/. Embryol. exp. Morph. Vol. 34, 3, pp. 531-557, 1975
Printed in Great Britain
531
Immunochemical analysis of
water-soluble antigens of chick retina in the
course of embryogenesis
By A. T. MIKHAILOY 1 AND V. M. BARABANOV 1
From the Laboratory of Embryology, Institute of Human
Morphology of the Academy of Medical Sciences of the USSR,
Moscow
SUMMARY
Water-soluble antigens of chick retina were investigated using rabbit antisera to total
extract and to individual electrophoretic fractions of retinal extract by methods of immunoelectrophoresis and Ouchterlony precipitation test. In the retina of the adult chick six serum
and eleven tissue antigens were demonstrated. The tissue antigens of the retina comprised
one organ-specific antigen and ten inter-organ antigens which were characterized by nonuniform distribution in tissues and organs of adult chick. Three antigens out of these were
found only in tissues of the eye (retina, iris) and in the brain - inter-organ antigens of
'narrow' specificity. The other seven inter-organ antigens were found in tissues of brain
and eye, as well as in various tissues and organs of hens - inter-organ antigens of 'broad'
specificity.
A high degree of antigen similarity between retina and iris was observed. Anti-retina sera
in chick lens could detect only inter-organ antigens of 'broad' specificity.
In the course of embryogenesis the first to appear in the developing retina were interorgan antigens of 'broad' specificity (on 3rd day of incubation). Formation of antigens of
this group was completed by the 9th day of incubation. On the contrary, inter-organ antigens
of 'narrow' specificity appeared later, in the period of histogenesis of retina (from 5 to 18
days of incubation). The organ-specific antigen of retina was found by 7th day of incubation.
One of the inter-organ retinal antigens of'narrow' specificity (retina-iris-brain) appeared
in the developing chick brain at the same time as in retina - on 10th—11th day of incubation.
Using the indirect immunofluorescence antibody technique this antigen was identified in the
cytoplasm of retinal cells and brain neurones, but was not detected in the nerve fibres.
INTRODUCTION
The developing eye rudiment in vertebrates represents a convenient model for
investigating tissue interactions in the course of organogenesis. The interrelations between the developing lens and retina have always attracted many
investigators (Lopashov, 1961, 1963; Lopashov & Stroeva, 1963; Muthukkaruppan, 1965; Philpott & Coulombre, 1968; Lopashov & Hoperskaya,
1970). New possibilities for gaining deeper insight into the problem of changes
1
Authors' address: Institute of Human Morphology, Academy of Medical Sciences
of USSR, Tsuryupa St., 3, Moscow, 117469. USSR.
34
EMB
34
532
A. T. MIKHAILOV AND V. M. BARABANOV
in retina and lens during differentiation arose through application of immunological methods to experimental embryology (Woerdeman, 1962; Vyazov,
1962). It should be emphasized that immunoembryological investigations of the
vertebrate eye have concentrated on studies of lens antigens, whereas antigenic
properties of retina have been studied insufficiently.
Various immunological methods have been used to analyse retinal influences
on lens: immunofluorescent method (Nace & Clarke, 1958; Fowler & Clarke,
1959), precipitation technique (Woerdeman, 1953, 1962) and cytotoxic test
(Clarke & Fowler, 1960; Langman & Maisel, 1962; Langman, Maisel & Squires,
1962). Insufficient attention in previous studies was paid to the problem of antigenic structure of the retina itself during the early stages of development. These
studies failed to ascertain the number and tissue specificity of antigens present in the presumptive retina. No data were available on the process of
antigenic differentiation of retina during embryogenesis. Moreover, the view
advocated by a number of authors (Fowler & Clarke, 1959) that lens antigens
may be detected in the forming optic cup is disputable and needs further
verification.
In adult animals the antigenic structure of retina in contrast to the lens has
been studied insufficiently. Some data on antigenic properties of the retina in
vertebrates were obtained mainly in studies of the role of retinal antigens in
autoimmune damage of the eye tissues (Wacker & Lipton, 1965,1967, 1968 a, b;
Wacker & Barbee, 1968; Lerner, Stone, Meyers &von Sallmann, 1968; Aronson,
1968; Wacker, Barbee & Macdonald, 1969; Wacker & Lipton, 1971; Kalsow &
Wacker, 1973). Indirect data on the retinal antigens common to other tissues
may be found in studies where retina was used as a test-antigen in order to
determine the organ specificity of antigens of choroid coat (Nozaki, Foster &
Sery, 1964), of lens (Langman & Prescott, 1959; Maisel, 1962, 1963; Clayton,
Campbell & Truman, 1968) and of brain (McCalion & Langman, 1964; Wenger
& Friedman, 1970). Only a few investigations have been devoted to immunochemistry of the retina proper (Hess & Romer, 1906; Perkins & Woods, 1963;
Tilgner, Meyer, Hempal & Schroder, 1973), and particularly to the antigenic
structure of its photoreceptor layer (Dewey, Davis, Blarie & Barr, 1969;
Rahi, 1970; Schillinger & Homberg, 1972). All these works were carried out on
various species using different conditions of experimentation. This impedes a
comparison of data obtained by different authors and prevents formulation of
a general concept concerning the spectrum of retinal antigens and their tissue
specificity.
As is known, the retina is a derivative of the brain and in structure and organization is similar to it (Zavarzin, 1941; Vinnikov, 1947). Therefore, it could be
assumed that there should exist antigenic similarity between retina and brain.
If so, an analysis of antigenic composition and differentiation of retina will make
it possible to identify certain common stages in the development of the nervous
tissue during embryogenesis.
Analysis of water-soluble antigens of chick retina
533
The present study was dedicated to investigation of antigenic differentiation
of chick retina during embryogenesis. The objectives of the work were: (a) to
analyse the antigenic composition of chick retina and organ specificity of its
antigens; (b) to investigate formation of antigens specific for adult chick retina in
embryogenesis; (c) to compare the antigenic properties of chick retina and
brain and to establish the pattern of antigenic formation of these tissues in
embryogenesis.
