THE EFFECTS OF ADULT TISSUE HoMossuAns, ‘
AND OF ANTlSERA AGAlNST ADULT TISSUES.
ON EMBRYONIC DEVELOPMENT m THE FROG

”E‘hosishfiobogreqofl’h.by
MECHEGAN STATE UNIVERSKTY
Wiilis Caivin Owens
1.960

 

 

 

 

This is to certify that the

thesis entitled
The effects of adult tissue homogenates,

and of antisera prepared against
'adult tissues, on embryonic
development in the frog.
presented by

Willis C. Owens

has been accepted towards fulfillment
of the requirements for

_Eh..D.._ degree in ELI-08L

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Major professor

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Michigan State
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THE EFFECTS OF ADULT TISSUE HOMOGENATES, AND OF ANTISERA
AGAINST ADULT TISSUES, ON EMBRYONIC DEVELOPMENT

IN THE FROG

by

Willis Calvin Owens

A THESIS

Submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Sciences
in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1960

Willis C. Owens

ABSTRACT

A study was made of the relative effects of tissue fractions of
adult organs of‘ggg§,pipiens and of antisera prepared against homogenates
of the same organs on the differentiation of homologous organs in embryos
of this species. Experimental treatment was performed in 2 ways: cul-
turing embryos in the presence of the tissue fractions or antisera, and
injecting embryos with these same substances.

Most of the work with tissue fractions was done with the nervous
system. The results of these studies suggest that: (1) the nervous
system is rather poor for such studies since it is easily affected by a
variety of treatments and the types of effects produced on this system
are not easily interpreted, (2) the injection technique is superior to
the culturing method for producing specific effects on the developing
nervous system, and (3) the fraction of cytoplasmic granules produces
more consistent effects on the development of the nervous system than
other cytoplasmic fractions.

The most consistent effect on embryonic development of antisera
prepared against adult organs was a marked-inhibition of heart develop-
ment by anti-heart serum.~ Thirty per cent of all embryos which were
injected with anti-heart serum showed this inhibitory effect. Anti-
.kidney serum appeared to have some inhibitory effect on the aMbryonic
heart while antidbrain serum.had no inhibitory effect on the developing
heart at all.

.m. .Analysis of antisera prepared against adult brain, heart, and
kidney of frog showed that these organs have several common components.

The antigenic properties of kidney and heart are quite similar and these

Willis C. Owens
Abstract, Page 2

differ considerably from the antigenic properties of brain. Using adsorp-
tion techniques it was possible to demonstrate specific components in most
of the antisera.

Preliminary studies in which embryos were injected with antisera
prepared against protein extracts (if heart and skeletal muscle were

somewhat inconclusive although some heart inhibition was noted.

CURRICULUM VITAE

Final Examination: August 3, 1960

Dissertation: The effects of adult tissue homogenates, and of antisera
against adult tissues, on embryonic development in the
frog.

Biographical Items:
Born: -December 4, 1926, Nashville, Tennessee
Undergraduate Studies: David Lipscomb College, 1949-1953

Graduate Studies: George Peabody College for Teachers, 1953-1954,
Michigan State University, 1956-1960

Experience: High School Science Teacher, 1953-1954, Instructor in
Biology, David Lipscomb College, 1954-1956, Graduate
Assistant, Department of Zoology, Machigan State
University, 1956-1957, Predoctoral Fellow of the
National Cancer Institute, 1958-1960

Professional Membership: Society of Sigma Xi, Tennessee Academy of
Science .

iii

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to Dr. John R. Shaver
for his guidance and interest during the course of this investigation.
Sincere thanks are also due Dr. Allen S. Fox, Dr. Richard A. Fennell,
and Dr. Robert S. Bandurski for their helpful suggestions and advice
while serving on the author's guidance committee. The assistance of
the Department of Zoology at Michigan State university in supplying
space and materials for this study is likewise gratefully acknowledged.

The author is especially appreciative of the financial support
provided by a predoctoral fellowship (CF 8193) from the National Cancer

Institute of the National Institutes of Health.

iv

TABLE OF CONTENTS

, Page
LIST OF TABLES O O O O I O O I O O O O O O O O O O O O O O O O O O O 0 Vi

LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . ... . . . . vii
(LIST OF TEXT FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . viii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . . . 14
RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

A. Effects of adult tissue fractions on embryonic
development 0 O O O O O O O O O O O C O O O O O O O O O l O O 21

B. Effects of antisera prepared against adult organs
on the differentiation of embryonic tissues. . . . . . . . . 34

1 O An‘lys 18 Of antisera O O O O O O O O O O I O O O O O O O 34
2. Effects of antisera on differentiation of
embryonic tissues. . . . . . . . . . . . . . . . . . . . 36

a. External manifestations of treatment

with antisera. . . . . . . . . . . . . . . . . . . . 36
b. Histological analysis of embryos

treated with antisera. . . . . . . . . . . . . . . . 40

C. Effects of antisera prepared against muscle
proteins on embryonic development. . . . . . . . . . . . . . 45

DISCUSSION 0 O O O O I O C O O O O O O O O O O O O O O O p O C O O O O O 67

A. Effects of adult tissue fractions on embryonic
deve 1 Opment o o O o o O o o O o O o o o o o O o o o o o o o o 6 7

B. Effects of antisera prepared against adult organs
on the differentiation of embryonic tissues. . . . . . . . . 70

C. The effects of antisera prepared against muscle
proteins on embryonic development. . . . . . . . . . . . . . 76

SWY. . I O O C O O O O O O C O O O O O O O O O O O O O O O O O O O 78

LITERATURE-CITED...ooooooeeoooooooo0000's... 81

LIST OF TABLES

Table Page
1 Effects of tissue fractions of adult brain on the
developing nervous system . . . . . . . . . . . . . . . . . . . 22
2 Notochord size (diameters in microns) of individual
embryos treated with adult brain tissue fractions . . . . . . . 24
3 Effects of tissue fractions of adult brain and heart

on the differentiation of the embryonic brain and heart . . . . 25

4. Abnormalities of the nervous system produced by
treatment of embryos with antisera prepared against
adu1 t organs 0 O O O O O O O O O O O O O O O C O I O O O O O O O 25

5 The presence of specific factors in anti-organ sera
as determined by the technique of adsorption. . . . . . . . . . 37

6 Edema in embryos treated with adult organ antisera. . . . . . . 38
7 ' Curvature of the spine as caused by culture in heart
antiserum o a o o o e. e o o o o o o o o o e ' o o o o o o o o o o 39

8 Inhibition of heart development in embryos injected

with antisera prepared against adult organs . . . . . . . . . . 43
9 Inhibition of heart development by antisera prepared
against adult muscle proteins . . . . . . . . . . . . . . . . . 48

vi

Plate

10

11

12

LIST OF PLATES

Examples of "solid" mid-brains as produced by injection
of embryos with tissue fractions of adult frog brain. . .

Examples of "solid" hind-brains as produced by injection
of embryos with tissue fractions of adult frog brain. .

Abnormalities of the spinal cord and notochord as 4

produced by injection of embryos with
of adult frog brain . . . . . . . . .

Analysis of antisera prepared against
AnalySis of antisera prepared against
Analysis of antisera prepared against

Analysis of antisera prepared against
adult organs I. . . . . . . . . . . .

Analysis of antisera prepared against
adult organs II . . . . . . . . . . .

Analysis of antisera prepared against
adult organs III. . . . . . . . . . .

Analysis of antisera prepared against
adult organs IV . . . . . . . . . . .

tissue fractions

muscle proteins I .
muscle proteins II.
muscle proteins III

homogenates of

homogenates of

homogenates of

homogenates of

O O O O O O O O O O

Inhibition of heart development resulting from
treatment with antisera prepared against adult organs

of the frog . . . . . . . . . . . . .

Heart inhibition and other abnormalities as produced

by treatment of embryos with antisera

prepared against

homogenates of adult organs and protein extracts of

skeletal and heart muscle . . . . . .

vii

Page

28

30

32

49

51

53

55

56

59

61

63

65

 

 

LIST OF TEXT FIGURES

Figure Page

1 Inhibitory effect of organ antisera on heart
development . . . . . . . . . . . . . . . . . . . . . . . . . . 44

viii

INTRODUCTION

In recent years there has been considerable discussion‘concerning
the effects of substances from differentiated cells on the differentia-
tion of homologous embryonic cell types. Some investigators claim to
have demonstrated that these substances have an inhibitory effect on the
differentiation of embryonic cells, whereas others believe they have
shown that growth processes are stimulated in the affected cells. Reports
that antisera produced against various types of differentiated cells
have an inhibitory effect upon the differentiation of homologous embryonic
cell-types, would lead to the conclusion that the complementary substances
in adult tissues could not possibly have an inhibitory effect, since
antigens and antisera should have opposite effects ( see Weiss 1942,

Ebert 1955).. Perhaps one of the major reasons that this controversy

has not been settled is that investigators holding opposing views have
worked with different experimental systems and have used widely different
experimental procedures. The present investigation was begun with the
idea that a meaningful test of these opposing hypotheses might be made
by subjecting the same differentiation system to substances from fully
differentiated tissue, and to antisera prepared.against these tissues.

A short discussion of the development of these different points of
view is in order here. The hypothesis that substances from differentiated
cells can inhibit the differentiation of undetermined cells with the same
prospective fate, is based on a proposal by Rose (1952) that differentia-
tion is caused by a hierarchy of self-limdting reaction systems. He sug-
gests that differentiation depends upon a series of gene-initiated reac-

tion systems which are successively inhibited by the collection of

specific inhibitors. According to this theory, one differentiating
reaction system, assumed to have an initial advantage, produces meta-
bolites, the concentrations of which are self-limdting. When the con-
centrations of these substances reach a critical level, differentiation
in this system ceases, and the next differentiating system in the hier-
archal series has the advantage and the differentiation of another tis-
sue is thereby initiated.

The evidence adduced for the theory mentioned above has been derived
from experiments in which tissues are allowed to differentiate in the
presence of adult or fully developed tissues. Experimental tests have
been performed in two general areas: namely, regeneration and embryonic
development. Extensive work has been done with the regenerating system
of the coelenterate, Tubulariagi In.TUbu1aria, any portion of the organism
proximal to the hydranth can, if isolated, reorganize and regenerate
hydranths. Rose and Rose (1941) found that when isolates were grown in
sea water which had previously been subjected to adult hydranths for
12-24 hours, regeneration of the isolates was inhibited. This inhibition
was attributed to the action of a diffusible substance produced in the
hydranths which in the normal situation diffuses down the stem and pre-
vents more proximal parts from forming distal portions._ Thus, the pre-
sence of this substance in the 'inhibitory water' produced the same results
as if an intact hydranth had been on the isolated portions. Consequently,
no regeneration occurred. Steinberg (1954) studied this phenomenon in
great detail and substantiated the findings mentioned above. However,
Fulton (1959) has re-examined the whole problem and has shown rather

conclusively that the inhibition is due entirely to bacterial growth in

the cultures.

Using grafting techniques, Rose (1955, 1957) has shown that regen-
eration of distal structures in an isolate can be inhibited in the pre-
sence of older distal structures. Hejproposes that differentiation in

Tubularia depends upon a specific inhibitor which acts in a distoproximal

 

direction. Thus, more proximal regions never form distal structures as
long as normal distal structures are present.

‘Lender (1956) has demonstrated a similar situation in the regenera-
tion of the flatworms, Polycelis giggg (Ehrb.) and Dugesia lugubris (O. Schm.).
He cut the heads from these organisms and allowed them to regenerate in the
presence of homogenates of other individuals of the same species. Under the
influence of raw homogenates of heads, the brain fails to regenerate to the
normal extent, although the eyes and other tissues regenerate normally,.
Similar homogenates of tails, on the other hand, had little or no inhibitory
effect. Centrifugation of the homogenates showed the inhibitory effect to
be in the supernatant fluid causing Lender to propose that the inhibitor is
a diffusible chemical substance, derived from the brain of planarians.

