/ . Embryo/, exp. Morph. Vol. 23, 2, pp. 289-97, 1970
289
Printed in Great Britain
Accumulation of an organ-specific protein during
development of the embryonic chick brain
By H A R V E Y P. F R I E D M A N 1 and B Y R O N S. W E N G E R 2
From the Department of Biology, University of Missouri at St Louis
and the Department of Biochemistry, University of Kansas, Lawrence
In an earlier investigation by the present authors (Friedman & Wenger, 1965)
the quantitative development of brain-specific antigens in chick embryos was
studied using antisera against whole brain homogenates. The antisera employed
were made brain specific by absorption with homogenates of various chicken
tissues. Based on this study, the period between 5£ and 12 days of incubation,
stages 28 and 38 of the Hamburger & Hamilton (1951) stage series, was considered to be critical for neural differentiation.
Previous studies of the development of brain-specific antigens had generally
employed qualitative precipitin tests or specific inhibition of differentiation by
antisera and did not measure the accumulation patterns of these antigens
(Schechtman, 1948; Ebert, 1950; Flickinger, 1958; McCallion & Langman,
1964).
An opportunity to study the differentiation of the brain with respect to a
single, well-characterized tissue-specific protein was presented by the isolation
from beef brain of a unique protein termed 'S-100' because of its solubility in
100 % saturated ammonium sulfate (Moore & McGregor, 1965; Moore, 1965).
This highly acidic protein becomes immunogenic when complexed with methylated bovine serum albumin (Levine & Moore, 1965) thereby providing the
means for such a study.
It had been shown that S-100 protein exhibited relatively little serological
variation among a wide range of vertebrate species (Levine & Moore, 1965;
Kessler, Levine & Fasman, 1968). This permitted use of antisera against bovine
brain S-100 in an assay for the presence of a similar protein in embryonic
chicken brain.
The present study, employing antisera against a single brain-specific protein
and a highly sensitive, quantitative microcomplement-fixation test, confirms and
extends earlier results obtained with absorbed antisera against crude homogenates.
1
Author's address: Department of Biology, University of Missouri at St Louis, St Louis,
Missouri 63121, U.S.A.
2
Author's address: Department of Anatomy, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada.
19
E M B 23
290
H. P. FRIEDMAN AND B. S. WENGER
MATERIALS AND METHODS
New Zealand White rabbits weighing 2-3 kg were immunized with a total of
2 mg each of purified beef S-100 protein, kindly supplied by Dr Blake Moore.
The protein was complexed with methylated bovine serum albumin (MBSA)
according to the procedure of Plescia, Braun & Palczuk (1964) and emulsified
with an equal volume of incomplete Freund's adjuvant before injection into the
hind foot pads of the rabbits. Injections were given 3 times weekly for 3 weeks,
the first two injections consisting of 125/*g S-100 complexed with MBSA and
all subsequent injections containing 250 /tg S-100 in the complex.
The rabbits were bled from the marginal ear vein prior to the 4th and 7th
injections and by cardiac puncture 8 to 10 days after the last injection.
Microcomplement-fixation tests were modifications of the quantitative
methods of Wasserman & Levine (1961) and Moore & Perez (1966). All stock
reagents were diluted for the tests in isotonic N-tris (hydroxymethyl) methyl-2aminoethane sulfonic acid (TES, Calbiochem) buffer pH 7-4, containing 0-01
M-TES, 5x 10-4 M-MgCl2, 1-5 x 10~4 M-CaCl2, 0-1 % human serum albumin and
0-14M-NaCl.
For a highly reproducible source of complement, lyophilized guinea-pig
serum (Courtland Laboratories) was reconstituted to its original volume with
cold triple distilled water and stored as a stock solution at - 80 °C in aliquots
of ca. 30 ji\. The complement used in each test had been frozen and thawed only
once. Rabbit anti-sheep erythrocyte serum (Colorado Serum Co.) was diluted
1 : 3 with cold triple distilled water and stored as stock in small aliquots at
-80°C.
Sheep erythrocytes (Colorado Serum Company), in either Kolmer's saline or
preserved in Alsever's solution, were washed in buffer and spectrophotometrically standardized to contain 2 x 109 cells/ml by the method of Kabat & Mayer
(1961). The cells were then sensitized and diluted so that each tube in the
complement-fixation test would receive 5 x 106 sensitized cells.
The tests were performed in siliconized tubes (6 x 50 mm) prepared by rinsing
in 'Siliclad' (Clay-Adams, Inc.) and oven-drying. Each tube received 4/d of
antigen followed by 40 /Â of a complement-antiserum solution containing
enough complement to give ca. 50 % hemolysis. Antisera were inactivated at
56 °C for 30 min before use. Control tubes containing buffer, antigen and
complement as well as controls containing buffer, antiserum, and complement
were always included. All operations were performed in an ice bath using cold
reagents; the contents of each tube were mixed after each addition. The tubes
were then incubated for 18 h at 5°C.