MATERIAL AND METHODS
The studies were conducted on hens of Russian white breed. Chick embryos
of 1-20 days of incubation and 1- to 3-day-old chickens were used.
Experimental material
The retina of adult hens and chick embryos was isolated from the posterior
parts of the eye only and contained therefore the most differentiated cellular
elements (Coulombre, 1955; Shen, Greenfield & Boell, 1956). In the process of
retinal isolation optic nerve and pecten were removed. Retinae were isolated
simultaneously from 300-400 eyes.
The retina was homogenized in five volume equivalents of distilled water
brought to pH 8-6 with ammonia solution. Extraction was carried out within
24 h at 4 °C. The retinal extract was centrifuged at 6000 g for 60 min and then
it was lyophilized. The yield of lyophilizate was 4-5-6-0% of the wet weight of
the tissue. The lyophilizate was dissolved in 0-1 M of tris-buffer (pH 8-6) up to a
concentration 40-50 mg/ml. Extracts from other tissues and organs of hens
(iris, lens, brain, liver, kidney, lung, spleen, cardiac and muscular tissues) were
prepared. In the same manner adult chick serum was also obtained and was used
either in lyophilized or native forms.
Embryos at different stages of development were obtained by incubation of
fertilized eggs in a laboratory incubator at 37-5-38-0 °C and relative humidity
60-70%. Stages of embryos were controlled according to Hamburger &
Hamilton (1951).
Retinae from embryos of 5 and more days of incubation were isolated without
pigmented epithelium. From embryos younger than 5 days of incubation the
primordial eye as a whole was taken, but in a number of experiments the optic
cup was separated from the lens. Heads and trunks of embryos 24 h old were
separated. The brain was removed from 10-, 11-, 12-, 15- and 18-day-old
embryos and anterior, middle and posterior segments of it were separated. For
obtaining embryonic tissues 4400 embryos were used.
Extracts from embryonic tissues were prepared in tris-buffer (0-1 M, pH 8-6),
the ratio of wet tissue weight to buffer being 1:3.
Concentration of protein in extracts was determined according to Lowry,
Rosebrough, Farr & Randall (1951).
34-2
534
A. T. MIKHATLOV AND V. M. BARABANOV
Preparation of immune sera
Total extract of adult chick retina was used for immunization of four rabbits
during 3-6 months; 1-5 ml of retinal extract were injected into animals intravenously, intraperitoneally and subcutaneously with Freund's adjuvant (Barabanov & Mikhailov, 1970; Mikhailov, 1973a).
Another group of rabbits (11 animals) were immunized with fractions of
retinal extract from adult chick. The fractions were obtained by means of preparative electrophoresis in agar gel (Barabanov & Mikhailov, 1970, 1971;
Mikhailov, 1973 a, b). Strips of agar with corresponding fractions were mixed
with Freund's complete adjuvant (Freund, 1947) and were injected subcutaneously into rabbits 2-4 times; the intervals between injections being 3-4
weeks. The technique of immunization by fractions of tissue extracts was
used also for obtaining antisera to antigens of anode fractions of chick
brain (two rabbits) and to antigens of retina of 5-day-old chick embryos (four
rabbits).
To obtain antisera of narrow specificity to individual retinal antigens, the
third group of rabbits (four animals) were immunized by the precipitate. The
precipitate was isolated in immunoelectrophoresis (Smith, Gallop & Tozer,
1964; Mikhailov, 1973a). Following 3-day washing in tris-buffer (0-1 M,
pH 8-6), agar stripes containing the precipitate of a corresponding retinal
antigen were emulgated with Freund's complete adjuvant and injected subcutaneously into rabbits once a week with 3-week interval between injections.
Antisera were obtained after two or three injections of the precipitate.
In this work 40 antisera from 25 rabbits were used.
Immunochemical methods of investigation of chick retinal antigens
Analysis of retinal antigens was carried out by immunoelectrophoresis in
1-25% agar gel (Bacto-Agar 'Difco') on plates 9 x 12 cm (Abelev & Tsvetkov,
1960; Kakpakov, Lysenko & Bashkaev, 1965) in tris-buffer system (0-1 M,
pH 8-6), with a gradient of voltage 4-6 V/cm and duration of electrophoresis
90-100 min. The relative electrophoretic mobilities of retinal antigens (mr)
were determined according to Zwaan (1963), assuming mobilities of albumin
and siderophilin of the human blood serum as 100 and 50 of arbitrary units
respectively.1
Another method used was the precipitation reaction in agar gel described by
Ouchterlony (1958). The modification of this method - double diffusion technique in agar gel with dilutions of antigens (Zilber & Abelev, 1962; Gusev,
1968) - was also applied. Dilutions of extracts from definitive tissues were prepared within the range of concentrations of lyophilizate from 40 to 0-035 mg/ml.
1
Mobility of antigens was calculated according to Zwaan (1963) mxr = mf-\mr
-
mfKAX/AS), where nfr is the mobility of retinal antigen, m* the mobility of albumin, mf
the mobility of siderophilin, AX the distance between albumin and retinal antigen mobilities and AS the distance between albumin and siderophilin mobilities.
Analysis of water-soluble antigens of chick retina
535
0
ANTI-I
ANTI-II
ANTI-III
ANTI-IV
ANTI-V
ANTI-VI
ANTI-VII
Fig. 1. Immunoelectrophoresis of adult chick retinal extract with antisera to
individual fractions of retinal extract. Extract of adult chick retina was introduced
before electrophoresis into all wells; in troughs - antisera to seven electrophoretic
fractions of adult chick retinal extract (ANTI-I-VII).
When embryonic tissues were used, dilutions were prepared from native fresh
extracts.
Determination of organ specificity of retinal antigens was carried out by
absorption of anti-retina sera (in immunoelectrophoresis) with extracts from
parenchymatous organs of adult chick after Bjorklund (1952), as well as by the
Ossermann immunoelectrophoretic technique (Ossermann, 1960).