Recently, Tucker (1959) has done a similar and very extensive study
with the nemerthan: worm, Lineus vegetus. Under normal conditions, sections
removed from anterior, posterior, or mid-body regions of the worm will regen-
erate both head and tail. Tucker studied the effects of homogenates of
heads, mid-body, and tails on the regeneration of pieces isolated from these
same regions. She found that head homogenates inhibit head regeneration in
isolates from all regions; mid-body homogenates inhibited head regeneration
in mid-body and posterior but not in anterior isolates; and tail homogenates
inhibited head regeneration only in isolates from the posterior region. Con-

versely, tail homogenates inhibited tail regeneration in all isolates; mid—body

4
homogenates inhibited tail regeneration in mid-body and posterior isolates;
and head homogenates inhibited tail regeneration only in anterior isolates.
Similar homogenates of Cerebratulus californiensis, another nemertean worm
of the same family, produced about the same effect on regeneration of Lineus
vegetus as did the homogenates of the latter species. However, homogenates
of Amphiporus formidibalis, a worm belonging to a different order, had no
effect upon regeneration in‘L. vegetus. Tucker proposes that anterior regen-
eration in L. vegetus is governed by a hierarchy of regions extending from
the head to the tail. Posterior regeneration, on the oEfiEr hand, is governed
by a similar hierarchy extending from tail to head.

Saetren (1956) has studied the effects of homologous organs in the
regeneration of liver and kidney in the rat. Following removal of portions
of the liver and kidney of the same animal, the abdominal Cavity was in-
jected with macerates of either kidney or liver. The mitotic count in the
homologous organ was greatly decreased whereas the heterologous organ.was
not affected. This worked equally well with both kidney and liver.

Using micro-dissection and grafting techniques, Tartar (1958) has
shown that oral structures will inhibit the regeneration of new oral struc-
tures in Stentor (Ciliata, Protozoa).l

Thus, it appears that substances from differentiated cells or regions
of cells have an inhibitory effect on:homologous differentiating systems in
regenerating cells or tissues. A

The results of experiments on differentiating systems in embryos
have not proven as satisfactory as those on regenerating organisms. Weiss
(1952) cultured embryonic chick organs in tissue media prepared with ex-

tracts of whole embryos or of embryos from which the homologous organ had

 

 

 

 

been removed. Using heart fragments, it was observed that 2 out of 333
pulsated after 4 days in full extract, whereas 129 out of 349 pulsated
after 4 days in extract of embryos lacking hearts. With kidney fragments
(metanephros), 74 out of 1007 differentiated new tubules in full extract
whereas 176 out of 1006 did so in medium lacking kidney. Weiss proposed
that these results indicated that differentiation is reduced markedly in
the presence of a homologous organ.

Rose (1955) reported on experiments in which he cultured embryos
of Rana pipiens in the presence of adult tissues. Dejellied, fertilized
eggs were placed in culture dishes containing bits of adult tissue and
allowed to develop. In a small number of experiments Rose was able to
demonstrate some specific effects. The effects were twoffold; retarda-
tion of development at a specific stage and specific malformations of
homologous embryonic structures. Brain-treated embryos were delayed at
the neural plate stage, heart-treated embryos at the neural tube and
early tailbud stages, and blood-treated embryos were delayed following
the tailbud stage. Specific effects on the nervous system in embryos
treated with adult brain were: poor morphology of the central nervous
system, neural canal incomplete or missing, and a random arrangement of
cells in the neural tube. Heart treatment resulted in a reduction in
size, in varying degrees, of the heart. This was accompanied by a fail-
ure of outgrowth of the external gills and the tail. The most striking
effect in the blood-treated embryos was the absence of blood cells, even
though a good functional heart was present. These results seem quite
convincing except for the fact that only 4 of a total of 26 experiments

produced such results. Rose feels that the lack of consistent results

from one experiment to another was probably due to a number of uncon-
trolled factors such as seasonal variation and general condition of the
eggs employed in the experiments.

Recently, Clarke and McCallion (1959a) repeated Rose's experiments

on embryos of Rana pipiens using homogenates of brain and heart of this

 

species. They claim to have obtained specific effects on the nervous
system with brain homogenates. Heart-treated embryos did not show any
neural defects but 2 out of 124 embryos did show hearts which were re-
duced in size. They interpret their results to be in keeping with
Rose's theory.

It might be noted here that Spiegel (personal communication) has
performed experiments of the same nature as those of Clarke and'McCal-
1ion. When frog embryos were cultured with breis and extracts of brain,
spinal cord, heart, liver, lung, blood, skeletal muscle, kidney, and
ovary, no cases of specific inhibition of the homologous tissues were
noted. He did obtain some evidence of retardation of deve10pment, as
did Rose, but found that he could duplicate these results with rabbit
serum albumin. Thus he believes the retardation is not a specific
effect but is probably due to differences in concentration of protein
in the tissue fractions employed as "inhibitors."

Shaver (1954) injected embryos of‘gggg'pipiens with fractions of
adult.§, pipiens brain. He was able to show that Rose's results with
whole brain tissue could be duplicated with both cytoplasmic granules
and supernatant fluid of brain, insofar as external morphogenesis of the
nervous system was concerned. Cytoplasmic granules appeared to be more

effective than whole brain tissue in producing retardation during

neuralation. The present investigation was begun with a thorough histo-
logical analysis of this material, the results of which will be presented
later.

Recently, Braverman (1958 a and b) reported briefly on work with
the chick in which extracts of adult organs were injected beneath the
blastoderm of 1-day-old embryos. These preliminary reports state that
brain affected the developing nervous system and mesodermal derivatives
affected mesodermal structures, such as somites. Further statements
concerning this work must await a complete and detailed report. Clarke
and McCallion (1959b) have also reported briefly on experiments in which
homogenates of adult tissues of the chick were injected into the yolk of
early embryos. They also report specific effects on the nervous system
with homogenates of adult brain.

.mEvidence for the alternative hypothesis, that substances from
fully differentiated cells stimulate growth processes in differentiating
systems, comes mostly from work with grafts of adult tissues onto the
chorioallantoic membrane (d? the chick. Murphy (1916) observed that
chorioallantoic grafts of adult chicken spleen, liver and bone marrow
induced enlargement of homologous organs of host embryos. This was es-
pecially striking in the case of spleen grafts. This spleen hypertrophy
was attributed to an increased infiltration of small lymphocytes. These
observations were confirmed by Danchakoff (1916), although she attributed
the hypertrophy to increased proliferation of lymphoid hemocytoblasts.
The results of Sandstrom (1932) also demonstrated host spleen hypertrophy.
‘Minoura (1921) transplanted ovarian and testicular tissue of the chick to

the chorioallantoic membrane and observed that development and differen-

 

 

 

 

tiation of one sex was stimulated by grafts of the gonads of the same
sex and inhibited by grafts of the gonads of the opposite sex.

An extensive investigation of the problem of the effects of chorio-
allantoic grafts of adult chicken tissue on homologous tissue of the
embryo was carried out by Ebert (1951, 1954, 1955). He observed a very
marked spleen hypertrophy in host chicks following grafts of adult chick
spleen (Ebert, 1951). In other experiments, Ebert (1954) noted that the
nitrogen content of enlarged spleen was increased and he concluded that
spleen enlargement was directly related to increased protein content but
that increase in DNA content was not correlated with the increase in pro-
tein. Because of these findings, Ebert believes that hypertrophy of the
host spleen represents a true increase of spleen tissue and.not merely an
increase of certain lymphocytes as was suggested by some earlier investi~
gators. As a result of using grafts containing $35-1abeléed methionine,
Ebert further concluded that the hypertrophy was due in large part to a
"selective incorporation into the proteins of the host tissue of tissue-
specific components from the transplant.".

The results of the chorioallantoic graft experiments support in
general the idea that substances from differentiated cells have a stimu-
latory effect on homologous differentiating systems. Unfortunately, most
of this work has been done with the spleen which is not believed to be a
' typical organ by many workers. For example, Van Alten and Fennell (1959)
made the following observations from their work with chorioallantoic grafts:
(1) the host spleen was enlarged following grafts of adult spleen, duo-
denum, brain, and skin, and (2) the host liver and heart were enlarged

following spleen, liver and duodenal grafts. It appears from this study

that the stimulatory effect of chorioallantoic grafts is not tissue-
specific as had been suggested by Ebert.

A slightly different approach to this problem was taken by Andres
(1955) who injected suspensions of chick mesonephros and liver into
6-day chick embryos, intravenously. With mesonephros, host mesonephros
exhibited increases in mitotic indices. Liver showed some increase but
not as great as that of mesonephros. When liver was injected, the host
liver had a slight increase in mitotic index but mesonephros had a slight
decrease. Andres proposes that adult tissues have a stimulatory effect
on homologous embryonic tissues in the chick.

In the last few years, an increasing number of investigators have
used various immunological techniques in studying embryonic development.
Most of these studies have been concerned with the embryonic appearance
of molecules similar to those present in the adult. Nace (1955) and'
Tyler (1955, 1957) give excellent reviews of the work in this area. A
relatively small number of investigators have studied the effects of
antisera on the development of the embryo.

.Burke, 25 21. (1944) injected chick lens antiserum into chick
embryos and studied its effects upon differentiation of the lens and
other eye structures. The age of the embryos injected ranged from
146 hours to 337 hours. Using adult lens antisera, it was noted that
the lenses were affected in embryos ranging in age from 146 to 192 hours-
atthe time of injection.' The authors attributed failure to affect lens
beyond this age as due to the hardness already attained by the lens. In
addition to the effect on the lens, it was observed that some of the

neighboring tissues, such as the lens epithelium and the retina, were not

15
completely normal. Using antiserum against 160-hour lens, the authors
could not detect any morphological defects in the lens or other eye
structures.

Parkes (1946) studied the effects of amphibian larval antiserum
on the normal amphibian larva. Antisera were prepared against 2 species
of frogs, Xenopus laevis and Rana temporaria, and 1 species of toad,
Discoglossus pictus. It was found that with concentrations of the Xenogus
antiserum.greater than 1:50 all larvae of this species died within a
short while. The same larvae lived well in normal rabbit serum oi'con-
centrations up to 1:25. Decreasing the concentration increased the time
the larvae would live in the antiserum. If the antiserum was absorbed
with tadpole extract, no adverse effect was noticed. Parkes was also able
to produce some lethal effects on the Xenopus larvae with heterologous
antisera, but of somewhat lesser intensity than was noted with the homo-
logous antiserum- Flickinger and Nace (1952) found that an antiserum
prepared against the supernatant fluid resulting from centrifugation at
18,000 x g of homogenates of hatchedigggg'fuggg larvae (Shumway stage 20)
caused a cytolysis of the lateral epidermis of the same stage when demem-
branated embryos were grown in the antiserum. Grundwaldt (1949) studied
the effects of antisera against 3 separate fractions of newly hatched
chicken brain on growth in culture of nerve cord from 9-day and 13-day-
old chick embryos. The 3 fractions consisted of a saline-soluble fraction,
an alcohol-soluble fraction, and an alcohol-insoluble fraction. Antisera
prepared against the saline-soluble and the alcohol-insoluble fractions
inhibited outgrowth in cultures of both 9-day and 13-day-old cord. 0n the

other hand, the alcohol-soluble fraction affected only l3-day-old cord.

11
Grundwaldt concluded that 2 different antigens were produced during devel-
opment of the chick brain; one, an alcohol-insoluble antigen appears as
early as the ninth day; the second, which is alcohol-soluble, develops
between the ninth and thirteenth day of incubation.

Lippman, £5 31, (1950), studied the effects of rat anti-kidney
serum on tissue culture explants. A large number of embryonic tissues
from the rat and a few from the chick were used to test the antiserum.

The antiserum was toxic to explants of rat kidney, heart, and brain. It
was also toxic to some of the chick explants which were tested.

Ebert (1950) has done extensive work in connection with the effects
of antisera on development. He used antisera prepared against heart,
spleen, and brain of the chick. Embryos to be tested with the antiserum
were explanted according to the method of Spratt (1947). Normal devel-
opment could be attained by growing embryos on culture media containing
adult blood serum of the chick. These explanted embryos served as con-
trols. The antisera had very pronounced effects on the embryos. At
high concentrations, the antisera were all lethal or produced extremely
abnormal effects on-the embryos. At lower concentrations, Ebert noticed.
that differentiation proceeded in the complete absence of growth. This
was a non-specific reaction since it was produced by certain concentra-
tions of all 3 antisera.

With concentrations of the antisera lower than those producing the
non-specific reactions, growth continued but specific defects were noted.
Antiespleen and anti-heart sera affected mesodermal structures, whereas
anti-brain serum affected nervous tissue. The anti-heart and anti-spleen
sera caused a blockage in somite and lateral plate mesoderm differentiation.

The morphogenesis of the nervous system in these antisera was basically

 

 

 

 

 

12
complete, but the central nervous system was somewhat abnormal. Embryos
cultivated in anti-heart serum never developed pulsating hearts, While
those in anti-spleen serum did. Embryos treated with anti-brain serum
had specific defects in the forebrain region. Ebert thinks this may
'reflect a general effect on nervous tissue, but is expressed only in
the anterior end of the embryo due to some type of antero-posterior
gradient.

