J. Embryo!, exp. Morph. Vol. 65, pp. 27-39, 1981
Printed in Great Britain © Company of Biologists Limited 1981
27
Effects of concanavalin A on developing ganglion
cells in the retina of chick embryos
By K. MELLER 1
From the Institut fur Anatomie, Arbeitsgruppe fur Experimented Cytologie
Ruhr-Universitdt Bochum
SUMMARY
The administration of concanavalin A (Con A) (50-200 /^g/egg) to chick embryos between
the third and the seventh day of incubation has the following effects on the retina:
(1) Con A causes the degeneration of a large number of ganglion cells and consequently
the layer that should be formed by these cells is not present or is constituted only by a small
number of ganglion cells.
(2) The lectin seems to be effective only when it is administered during the postmitotic
phase of the ganglion cells.
(3) The degenerated cells are phagocytosed by the Muller cells in a manner similar to that
occurring during the natural cell death in normal retinal development.
(4) The differentiation of other retinal elements (photoreceptors, bipolar, amacrine and
Muller cells) is not affected by the lectin administration.
(5) The administration of Con A in later stages of development, even at ten times higher
dosages (2000 /tg/egg), fails to affect retinal neurogenesis.
It is suggested that Con A binding to receptor sites of the cell membrane affects the distribution or mobility of surface components producing an alteration in the mechanism by which
the developing cells regulate positional information during retinal neurogenesis.
INTRODUCTION
The neurogenesis of the nervous system includes a number of events distinguishable as cell proliferation, migration, cytodifFerentiation and the establishment of specific connexions (Cowan, 1979).
All these processes occur during periods of development characteristic for
each of the different regions of the nervous system. Thus the neurogenetic
sequence begins in the medulla earlier than in the retina and the development
of the cerebellum in the chick embryo takes place in the later stages of incubation
and continues through the first days after hatching. The major events of cell
proliferation occur in the chick retina during the first week of incubation. The
postmitotic ganglion cells reach their final position or layer beneath the vitreal
surface on the seventh to eighth day of incubation and the same time the inner
plexiform layer becomes discernable (Meller, 1968; Kahn, 1973, 1974; Meller &
1
Author's address: Institut fur Anatomie, Arbeitsgruppe fur Experimentelle Cytologie,
Ruhr-Universitat Bochum, Universitatsstr. 150, 4630 Bochum 1, Federal Republic of
Germany.
28
*
*
Con A effects on developing chick retina
29
Tetzlaff, 1976). During these processes the developing retinal cells are organized
in columns and this fact suggested that the cell-to-cell contacts in a given column
facilitate the transfer of the necessary positional information for correct
migration so that the cells can reach their destination at the right time (Meller,
1979a). Cell migration, cell recognition and the formation of specific contacts
are probably dependent upon some properties of the cell membrane (Roseman,
1970; Roth, McGuire & Roseman, 1971; Roth, 1973). Some authors (Brunngraber, 1969; Barondes, 1970; Gombos et al, 1978) have proposed that glycoprotein molecules situated on the cell membrane act as information molecules in
contact phenomena that take place in the developing nervous system especially
at the time of migration and synaptogenesis. In addition, the localization of such
recognition molecules changes during development preparing the cell membrane
for specific processes of cell recognition. Plant lectins, such as Con A, binding to
sugar molecules of the cell membrane caused aggregation of embryonic cells
depending on their stage of differentiation (Moscona, 1971; 1974). Various
types of lectins show a binding specificity of cells of different brain regions
(Hatten & Sidman, 1978). Furthermore, the linkage of lectins to the cell membrane produces alterations in the migration of cells as described by Moran
(1974a, b) in the developing neural crest, cessation of morphogenetic movements in amphibian embryos (Boucaut et al. 1979) and the inhibition of the
closure of the neural tube (O'Dell, Tencer, Monroy & Brachet, 1974; Lee,
1976).
The present work studies the effects of the lectin Con A on the development
of the chick retina. Con A causes degeneration of ganglion cells in an early
stage of morphological differentiation when it has been administered during the
postmitotic period. Differentiation processes in other retinal cells, however, are
not affected.
MATERIALS AND METHODS
Concanavalin A (Sigma, Miinchen) (50-200, and 2000 /*g/egg in Hanks) was
injected into the amnionic cavity of 3- to 15-day-old chick embryos. After
various incubation times, the embryos were fixed either in a glutaraldehyde/
paraformaldehyde or a 1 % osmium tetroxide solution for electron microscopy
or in Bouin's fixative for light microscopy. Embryos older than 14 days were
perfused through the heart. Paraffin embedding and routine electron microscopical techniques (Alcohol dehydration, Epon embedding) were used. A total
of ca. 600 eggs were injected for this investigation. Less than 50 % of the
Fig. 1. (a) Semithin section of the retina of a 14-day-old embryo (x 1000). (b, c, d)
Retina of a 14-day-old embryo that received a dose of 200 /tg Con A on the seventh
day of incubation. Observe the malformation of the ganglion cell layer and the
necrotic cells in the bipolar layer (x 1000). (e) Oil immersion of the vitreal portion
of the retina of a 14-day-old embryo, treated as in b, c, d. Observe the phagocytosed
material in the Muller cell feet (x 2000).