Localization of retinal antigens in adult chick eye and brain was determined
by the indirect immunofluorescence antibody technique (Weller & Coons, 1954;
Engelhardt, 1968). Paraffin sections were prepared by the Sainte-Marie method
(1962) from adult chick retina, iris, great hemispheres, optic lobes, cerebellum,
medulla oblongata and spinal cord. Fluorescein isothiocyanate-conjugated
donkey antisera to rabbit y-globulin were used.
536
A. T. MIKHAILOV AND V. M. BARABANOV
Fig. 2. Double diffusion precipitation reactions of adult chick retinal extract with
antisera to individual electrophoretic fractions of retinal extract. In peripheral
wells - dilutions of adult chick retinal extract (40, 20, 10, 5, 2-5, 1-25 mg of lyophilizate in 1 ml); in central wells - antisera to seven electrophoretic fractions of
adult chick retina extract: (a) ANTI-I; (b) ANTI-II; (c) ANTI-III; (d) ANTI-IV;
(e) ANTI-V; (/) ANTI-VI; (g) ANTI-VII.
EXPERIMENTAL RESULTS
Antigenic spectrum of adult chick retina
The extract of chick retina reacting with antisera to the total extract formed in
lmmunoelectrophoresis from four to ten lines of precipitation. However, following the absorption of these antisera by chick serum after Bjorklund (1952) only
1-4 arches of low intensity in the anode and the middle zone of lmmunoelectrophoresis were retained. Thus, antisera to the total extract of chick retina
537
Analysis of water-soluble antigens of chick retina
Ib Ia
46
4a
\b
2
5
6
7
(a)
(b)
ANT1-II
(c)
Set of
antibodies
1 a, 1 b
2,4 a
ANTI-III ANTI-IV ANTI-V ANTI-VI ANTIVII
3,46,6
5,6
5,6,8
7,8
7,8,9
Fig. 3. Spectrum of adult chick retinal antigens identified with antisera to electrophoretic fractions of retinal extract, (a) Immunoelectrophoretic spectrum of
retinal antigens, (b) Electrophoresis pattern of total retinal extract, (c) Set of antibodies in antisera to seven electrophoretic fractions of retinal extract.
contained antibodies predominantly to serum antigens and a small set of antibodies to tissue retinal antigens.
A more diverse spectrum of tissue antigens was revealed in adult chick
retina with antisera to electrophoretic fractions of the retinal extract. Each of
these antisera contained a small set of antibodies and formed in immunoelectrophoresis (Fig. 1) and Ouchterlony precipitation reactions (Fig. 2) 2-3
distinct lines of precipitation. As may be seen from Fig. 1, some of the precipitation lines were similar as to their form and localization along the electrophoretic
axis, but they were formed in reactions with antisera to various fractions of the
retina (Fig. 1, sera ANTI-III-Y). This might indicate the presence of antibodies with similar specificity in different antisera. To characterize antibodies in
antisera a comparison between these antisera was made in Ouchterlony precipitation reactions under optimal retinal extract dilutions. These dilutions were
chosen in double diffusion precipitation reactions of retinal extract with antisera to individual electrophoretic retinal fractions (Fig. 2).
The results obtained showed that antisera to the anode fractions of retina
(ANTI-I, ANTI-II) contained no antibodies similar to those in other antisera.
On the contrary, antisera to the cathode fractions of the retina (ANTI-III—VII)
contained antibodies to a number of antigens of adjacent fractions. In adult
chick retina about eleven tissue antigens differing in mobility in electrophoresis
and in the form of precipitation lines were revealed (Fig. 3). The antigen with the
highest mobility was designated as antigen 1, and other retinal antigens were
numbered in accordance with the decrease of their electrophoretic mobility.
538
A. T. MIKHAILOV AND V. M. BARABANOV
Table 1. Relative electrophoretic mobilities (mr)
of chick retinal tissue antigens
Retinal tissue
antigens
Number of
measurements
la
lb
23
155-0 ±1-5
23
6
5
29
1550 ±1-5
115-7 ±0-4
79-5 ±0-4
113-1 ±1-0
1200 ±1-4
89-5±l-8\
85-1 ± 1-8X
43-9 ±0-6
35-6 ±0-2
25-4 ±0-8
161 ±0-4
91 ±0-2
Anode
Average
Cathode
2
3
4a
4b
4
12
24
5
18
6
45
9
37
10
7
8
9
Electrophoretic
mobility (M±m)
The data on relative electrophoretic mobilities (Table 1) of antigens show that
adult chick retina contains antigens with very low electrophoretic mobility
(antigen 9, m r = 9-1) as well as those with high speed of migration in electrophoresis, exceeding the mobility of blood serum albumin (antigen la, mr =
155-0, antigen 2, mr = 113-1). Retinal antigens with low and average electrophoretic mobilities formed in immunoelectrophoresis typical arches of precipitation. On the contrary, some of the antigens with high mobility (antigens
1 b, 2) formed lengthy precipitation lines of complex profile, which could indicate the presence in chick retina of electrophoretically heterogeneous populations of molecules with similar antigenic specificity. Thus, the precipitation
line of antigen 1 b consisted of three arches fusing at their ends, formed by
three electrophoretic retinal fractions with mobilities of 155, 115, and 80
arbitrary units respectively (Figs. 1,3).
The majority of retinal antigens, except antigen 1 a, differed from each other
in their electrophoretic mobility. The precipitation arch of antigen 1 a sometimes almost entirely merged with the anode arch of antigen 1 b, and an evidence
of the presence of antigen 1 a in the extract were small ' spurs' at the ends
of this arch. These antigens however were discernible in the homologous
precipitation reaction with dilutions of retinal extract (Fig. 2a). On this
basis we considered them as two independent antigens and designated them
as antigen \a and antigen \b. Another two antigens (4a and Ab) found in
retina also possessed similar electrophoretic mobilities, though according to
the data on crossprecipitation reactions they were not identical immunologically.
Tissue antigens described constituted the basis of the antigenic spectrum of
Analysis of water-soluble antigens of chick retina
539
Tissue extracts
Ami- serum
ANTI-I
1 a,
1 h
ANTI-II
ANTI-III
o
ANTI-1V
ANTI-V
5
***•»
6
ANTI-VI
Fig. 4. Cross-precipitation reactions (after Ouchterlony, 1958) between antisera to
electrophoretic fractions of retina and extracts of adult chick tissues and organs.