Johnson and Leone (1955) report that antiserum prepared against
actomyosin from adult chick hearts inhibits morphogenesis of the heart
in chick embryos, but the degree of specificity is not clear since there
is also general inhibition of development. Another example of what appears
to be a general lethal effect of antisera on developing chick embryos, is
the work of Nettership (1953).

Gluecksohn-Waelsch (1957) studied the effects of antisera on
‘development by immunizing virgin female mice and subsequently mating
them. The embryos from mice so treated were studied to determine any
effects which may have been produced. Two inbred strains of mice were
used for experimental animals whereas mice from a strain which was not
inbred were used for tissue donors. Virgin female mice were given doses
of mouse brain and heart antigens. Subsequently, samples of blood were
taken to determine whether an antiserum had been produced by the injected
mice. Then the mice were bred,‘andthe embryos were allowed to develop
for 10 days. The mothers were then sacrificed and the embryos were
studied for specific abnormalities. The embryos from mice injected with
brain emulsion were observed to have abnormal nervous systems in some

cases. These abnormalities consisted of suppression of differentiation

13
of nervous tissue, microcephaly, and abnormalities of closure of neural
folds. Embryos from.mice injected with heart emulsions showed no effects
of the treatment. These results are suggestive of a transmission of
molecules from the mother to the embryo which produce specific morpho-
genetic effects in the case of the nervous system, but it was not possi-
ble to demonstrate that the antibodies present in the mother were able
to enter the embryo.

Thorough reviews of the work on the effects of antisera on embryonic
development can be found in Nace (1955) and Tyler (1957). It is apparent
that evidence exists that substances from adult tissues and antisera
against these substances have an effect on homologous differentiating
systems. However, there is considerable confusion as to exactly what
these effects are.

The objectives of the present investigation were: (1) a detailed
analysis of some previous experiments (Shaver 1954) on the differentia-
tion of the nervous system of embryos of.§ag§’pipiens which had been
cultured in or injected with fractions of adult brain of.§. pipiens, (20
additional experiments in which early embryos of‘gs.pipiens were treated
with adult tissue fractions, employing additional tissue types, in an
. effort to secure more comparable data, (3) a study of the effects on the
differentiation of embryonic tissue of antisera prepared against homolo-
gous adult tissue, and (4) an investigation of the effects of antisera
prepared against specific proteins from the adult frog heart on the devel-

opment of the embryonic heart.

_ ____,:_._;

 

gar era-.3 ‘ws‘...’

 

 

14

MATERIALS AND METHODS

The leopard frog, Rana pipiens, served as the experimental animal
for all experiments. These frogs were obtained from commercial dealers
in Wisconsin and Vermont.

The adult tissues with which embryos were cultured or injected were
prepared in the following manner. Hearts and brains were removed from
adult frogs, weighed, and suspended in a 0.005 M.KH2PO4-Na2HPO4 buffer
solution at pH 7.4 in the ratio of 1 gram of tissue to 10 ml. of buffered
solution. The tissues were then homogenized at 4°C, either in a glass
homogenizer or a Virtis homogenizer, model 23. The homogenates were .
centrifuged at 2,500 x g at 4°C to remove connective tissue, unbroken
cells, and other debris. The resultant supernatant fluid was centrifuged
at 18,000 x g. The particles thrown down at this force are referred to
in the experiments as cytoplasmic granules. These were washed by centri-
fugation and resuspended in a volume of phosphate buffer equal to the
volume of the supernatant fluid. The supernatant fluid from the centri-
fugation at 18,000 x g was used without further dilution. In the experi-
ments reported by Shaver ( L954), the histological analyses of which are
reported below, the procedure for obtaining fractions of tissue homogen-
ates was identical to that described above except that Holtfreter's solu-
tion was employed as the homogenization medium. Holtfreter's solution
is a dilute Ringer's solution, buffered with NaHCO3 at pH 7.2.

The eggs employed were obtained after artificially induced ovula-
tion resulting from injection of pituitary glands of frogs, (Rugh 1934).
Sperm suspensions for inseminating the eggs were prepared in the following

way. Testes were removed from pithed male frogs and crushed in 10 per cent

 

 

my slur

 

 

15
full strength Holtfreter's solution, 5-7 ml. per testis. The suspension
was then filtered through glass wool to'remove excess testicular tissue.
Eggs were placed on 1 x 3 inch glass slides and flooded with sperm suspen-
sion. After 10 minutes the excess sperm suspension was poured off, the
eggs placed in a finger bowl filled with aerated tap water, and allowed to
develop at room temperature to the 32-128 cell stage (Shumway stages 748)
at which time treatment was begun. This stage was found to be most suit-
able for 2 reasons: (1) injection of any material usually produces a
large percentage of gross abnormalities in earlier stages, and (2) treat-
ment at later stages does not appear to affect early morphogenesis signi-
ficantly.

Embryos were treated with tissue fractions or antisera in 2 ways.
In some experiments, embryos were dejellied and cultured in the presence
of the tissue fractions or antisera. In these experiments the developing
embryos were kept at about 12°C and fresh tissue fractions or antisera
were added daily. Following gastrulation, the developing embryos were
placed in 10 per cent full strength Holtfreter's solution and kept at
20°C. In experiments of Shaver (1954)-and. personal comunication), it
had been noted that there were no apparent differences in bacterial growth,
under these conditions, and when an antibiotic (chloromycetin) was used.
Due to this fact, and because antibiotics are generally quite toxic to,
embryos, they were not employed in these experiments.

In other experiments the tissue fractions or antisera were injected
into the embryo at Shumway stage 7 or 8. Injection of tissue fractions
6r antisera into embryos was performed with a micro-injection apparatus,

consisting of a 10 cc. syringe to which polyethylene tubing of small dia-

16
meter was attached by a special adapter. A.micropipette (tip diameter
approximately 0.02 to 0.05 mm.) made by drawing out a capillary tube of
1 mm. outside diameter over a small flame, is inserted in the free end
of the tube. The syringe was filled with water prior to the insertion
of the pipette, an air space being left to keep the material being in-
jected from coming in contact with the water. An attempt was made to
inject the material into the small blastocoele which was beginning to
form when the embryos were injected. In all cases injections were made
in the animal hemisphere. The embryos injected with tissue fractions
were kept at 12°C for 2 days and then allowed to develop at 20°C." The
2-day cold period was eliminated in the experiments in which antisera
were injected into embryos. '

The embryos were observed periodically and all changes and abnor-
malities were recorded. Embryos subjected to fractions of brain’were
fixed at the early tailbud stage (Shumway stages 17-19), while embryos
which had developed in the presence of fractions of heart were allowed
to develop into swimming larvae (Shumway stages 21-24) before being.
fixed. Embryos in early stages (Shumway stages 17-19) were fixed in
Smith's modification of Tellyesnicky's fluid, while those in later stages
were fixed in Smith's modification of Bouin's fluid. The embryos were‘
embedded, serially sectioned at 13 microns, mounted, stained in hematoxy-
lin and eosin, and examined for abnormalities. In the experiments where
the embryos appeared to have enlarged notochords, the diameter of the
notochord of each embryo was measured with an ocular micrometer. .

Organs which were to be used as antigens were removed, weighed,

and homogenized in 0.85% NaCl buffered at pH 7.4 with 0.005 M phosphate

 

- ’J.‘ '

-3, AZ*_3_"~ . -‘ '.

 

 

l7
buffer. The homogenate was centrifuged at 1200 x g and the supernatant
fluid was used for injection into rabbits.

The first antisera prepared against these antigens were produced
by giving a male rabbit 5 intravenous injections during a lO-day period,

1 every other day. The first 2 injections were 0.5 m1. of antigen each;
the third 1 ml.; the fourth 1.5 ml.; and the fifth was 2 ml. . Two weeks
following these injections each animal was given an anamnestic injection
of 2 ml. of antigen intravenously. Seven days later the rabbit was bled
from the ear and a precipitin titer was run. 'It the titer was at least
1:64 the rabbit was bled as soon as possible by heart puncture. If the
concentration of antibodies was not high enough, the rabbit was given
another 2 m1. injection of antigen and a titer was run again in 1 week.
This procedure was carried out until the desired concentration of anti-
bodies was obtained. .

Most of the antisera used were prepared by using the Freund adju-
vant technique. The adjuvant mixtures, available from Difco Laboratories,
Detroit, Michigan, are of 2 types, complete and incomplete. The complete
adjuvant mixture is composed of mannide monooleate, 1.5 ml.; paraffin oil,
8.5 ml.; and 5 mg. of killed and dried Mycbbacterium butyricum. The incom-
plete adjuvant mixture lacks the Mycobacterium butyricum. Each rabbit
received 3/4 m1. under each scapula of an emulsion consisting of equal
parts of antigen and complete adjuvant mixture. A week later an injection
using incomplete instead of complete adjuvant mixture was similarly made.
From 3 to 4 weeks later, the rabbit was bled from the ear and the anti-
serum was analyzed using the Ouchterlony technique as modified by Fox (1959).

If the analysis indicated the presence of antibodies, the rabbit was exsan-

l8

guinated by cardiac puncture as soon as possible. Otherwise, the animal
was given a second injection with the incomplete adjuvant-antigen mixture.

The Ouchterlony technique was performed in the following way. A
2% agar solution in 0.85% NaCl buffered with 0.005 M phosphate buffer at
pH 7.4 was prepared, filtered, and measured out in 30 ml. quantities.
Eight 1 5/8 x 1/2 inch strips of filter paper (Whatman #1) were arranged
equidistantly around the lip of the male half of a Petri dish with a small
portion lying on the bottom of the dish. The dish was then sterilized
by dry heat at 120°C for 12 hours. Following sterilization, 30 ml. of
sterile agar were poured into the dish and the agar was allowed to harden.
After hardening, a template was placed over the agar, wells were cut and
the pieces of agar left in the wells were removed. A drop of agar was
then placed in each well to seal the bottom. Each of the wells held 0.15 ml.
of solution. In most cases each plate was given a total of 10 doses of
0.15 ml. in each well. For most of the anaylses performed, the antiserum
being analyzed was placed in the center well (labelled An in Plate 4,
figurelzo, the homologous antigen was placed in well 2, and other antigens
being used for comparative purposes were placed in wells 1 and 3 (see
Plate 4, figure12). In some plates a comparison of 3 antiserawas accom-
plished by placing the antisera in wells 1, 2, and 3, while the antigen
was placed in the center well. For the analysis of complex systems invol-
ving numerous antigenic components, the Bjorklund (1952) specific inhibition
test was employed. In this procedure, the antigen which was used for inhi-
bition was placed in the antiserum well in doses of 0.15 ml. until the de~
sired number of inhibiting doses were absorbed. Subsequent to the last dose

of inhibiting antigen, the plates were run as usual.

19

Protein extractions from muscular tissues were carried out with a
modification of Mommaert's (1958) procedure for the preparation of myosin.
Extractions were made of heart and skeletal muscle from both adult chicken
and frog. Skeletal muscle and heart ventricles were removed from freshly
killed animals and placed immediately into ice water. After thorough cool-
ing, the muscle was weighed, mixed with 3 m1. of Guba-Straub solution
(0.3 M KCl, 0.10 M KH2P04, and 0.05 M KZHPOA) per gram of tissue, and minced
with the Virtis homogenizer. After extracting for 15 minutes with constant
stirring the material was centrifuged to remove the muscle residue. The
solution was diluted with 10 volumes of cold, deionized water (added slowly
with constant stirring) and left in the cold until the protein settled
to the bottom, usually about 3 hours. The supernatant fluid was siphoned
off as far as was practicable and the protein collected by centrifugation.
The protein was then dissolved in as small amount of 0.6 M KCl as possible.
This solution was diluted with 10 volumes of cold, deionized water in the
same manner as before. The protein was allowed to settle in the cold and
was collected by centrifugation after siphoning off the supernatant fluid.
The protein was then dissolved in a K01 solution, buffered at pH 6.5-6.8,
the concentration of which was 0.4 M.with respect to KCl and contained

KHZPO4 and K HPO4 in equimolar proportions, to bring the ionic strength

2
up to 0.5.
The procedure for preparation of antisera against the protein extracts
of muscular tissue was identical with that already described for the anti-
sera against homogenates of adult frog heart and brain. With the chicken

material, a concentration of 1 volume of packed protein to 5 volumes buf-

fered KCl was sufficient to elicit an immune response in the rabbit. How-

20
ever, a concentration of 1 volume of the protein from frog muscle to 2
volumes of buffered KCl was necessary to produce the desired immune
reaction which was analyzed with the Ouchterlony technique. The treat-
ment of embryos with the antisera against muscle protein was essentially
the same as that described for the antisera against adult frog heart and
brain except that no culturing experiments were performed. Fixation,
histological preparation, and microscopic examination of the embryos were
the same as that for the organ antisera.