Following incubation, 5x 106 sensitized erythrocytes in a volume of 40 /*1
was added to each tube. The hemolytic reaction was allowed to proceed for
60 min at 37-5 °C in a shaking water bath. The tubes were then placed in an ice
bath and 400 /A of cold buffer added to each to stop the reaction and dilute the
Brain-specific protein
291
contents for spectrophotometric determination of hemolysis. Released hemoglobin was measured at 413 mju, after centrifugation at 800 xg in a refrigerated
centrifuge to sediment unlysed cells.
Double diffusion in agar was performed according to the method of Ouchterlony (1958). Antisera diluted 1 : 2 with 0-85 % saline were tested against varying
concentrations of antigen.
Embryonic test antigens were prepared from New Hampshire Red eggs
incubated at 38-39 °C in a forced draft incubator. Embryos were removed at
various incubation times, floated in saline to remove adhering yolk and staged
according to the Hamburger & Hamilton (1951) stage series. Brains were
removed under a stereoscopic microscope using iridectomy scissors and watchmaker's forceps. Skin and mesenchyme were completely removed from older
embryonic brains, but this was not always possible for early embryos. Samples
were either homogenized immediately in triple distilled water for stock homogenates or frozen on dry ice for later homogenization. Protein determinations
were performed on each homogenate using the method of Lowry, Rosebrough,
Farr & Randall (1951). Tissue could not be homogenized directly in the TES
buffer since the latter was found to interfere with the protein determinations. In
a few cases, the sample brains were divided into two equal portions, one
homogenized in water for protein determinations and one in TES buffer for
complement-fixation.
RESULTS
The antisera obtained showed a single precipitation band when tested in
Ouchterlony double diffusion plates against S-100 protein at concentrations of
100 and 200/tg/ml (Fig. 1). No precipitation was obtained with BSA, saline, or
stage 34 (8-day) embryonic brain homogenate in the antigen wells. A slight
reaction with 7-day post-hatching chick brain homogenate was questionable,
being possibly due to fusion of the bands formed by S-100 placed in wells on
either side of it.
When tested by the micro procedure used, the antisera fixed complement with
as little as 1-25 x 10~ n g of the homologous beef S-100 protein (Fig. 2). Cross-
reactivity between the anti-beef protein sera and homogenates of brain of adult
New Hampshire Red chickens confirmed the presence in chickens of a protein
similar to bovine S-100 as noted by Levine & Moore (1965). The reactions of
the anti-beef protein sera with chicken brain homogenates were more variable
and less sensitive than those with homologous antigen. A curve exhibiting
inhibition in the zone of antigen excess was obtained (Fig. 3). Fixation occurred
with as little as 1 x 10~6 g of total brain protein, reached a maximum at about
6 x 10 -6 g and declined at higher concentrations.
The anti-beef S-100 sera were used in microcomplement-fixation tests against
homogenates of embryonic chick brains of various developmental stages. Since
the absolute amounts of S-100 protein in the homogenates were not known,
19-2
292
H. P. FRIEDMAN AND B. S. WENGER
Fig. 1. Photograph of agar-diffusion plate showing precipitin lines formed between
purified 'S-100' and anti-'S-100' serum. Antiserum in central well. Key to antigens
in peripheral wells: bsa = bovine serum albumin; s = saline; 1 = 100/tg/ml S-100;
2 = 200 /tg/ml S-100; 34 = stage 34 (8-day) embryonic chick brain; 7 = 7-day posthatching chick brain.
0-40 H
Chicken brain
0-35
0-4 -
+
0-30a. 0-3 H
E
Antigen
'5
antiserum
- „
+
c ä
ts 0-2 -
antiserum
0-25 v
0-20 H
•Sï 015 H
O. rtf
•S? 0-10 H
0-1 -
0-
a<
Control (no antiserum)
2-5
'S-100' protein (gx10 - 1 1 )
50
Control chicken brain
(no antiserum)
005 H
u
rrn
1-4
r
5-58
11-17
22-3
Total brain protein (gx10 - 6 )
Fig. 2. Complement-fixation by purified bovine 'S-100' protein and rabbit antiserum
against bovine 'S-100'.
Fig. 3. Complement-fixation by adult chicken brain homogenate and rabbit
antiserum against bovine 'S-100'. Note inhibition in zone of antigen excess.
Brain-specific
293
protein
results were related to total protein (Fig. 4) and plotted as total protein against
per cent of maximum fixation.