Results of four series of reactions between antisera I-VI and all the tissue extracts
are presented. Dotted line shows weak and additional precipitation lines.
adult chick retina. All these antigens were regularly found in retinal extracts
irrespective of methods, retinal extracts and antisera used.
Organ specificity of adult chick retinal antigens
To determine organ specificity of retinal antigens anti-retinal sera were
absorbed in immunoelectrophoresis after Bjorklund with extracts of brain,
liver, spleen, muscular, cardiac tissue and also with chick serum. The presence
of a retinal antigen in heterological extracts was identified by the disappearance
of a corresponding precipitation arch from the antigenic spectrum of the retina.
Parallel series of precipitation reactions with dilutions of antigens were
performed where each anti-retinal serum reacted with test-antigens from nine
tissues and organs of adult chick, including eye tissue. In cross reactions (after
Ouchterlony) similarity between antigens of retina and antigens detected in
other tissues was investigated (Fig. 4). As may be seen from Fig. 4 sera ANTJ-I,
ANTI-II reacted only with eye and brain tissues, whereas antisera which
contained antibodies to antigens of cathode retinal fractions formed precipitation lines with almost all tissue extracts.
540
A. T. MIKHAILOV AND V. M. BARABANOV
Table 2. Organ specificity of adult chick retinal antigens
Retinal antigens
Tissue and
organs
Retina
Brain
Iris
Lens
Liver
Spleen
Kidney
Muscle
Lung
Serum
,
la
A
lb
2
+
+
+
+
+
—
—
+
+
— - —
—
—
—
—
—
—
—
—
_
_
_
_
3
4a
4b
5
6
7
8
+
+
+
+
+
+
+
—
+
+
—
—
—
+
+
+
—
- _
_
+
—
—
—
—
—
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
_
*
_
*
9
+
+
+
+
+
_
_
+, Antigen is present in the extract.
—, Antigen is absent in the extract.
*, Retina antigens are similar to serum antigens.
., Experiments were not conducted.
The results of these experiments (Table 2) showed that tissue antigens detected
in retina were characterized by their differing distributions in other chick
tissues and organs. Only one antigen, 4b (mr = 85-1), among antigens revealed
in chick retina, could be considered organ-specific, since it was present in
retina and was absent in extracts of other tissues. The other ten antigens were
detected not only in retina but in various organs of adult chick as well. In
accordance with terminology accepted in our laboratory we called these ten
antigens inter-organ retinal antigens (Barabanov, 1966, 1967; Vyazov, Barabanov & Mikhailov, 1970, 1971). The majority of inter-organ retinal antigens
possessed an individual tissue specificity but they could be divided tentatively
into two groups. The first group consisted of inter-organ antigens la, lb, 2,
which represented a small but a very important part of the antigenic spectrum
of chick retina. These antigens, having a very high speed in electrophoresis
(mr in the range of 155-0-113-1), were found only in eye and brain tissues. Thus,
antigen la (mr = 155-0) was found in retina and brain; antigen lb (mr =
155-0-115-7-79-5) was found in retina, iris, brain. Antigen 2 (mr = 113-1) was
typical only of eye tissues, since its distribution was confined to retina and iris.
Based on these findings, we designated all these antigens as inter-organ antigens
of 'narrow' specificity to emphasize that these antigens reflect a qualitative
specificity for retina, iris and brain as a system of related tissues (Barabanov
& Mikhailov, 1970, 1971; Mikhailov, 1973 a, b). These three antigens were not
found in adult chick lens.
The second group of inter-organ antigens of retina included 3, 4a, 5, 6, 7, 8, 9
antigens, which were more widely distributed in chick tissues and organs. In
contrast to inter-organ retinal antigens of narrow specificity, almost all these
Analysis of water-soluble antigens of chick retina
541
Fig. 5. Immunoelectrophoretic analysis of tissue specificity of adult chick retinal
antigen 6 by Ossermann method. In wells: extract of adult chick retina; in troughs:
(a) adult chick serum (35 mg of protein/ml); (b) antisera to the IV fraction of adult
chick retina extract; (c) extract of adult chick retina (2-5 mg of protein/ml). The
arrows show antigen 6 precipitation arch.
antigens were characterized by moderate or low speeds of migration in electrophoresis (mr was in the range of 120-0-9-1). Some of them (antigens 5, 6, 7, 8)
were present in all tissues and organs of adult chick studied. All these antigens,
with the exception of antigen 6, were absent in blood serum, which allowed us
to define them as inter-organ tissue antigens of 'broad' specificity (Table 2).
The antigen 6 in a number of cross-precipitation reactions showed similarity to
serum antigens. Analogous data were obtained on specificity of antigen 6 in
immunoelectrophoresis by Ossermann method, where extract of chick retina
(2-5 mg of protein/ml) and chick serum (35 mg of protein/ml) served as testantigens. Both test-antigens formed in agar precipitation lines which merged
with the arch of antigen 6 (Fig. 5). However, the precipitation line formed by
retinal extract was more intensive than the serum line, although the retinal
extract contained lesser amounts of protein. This confirms the assumption that
retina, in contrast to serum, contains a higher concentration of antigen 6 and
therefore we considered it as a retinal tissue antigen of broad specificity.
Other retinal antigens, which were included in the group of inter-organ,
antigens of broad specificity (i.e. antigens 3, 4a), were found to have more
heterogeneous pattern of distribution in adult chick tissues. These antigens had
an 'intermediate' type of inter-organ specificity and could be detected in some
parenchymatous organs but not in all tissues of adult chick. So antigen 3 was
found in all organs with the exception of muscles; antigen 4a was found in
retina, kidney, liver and spleen (Table 2).
The results obtained show that the retina of adult chick is characterized by
a complex antigenic composition and contains serum antigens, inter-organ
antigens of broad specificity, antigens inherent only in eye and brain tissues,
and an organ-specific antigen.