In cataloging the results, all abnormalities were noted but special
attention was placed on the heart. Each embryo was scored as either hav-
ing a normal-sized heart or as possessing an inhibited or reduced heart.
At first, an attempt was made to catalog the hearts as normal, slightly
inhibited, and greatly inhibited. The "slightly inhibited" category was
later drapped after it was decided that this fine distinction could not

be made with any degree of accuracy.

   

 

21

RESULTS

A. Effects 2: Adult Tissue Fractions gngmbryonic Development.

Most of the results of treating developmental stages with adult
tissue fractions were obtained with frog brain. They represent in part
a microscopic examination of embryos produced in experiments performed
by Shaver (1954), in which embryos which were cultured in tissue fractions
of adult brain showed no consistent effect on“ the development of the
nervous system. 1

In experiments in which embryos were injected with fractions of
adult frog brain, however, the neurocoele was very much reduced or ab-
sent in many of the treated embryos. This condition, termed "solid ner-
vous system” (Plates 1-3, pages 29-33% extended throughout the entire
length of the brain and spinal cord in some embryos, but in others was
localized in a particular region of the nervous system. No correlation
could be made between the location of this abnormality and the tissue
fractions employed. However, the fraction of cytoplasmic granules pro-
duced the effect with greater frequency and with more consistency from
one experiment to another. In 3 of 4 experiments in which cytOplasmic
granules of adult brain were injected, from 28 to 37 per cent of the
embryos had solid nervous systems. A fourth experiment gave completely
negative results. The data from these experiments are given in Table l
. (page 22) .

It can be seen from Table 1 that the development of the nervous
systems of embryos treated with blood cells from adult frogs were normal.

However, the number of embryos so treated was extremely small.

22

 

 

o.o o.o -- -- o A A -- -- Hoouoomcav ooon
o.o o.o -- -- o m m -- -- Aewu=UHsuV soon
¢.mm o.wN 0.0m m.mm NH we n mN 0H noouoonoHv moHocouo onum
N.m o.oH 5.0 n.0H m Hm OH mH o AoououHoov moHscouo swoon
o.“ 0.0 k.o 0.0m m HA Hm om oH A.meav wasHm “assessmesm caste
N.mH o.o o.ON o.oN q NN N mH m A.uH50v ownHm oomuocuomom onum
o.w m.m o.o N.wH m mm mH HH HH owoum oHonz
-m.m o.o a.e 0.0 H o H c om NH mH H seamen osmeemoem
o.o o.o 0.0 0.0 o o o 0 NM H NN m Houuooo ooNNHHuuom
m N H m N H m N H
.24 mam mam mam mHMuOH mam mam mam mHmuoa nxm mam axm
maoumzm maoummm unoeumouH

womanfim mo
Honaoz Houoa

moo>uoz oHHom cqu
ochunam mo uCoU pom

moo>uoz oHHom HHHB
ochunEm mo nonaoz

 

 

EoumMm mso>uoz odekunam wcHooHo>on onu co swoon uHoo< mo moowuomum oommHH mo mucoHMMIn.H oHan

 

 

 

 

23

The only other effect on the embryos which was noted was an apparent
increase in the size of the notochord in embryos injected with supernatant
fluid of adult brain homogenates. This was very marked in Experiment 2
and was noted to a lesser extent in Experiment 3. In Experiment 2 the
mean diameter of the notochord in embryos injected with supernatant fluid
was 129.18 microns as compared to 97.86 microns for the controls. The
range of notochord diameters was 70-180 microns for the embryos injected
with supernatant fluid, whereas the range for the controls was 80-120
microns. Plate 3, figure 11 (page'33) shows an embryo.:w.i‘th an extremely
enlarged notochord. The values obtained from an analysis of individual
measurements (Table 2, page 24) suggest that the notochords were either
grossly affected or were not affected at all. It is possible that this
size difference may represent merely a general delay in development. Most
of the embryos with enlarged notochords still had unclosed neural folds,
indicating that their development was retarded. As the notochord differ-
entiates, its diameter becomes less and consequently a greater diameter
of the notochord may represent no more than general delay in development.

Additional experiments were performed by the writer during the
winter of 1958. The data from some of these experiments, given in Tables
3 and 4 (page 25), indicate that the specific inhibition of differentia-
tion of embryonic nervous tissue by adult brain may be more difficult to
demonstrate than the earlier experiments suggested, inasmuch as the embry-
onic nervous system can be shown to be affected by other agents.

In Table 3 data from experiments are presented in which a comparison
was made between the effects of tissue fractions from heart and brain of

adult frog on the development of the homologous embryonic tissues. In

 

"try—”1'1. 9.7.2.? -""" ' V"

‘

 

 

 

24

Table 2.--Experiment 2 Notochord Size (diameter in mocrons) of Individual
Embryos Treated With Adult Brain Tissue Fractions.

 

Fertilized Injected Injected Injected Cultured Cultured Whole
Control P04 Granules Supernate Granules Supernate Brain

(21 emb.) (13 emb.) (22 embi) (22 emb.) (13 emb.) (15 emb.) (10 emb.),

 

100 100 120 180 150 90 90
100 80 85 100 120 130 90
110 110 100 150 120 90 90
120 110 90 100 90 110 100 ‘
100 90 130 140 85 80 80
120 80 105 120 90 135 100
120 90 90 100 80 95 150
80 110 130 100 110 110 130
120 110 140 140 100 95 120
110 100 105 140 110 100 110
95 90 125 160 120 40
9O 80 100 120 105 90
90 160 80 90 65 130
90 105 120 100
90 120 110 80
80 110 70
80 110 170
100 100 170
100 100 100
80 ,80 120
80 120 180

110 140

X 97.86 100.77 107.14 128.18 103.93 98.33 106.00

P(.05

 

Table 3.--Effects of Tissue Fractions of Adult Brain and Heart on the
Differentiation of the Embryonic Brain and Heart

 

25

 

 

Total Number

Number of Embryos

Number of Embryos

 

 

of Embryos With Abnormal With Abnormal
Nervous Systems Hearts
Treatment
Exp 5 Exp 6 Exp 5 Exp 6 Exp 5 Exp 6

Fertilized Control 20 - 0 - 0 -
Phosphate Buffer (c) -- 6 - 0 - N
Phosphate Buffer (i) 18 15$ 0 3 0 2
Brain Granules (c) -— 6 - 0 - N
Brain Granules (i) 20 6 3 3 N N
Brain Supernate (c) -- 6 - l - N
Brain Supernate (i) -- 6 - 3 - N
_Heart Granules (i) 24 12 0 3 1 4
Heart Supernate (i) -- 7 - 3 - l

 

Table 4.--Abnormalities of the Nervous System Produced by Treatment of
Embryos with Antisera Prepared Against Adult Organs

 

Treatment

Total Number
of Embryos

Number of Embryos With
Abnormalities of the
Nervous System

 

Fertilized Control
Holtfreter's Solution (i)
Nonmal Rabbit Serum (c)
Normal Rabbit Serum (1)
Brain Antiserum #11 (c)
Brain Antiserum #11 (1)
Heart Antiserum #2 (c)
Heart Antiserum #2 (i)

24

5
21
17
10
12
ll
23

I—‘uI-‘UD-‘J-‘U'Iow

 

cultured
injected
no observation

26
Experiment 5 (Table 3) it appears that nervous tissue development is
affected by adult brain granules, but not by heart granules. However,
in Experiment 6 (Table 3) it can be seen that fractions of both heart
and brain affect the developing nervous system. Due to the fact that
embryos which were treated with fractions of adult brain were fixed
at earlier stages than the embryos treated with adult heart fractions,
it was not possible to assess the effects of adult brain on heart dif-
ferentiation, since at the stage when these embryos were fixed (Shumway
stages 17—19) heart development is just commencing. However, the effect
of heart granules on the developing heart in Experiment 6, and to a
lesser degree, in Experiment 5 was clearly inhibitory inasmuch as heart
development was greatly suppressed.

Table 4 (page 25) presents results of an experiment in which embryos
were treated with antisera against adult tissues. It will be noted that
both normal rabbit serum and antisera prepared against adult frog brain
and heart produced abnormalities of the developing nervous system. .These
abnormalities were of the same nature as those which were produced by the
tissue fractions of adult frog brain. Results such as those listed in
Table 4, showing effects on the developing nervous system, were observed
periodically in the experiments with antisera prepared against various
adult organs.

The results presented above, together with some suggestions from
preliminary experiments on the effects of antisera prepared against adult
organs, led to the re-orientation of the experimental program towards the
following objectives: (1) A study of the effect of experimental treat-

ments on the differentiation of the heart. This organ would seem to

“: v m”-

 

 

 

27

present the possibility for a more definite assessment of the experimen-

tal treatment, inasmuch as inhibition of its development can apparently
occur without concomitant effects on other organs, and (2) A study of

the effects of antisera prepared against various organs of adult frog on
embryonic development. Similar studies on the chick had demonstrated
specific inhibitions of organ differentiation, and it seemed desirable

to extend this type of approach to the frog, in view of the claims made
(Rose, etc.) that presence of the adult tissue in itself could produce

specific inhibition of differentiation.

28

PLATE 1

Examples of "Solid" Mid-brains as Produced by Injection of Embryos with

Tissue Fractions of Adult Brain.

Figure 1

Figure 2

Figure 3

Figure 4

x.s. through the mid-brain of a normal embryo from the
fertilized controls of Experiment 2

x.s. through the mid-brain of an embryo from Experiment 1
which had been injected with cytoplasmic granules of adult
frog brain '

x.s. through the mid-brain of an embryo from Experiment 3

which had been injected with cytoplasmic granules of adult
frog brain

x.s- through the mid-brain of an embryo from Experiment 1
which had been injected with supernatant fluid from adult
frog brain

 

 

c.”

 

30

PLATE 2

Examples of "Solid" Hind-Brains as Produced by Injecting Embryos With
Tissue Fractions of Adult Frog Brain

Figure 5 x.s. through the hind-brain of an embryo from the
fertilized controls of Experiment 2 (Same embryo as
in Figure 1)

Figure 6 Ix.s. through the hind-brain of an embryo from Experi-
ment 3 which had been injected with cytoplasmic gran-'
‘ules of adult brain

Figure 7 x.s. through the hind-brain of an embryo from Experi-
ment 1 which had been injected with supernatant fluid
of adult brain .

 

)1.

 

32

 

PLATE 3

Abnormalities of the Spinal Cord and Notochord as Produced by Injecting
Embryos with Tissue Fractions of Adult Frog Brain

Figure 8

Figure 9

Figurelfl

Figurell

x.s. through the mid-abdominal region of an embryo
from the fertilized controls of Experiment 2 (same
embryo as Figures 1 & 5).

x.s. through the mid-abdominal region of an embryo
from Experiment 1 which had been injected with super-
natant fluid from adult frog brains

x.s. through the mid-abdominal region of an embryo
from Experiment 1 which had been injected with cyto-
plasmic granules from adult frog brains

x.s. through the mid-abdominal region of an embryo
from Experiment 1 which had been injected with super-
natant fluid from adult frog brains. Note the size

of the notochord as compared to the control (Figure 8).

 

 

33

 

 

 

   

34

Effects 2f Antisera Prepared Against Adult Organs 23 the Differentia-
tion.g£ Embryonic Tissue. .

1. Analysis of Antisera

The analysis of the antisera prepared against adult brain,
kidney, and heart showed that these organs have several complemen-
tary factors in common (Plates 7-10). In fact, it was impossible
to determine with certainty that any of the antisera had specific
components without the use of adsorption techniques. From analyses
in which several antisera prepared against the same adult organ were
compared, it was seen that these antisera contained several common
components, but some antisera possessed components which were not
present in other antisera prepared against the same organ (Plate 10,
Figures 45 and 46). Presumably, this is due to the well-established
fact that the antibody-producing mechanisms of individual rabbits
react differently to the same antigen.

