the terminals or between two terminals are often to be seen, as well as
similar junctions between receptor perikarya.
A great number of axonal processes passes through the bipolar layer. The
bipolar cytoplasm still contains mitochondria, Golgi elements and ribosomes.
In the amacrine cells there are large endoplasmic lamellae contacting the nuclear
envelope. The large intercellular spaces are filled completely with small axons
Development of retina in Tilapia
3 days
253
6 days
Fig. 12. Schematic drawing of the differentiation of the functionally important
structures in the developing retina. On day 3 only ganglion cells produce dendrites
and neurites. On day 6 all the important structures are represented: neurites and
dendrites have been formed, but the synapses have not yet achieved their mature
structure. The receptor cells form inner and outer segments and synaptic vesicles.
Horizontal cells appear. On day 12 these structures have matured and multiplied,
the retina is' ready', additional formations are the twin cones and dendritic invaginations and synaptic ribbons in the receptor terminals.
a, Amacrine cell; b, bipolar cell; c, cone; g, ganglion cell; /?, horizontal cell;
r, rod; re, undifferentiated receptor cell. The mature synapses have been characterized by a black thickening in the synaptic cleft.
(Fig. 11) and synaptic structures. The outer plexiform layer where the number
of neural processes has increased, essentially consists of dendritic processes. In
the inner plexiform layer dendrites and neurites often occur in groups. There is
no change with respect to the preceding stages. The same holds for the ganglion
cells.
DISCUSSION
It is the general opinion that retinal maturation proceeds vitreo-sclerad. This
conclusion has been reached from light-microscopic and radioautographic
observations (for a recent example see Hollyfield, 1972). Olney (1968a, b), however, could show by electron microscopic and physiological studies, that at least
some functions in the mouse retina mature in the opposite direction. The present
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G. GRUN
study leaves little doubt that differentiation is directed vitreo-sclerad. The
ganglion cells are the first to contain endoplasmic reticulum and after day 6
they show no further change. They are the first to produce neurites and dendrites.
Up to day 5 the inner plexiform layer mainly consists of dendrites and it is only
after this day that the number of axons from the intermediate neuron layer
increases markedly. In the ganglion cells there appear ribosomal caps 12 h
before similar caps appear in the bipolar cells. Between days 6 and 8 the ribosomes and the mitochondria of the bipolar cells show a tendency to decrease,
maybe the embryonic period of these cells has finished. The scleral-lying horizontal cells are the last of the intermediate neurons to differentiate. The synapses
of the inner plexiform layer seem to be mature before those of the outer plexiform
layer. The receptor cells are the last cells to differentiate, although they start
rather early. The outer and inner segments, the twin cones, and the synaptic
infoldings and ribbons are the very last structures to be finished.
The outer plexiform layer, the intermediate neuron layer and the receptor
nuclear layer seem to differentiate more slowly than ganglion cells and they do
so over most of the embryonic period.
In spite of this vitreo-scleral direction it is difficult to establish a similar order
for the achievement of the different functions, at least by morphological criteria.
There are synaptic structures to be found rather early in the inner plexiform
layer, but they do not seem to be mature before day 8 or 9 (according to the
criteria given by Glees & Sheppard, 1964). Since there is a connexion between
the development of reflex response and synaptic ultrastructure, as was shown
by Bodian, Melby & Taylor (1968), one must assume that the synapses are not
able to function before day 8. According to Hamburger & Levi-Montalcini
(1950) synapses may be necessary for growth and differentiation of neurons. So
they could exist without necessarily indicating neurophysiological maturity.
While cell differentiation and nerve fibre formation occur during the whole
of development, the important landmarks, such as membrane discs and receptor
terminal synapses, develop only in a restricted time.
The receptor outer segments start their development between day 5 and 6
and the single membranes attain their final aspect within a few hours (Grim,
1974). But as there seems to be a complicated biochemical and structural system
to be formed, it might be supposed that the ability to function is achieved
only later, presumably not before day 9. The dendritic invaginations which
are the final connexion between the receptor cell and the rest of the retina do
not appear before day 10. This is the last step of functional development.
Further development is concerned with the multiplying of outer segment membranes, inner segment mitochondria, and synaptic vesicles in the terminal.
Similarly the non-neural part of the eye, the pigment layer, the lens, etc., seem
to have matured before day 12. So we come to the following sequence of development. (1) Ganglion cells; (2) over a longer period: intermediate neurons,
plexiform layers and receptor inner segments; (3) receptor outer segments,
Development of retina in Tilapia
255
receptor terminals and horizontal cells; (4) outer plexiform layer and receptor
synapses, twin cones and receptor mosaic.
This sequence, however, does not represent a strict temporal order, various
developmental processes occurring in different layers at any given time. Even
in the single receptor cell there are various stages of differentiation, the perikaryon, the inner segment, the outer segment and the terminal starting and
finishing at different times and overlapping for certain periods.
Therefore it is difficult to determine the time when the whole retina is able to
receive and transmit light impulses. The ability to see, as inferred from behaviour,
is not achieved before day 13. This is also the time of leaving the mother's mouth.
Perhaps the retina could already perform its task on day 9 and it is only the
tectum opticum which is not yet differentiated. But to quote Angevine (1970)
we 'have to assume that the final ability is not achieved before the receptor
synapses are fully differentiated'. Here biochemical and neurophysiological
studies are required. The early occurrence of neural processes from the ganglion
cells possibly might indicate that they exert an influence on the tectum opticum.
An influence of this kind has been reported for developmental stages by Eichler
(1971), Kelly & Cowan (1972) and Schmatolla (1972), to cite only some recent
papers. It has been shown, repeatedly, that protein and other molecules are
transported from the ganglion cells to the tectum opticum (Sjostrand, Karlsson
& Marchisio, 1973; Crossland, Cowan & Kelly, 1973). Jacobson (1968) has
shown that in Xenopus the polarization of the eye which underlies the phenomena
of neuronal specificity occurs early in development and it seems to be so that
the axons of the optic ganglion cells establish an early pattern of connexions
between the retina and the optic tectum.
The author wishes to thank Dr M. Brestowsky for adult Tilapia specimens and important
advice, as well as Miss A. Kastle and Miss M. Schneiders for skilful technical assistance.
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{Received 9 September 1974)