542
A. T. MIKHAILOV AND V. M. BARABANOV
Table 3. Formation of adult chick retinal tissue antigens
in embryogenesis
Retinal tissue antigens
Days of
incubation
K
,
N
la
lb
1
3
5
_
-
_
9
11
13
15
16
18
20-21
Adult
+
+
+
+
+
+
+
+
+
+
2
_
3
_
4a
_
4b
_
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
5
6
7
8
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+, Antigen is present.
—, Antigen is absent.
Formation of adult chick retinal tissue antigens in embryogenesis
Studies of antigenic differentiation of the retina were carried out on embryos
of 3-20 days of incubation and in some tests on embryos of 1-2 days of incubation. These periods of development covered both early stages of development
of the retina and the whole period of its histogenesis, which in chick embryos,
according to Coulombre (1955), begins on the 4th-5th day of incubation and is
complete in principle by the 15th day of incubation. Extracts of presumptive
retina from embryos of various stages of development were investigated with
immuno-electrophoresis and by Ouchterlony precipitation test with antisera
to every individual fraction of adult chick retina.
The results of these experiments (Table 3) show that formation of definite
retinal antigens proceeds for a long time and is complete by the end of embryogenesis. Moreover, certain relationships between the appearance of individual
antigens and the pattern of their organ specificity were observed. The first to
appear in the developing retina were inter-organ antigens of broad specificity
(antigens 5, 6, 7, 8). They were detected in eye rudiments at the stage when the
optic cup is almost completely formed - on the 3rd day of incubation. Furthermore, antigen 8 of this group could be detected as earlier stages of embryogenesis. Using monospecific antiserum, antigen 8 was detected in the head
segment of embryos 24 h old (Fig. 6). However, in presumptive retina antigen 8
was detected only on the 3rd day of incubation. Thus, tissue antigens of broad
inter-organ specificity originate in presumptive retina simultaneously, at early
stages of its development, before the histogenesis of this tissue. Antigens with
intermediate type of inter-organ specificity, antigens 3 and 4 a, were formed in the
Analysis of water-soluble antigens of chick retina
543
Fig. 6. Identification of antigen 8 in head part of chick embryos 24 h old. In upper
well - homogenate from tissues of the head part of embryos 24 h old; in lower
well-extract of adult chick retina (control); in trough -monospecific antiserum
to antigen 8.
developing retina later - on the 5th and 9th days of incubation respectively, which
coincided in time (Coulombre, 1955) with the beginning of the histogenesis of
retina and with the period of its division into layers. Consequently, by the 9th
day of incubation all inter-organ antigens of broad specificity were present in
the presumptive retina. On the other hand, antigens localized only in eye and
brain, as well as the retinal organ-specific antigen, appear at later stages of the
development (5-18th days of incubation) when histological differentiation of
chick retina occurs (Table 3). Antigen 2 (retina-iris), in particular, was detected
in retina at the initial phase of its histogenesis - on the 5th day of incubation.
Antigen \b (retina-iris-brain) was detected for the first time in retina of 10- to
11-day-old embryos, i.e. during the period of intensive histological differentiation, of retina, when a layer of future photoreceptor cells was being formed
(Coulombre, 1955; Vinnikov, 1964). Finally, inter-organ antigen la of narrow
specificity (retina-brain) was identified in retina on the 18th day of incubation,
Organ-specific antigen 4b could be revealed in the presumptive retina on the
7th day of incubation. This suggests, that by the moment of chick embryo
hatching, its retina already contains the entire spectrum of antigens typical of
definitive tissue (Table 3).
Analysis of antigenic differentiation of retina shows that at early stages
(3-5th days of incubation) the presumptive retina contains mostly inter-organ
antigens of broad specificity. Inter-organ antigens of narrow specificity and
organ-specific antigen are formed during the period of histogenesis of retina.
Consequently, they cannot determine the lens-inducing capacity of the optic
vesicle. According to the data reported by some authors (Nace & Clarke,
1958), the lens-inducing capacity of the retina of the vertebrate eye may be
associated with so-called 'transitory' antigens that disappear from the eye at
later stages of the development.
Thus, the spectrum of retinal antigens at the initial period of retinal histogenesis has been studied with antisera to embryonic retina. By immunoelectrophoresis with antisera to four electrophoretic fractions of 5-day-old chick
retinal extract it was established that the retina of 5-day-old chick embryo contained up to eight tissue antigens. These antigens were numbered in accordance
with the speed of their electrophoretic mobility (Fig. 7). The comparison
544
A. T. MIKHAILOV AND V. M. BARABANOV
1
2
3
4
p
r~ P p
©
41
100
5
P
£
6
P
7
8
r~
1
0
1
50
Fig. 7. Scheme of immunoelectrophoretic pattern of retinal antigens of 5-day-old
chick embryos. These results were obtained with antisera to four individual
electrophoretic fractions of retinal extract of 5-day-old chick embryo.
Table 4. Distribution of retinal antigens of 5-day-old chick
embryos in retina and other tissues of adult chick
Tissue and
organs of
adult chick
Retina
Brain
Liver
Spleen
Serum
Antigens of the retina of 5-day-old
chick embryos
1
2
3
4
5
6
7
8
+
+
_
_
—
_
*
_
+, Antigen is present.
—, Antigen is absent.
*, Immunological similarity between retinal and serum antigens.
between embryo antigens and adult chick retinal antigens showed that among
the eight antigens of the 5-day-old embryo retina there were inter-organ antigens
of narrow specificity (antigen 2) and of broad specificity (antigens 5, 6 and 8).
However, the total number of antigens revealed in the 5-day-old embryo
retina exceeded the number of antigens found using antisera to the definitive
retina in chick embryos of the same stage of development (Table 3, six antigens).
This suggested that the retina of 5-day-old chick embryos contained antigens
absent in the definitive tissue. To elucidate this question, absorption of antisera
to the presumptive retina (in immunoelectrophoresis) with extracts of definitive
tissues (Table 4) was carried out. The results obtained showed that all eight
antigens detected in the 5-day-old embryo retina were also found in the retina
and in a number of tissues of adult chick. Thus, in the presumptive retina at
early stages of its histogenesis we could identify only antigens of definitive
tissue.