2
EMB 65
К. MELLER
10/um
а
Con A effects on developing chick retina
31
embryos injected with Con A in saline solution survived. Lethality was independent of the dose employed and the stage of development at the moment of injection. Only 30 % of the injected surviving embryos treated in the first week of
incubation exhibited Con A effects in the retina. No effects on the retina were
observed in the surviving embryos injected in the second (8-15) week of incubation. No macroscopical malformations were observed.
RESULTS
The most important events of cell proliferation and migration in the chick
retina occur during the first week of incubation. The formation of the inner
plexiform layer takes place between the seventh and the eighth days as a consequence of the migration of the ganglion cells. Synaptic development begins on
the 14th day and continues until hatching. The morphological differentiation of
receptors, bipolar, amacrine and Miiller cells takes place during the final third of
incubation (for details see Meller, 1964; Meller & Breipohl, 1965; Meller,
1968; Meller & Tetzlaff, 1976).
The effects of Concanavalin A
Figure 1 b shows an overview of a portion of a retina of a 14-day-old chick
embryo that was treated on the seventh day of incubation with a dose of 200 jug
Con A. A comparison with the control (Fig. 1 a) clearly demonstrates the following effects:
(I) Abundant cell degeneration localized in the basal portion of the bipolar
cell layer. Numerous pycnotic nuclei are situated between the bipolar cells
(Figs. 1 b, c).
(II) Ganglion cells are not present in the habitual position and normal
number. Examination of the whole retina in serial sections shows that this is
true for most of it, although single or groups of ganglion cells are visible in their
habitual position. The finding of isolated emigrated ganglion cells seems to be
fortuitous. They are found in irregularly distributed areas.
(III) The development of bipolar cells seems to be unaffected. Photoreceptor
cells (Fig. Id) developed inner segments characteristic of this stage.
(IV) In further stages of development prior to hatching (Fig. 2), the necrotic
material can only be observed in the basal prolongations of the Miiller cells or in
the extracellular spaces near the vitreal surface (Fig. 1 e). Ganglion cell layer and
fiber layer are missing (Fig. 2).
The electron microscopical investigation confirms the main aspects of these
light microscopical observations. Figs. 3 and 4 show overviews of the retina in
Fig. 2. (a) Paraffin section of a retina of an 18-day-old embryo, control
(x 1200). (b) Semithin section of a retina of an 18-day-old embryo injected on the
third day of incubation with 200/^g Con A. The ganglion cell layer is missing
(xl200).
2-2
32
K. MELLER
;
\f.
"V/ * •{ . *•• '"\\
Con A effects on developing chick retina
33
the stages of 12 and 17 days of incubation, respectively. Both embryos were
injected on the seventh day of incubation with a dose of 200 fi% Con A. One can
observe the absence of a ganglion layer. A considerable number of pycnotic
nuclei, fragments of cell cytoplasm of degenerated cells and osmophilic bodies of
unidentifiable material are present at the 12-day stage (5 days after injection)
between the bipolar cells (Fig. 5 a). Figures 5(6), (c) demonstrate the necrotic
material phagocytosed by the prolongations of the Miiller cells and accumulated in their basal feet. The development of the outer plexiform layer, especially
of the synapses between receptor and bipolar cells is similar to that in controls,
whereas the synaptic organization of the inner plexiform layer is obviously
altered by the absence of most of the ganglion cells. These alterations will be the
subject of a separate study. Con A did not affect the ganglion cells, even at a
ten times higher dose (2000 /*g/ml), when it was administered after the 8th day
of incubation.
DISCUSSION
Con A administration causes the degeneration of developing ganglion cells
in the retina and consequently the malformation of the ganglion cell layer with a
high degree of cell loss. As a result the fibre layer is also poorly developed.