With adsorption techniques, it was seen that the antigenic
properties of heart and kidney are similar and that these differ
considerably from the antigenic properties of brain (Plates 7-10).
This seems to be largely due to the presence of more factors in
antisera prepared against heart and kidney than are present in anti-
brain sera., For instance, when anti-kidney or anti-heart sera were
adsdrbed with brain antigen, these antisera still possessed several
components against homogenates of heart and kidney (see Plate 8,
Figures 28, 31 and 34). On the other hand, when anti-brain sera
were adsorbed with heart or kidney, no more than one component could

be demonstrated in these antisera. (Plate 10, Figure 43).

 

35

0f the 4 antiaheart sera tested, 2 (#10 and #52) showed single
specific components when adsorbed with kidney. Anti-heart serum #53
appeared to have a specific component when adsorbed with brain but
this disappeared when this serum was adsorbed with kidney antigen
(see Plate 8, Figures 31 and 32). It is interesting to note that
antiserum #53 produced less heart inhibition than any of the other
anti-heart sera employed. However, anti-heart serum #49 produced
the highest per cent of inhibition (36.8%) of any of the anti-heart.l
sera used and it showed no specific line in the Ouchterlony plate
when adsorbed with kidney.

All of the anti-kidney sera seems to have specific components
when adsorbed with brain (see Plate 9, Figures 37 and 40; Plate8,
Figure 34). However, when heart was used for adsorption, only 2 of
the anti-kidney sera (# l4 and #26) seemed tdnhave a specific compo-
nent. This may be due to the fact that the heart homogenate was a
very poor material for adsorption tests. In nearly every test which
was made the heart homogenates that were used for adsorption clogged
the antiserum well to the extent that the antiserum being tested did
not diffuse into the agar nearly as rapidly as the antigens. In many
instances the tests were discontinued before completion because the
antisera would not diffuse at all. Attempts to overcome this diffi-
culty by centrifuging the homogenates at greater forces and using the
supernatant fluid for adsorptions were not successful: For these
reasons, it is felt that the negative tests involving adsorption with

heart antigen may not necessarily represent the true situation.

 

 

36

Table 5 (Page 37) gives a complete list of all the adsorption
tests performed. It can be seen that most antisera seemed to have
some degree of specificity. However, most plates were given only
6 doses of inhibiting antigen, while a total of 5 doses of the same
antigen was given to the plate following adsorption. Consequently,
it is very likely that the specific lines which appeared in the
Ouchterlony plates represent no more than quantitative differences
of antigenic substances in the various organs employed in the tests.
2. Effects of Antisera on Differentiation of Embryonic Tissues

a. External Manifestations of Treatment with Antisera

Embryos treated with antisera prepared against homogenates of
adult organs were observed from the time of treatment (late cleavage
to early blastula, Shumway stages 7-8) until they were fixed (5-6 days
following treatment). In most experiments, all the embryos, including
those treated with phosphate buffer and control sera, showed some gen-
eral abnormalities, but nothing specific was observed. However, there
were a few cases in which certain characteristic abnormalities were
noted in the embryos treated with antisera prepared against adult
organs.

In Experiment 20, about 57% of embryos injected with a 1:8 con-
centration of kidney antiserum #14 were extremely edematous. Control
serum, antiserum against adult brain, and antiserum against adult
heart did not produce this effect at all. This syndrome is charact-
erized by extreme distention of the central body wall due to the pre-
sence of accumulated fluid in the body cavity. Although antiserum

#14 was used in a total of 5 experiments, Experiment 20 was the only

37

Table 5.--The Presence of Specific Factors in Anti-Organ Sera as Deterr
mined by the Technique of Adsorption

 

Adsorbed With

 

 

Antiserum p‘
u m C.‘ '08
5.4 c: ”-1 021
m w: m on
m -a u r40
.11 M an new
Heart Antiserum #2 - 0 + 0
Heart Antiserum #10 - + + -
Heart Antiserum #49 0 - 0 0
Heart Antiserum #52 - + + 0
Heart Antiserum #53 - - + 0
Kidney Antiserum #14 + - + -
Kidney Antiserum #26 + - + 0
Kidney Antiserum #47 - - + 0
Kidney Antiserum #57 - - + 0
Brain Antiserum #11 + 0 - 0
Brain Antiserum #17 + + - +
Brain Antiserum #56 - - - 0
+ = Presence of lines in the Ouchterlony
plate which appeared specific
- = Absence of specific lines in the Ouch-
terlony plate
0 = No test made

 

 

 

 

 

 

38

one in which the edematous condition was observed. Three other kidney-
antisera (#26, #47, and #57) were used in other experiments, and none
of these produced this condition in any experiment. It is possible
that the particular embryos used in Experiment 20 were extremely sensi-
tive to antiserum prepared against adult kidney.

Histological examination of the edematous embryos of Experiment
20 showed that the pronephros was present. Unfortunately, the extreme
edema had either distorted the tissues or had resulted in such poor
fixation that a thorough histological examination was impossible. In
some of the embryos, the cells comprising the walls of the pronephric
tubules seemed loosely arranged and the lumen of the tubule appeared
enlarged. Plate 12, Figure 62 shows a cross-section through the
pronephros of one of the edematous embryos. In many of these embryos,
the heart was very much reduced in size, as well. This appears to be
a secondary effect of the edema, sinCe the heart was not affected in
any embryo of this group that was not edematous. Also the heart was
normal in edematous embryos if the edema was confined to the regions
of the abdomen posterior to the pericardial space. Table 6 below
gives the data from Experiment 20 in regard to the edematous condition.

Table 6.--Experiment 20. Edema in Embryos Treated With Adult Organ
Antisera.

 

Treatment Total Number Number of Embryos % of Embryos

 

of Embryos with Edema with Edema
Fertilized Controls 52 0 0.0
Phosphate Buffer 49 3 6.5
Control Serum #10 2 0 0.0
Anti-heart Serum #10 27 0 0.0
Anti-heart Serum #17 19 0 0.0
Anti-kidney Serum #14 40 23 57.5

 

 

 

 

 

 

39

Another type of characteristic abnormality which was externally
identifiable was noted in larvae of 2 experiments (10 and 12) in which
embryos were cultured in and injected with a 1:8 concentration of an
antiserum prepared against adult frog heart (Anti-heart serum #2). A
high percentage of embryos cultured in this antiserum showed a very
marked abnormality of the spine. The curvature of the spine was so
abnormal that the tip of the tail was located directly beneath the
abdomen. Unfortunately, no pictures were taken of the external appear-
ance of these embryos. Histological examination of the abnormal embryos
in Experiment 10 showed that most of them had a very specific defect in
the central nervous system. At the juncture of the hind-brain and neural
tube, the nervous tissue was greatly enlarged and considerably disorgan-
ized. There were often 2 or more neurocoels throughout the affected
region. Examples of this abnormality are shown in Plate 12, Figures 59
and 60. Embryos in Experiment 12 were not analyzed because poor fixa-
tion made sectioning impossible. The data from these 2 experiments are
summarized in Table 7 below.

Table 7.--Curvature of the Spine as Caused by Culture in Heart Anti-
serum. (data from Experiments 10 and 12)

Total Number of Number of Embryos with
Treatment Embryos Abnormal Spines
(external appearance)

Fertilized Controls 22 0
Control Serum. 34 2
Holtfreter's Solution 12 0
Heart Antiserum #2 31 13

 

In another experiment (11), embryos were cultured in dilutions of

the same antiserum (#2) of 1:16 and 1:32. The effect noted above was .

--w-:‘v‘—" we’d-'3? '3 Wm " .- k4

 

 

 

 

40

not observed in.this experiment at all. Nothing can be said of the
specificity of this effect since antiserum prepared against heart was
the only one used in these experiments. It is entirely possible that
most antisera prepared against other adult organs would affect the
nervous system in this way, under similar conditions.
b. Histological Analysis of Embryos Treated with Antisera

“The most consistent and significant morphogenetic effect noted
in the histological analysis of larvae developed from embryos into
which antisera against adult organs were injected, was the inhibition
of the development of the heart by the antisera prepared against homo-
genates of adult frog heart. This inhibitory effect ranged from a
slight decrease in size of the heart to ancextreme condition in which
the heart was completely absent (see Plate 11). Usually, all other
internal structures, including the pericardial space, were normal.
Externally, these embryos showed stunted gills, varying degrees of
failure of outgrowth of the tail, and in some cases a very rough epi-
dermis. In addition, there seemed to be a greater tendency among these
embryos to be slightly edematous. However, these external abnormal-
ities did not prove to be reliable criteria of heart abnormality, since
they were noted in embryos in which the heart was essentially normal.

As was mentioned earlier, an attempt was made at first to classify
the affected embryos into 2 groups, slightly or extremely inhibited.
Due to the lack on an adequate criterion upon which to make this dis-
tinction, the "slightly inhibited" group was dropped and these embryos
(were classified as normal. Consequently, in all the results which are
listed below only those embryos in which the heart was either drasti-

cally reduced in size or absent are classified as being affected.

 

 

 

 

41

In these experiments, dilutions of the antisera ranging from
1:4 to 1:32 were employed. From preliminary experiments it was thought
that this would be sufficient to show a dilution effect. However,
when the data were tabulated and analyzed with the chi-square test for
heterogeneity, it was found that there were no significant differences
in the percentages of inhibition produced by the dilutions of heart
antisera in the range given above. Thus, in Table 8 all embryos which
were treated with the same antiserum are listed together, the results
of treatment with the various dilutions having been pooled. In some
experiments only one concentration of the several employed was used.
In Experiments 10, 13, and 14 a concentration of 1:8 was used, while
in Experiments 18 and 19 a dilution of 1:6 was employed. It will be
noted in Experiment 17 (Table 8) that 5 of 32 embryos treated with
kidney antiserum #14 had inhibited hearts. All but one of these 5
were in the group treated with a 1:4 concentration. Weaker concentra-
tions of this antiserum apparently were ineffective in producing heart
inhibition. This was the only case noted in the experiments of a de-
crease in inhibitory effect with a decrease in concentration of the A
antiserum.

From Table 8 it will be noted that of 8 experiments, data from
4 experiments (10, 15, 18, and 19) were statistically significant be-
yond the 0.01 level; data from 2 experiments (16 aaui 17) were signi-
ficant beyond the 0.05 level; and data from 2 experiments (13 and 14)
were not statistically significant when the chi-square test for hetero-
geneity was used. It can be seen (Text figure 1, page‘44) that the

developing hearts in 30% of all embryos which had been injected with

 

42

heart antisera were inhibited as compared to the hearts of embryos
injected with control sera (8.6% inhibition). Kidney antisera had a
slight inhibitory effect (12.5%) on the development of the heart,

while brain antisera appeared to have no inhibitory effect on the

heart at all. Phosphate buffer, when injected alone, seemed to be
inhibitory to the developing heart to a very slight extent. However,
this inhibition appears to be due to a general cytotoxic effect, since
many of the embryos were extremely abnormal and many died. The addition
of a tissue homogenate or rabbit blood serum tended to decrease this
apparent toxicity of the phosphate buffer.

Apparently, the period at which embryos were treated was very
critical in the experiments involving injection of antisera into
embryos. Several experiments were performed in which embryos were
injected from late blastula through early tailbud stages. No inhibi-
tion of organ structure or function was noted in these experiments.