Homogenates of brains at stage 24 (4 days of incubation) reacted variably
and only at a total protein content greater than 0-4 fig. 12 different brains were
tested, of which 7 fixed complement. Stage 26 brain homogenates (4f days of
incubation) reacted more consistently and gave reactions with as little as 012 fig
total protein. By stage 29 (6 days of incubation) 0028fig of protein was
sufficient for fixation. From stages 29 to 39, reactivity gradually rose with
reactions occurring with 0-016 fig of protein at stages 39 and 42 (13 and 16 days
of incubation respectively). With homogenates of adult brain complementfixation occurred with 0008-002 fig of protein.
005
0-15
0-25
0-35
0-45
Total brain protein (gx10 _t )
0-55
0-65
Fig. 4. Complement-fixation by homogenates of embryonic chicken brains of various
ages tested with rabbit anti-bovine 'S-100'. Results plotted as percentage of maximumfixationfor each stage.
DISCUSSION
These results show rapid accumulation of a protein antigenically similar to
bovine S-100 in brains of chick embryos between stages 24 and 29 (4-6 days of
incubation) and a gradual rise to adult concentrations by stage 39 (13 days).
The microcomplement-fixation test employing antiserum to bovine S-100 was
capable of measuring ca. 1 x 10 -11 g of purified bovine S-100. In cross-reaction
with adult chicken brain homogenates, 1 x 10~6 g of total protein gave fixation.
Moore, Perez & Gehring (1968) have found about 200 fig of S-100/g of wet
bovine brain. Assuming a content of 100 g protein/kg fresh bovine brain (Long,
1961), we calculate a concentration of S-100 of 2x 10 _3 g/g of protein. Our
294
H. P. FRIEDMAN AND B. S. WENGER
assay detected 1-25 x 10 -11 g of purified S-100 which is equivalent to ca. 6 x 10~8
g of total beef brain protein. Using chicken brain homogenates, fixation
occurred with 1 x 10 -6 g of total brain protein. Thus 170 times as much chicken
brain protein is needed to fix complement as is needed with beef brain protein.
The difference may be accounted for either by differences in relative concentrations of S-100 in beef and chicken brain or by differences in structure of the
protein or both. In any case our assay measures a chicken protein similar to
beef S-100 in amounts of the order of magnitude of 10~9 g or less.
Adult
Age of embryo
Fig. 5. Accumulation of brain antigens during development. Results plotted as age of
embryo versus percentage of maximum fixation. Dashed line = crude brain homogenates versus antiserum against adult crude brain homogenate. Solid line =
crude brain homogenates versus antiserum against purified bovine 'S-100'.
The results of this investigation are generally similar to those of an earlier
study of the development of brain-specific antigens using antisera prepared
against crude brain homogenates (Friedman & Wenger, 1965). This suggests
either that antibodies to S-100 constituted a major portion of those present in
our previous sera or that a number of different proteins display similar patterns
of accumulation during this period of rapid differentiation. We previously
reported the appearance of brain specific proteins between 5£ and 6£ days of
incubation (stages 28, 30). Our present data showing the appearance of the
S-100 antigen somewhat earlier, e.g. 4-6 days (stages 24, 29) may be due to the
greater sensitivity of the microcomplement-fixation test presently used. The
general accumulation patterns observed in both studies, however, are quite
similar (Fig. 5). Whether one considers the two curves in Fig. 5 to represent the
Brain-specific protein
295
same antigen or different antigens with similar accumulation patterns, the
present data provide additional support for the hypothesis of Burt & Wenger
(1961) that an increase in glucose-6-phosphate dehydrogenase (G6PDH)
activity occurring between 4£ and 6 days and persisting through 8 days of
incubation is associated with formation of the RNA involved in the synthesis
of specific proteins of the differentiating nervous system. Both of our antigen
accumulation curves follow a pattern consistent with this hypothesis.
The rapid accumulation of the protein similar to S-100 occurs just after a
transient peak in mitotic index (Kallen, 1955) and coincides with a spurt in
(G6PDH) activity. Such a temporal sequence would be expected if G6PDH
were active in providing ribose, through the pentose cycle, for synthesis of RNA
(Horecker & Hiatt, 1958).
Additional support for the hypothesis has been provided by the recent
demonstration, using the technique of DNA hybridization, of the appearance
and accumulation of a new species of 6-16 S RNA in the brain of the embryonic
chick between 4 and 7 days of incubation (Johnson, Newmark & Wenger, 1969).
This same fraction of heterogeneous RNA has further been shown to provide
the synthesis in a cell-free system of an antigen immunochemically identical to
S-100 protein (Johnson, 1969).
SUMMARY
1. In an earlier study the development of brain-specific antigens was demonstrated using anti-whole brain sera absorbed with homogenates of other tissues.