This effect is brought about when the lectin is administered at a determined
stage of development, in this case the postmitotic period in the differentiation of
these cells. Histoautoradiographic (Kahn, 1974) and morphological data
(Meller & Tetzlaff, 1976) show that the transition from the proliferation phase
to the postmitotic phase occurs in the ganglion cells between the third and the
fifth day of incubation and the inner plexiform layer, a consequence of ganglion
cell migration, becomes discernible between the seventh and the eighth day of
incubation. Only during this limited period of time does Con A cause the
degeneration of developing ganglion cells. The fact that the retinae of only 30 %
of the surviving embryos are affected cannot yet be explained. A precise understanding of the mode of Con A incorporation in the living embryo requires
further investigation. Inhibiting effects of Con A on morphogenesis have been
reported in early stages of neurogenesis. Moran (1974a, b) described an inhibition of gastrulation and neurulation in amphibian embryos depending on
the concentration of the lectin. Such inhibition of neuronal differentiation was
also found after Con A administration by Lamon & Duprat (1976) although
other cells such as myoblasts, epithelial cells and melanophores are less affected
by the lectin. Con A blocked neural tube formation in explanted chick embryos
but somite and heart development and blood island formation were usually
unaffected (Lee, 1976). Similar results by Boucaut. et al. (1979) in amphibian
Fig. 3. Electron microscopical overview of the retina of a 12-day-old embryo
injected on the seventh day of incubation with a dose of 200 fig Con A (x 1600).
34
K. MELLER
Fig. 4. Electron microscopical overview of the retina of a 17-day-old embryo
injected on the seventh day of incubation with a dose of 200 /<g Con A (x 1200).
Con A effects on developing chick retina
35
embryos were interpreted as a consequence of a masking by the lectin of the
glycoproteins related with cell recognition and migratory activity.
In all of these experiments, the apparently higher degree of sensitivity of the
developing nervous structures to the action of the lectin still remains inexplicable. Kleinschuster & Moscona (1972) postulated the existence of different
receptor sites for lectins during development. The receptor sites might be masked
during maturation with a trypsin-sensitive material making them less accessible
for interaction with Con A. In this case the changes in the membrane surface
would be correlated with the developmental process. Subsequently Martinozzi &
Moscona (1975) suggested that the most essential feature distinguishing
embryonic cells from adult ones is not the masking of receptors but changes in
the fluidity of the cell membrane affecting the mobility and topographical
distribution of the receptors. Krach, Green, Nicolson & Oppenheimer (1974)
who worked with agglutination assays, proposed that receptor binding sites
change during development and maturation and their different topography
would affect the social behaviour of the cells during organogenesis. Evidence of
different responses of diverse cells of the neural crest to lectin, depending on the
stage of differentiation, were also detected by Sieber-Blum & Cohen (1978).
Some authors (Pfenninger & Maylie-Pfenninger, 1975; Pfenninger & Rees,
1976; Vaughn, Henrickson & Wood, 1976) described changes in the glycoprotein composition of the cell membrane in developing neuroblasts and in the
portion of the cell membrane in the receptor cells that forms synaptic contacts
(McLaughlin & Wood, 1977) and during brain development (Margolis &
Gomez, 1974). Changes in the glycoprotein patterns were also detected with
other lectins such as wheat germ agglutinin (WGA) and ricin (RCA) during the
development of the chick retina between the seventh and thirteenth day of
incubation (Mintz & Glasser, 1978). Despite these investigations, the question of
whether Con A receptor sites are directly responsible for cell adhesion remains
open (Steinberg & Gepner, 1973). In this connexion Zanetta et al. (1978)
demonstrated an affinity of the parallel fibres to Con A during the major events
of cerebellar development and suggested that the 'recognition molecules'
contribute to form a preliminary type of contact between complementary
neuronal surfaces. The presence of temporary gap junctions during the early
stages of retinal neurogenesis has been interpreted as the morphological basis of
a mechanism whereby the developing cells reorganize or regulate positional
information during migration (Sheffield & Fischman, 1970; Dixon & CronlyDillon, 1972; Jacobson, 1976; Fujisawa, Morioka, Watanabe & Nakamura,
1976). Possibly the uncoupling of these contacts between the developing ganglion cells under the influence of Con A leads to the degeneration of the cells.
This remains to be verified in vivo because, as demonstrated in vitro, Con A
partially inhibits the formation of specific contacts such as gap or tight junctions
(Meller, 1979 b). From the data provided by the studies with lectins and the
present results it can be said that the macromolecular characteristics of the cell
36
K. MELLER
Con A effects on developing chick retina
37
membrane, especially the glycoproteins in the membrane play a decisive role
during the neurogenesis.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Me 276/9),
from the Stiftung Volkswagenwerk (Az 11 2977) and from the Ministerium fur Wissenschaft
und Forschung des Landes NordrheinWestfalen (II B 5-FA 7470). The author thanks R.
Maeder and E. Descher for the excellent technical assistance and K. Donberg for preparing
the photographs. I also thank K. Rascher for her help with the English translation and C.
Bloch for typing the manuscript.
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(Received 11 November 1980, revised 12 April 1981)