The antisera were adsorbed with homologous antigens in an attempt
to remove the inhibitory effect. Unfortunately, in 2 experiments in
which embryos were treated with adsorbed antisera, the unadsorbed antiL
sera did not produce any appreciable inhibition of heart development.
Consequently, it was impossible to show that the inhibitory activity
could be removed by adsorption. However, in these experiments with
adsorbed antisera, heart inhibition was noted in a few embryos treated
with heart antisera which had been adsorbed with brain. On theoother
hand, there were no cases of inhibition among the embryos treated with
anti-heart serum adsorbed with heart. From these preliminary studies,
it appears that adsorption of the heart antisera with heart antigen

removes the inhibitory effect but adsorption with brain antigen does not.

abummHuq< unmow u m<m
23 abuomauqd hoavfix u m<M
A. muummm vmanHnaH nufi3 womanam mo ucmo mama n N asuomHua< chum u m<m
muumom pmananH nuwa moNHABm mo Honasz u z Enumm Houucou n mo
achunam mo Honasz Hmuoa n H mommam ouwndmosm u «om

mHouuaoo wouNHHuuom n on

 

Ho.v Ho.v Ho.v no. v mo.v Ho.v 8. om. How «H
H q mN ------- a.NH a mN -------- -------- -------- ------- ------- ------- mmam<m
N m on ------- N.NN w on -------- -------- -------- ------- ------- ------- NmNmam
m «H mm m.mm o NH H.wm m HN -------- -------- -------- ------- ------- ------- aqam<m
m mm.oHH ------- ------- ¢.om NH mm N.NN NH Ne «.mm oH om ------- ------- ------- oHNm<=
m NN ma ------- ------- N.mm NH an -------- -------- N.©H N NH m.oN m qN .mm a NN N4m<m
H

H

H

mm ------- N.mH m mm -------- -------- -------- ------- ------- ------- Nmamau
Nm w.wH m 0H H.HH a on -------- -------- -------- ------- ------- ------- NHNN<H
mm H.N H qH m.w N 0N -------- -------- -------- ------- ------- ------- oNamax
Nm ------- -- ..... o.mH m Nm -------- -------- ------- ------- ------- ¢H*m4x
an ..... -- N.N H «m -------- -------- -------- ------- ------- ------- omamam
NN ------- ....... o.o 0 HH -------- o.a H HH ------- ------- ------- NH4m<m
NN -- ..... ------- ..... --- -------- -------- o.o o NH o.o 0 OH ------- HHNm<m
NN ------- m.o N,NN -------- -------- -------- ------- ------- ------- Nmamo
mm ...... - N.N m an -------- -------- -------- ------- ------- ------- mmfimo
om ------- o.oH m on -------- -------- -------- ------- ------- ------- Nsamo
NH «.mH N NH ------- -------- -------- -------- ------- ------- ------- oNamo
ea ------- ------- N.©H N NH m.NH m as m.¢ N «a ------- ------- ------- oHamo
m.» H NH «.0 N Nm o.o o m NNNU
NN 0.0 0 NH o.o o N m.m H NH N.NN m HH o.a H HH m.w H NH N.mH N HH ------- Hem
NNH o.q H mN m.N m as m.m H NH o.o o NH m.w H NH o.o o NH m.m H NH o.o o NH on

2 H N z B N z B N 2 H N z B N z B N z B N z B N z B

mHmHOH mH wH NH oH mH «H mH oH acme
nummua

as ------- ------- -------- -------- --------

CQ’HQ'Q‘OO‘QOWO\OO\MNN®®N<T
MOOGWONOOQ'NWNMMHHONN

[\mMO‘NMMNOHHMMNl-fi

 

B\°

Honabz unmaaummxm

 

 

momwuo uHsv< umchw< muomHun< Maw: pouoowdm,m0hun8m aH unoaaoHo>oo unmom mo :oHuNHNncHnu.w «Home

 

Per Cent of Embryos with Inhibited Hearts

TEXT FIGURE 1

 

Inhibitory Effect of Organ Antisera On Heart Development
Accumulation of data from all experiments and all

 

antisera which were employed

 

 

 

 

 

 

 

 

 

 

 

 

 

3o!
30%
l
25
20
15
12.5%F
J.lQa&Z
10 1
8.6% _
w...——J:.
5 + f
2.8%
4rL
Fertilized Phosphate Control Brain Kidney wHeart

Control Buffer Serum Antisera

Antisera Antisera

 

 

'45

Effects pf Antisera Prepared Against Muscle Proteins 23 Embryonic
Development.

 

 

It was thought at the beginning of the investigation that it
would be possible to extract myosin from frog heart and skeletal mus-
cle by the same procedure as was used for extraction of myosin from
mammalian muscle. The original procedure used for protein extraction
was one suggested by Dr. Karl Guthe, University of Michigan. Accord-
ing to Guthe, this procedure produces myosin from rabbit muscle which
is 98% pure without employing any special procedures for removing
contaminants such as actin and actomyosin. However, when a method for
removing actomyosin was added to this procedure, practically all mat-
erial resulting from the protein extraction of frog muscle was removed,
suggesting that this protein was essentially actomyosin. Recent in-
vestigations (deVillafrance, 1956; Laufer, 1959) have shown that stand-
ard procedures for the isolation of myosin from mammalian muscle pro-
duce mostly actomyosin when applied to frogs and salamanders. Due to
the uncertain identification of the materials extracted from the muscle
tissues employed, the antigens are referred to below as "muscle protein."

Analysis of the antisera prepared against the muscle proteins
showed that all of the antisera contained more than a single comple-
mentary component in common with protein extracts of muscle or with
homogenates of organs. For example, antiserum #20, prepared against
protein extracted from heart muscle of the frog, was seen to have only
a single strong component against frog heart muscle protein and exhi-
bited no reaction with frog skeletal muscle protein and chick heart
muscle protein (Plate 5, Figure 15). However, when other tissues from

the frog, such as brain and kidney, were tested against antiserum #20,

 

46

there was some reaction between the antiserum and these organs (Plate
5, Figure 16).

Antiserum #15, prepared against frog skeletal muscle protein,
exhibited 2 complementary components against skeletal muscle protein
of the frog (Plate 4, Figure 13) and 1 against frog kidney (Plate 4,
Figure 14). The antisera prepared against proteins extracted from
chick heart and skeletal muscle contained numerous complementary comr
ponents to their respective antigens as well as to chick liver and
skeletal muscle homogenates (Plate 5, Figure 17; Plate 6, Figures 18-20).

Although there were no cross reactions between the antigens and
antisera from frog heart and skeletal muscle proteins, such cross
reactions were observed between the antigens and antisera from the
chick heart and skeletal muscle proteins. No cross reactions of anti-
gens and antisera of the frog with the antigens and antisera of chick
were ever observed. However, a comparison was made of extractions of
proteins from skeletal muscle from 3 species of frogs, Rana pipiens,

.3. clamitans, andIR. sylvatica, and these contained identical comple-
mentary components to antiserum #15 prepared against skeletal muscle
protein of‘g. pipiens (no photograph was made of this observation).

It would appear from this result that the proteins extracted from

these species of the genus Répg are very similar in antigenic proper-
ties. However, proteins extracted from the skeletal and cardiac muscle
of Rana pipiens are different in nature from chick proteins of similar
origin.

Embryos treated with the antisera prepared against muscle proteins

showed no detectable external abnormalities which were not observed.in

 

 

 

 

 

 

 

47
the controls. Microscopic examination of the embryos treated with
these antisera showed abnormalities similar to those which were observed
in embryos which were treated with antisera prepared against heart.
The most consistent of the abnormalities were varying degrees of inhi-
bition of heart development. Four experiments (#21-#24) were performed
in which the effects of the antisera prepared against muscle protein
on the developing heart were studied. Table 9 lists the number of
embryos with inhibited heart development which were observed in these
experiments. It will be seen from the table that there was little
consistency from one experiment to another. For instance, in Experi-
ment 21 antiserum #20, prepared against frog heart muscle protein, pro-
duced 34.5% inhibition of the heart but in other experiments this anti-
serum showed no appreciable effect upon the developing heart. When the
data were analyzed by the chi-square test for heterogeneity, the results
were significant beyond the 5 per cent level in only one experiment (#24).
Even when the data were pooled (Table 8, column marked "totals"), the

results were not statistically significant.

48

cNmuoum oHomDE ammo: Howso u 230
cHououm oHomse undo: woum n 2mm
cHououa mHomDE Hauonxm onno u Emu
:Nmuoum oHomss kuonxm monk n 2mm

wouNanaH muumon HUN3 moxunam mo uaoo pom u N
vmananH muumon :uH3 moNHHEo mo Monasz
moxunEm mo Honsbc Hauoe u H

ll
2

 

 

q.wH wH mm o.mN o ¢N N.oH q «N N.m N mN N.NN o NN Azmov mNN asumm
«.3 HH 3 o.o o «N Na H N -- - .. 0% 2 N cat 8* 58%
N.mH «H No m.w N qN o.mH m mN N.NH ¢ NN N.NN m NN AZmov wHN Epsom
m4: .2 an HON m 1N -- - -- -- - .. NEH m om 293 2* 53%
n.w N mN 0.0 o m N.NN N m o.o o m :- u I: abumm Houucoo
m.NH H w u- u .. nu- . u- u- u -u m.NH H w cOHusHom m.uoumumuHom
q.o m Nm 0.0 0 HH o.o 0 HH o.om m OH I. u .. Hommsm mumnmmonm
0.0 o Nq 0.0 o NH o.o 0 HH o.o o NH o.o o NH Houucoo umNHHNuumm
N z B N z B N z B N z B N z H
mHmuoH ¢N mN . .NN HN
wmnEDZ ucoafiuoaxm acoaumouh

 

 

.mchuoum oHomnz ust< uchmw< woumamum whomwua< Np uaofiaon>ma uummm mo aoHananHuu.a mHHmH

49

 

PLATE 4

g'Analysis.Of;AntiseragPrepgredsAgainst Muscle Proteins I

Figure 12

Figure 13

Figure 14

Geometry of the Ouchterlony Plates used in these
analyses, (after Fox, 1959).

Ouchterlony Plate No. 11
Antiserum #15-prepared against protein extract from
skeletal muscle of the frog

Well 1--Frogdheart muscle protein
Well 2--Frog skeletal muscle protein
Well 3--Chick skeletal muscle protein

Ouchterlony Plate No. 10
Antiserum #15-prepared against protein extract from
skeletal muscle of the frog

Well l--Frog brain Homogenate
Well 2--Frog skeletal muscle protein
Well 3--Frog kidney homogenate

 

 

 

 

 

 

 

 

 

r9 ..

, 6..

51

2 b .":’¢" J I" “3.".-

PLATE 5

Analysis of Antisera Prepared Against Muscle Proteins II.

Figure 15 Ouchterlony Plate No. 15

Figure 16

Figure 17

Antiserum #20-Prepared against frog heart muscle protein

Well l--Frog skeletal muscle protein
Well 2--Frog heart muscle protein
Well 3--Chick heart muscle protein

Ouchterlony Plate No. 14
Antiserum #ZO-Prepared against frog heart muscle protein

Well l--Frog brain homogenate
Well 2--Frog heart muscle protein
Well 3--Frog kidney homogenate

Ouchterlony Plate No. 13
Antiserum #18-Prepared against chick skeletal muscle protein

Well l--Chick heart muscle protein
Well 2--Ch1ck Skeletal muscle prOtein
Well 3--Fr6g skeletal muscle protein

 

 

 

 

 

 

 

 

53

‘3“‘“

PLATE 6

Analysis of Antisera Prepared Against Muscle Proteins III.

Figure 18

Figure 19

Figure 20

Ouchterlony Plate No. 12
Antiserum #18-Prepared against chick skeletal muscle protein

Well 1--Chick skeletal muscle homogenate
Well 2--Chick skeletal muscle protein
Well 3--Chick liver homogenate

Ouchterlony Plate No. 17
Antiserum #23-Prepared against chick heart muscle protein

Well l--Chick skeletal muscle protein
Well 2--Chick heart muscle protein
Well 3--Frog heart muscle protein

Ouchterlony Plate No. 16
Antiserum #23-Prepared against chick heart muscle protein

Well 1--Chick skeletal muscle homogenate
Well 2--Chick heart muscle protein
Well 3--Chick liver homogenate

 

 

.._._.__ _-—.._..._......__

 

 

 

— ~— —~ M" M.‘_.;_‘.:.

. 3"

 

 

'55

PLATE 7

Analysis of Antisera Prepared Against Homogenates of Adult Organs I

Figure

Figure

Figure

Figure

Figure

Figure

21

'22

23

24

25

26

Ouchterlony Plate III-A
Antiserum #ll-Prepared against frog brain homogenate

Well l--Frog heart homogenate
Well 2--Frog brain homogenate
Well 3--Frog liver homogenate

Ouchterlony Plate I-A
Antiserum #2-Prepared against frog heart homogenate

Well 1--Frog heart homogenate
Well 2--Frog brain homogenate
Well 3--Frog liver homogenate

Ouchterlony Plate No. 53
Antiserum #10-Prepared against frog heart homogenate

Well 1--Frog brain homogenate '
Well 2--Frog heart homogenate
Well 3--Frog kidney homogenate

Ouchterlony Plate No. 38

Same as Plate No. 53 (Figure 23) except the antiserum well
received inhibiting doses of brain homogenate prior to
testing the antiserum.

Ouchterlony Plate No.43

Same as Plate No. 53 (Figure 23) except the antiserum well
received inhibiting doses of kidney homogenate prior to
testing the antiserum.