Studies of antigenic properties of the retina of chick embryos show that all
antigens typical of definitive retina are formed already in the course of embryogenesis. It should be noted that the retina at early stages of development contains inter-organ antigens of broad specificity. On the contrary, the phase of
Analysis of water-soluble antigens of chick retina
545
Fig. 8. Immunoelectrophoresis of adult chick retina and brain extracts with antiserum to II fraction of brain extract, (a) Adult chick brain extract in reaction with
native antiserum. (b) Adu t chick retinal extract in reaction with native antiserum.
(c) Adult chick brain extlract in reaction with antiserum absorbed with retinal
extract in trough by the Bjorklund method.
retinal histogenesis is characterized by formation of both an organ-specific
antigen and inter-organ antigens of narrow specificity.
This pattern of antigenic differentiation of chick retina is in accord with the
dynamics of formation of the antigenic structure of brain tissue in embryogenesis. Chick brain organ-specific antigens and antigens specific only for the
retina, brain and spinal cord, are formed during histogenesis or at the end
of the development (McCallion & Langman, 1964; McCallion & Trott, 1964).
In this connexion it would be of interest to investigate the similarity of the
antigenic structure and antigenic differentiation between chick retina and brain.
Comparative analysis of antigenic properties
of chick retina and brain
Evidence of antigenic similarity between the retina and brain in adult chick
was obtained in the course of investigation of organ specificity of retinal
antigens (Table 2). The results of these experiments show that the retinal
antigenic structure is more similar to that of the nervous tissue than to any
other tissues of adult chick. Out of eleven antigens detected in retina, eight
antigens (antigens la, \b, 3, 5, 6, 7, 8, 9) were also in brain.
It should be emphasized that among these eight antigens there were two
antigens (antigens la, \b) specific only for brain and neural tissue of eye
(retina, iris).
Another six retinal antigens (antigens 3, 5, 6, 7, 8, 9) manifested the similarity between these tissues in a lesser degree, since they were found also in
many other tissues of adult chick (Table 2).
On the basis of the data obtained it may be concluded that chick retina and
brain are characterized by a great similarity in their antigenic composition. We
546
A. T. MIKHAILOV AND V. M. BARABANOV
Table 5. Appearance of antigen lb in the retina and brain of10- to
18-day-old chick embryos according to data on precipitation
reactions {after Ouchterlony)
Tissues of chick embryos
c
\
Brain segments
Days of
incubation
Retina
Anterior
Middle
Posterior
10
11
12
13
15
18
±
+
+
+
+
+
—
—
—
±
+
—
—
±
+
+
±
±
±
+
+
+
4-, Obvious reaction.
±, Weak reaction.
—, No reaction.
considered the common antigens 1 a and 1 b to be specific for the central nervous
system.
These data were confirmed also by the analysis of antigenic properties of
retina and brain with antisera to anode fractions of adult chick brain extract.
In immunoelectrophoresis these antisera revealed a number of antigens in
brain, but following their absorption with retinal extract no precipitation lines
appeared in agar (Fig. 8). These observations indicate the serological identity of
antigens of the anode fractions of adult chick brain and retina. Moreover, in
cross-precipitation reactions it was shown that one of the brain antigens
detected with antiserum to the first brain fraction was identical to retina antigen lb.
Analysis of the retinal antigenic differentiation showed (Table 3) that synthesis of antigens 1 a and 1 b coincided in time with the phase of advanced
histogenesis of retina but not with the stage of vesicle formation. The questions
to be answered were as follows: when do these antigens arise in the developing
brain, and with differentiation of which segments of brain their synthesis in
embryogenesis is associated ?
In this connexion an analysis (by Ouchterlony precipitation test) was carried
out for antigen \b on 10- to 18-day-old chick embryos. It was established that
antigen 1 b appeared in the brain posterior segment at the same time as in the
retina, i.e. on the 10th - 1 Ith days of incubation. In the presumptive brain
this antigen was synthesized asynchronously: in the middle segment it was
detected on the 13th day, but in the anterior brain segment on the 15th day of
incubation (Table 5). Consequently, it may be assumed that \b antigen formation occurs from posterior to anterior parts of the developing chick brain.
Analysis of water-soluble antigens of chick retina
547
Fig. 9. Section of adult chick retina and other eye tissues treated with antiserum to
antigen 1 b and with fluorescein-conjugated antiserum to globulin fraction of
rabbit serum, (a) Test: section + antiserum to antigen 1 b + fluorescein-conjugated
antiserum. Fluorescence only in retina (R); no fluorescence in pigmented epithelium (PE), nor in vascular (VC) and scleral (SC) coats, (b) Control: section+
normal rabbit serum + fluorescein-conjugated antiserum. No fluorescence on
section, x 30.
The formation of antigen 1 b in retina and in brain at relatively late stages
of their histological differentiation indicates that, in the antigenic structure of
these tissues, identical changes occur and that these changes take place synchronously.
By the indirect fluorescence antibody technique localization of antigen \b in
the eye tissues (retina, iris) and in various parts of brain and spinal cord of adult
chick was studied. Antigen 1 b was localized in retina and was absent in pigmented
epithelium, vascular and scleral coats (Fig. 9). Antigen 16was distributed predominantly in inner segments of photoreceptors of the retina where the most
intensive fluorescence was observed. The bordering areas of the inner nuclear
and inner plexiform layers also had specific fluorescence. Intensive specific
fluorescence was observed also in the layer of retinal ganglionic cells (Fig. 10).
In the iris the distribution of antigen 1 b was confined to the outer part of its
neuro-epithelial layer. Antigen 1 b was not revealed in the stroma layer nor
in the inner part of the neuro-epithelial layer of iris (Fig. 11).