Moore's isolation of a protein unique to nervous system allowed us to study
differentiation of chick brain with respect to a single, well characterized, tissuespecific protein.
2. Rabbits were immunized with a total of 2 mg each of purified beef 'S-100'
protein-methylated BSA complex in incomplete Freund's adjuvant. Antisera
showed single precipitation bands in gel diffusion against homologous antigen.
As little as 1-25 xlO - 1 1 g of 'S-100' fixed complement with these antisera.
Antisera against beef 'S-100' protein showed cross-reactivity with adult New
Hampshire Red chicken brain homogenates, indicating the presence of a similar
protein in this breed of chicken. Reactions of anti-beef'S-100' sera with chicken
brain extracts were more variable and less sensitive than with homologous
antigen.
3. Anti-beef 'S-100' sera were used in microcomplement-fixation tests against
homogenates of chicken brains of various developmental stages. HamburgerHamilton stage 24 brain homogenates react only variably and only at total
protein content greater than 0-4 fig. Stage 26 brains react more consistently and
at protein content as low as 0-12 pig. At stage 29,0-028 jug protein yields positive
complement-fixation reactions. From stages 29 to 39, reactivity gradually rises
with reactions occurring with 0016fig protein at stages 39 and 42. Adult brain
reacts at protein content ranging from 0-008 to 0020fig.
296
H. P. FRIEDMAN AND B. S. WENGER
4. These results show rapid accumulation of a protein antigenically similar
to 'S-100' between stages 24 and 29 and a gradual rise to adult concentration
by stage 39. They suggest the presence of antibodies against 'S-100' in previously used antisera prepared against crude brain homogenates.
RÉSUMÉ
Accumulation d'une protéine spécifique d'organe pendant le développement du
cerveau embryonnaire de poulet
1. Dans une étude précédente, le développement d'antigènes spécifiques de
cerveau a été démontré en utilisant des immunsérums anti-cerveau total,
absorbés par des homogénats d'autres tissus. L'isolement, par Moore, d'une
protéine spéciale au système nerveux nous a permis d'étudier la différenciation
du cerveau de poulet, en ne considérant qu'une seule protéine tissulaire spécifique
bien caractérisée.
.2. Des lapins ont été immunisés avec une quantité individuelle totale de 2 mg
de protéine 'S 100' de bœuf purifiée, BSA, méthylée, associée à de l'adjuvant
incomplet de Freund. Les immunsérums ont montré une seule bande de précipitation par diffusion en gélose, contre l'antigène homologue. Une quantité aussi
faible que 1,25 x 10 -11 g de 'S 100' suffit à fixer le complément avec ces antiserums. Les antiserums anti-'S 100' de bœuf ont montré une réactivité croisée avec
les homogénats de cerveau de poulet New Hampshire Red, ce qui indique la
présence d'une protéine semblable dans cette souche de poulet. Les réactions
de l'anti 'S 100' de bœuf avec les extraits de cerveau de poulet se sont montrées
plus variables et moins sensibles qu'avec l'antigène homologue.
3. Les sérums anti-'S 100' de bœuf furent utilisés pour des micro-réactions
de fixation du complément par les homogénats de cerveaux de poulet à différents
stades de développement. Au stade 24 de la table de développement de Hamburger et Hamilton, les homogénats de cerveau ne réagissent que de façon
variable et seulement pour une quantité totale de protéines supérieure à 0,4 pig.
Les cerveaux au stade 26 réagissent de façon plus importante et pour une
quantité de protéines de 0,12 ptg. Au stade 29, 0,028 peg de protéines donnent
une réaction de fixation du complément positive. Du stade 29 au stade 39 la
réactivité augmente graduellement avec des réactions positives pour 0,016 ptg de
protéines aux stades 39 et 42. Le cerveau adulte réagit pour des quantités de
protéines de l'ordre de 0,008 à 0,020 ptg.
4. Ces résultats mettent en évidence une accumulation rapide d'une protéine
antigéniquement semblable à la ' S 100' entre les stades 24 et 29 et son augmentation graduelle jusqu'à la concentration de l'adulte, atteinte au stade 39. Ils
suggèrent la présence d'anticorps anti-'S 100' dans les immunsérums préparés,
au cours d'expériences précédentes, contre des homogénats de cerveau entier.
Brain-specific protein
297
This investigation was supported by Public Health Service Research Grant 2R01 NB 07141,
from the National Institute of Neurological Diseases and Blindness and H D 03474 from the
National Institute of Child Health and Human Development. The expert assistance of Miss
Mary Ruscha and Mrs Jeanne Ellermeier is gratefully acknowledged.
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(Manuscript received 5 June 1969)