Ouchterlony Plate No. 82
Antiserum #49-Prepared against adult frog heart

Well l--Frog kidney homogenate
Well 2--Frog heart homogenate
Well 3--Frog skeletal muscle homogenate

 

 

56

 

57

 

 

.' " ‘“"!‘{"7".‘.*” .H7

PLATE 8

Analysis of Antisera Prepared Against Homogenates of Adult Frog Organs II

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

27

28

29

30

31

32

33

34

Ouchterlony Plate No. 37

Antiserum #52-Prepared against frog heart homogenate
Well l--Frog brain homogenate

Well 2--Frog heart homogenate

Well 3--Frog kidney homogenate

Ouchterlony Plate No. 40

Same as Plate No. 37 (Figure 27) except the antiserum well
received inhibiting doses of brain homogenate prior to test-
ing the antiserum

Ouchterlony Plate No. 46

Same as Plate No. 37 (Figure 27) except the antiserum well
received inhibiting doses of kidney homogenate prior to test-
ing the antiserum. Although difficult to see on the photo-
graph, there is a single line between the antiserum well and
well 2.

Ouchterlony Plate No. 30

Antiserum #53-Prepared against frog heart homogenate
Well l--Frog brain homogenate

Well 2--Frog heart homogenate

Well 3--Frog kidney homogenate

Ouchterlony Plate No. 25

Same as Plate No. 30 (Figure 30) except that the antiserum
well received inhibiting doses of brain homogenate prior to
testing the antiserum. Note the line between the antiserum
well and well 2 which appears to be specific.

Ouchterlony Plate No. 44 -

Same as Plate No. 30 (Figure 30) except the antiserum well
received inhibiting doses of kidney homogenate prior to
testing.the antiserum. There were no lines in this plate.

Ouchterlony Plate No. 34

Antiserum #47-Prepared against frog kidney homogenate
Well l--Frog brain homogenate

Well 2--Frog kidney homogenate

Well 3--Frog heart homogenate

Ouchterlony Plate No. 41

Same as Plate No. 34 (Figure 33) except that the antiserum
well received inhibiting doses of brain homogenate prior to
testing the antiserum.

 

 

 

 

s {H

ctr

 

59

‘ 7"". ”‘WQ .——.— _ *—

PLATE 9

Analysis of Antisera Prepared Against Homogenates of Adult Frog

Figure

Figure

Figure

Figure

Figure

Figure

35

36

37

38

39

40

Organs III

Ouchterlony Plate No. 33

Well l--Frog brain homogenate
Well 2--Frog kidney homogenate
Well 3--Frog heart hOmogenate

Ouchterlony Plate No. 26

Same as Plate No. 33 (Figure 35) except the antiserum
well received inhibiting doses of brain homogenate
prior to testing the antiserum.

Ouchterlony Plate No. 28

Same as Plate No. 33 (Figure 35) except the antiserum
well received inhibiting doses of heart homogenate
prior to testing the antiserum. Notice the line which
appears to be specific for kidney.

Ouchterlony Plate No. 20

Antiserum #14-Prepared against frog kidney homogenate
Well l--Frog brain homogenate

Well 2--Frog kidney homogenate

Well 3--Frog heart homogenate

Ouchterlony Plate No. 35

Antiserum #57-Prepared against frog kidney homogenate
Well 1--Frog brain homogenate

Well 2--Frog kidney homogenate

Well 3--Frog heart homogenate

Ouchterlony Plate No. 42

Same of Plate No. 35 (Figure 39) except the antiserum
well received inhibiting doses of brain homogenate
prior to testing the antiserum.

 

 

 

.,

V. a... .vzas‘! : ,

$Ap.~~u‘ .

 

’\

‘c- -. .

q H... ‘ 'hfi“fl_ 1e

 

 

61

PLATE 10

Analysis of Antisera Prepared Against Homogenates of Adult Frog

Figure

Figure

Figure

Figure

- Figure

Figure

41

42

43

44

45

46

Organs IV
Ouchterlony Plate No. 22
Antiserum #17-Prepared against frog brain homogenate
Well l--Frog heart homogenate
Well 2--Frog brain homogenate
Well 3--Frog blood serum

Ouchterlony Plate No. 23

Same as Plate No. 22 (Figure 41) except that the anti-
serum well received inhibiting doses of frog blood serum
prior to testing the antiserum.

‘Ouchterlony Plate No. 48

Same as Plate No. 22 (Figure 41) except that the anti-
serum well received inhibiting doses of heart homogenate
prior to testing the antiserum. Also well 3 contained
kidney homogenate instead of blood serum. Although dif-
ficult to see there is a line between the antiserum well
and well 2.

Ouchterlony Plate No. 36

Antiserum #56-Prepared against frog brain homogenate
Well l--Frog kidney homogenate

Well 2--Frog brain homogenate

Well 3--Frog heart homogenate

Ouchterlony Plate No. 32

Center well--frog heart homogenate
Well l--Anti-heart serum #10

Well 2--Anti-heart serum #52

Well 3--Anti-heart serum #53

Ouchterlony Plate No. 31

Center well--frog kidney homogenate
Well 1--Anti-kidney serum #14

Well 2-4Anti-kidney serum #26

Well 3--Anti-kidney serum #47

 

 

 

 

 

 

5"

 

 

 

63
PLATE 11

Inhibition of Heart Development Resulting From Treatment of Embryos
with Antisera Prepared Against Adult Organs of the Frog

Figure 47 x.s. through the heart of a control embryo from Exp. 19

Figure 48 x.s. through the heart region of an embryo from Exp. 15
which was injected with anti-heart serum #10

Figure 49 x.s. through the heart region of an embryo from Exp. 10
which was injected with anti-heart serum #2

Figure 50 x.s. through the heart region of an embryo from Exp. 19
which was injected with anti-heart serum #49

Figure 51 x.s. through the heart region of an embryo from Exp. 15
which was injected with anti-heart serum #10

Figure 52 x.s. through the heart region of an embryo from Exp. 19.
which was injected with anti-heart serum #49

Figure 53 x.s. through the heart region of an embryo from Exp. 10
which was injected with anti-heart serum #2

Figure 54 x.s. through the heart region of an embryo from Exp. 19
which was injected with anti-kidney serum #47

 

 

aw: a" q

 

65

PLATE 12

Heart Inhibition and Other Abnormalities as Produced by Treatment of
Embryos with Antisera Prepared Against Homogenates of Adult Organs
and Protein Extracts of Skeletal and Heart Muscle

Figure

Figure

Figure

Figure

Figure

Figure

. Figure

Figure

55

56

57

58

59

60

61

62

x.s. through the heart region of a normal control from
Exp. 21

x.s. through the heart region of an embryo from Exp. 21
which was injected with antiserum #15 prepared against
frog skeletal muscle protein

x.s. through the heart region:of an embryo from Exp. 21
which was injected with antiserum #20 prepared against
frog heart muscle protein

x.s. through the heart region of an embryo from Exp. 21
which was injected with antiserum #20 prepared against
frog heart muscle protein. Note the double heart.
Slightly anterior to this section the pericardial cavity
was divided.

x.s. through the hind-brain ofi'an embryo from Exp. 10
which had been cultured in anti-heart serum #10. Note
the dogble neurocoele. '

x.s. through another embryo which had been treated in
the same manner as the embryo in Figure 59. Note once
again the extra neurocoels.

x.s. through the pronephros of a control embryo from
Exp. 20

x.s. through the pronephros of an edematous embryo from
Exp. 20 which was treated with anti-kidney serum #14.
The pronephric tubules seemed to be enlarged.

 

(.6

6
5

 

 

P.1v—'

’4 , .4. :S’fi~.,fi.¢g..p,

"‘JM

 

67
DISCUSSION

Effects gprdult Tissue Fractions 23 Embryonic Deve10pment

As has been pointed out in the introductory section, one group
of investigators believes that substances from adult tissues stimulate
the growth of homologous embryonic tissue, whereas others feel that
such substances have inhibitory effects on the differentiation of the
corresponding developing system. The bulk of the evidence which has
been used to support the former idea has been derived' from experiments
in which chicken spleen was grafted onto chorioallantoic membranes of
the chick embryo. There are many earlier observations which strongly

suggest that spleen is not a typical differentiating tissue, due to

'its mixed content of cells. Furthermore, it appears that almost any

tissue, when placed on the chorioallantoic membraneg,can.produce'
spleen hypertrophy in the host chick embryo (Van Alten & Fennell, 1959).
Experiments which have been devised to corroborate the alternative
hypothesis, namely that substances from adult tissues may be inhibitory
to homologous differentiating tissue, also appear to suffer from a lack
of sufficient demonstration of specific reactions. This lack of speci-
ficity can be seen in the results of attempts to inhibit the morpho-
genesis of the nervous system. In the first place, the developing ner-
vous system is affected by various types of treatments, as has been
shown by experiments described in this paper, making it difficult to
ascribe any specificity to effects upon the nervous system of materials
from adult nervous tissue. Furthermore, the types of abnormalities des-
cribed in the reports of Rose (1955), Clarke and McCallion (1959 a and b),

Braverman (1959a), and the analyses described earlier in this paper are

68
difficult to interpret. One of the common abnormalities described
in most of this work is a condition in which the neurocoele is greatly
reduced or lackigg. Whether this abnormality is the result of an inhi-
bition of differentiation of nervous tissue or is actually a prolifera-
tion of additional nervous tissue is difficult to determine. Rather
than being an example of specific inhibition, the increase in cells
which results in "solid nervous system" might as well serve as an exam-
ple of hypertrophic growth. To evaluate properly the results from ex-
periments involving the effects of substances from adult tissue upon:
homologous differentiating tissues, it is necessary to be able to dis-
tinguish between the processes that initiate the differentiation of an
organ and the ensuing processes which result in its growth.

For a number of reasons, therefore, the developing nervous system
appears to be a very poor system to use in attempting to determine the
effects of substances from adult tissues on homologous embryonic tissues.
Other differentiating systems would appear to provide more definite
criteria for such studies, as for example, the developing heart.

In connection with the question of substances from adult tissues
affecting embryonic differentiation, some conclusions may be drawn from
the experiments reported in this paper which concern both the method of
treating embryos and the particular fraction of the tissue cells which
may be more effective. First of all, it appears that injection of adult
tissue material into developing embryos is much more effective than
culturing embryos in the presence of the same substances. Although Rose
(1955) and Clarke and McCallion (1959a) claim to have obtained "specific

inhibition" by culturing developing frog embryos in the presence of adult

 

 

 

 

   

69
tissues of the frog, Spiegel (personal communication), Shaver (unpub-
lished), and the writer have not been able to demonstrate any specific
effect upon the developing embryos when employing culturing methods.
However, the results of Shaver's (1954) experiments, the microscopic
analysis of which is given in this paper, show that injection of the sub-
stances of adult tissues into deve10ping embryos has a very pronounced
effect in the case of the nervous system. Also, in the work with anti-
sera against adult tissues, it was found that the injection procedure
was superior to the culturing technique. It is possible that the sub-
stances from adult tissues may never enter the embryo when the culturing
method is employed, since it is known that the surface of the developing
frog embryo is highly impermeable to most substances (see Holtfreter,
1943). Recent investigations concerning treatment of frog embryos with
various chemicals have also demonstrated that the injection technique is
superior to the culturing procedure (Grant, 1960).

Another interesting observation from the present work with nervous
tissue is that the fraction of cytoplasmic granules of adult brain was
more effective in producing specific abnormalities on the differentiating
nervous system than any of the other centrifugal fractions employed.
Several explanations of this observation are possible. The cytoplasmic
granule fraction may have a higher content of effective materials, either
in their native state or through adsorption in the homogenization process.
Possibly, the particulate fraction may release materials such as enzymes
of the respiratory type, which could change the normal metabolic pro~
cesses of the early embryo. Thus the problem of what effect substances
from adult tissues have on homologous differentiating tissues in the

embryo, as well as the mode of action of such substances, remains unsolved.

 

‘l' “‘1’"? .1- ‘

 

B.

 

70

Effects pf Antisera Prepared Against Adult Organs gg_the Differentiation
.2: Embryonic Tissues

The analysis of the antisera prepared against adult organs of the
frog suggests that there is not asnmuch antigenic difference between
organs from the same organism as would be desirable for studies con-
cerned with the effects of organ antisera on embryonic development. As
has been reported earlier in the paper, antisera prepared against adult
frog brain, kidney, and heart exhibit several common components. It
appears that there is little difficulty in showing differences between
antigenic properties between an ectodermal derivative such as brain and
a mesodermal derivative. However, considerable difficulty is encountered
when one attempts to show specific differences in antigenic properties
between 2 mesodermal derivatives such as heart and kidney. Such differ-
ences have been demonstrated in this study but the technique employed
does not rule out the possibility that these differences could be quanti~
tative rather than qualitative. On the other hand, the inability to dem-
onstrate specific differences in antigenic properties does not conclur
sively prove that there are no differences in biochemical constitution
of these organs. It is possible that the antibody-producing mechanism
of the rabbit is not sensitive to the substances involved in specific
chemical differences in organ composition or if specific differences do
occur, the substances responsible for these differences may be present in
amounts too small to elicit an immune response in the rabbit. When the
frog embryo is treated with the antisera prepared against adult organs,
however, it responds differently to the various antisera, indicating that
there are either quantitative or qualitative differences, or both, in the

antisera.