Analysis of 1 b antigen localization was carried out on sections from spinal
cord, medulla oblongata, cerebellum, optic lobe, and great hemispheres of
adult chick brain. In medulla oblongata antigen \b was observed only
in cells (Fig. 12). Moreover, intracellular distribution of this antigen was
identical in neurons of different types. It was always found in cytoplasm of
cells and was never revealed in nerve fibres. Analogous results were obtained
35
E M B 34
548
A. T. MIKHAILOV AND V. M. BARABANOV
— /PL
GC
Fig. 10. Section of adult chick retina treated with antiserum to antigen 1 b and
with fluorescein-conjugated antiserum to globulin fraction of rabbit serum. PE,
Pigmented epithelium; OS, outer segments of photoreceptors; IS, inner segments
of photoreceptors; ELM, external limiting membrane; ONL, outer nuclear layer;
OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer;
GC, ganglionic cells. Fluorescence in IS of photoreceptors, on the border of INL
and IPL and in GC. x 270.
Analysis of water-soluble antigens of chick retina
549
Fig. 11. Sections of adult chick iris treated with antiserum to antigen 1 b and with
fluorescein-conjugated antiserum to globulin fraction of rabbit serum, (a) Test:
section + antiserum to antigen 16 +fluorescein-conjugatedantiserum. Fluorescence
only in the outer part (OP) of iris neuro-epithelial layer; no fluorescence in the inner
part (IP) of iris neuro-epithelial and stroma (S) layers, (b) Control: section + normal rabbit serum + fluorescein-conjugated antiserum. No fluorescence on section.
x50.
a
Fig. 12. Sections of adult chick medulla oblongata treated with antiserum to antigen
1 b and withfluorescein-conjugatedantiserum to globulin fraction of rabbit serum.
a, b, c, Various neurons of chick medulla oblongata. Fluorescence only in cytoplasm of neurons, x 270.
on sections from chick spinal cord where specific antigen \b fluorescence
could be observed only in grey matter. In the anterior cerebral structures of
adult chick brain an intensive background fluorescence was observed and
specific fluorescence in individual cells could not be identified. It is likely that
the number of cells containing antigen 1 b constitute a negligible percentage of
the total cellular population of these parts of brain.
Analysis of these data leads to a conclusion that antigen 1 b is localized in
cytoplasm of retina and brain cells. This antigen was not observed in nerve
fibres.
35-2
550
A. T. MIKHAILOV AND V. M. BARABANOV
DISCUSSION
Analysis of antigenic differentiation of tissues represents one of the new
lines of approach to the problems of experimental embryology. The processes
of organo- and histogenesis are reflected in the synthesis of specific proteins
which could be identified by immunological methods. In this connexion,
studies of antigenic formation in rudiments of various organs may yield
valuable information on the sequence of activation (derepression) of corresponding structural genes (Vyazov, 1962; Vyazov, Barabanov & Mikhailov,
1970, 1971).
A classical object of experimental embryology, widely used in studies of
differentiation, embryonic induction, and processes of metaplasia, is the
system of tissues of developing vertebrate eye. Therefore, a great number of
immunoembryological investigations were devoted to the analysis of antigenic
properties of eye tissues. Investigations of the lens antigen structure were
particularly numerous (Rabaey, 1962; Zwaan, 1963; Ikeda & Zwaan, 1966;
Barabanov, 1966, 1967; Clayton, 1970; Barabanov, 1973; McDevitt & Brahma,
1973; Katoh & Yoshida, 1973; van de Kamp & Zwaan, 1973). The antigenic
composition and sequence formation of iris antigens were studied to a lesser
degree (Maisel & Harmison, 1963; Brahma, Bours & van Doorenmaalen,
1971; Bours, 1973, 1974).
The present work was aimed at the analysis of antigenic differentiation of
chick retina which previously had been insufficiently studied.
Using antisera to total retinal extract a number of tissue antigens, as well as
serum antigens, were revealed in chick retina. The presence of serum antigens
may be explained by the proximity of chick retina to eye choroid coat.
On the other hand, using antisera to electrophoretic fractions of retinal
extract we were able to identify in adult chick retina up to 11 individual tissue
antigens. In immunoelectrophoresis these antigens were distributed in a wide
zone of electrophoretic mobilities. In agar some of the retinal antigens formed
complex precipitation lines localized in zones of several fractions. Antigens
with identical electrophoretic mobilities, though immunologically non-identical,
were also found in chick retina. On the basis of the data obtained it became
clear that the immunoelectrophoretic spectrum of retinal antigens differed
considerably from that of antigens of adult chick lens (Zwaan, 1963) and iris
(Maisel & Harmison, 1963). At the same time, it was shown that the vertebrate retina contains antigens similar to those of eye choroid coat (van Alphen
& Robinette, 1961; Aronson, 1968), of lens (Maisel, 1962, 1963; Clayton et
al. 1968), and of brain (McCallion & Langman, 1964; Wenger & Friedman,
1970).
Results of our experiments also showed that chick retina contained mainly
inter-organ antigens that were present in other organs. It should be noted that in
this respect retina differed considerably from lens where organ-specific anti-
Analysis of water-soluble antigens of chick retina
551
gens constituted the main part of total lens protein (Zwaan, 1963; Barabanov,
1967). Only one of the retinal antigens may be considered organ-specific,
since it was found exclusively in chick retina (antigen 4b). The likelihood of the
presence of organ-specific antigens in vertebrate retina has been suggested by
some authors (Hess & Romer, 1906; Wacker & Lipton, 1968&; Schillinger &
Homberg, 1972). Some of the retinal inter-organ antigens, identified in the present
study were of narrow specificity. These antigens were found only in eye (retina,
iris) and brain tissues. The presence of such antigens in chick retina suggest the
similarity in chemical nature of neural derivatives of eye and brain. The other
inter-organ antigens of chick retina were present in different tissues of adult
chick. Most of them were present in all organs and tissues studied, and therefore they were referred to as inter-organ antigens of broad specificity, or,
according to other authors, as 'common-organ' (Schechtman, 1948), or as
'hetero-organ' (Olenov, 1970) antigens. The pattern of inter-organ specificity
of these antigens may suggest that they belong to the group of substances
universal for all tissues, irrespective of types of their specialization. As well, we
identified retinal antigens with an intermediate type of inter-organ specificity
(antigen 3, 4 a). These were common to retina and to a number of extraeye tissues. This indicates their similarity to inter-organ antigens of broad
specificity. Nevertheless, it seems possible that the presence of these antigens in
retina and in some parenchymatous organs (kidney, liver, spleen) reflects
common features in structure and metabolism of such different tissues and
organs.