 

71

The most interesting observation made in the studies reported in
this paper was that concerning the inhibition of heart development.
This work bears out the earlier work of Ebert (1950) who found that anti-
sera prepared against adult chick heart inhibited the development of the
heart in explanted chick embryos. The work with the frog has an added
advantage since the embryos can be grown in their normal environment.

Although the results of the experiments in which embryos were
treated with antisera prepared against adult organs are highly signifi-
.cant statistically inasfar as inhibition of heart development was con-
cerned, the question arises as to why the response is not the same in
embryos developed from eggs of the same female or in embryos originating
from eggs of different females. >There are a number of possible explana-
tions for this.. First of all, the experimental proceduresamay account
for a considerable amount of the variation. With the injection procedure
employed, it was not possible to inject the same amount of material into=”
each embryo and even if it were, there was no way to determine how much
of the injected material remained in the embryo. The other possibkéf
source of individual differences in response to treatment withfantisera
,is that the embryos may differ genetically and this heritable difference
may cause a variation in the response of the embryos. Many workers have
found that eggs from different females respond differently to the same
experimental treatment. Whether this is due to genetic differences or
to differences in uncontrolled environmental variables is not apparent.

In the experiments with organ antisera it would be desirable to

know exactly what factor or factors in the antisera were producing the

inhibition of heart development. Although factors specific for heart

 

‘72
were demonstrated to be present in some of the heart antisera, attempts
to correlate heart inhibition with these factors were not successful.
Furthermore, specific factors were not demonstrated in same of the
heart antisera which were very effective in producing heart inhibition.
There is the possibility that specific factors in some antisera may be
non-precipitating and consequently would not be seen with Ouchterlony
technique.

With the techniques employed in the experiments with organ anti-
sera, it was not possible to determine what was the primary effect on
the embryo which led to heart inhibition. Obviously, either processes
of growth or differentiation, or both, were being affected by the
treatment. If the processes of growth and differentiation were more
fully understood, an evaluation of the experiments involving the
effects of adult antigens or antisera on embryonic development might
be more easily made.

A number of theories have been proposed within the last 20 -fi;
years to account for embryonic differentiation, morphogenesis, and
growth. Prominent among these proposals are the template-antitemplate
theory of Weiss (1947, 1953), the auto-antibody concept of Tyler (1947,4
1955), the specific inhibition theory of Rose (1952, 1957), and the
"building block" hypothesis of Ebert (1954). Whether the above con-
cepts can adequately explain differentiation and growth remains to be
seen, but it seems pertinent to discuss the present work with organ
antisera in connection with these ideas.

The template-antitemplate theory of Weiss states that growth and

differentiation are controlled by paired substances, an intracellular

 

73

"template" for further reproduction of cells, and an accessory diffu-
sible "anti-template" capable of inactivating the former. As the anti-
templates accumulate in the surrounding medium, they reach a critical
concentration and growth ceases. The evidence used in support of this
theory comes mostly from studies involving regeneration of liver in
adult organisms. If such a mechanism is responsible for growth of
embryonic organs, it would seem most likely that an antiserum prepared
'against adult tissues would contain antibodies against the intracellu-
lar templates. These antibodies, when introduced into the embryo, could
inactivate the templates and produce an end result comparable to the
action of the normally occurring anti-templates. Consequently, cessa-
tion of growth processes would occur long before the organ involved
had reached its normal size. Unfortunately, the hypothesis as presented
by Wéiss is based upon a set of circumstances which may have little or
nothing to do with primary differentiation processes.

The auto-antibody concept of Tyler is in many respects similar
to the above‘mentioned theory but it goes further to specify that the
paired substances function as antigen and antibody.- Tyler's extensive
work with interacting substances in the egg and sperm of sea urchins,
as well as indications from work with cellular aggregation and venin-
antivenin systems, prompted the proposal of this theory. If growth is
controlled by the production of auto-antibodies, as Tyler suggests,
treatment of embryos with an exogenous source of similar antibodies
would consequently cause a premature cessation of growth and differen-
tiation.‘ This would constitute one of the simplest explanations for

the mode of action of the antisera. However, as is true with all of

74

these theories, there is little experimental evidence to substantiate
the auto-antibody concept as specifically applicable to histogenesis
of tissue cells.

At first sight, the "specific inhibition" theory of Rose seems
to be controverted by the results obtained by treatment of embryos
with antisera prepared against substances from adult tissues. Accord~
ing to this theory, the presence of substances from adult tissues should
inhibit the development of homologous embryonic tissue and it would
logically follow that antisera prepared against these same adult sub-
stances should stimulate growth in the embryonic tissue. One would
expect the antisera to contain complementary factors to the inhibitors
from adult tissues, and these factors would be expected to inactivate
the inhibitors in the embryo, resulting in hypertrophic growth. How-
ever, it is possible that cells in adult tissues contain substances
which have antigenic sites in common with similar substances in pre-
cursor cells of the same tissue. Thus, the antiserum might inactivate
these precursor cells and this tissue would fail to differentiate.

In considering the general theory of Rose in connection with ex-
periments of the type described by him and by others (see above) in
which frog embryos exposed to adult tissues are "specifically inhibited,"
a basic contradiction appears to exist when these results are made to
appear as correlative with results of experiments where an obvious
gradient system is involved. That is, the effects noted on the regen-
eration of "dominant" structures in the gradient system of such forms
as Tubularia and Lineus, caused by the presence of additional "dominant"

structure material, seems to have little connection with the type of

75
inhibition noted by Rose and others on the nervous or other systems
in frog embryos treated with substances from homologous adult systems.
For example, one would presumably predict, on the basis of Rose's gen-
eral theory, that the fore-brain and other anterior neural structures
of the embryo, would be selectively inhibited by adult brain. This is
not the case, either in Rose's experiments or in the tests made by other
workers. Thus, to attribute the differentiation of the nervous system,
and other systems, in the frog to the same type of gradient response
as may exist in Tubularia of Lineus, seems to be an oversimplification
of the actual facts. However, Braverman (1958a) in a short report,
states that a gradient effect was noted in the inhibition of chick
nervous system after treatment with extracts of adult nervous system
from different levels of the brain and spinal cord. There has been no
subsequent confirmation of this.

Ebert's "building block" hypothesis, derived from his experiments
with chorioallantoic grafts, suggests that cells in developing embryos
can either incorporate already-formed proteins into their cytoplasmic
structure or can utilize these proteins in the synthesis of new pro-
teins. According to Ebert, this accounts for the hypertrophic growth
observed in spleens of the host chick embryo following grafts of adult
chick spleen. Perhaps, antisera against adult tissues contain anti-
bodies that have reactive sites complementary to tissue precursors pre-
sent in the yolk. In this case, the mobilization of specific tissue
substances from the yolk might be blocked.

At the present time, it is impossible to ascribe the inhibition

of heart development in embryos treated with anti-heart serum to any

 

 

 

 

 

C.

76

particular mode of action. However, due to the early stage at which
the embryos had to be treated to produce heart inhibition, it seems
that some very early process of differentiation is involved. If some
general growth reaction only was involved in the inhibition, it would
seem that the embryos could be affected over a much wider period of
development than was demonstrated in the experiments described in this
paper. Furthermore, a few embryos showed no signs of heart development
though a normal pericardial space was present, indicating that morpholo-
gical differentiation was never initiated. This would lead to the con-
clusion, then, that the antisera prepared against adult heart were in
some way affecting the early biochemical events which ultimately result
in the morphological differentiation of the heart.

Th3 Effects 2f Antisera Prepared Against Muscle Proteins 23 Embryonic
Development“

One of the major shortcomings of the immunological approach to
studying embryonic development is that most of the work has been done
with highly heterogeneous antigens which are not easily analyzed.
Consequently, it was believed that muscle proteins from the heart would
be a source 10f substances which could be easily extracted in a rather
pure form. Thus, the study of the effects of antisera prepared against
muscle proteins on embryonic development was initiated. Unfortunately,
the methods used for extraction did not produce the degree of specifi-
city and purity desired. Therefore, the work presented in this paper
failed to overcome the basic problem of heterogeneous antigens. How-
ever, it is felt that the preliminary results with antisera prepared

against muscle proteins should not discourage further investigations in

 

 

 

 

 

this area. With procedures which have recently been worked out for
the extraction of muscle proteins from the frog, it should now be
possible to do a thorough analysis of the effects of antisera pre-

pared against purified muscle protein.

77

 

 

78

SUMMARY

1. Although numerous studies have been made concerning the effects
of substances from differentiated tissues, and of antisera against these
substances, on the differentiation of homologous embryonic tissues, there
is still considerable confusion concerning the results of these experi-
ments. This is due in part to the fact that different organisms were em-
ployed for the study of the effects of the tissue substances and of the
antisera prepared against them. The purpose of this paper is to attempt
a clarification of this problem by treating the same differentiating
system with substances from the homologous adult tissue as well as antisera
prepared against these substances.

2. The experimental approach was to treat early embryos of 3333
pipiens with tissue fractions of adult organs and with antisera prepared
against homogenates of adult organs of the same species. Two methods of
treatment were employed: injection of embryos with the tissue fractions
and organ antisera, and culture of embryos in the presence of these sub-
stances.

3. The results of the work with fractions of adult tissues were
somewhat variable but the following conclusions may be drawn: (a) It
appears from these studies that the nervous system, with which most of
the studies in this area have been made, is a rather poor system for demon-
strating specific effects, since a variety of treatments seem to affect
this system;(b)fihe. method of injecting substances proved to be more effec-
tive than the culturing technique in producing specific effects upon the
developing nervous systemsand (c) a final observation from this work is
that the fraction of cytoplasmic granules produced more consistent defects

in the embryonic nervous system than any other centrifugal fraction employed.

was?“ yrs WV“1"“‘"_."—~ .. .

 

 

79

4. Treatment of embryos with antisera prepared against adult heart
produced the most specific inhibitory effects in the developing embryo
of all the experimental procedures employed. These antisera produced a
marked inhibition of heart development in 30% of all embryos injected with
them. Control sera, phosphate buffer, and antisera prepared against adult
brain and kidney also produced this effect but in very much lower percen-
tages.

5. Other effects of antisera prepared against adult organs were
observed in embryos in some experiments. Embryos cultured in heart anti-
serum were observed to have a specific defect of the central nervous
system in 2 experiments. In another experiment, a large per cent of the
embryos which were injected with an antiserum prepared against adult
kidney showed extreme edema.

6. Analysis of the antisera prepared against adult kidney, brain,
and heart showed that these organs have complementary factors in common.
The antigenic properties of kidney and heart were found to be quite similar.
On the other hand, when antigenic pr0perties of heart and kidney were com-
pared with those of brain, considerable differences could be detected.
Using adsorption techniques, most of the antisera prepared against adult
organs could be shown to have specific components. However, attempts to
correlate heart inhibition with specific components in the antisera were
unsuccessful.

7. Preliminary experiments were performed to determine the effects
of antisera prepared against protein extracts of heart and skeletal muscle
of frog and chick on the development of the frog heart. Unfortunately,

the extracted proteins lacked the desired characteristics of homogeneity

80
and purity. When embryos were treated with antisera prepared against
these proteins, some heart inhibition was noted, but this inhibition was
not significantly greater than that observed in embryos treated with
control sera.

8. Analysis of the antisera prepared against protein extracts of
skeletal and heart muscle showed the following: (a) there were cross
reactions between these antisera and antigens from body organs such as
kidney;(b) there were no cross reactions between the antigens or anti-
sera-of frog heart muscle proteins with the antigens or antisera of
frog skeletal muscle proteins;(c) the antigens and antisera of chick
heart muscle proteins and chick skeletal muscle proteins did exhibit
cross reactions;and (d) there were never any cross reactions of the
antigens and antisera of the frog muscle proteins with the antigens and
antisera of the chick material.

9. The results of these studies are discussed in connection with

current theories of differentiation and growth.

 

 

 

 

81

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