It should be emphasized that the results obtained allow us also to estimate
the degree of antigenic similarity between retina and other tissues. With the
help of anti-retina sera the maximum number of common antigens was revealed in retina, iris and brain, including antigens of narrow inter-organ
specificity. On the other hand, the antigenic similarity between retina and lens
was confined only to a group of inter-organ antigens of broad specificity. Chick
lens contains no retinal antigens of narrow specificity, and, consequently, with
respect to retina, it is as 'alien' as parenchymatous organs.
The question concerning the presence of common antigens in lens and retina
has been investigated by a number of authors. Thus, with the help of anti-lens
sera organ-specific chick lens antigens were revealed in the pigmented epithelium
(Langman & Prescott, 1959; Maisel, 1962, 1963) and in the retina itself (Clayton et al. 1968; Clayton, 1970). In our experiments with anti-retina sera no
antigens identical to organ-specific lens antigens were detected either in retina
or in lens. Moreover, it is known that organ-specific lens antigens of chick are
characterized by a high degree of immunogenecity with respect to rabbits
(Zwaan, 1963). According to the data of Clayton (1970) and the others (Bours
& van Doorenmaalen, 1972), chick retina contains organ-specific lens antigens.
But it remains unclear why our antisera to chick retina contained no antibodies to lens organ-specific antigens. We suggest two possible explanations:
552
A. T. MIKHAILOV AND V. M. BARABANOV
(1) organ-specific lens antigens are absent in retina; or (2) they are present
in retina in negligible amounts insufficient to induce production of antibodies
in rabbits.
Thus, adult chick retina and lens differ qualitatively in properties of their
specific antigens. The significance of this fact becomes more apparent in analysis
of dynamics of retinal antigen formation in chick embryos 1-20 days old. It was
established that the first to appear in the developing retina were inter-organ
antigens of broad specificity (the 3rd day of incubation). An organ-specific antigen
and inter-organ retinal antigens of narrow specificity were found at later stages of
development, which coincided with retinal histogenesis (the 5th to 18th day of
incubation). It seems improbable that these antigens could participate in the
process of lens induction. Moreover, the results obtained with antisera to retina
of 5-day-old chick embryos showed that presumptive retina contained only
antigens specific for definitive retina. Stage-specific (transitory) antigens which
could participate in lens induction (Nace & Clarke, 1958) were not revealed.
These data indicate that during histological differentiation of retina and formation therein of tissue antigens of narrow specificity, the developing retina seems
to lose the lens-inducing capacity, which agrees with experimental findings on
triton embryos (Takeuchi, 1963).
Formation of retinal specific antigens takes place at advanced stages of its
histogenesis, in contrast to chick lens where main types of specific antigens
(crystallins) were found at early stages of development (Zwaan, 1963; Clayton,
1970). This shows that there are no common phases in antigenic differentiation
of these eye tissues. On the other hand, some data suggest that there exist a
considerable degree of antigenic similarity between retina and brain. Retina
and brain in adult chick have up to eight common tissue antigens. The spectrum
of antigens common to brain and retina contains two inter-organ retinal
antigens of narrow specificity typical of the central nervous system tissues. This
is confirmed by the data of McCallion & Langman (1964) who, using antisera
to adult chick brain, detected up to 11 common antigens in chick brain and
retina. Some of these antigens were specific only for nervous tissue. Formation
of one such antigen (retinal antigen 1 b) in some parts of the developing chick
brain was investigated. It was established that this antigen appeared in retina
and in brain posterior segments simultaneously - on the 10th—11th days of
incubation. By indirect fluorescent antibody technique antigen 1 b was identified
in retina (mostly in photoreceptors) and in the outer part of iris neuro-epithelial
layer. In brain it was found only in cytoplasm of neurons, but not in nerve
fibres. Thus, on the basis of these data, connection between antigen 1 b formation
and synthesis of myelin in nervous tissue seems to be improbable. The synchronous appearance of specific antigens at advanced stages of retina and brain
differentiation indicates the existence of common stages, phases in the development of different nervous system tissues.
It is worth while to consider possible mechanisms of synchronization of
Analysis of water-soluble antigens of chick retina
553
antigen 1 b synthesis in retina and brain. Probably the appearance of antigen
16 in these tissues was predetermined at earlier stages of embryogenesis. If so,
the synthesis of antigen 1 b should take place in retina and brain independently,
like a 'clock' which is 'wound up' in these tissues at the same moment in the
development. Also it can be suggested that retina and brain contain substances
regulating their own differentiation. In that case simultaneous \b antigen
synthesis in brain and retina may be activated by the substances acting on the
basis of a feed-back mechanism. The role of such agent-activators could be
attributed to substances originating outside the central nervous system. In this
case antigenic differentiation of retina and brain would be controlled by
humoral agent/s. Such a mechanism for regulation of retina and brain differentiation has been demonstrated by Moscona, Frenkel & Moscona (1972)
regarding the synthesis of glutamine-synthetase enzyme in chick nervous
tissue. This enzyme is synthesized in chick embryonic retina and brain during
definite periods of development, dependent upon the influence of hydrocortisone (Moscona, 1972).
All these suggestions may be accepted as speculations and at present it is
difficult to give any preference to either of the mechanisms of regulation of 1 b
antigen synthesis in chick retina and brain. To elucidate these mechanisms
further special investigations are needed.
Appreciation is extended to Professor O. E. Vayzov (The Institute of Human Morphology)
for guidance in the implementation of the present investigation. The authors also wish to
express their thanks to Professor G. V. Lopashov and to Dr Olgo Hoperskaya (The Institute
of Biology Developmental) for their useful critical advice and help in preparation of the
present manuscript.
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