THE EFFECT OF CHORIOALLANTOIC GRAFTS
ON THE DEVELOPING CHICK EMBRYO WITH
SPECIAL REFERENCE TO THE DEVELOPMENT
OF ANTIGENS IN THE DUODENUM

By
Pierson J. Van Alten

AN ABSTRACT
Submitted to the School for Advanced Graduate Studies
Michigan State University of Agriculture and
applied Science in partial, fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
Department of Zoology
1958
Approved______ il'

L (...

Pierson J. Van Alten

THESIS ABSTRACT
Various tissues from adult chickens (duodenum, liver,
spleen, heart, brain and skin) and embryonic duodena (20-,
18-, 17-, 16-, and 15-day) were grafted to the chorio­
allantoic membrane (CAM) of 9-day chick embryos and the
whole chick, spleen, liver, heart, intestine and duodenum
were weighed on the 18th day of incubation.

Following duo

denal grafts the weight of the whole chick decreased while
the weight of the spleen, liver and heart significantly
increased in weight.

The weight of the liver and heart

were found to be increased subsequent to liver grafts.
When heart was grafted no weight increases were found in
the host heart, spleen, liver and intestine.

Following

grafts of adult spleen the spleen and heart were found to
be enlarged.

The spleen, liver and heart were enlarged

following grafts of brain tissue.

When skin was grafted

to the CAM the spleen, liver and duodenum were enlarged.
Further, when embryonic duodenum was grafted no signifi­
cant changes were observed.

The degree of enlargement of

the various host organs was significantly greater follow­
ing grafts of adult duodenum than all other treatments.
The relative weight of the duodenum was found to be in­
creased following duodenal grafts.

2
Pierson J. Van Alten
Grafting of adult duodenum caused acceleration of
tissue differentiation of the host duodenum*

Following

grafting of duodenum, spleen and skin the host spleen ex­
hibited a marked increase in granuloblasts and granulo­
cytes.

The heart and liver following grafts were essential­

ly like those in control embryos.

The polysaccharides in

the connective tissue and goblet cells of the duodenum
following CA grafts of adult duodenum, differentiated at
least 24 hours earlier than in control chicks.
An immunological study of the embryonic duodenum from
11 through 21 days of incubation was carried out using
the Ouchterlony agar diffusion plate technique.

Antiserum

against adult chicken duodenum was prepared and saline
soluble antigens of the various ages of duodena were test­
ed.

Two lines were observed in 11-, 12- and 13-day duo­

dena; a third line in the 14-day duodenum; four lines in
15-9 16- and 17-day duodena; with only three lines in 18-,
19- and 20-day duodena and by the 21st day there were four
lines.

The BJorklund inhibition technique was used to dem­

onstrate that the fourth line in the 15-, 16-, 17-day and
in the 21-day duodena was not found in the 18-day duo­
denum.

Further, this method was also used to demonstrate

that the three lines in 20-day and three of the four lines
in 21-day duodena were not serum antigens.
The agar diffusion procedure was also used to dem­
onstrate changes in the antigenic pattern of the 17-day

3
Pierson J. Van Alten
embryonic duodenum and spleen following CA grafts of adult
duodenum and spleen.

Following spleen grafts no quali­

tative changes were observed in the spleen but an extra
line was observed in the duodenum.

One extra line was

observed in the spleen and three in the duodenum sub­
sequent to grafts of adult duodenum.

THE EFFECT OF CHORIOALLANTOIC GRAFTS
ON THE DEVELOPING CHICK EMBRYO WITH
SPECIAL REFERENCE TO THE DEVELOPMENT
OF ANTIGENS IN THE DUODENUM

By P
Pierson J* Van Alten

A THESIS
Submitted to the School for Advanced Graduate Studies
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY

Department of Zoology
1958

ProQuest Number: 10008581

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ACKNOWLEDGEMENTS
The author wishes to express his sincere thanks to
Dr* R. A* Fennell, as chairman of the committee for his con­
stant assistance, direction and interest during the course
of this investigation*
Grateful thanks is also due to Dr* A* S* Fox and Dr*
Sei-Byung Toon for their guidance in the immunological
studies•
The author is also indebted to Drs* Lois Calhoun,
A* S. Fox, J* R* Shaver, G. P* Steinbauer, and L. P*
Wolterink for their able assistance as committee members.
Thanks is due to Charles G* Mead and P* G. Coleman for
photographic work and Dr* P. J. Clark who has been most help­
ful with the statistical analysis.
Acknowledgement is also made to my wife, Lucille, with­
out whose competent help, advice and encouragement this study
could not have been conducted.
Final acknowledgement is made to the Cancer Division of
the National Institutes of Health for the much appreciated
predoctoral fellowship # CF-6731*

TABLE OE CONTENTS
Section
I

INTRODUCTION.......................

1

II

MATERIALS AND METHODS........................ 11

III

RESULTS..................... . .............. 22
1. The effect ofchorioallantoic transplants
of adult and embryonic chicken tissues on
the weight of homologous and heterologous
tissues of the host embryo.............. 22
2. The effect of soluble antigens of adult
and embryonic chicken organs on the host
embryo when injected into the yolk sac. . 36
3* Morphology of chick embryos and various
homologous and heterologous organs sub­
sequent to chorioallantoic transplants
of adult and embryonic chicken tissues. . 37
4. The appearance of adult duodenal anti­
gens in the developing embryonic duo­
denum of the chick.........
5*

IV

46

The effect of chorioallantoic transplants
of adult chicken duodenum and spleen on
the development of adult duodenal, and
spleen antigens in the duodenum and spleen
of I?-day embryos
.................... 30

DISCUSSION

............................. 34

1.

Weight and morphological changes on host
organs following CA grafts . . . . . . . .54

2.

Immunologicalstudies

..............62

V

SUMMARY......................................67

VI

LITERATURE CITED............................. 70

LIST OF TABLES
Table
1

Multiple Range Test ................

18

2

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic Transplants .............

21

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing chorioallantoic Transplants .............

22

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic Transplants

24

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic.Transplants...........

25

3

4

5

6

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic Transplants
.............26

7

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic Transplants . ...........

28

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic Transplants.. ............

29

8

9

10
11

Fresh and Dry Weights of Homologous and
Heterologous Organs Following Chorio­
allantoic Transplants.........................30
Covariance Test on The Weight of The Duo­
denum to The Weight of The Whole Embryo . . . .

32

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic Transplants
............

34

LIST OF TABLES (Cont.)
Table
12

Fresh Weights of Whole Embryos and of
Homologous and Heterologous Organs Follow­
ing Chorioallantoic Transplants . ...........

35

13

The Effect of Soluble Antigens on The Host
Embryo When Injected into The Yolk Sac . . . . . 37

14

The Effect of Chorioallantoic Grafts on The
Development of Goblet Cells in The Chick
Duodenum....... . .................... ♦ . . . 3 9

15

The Differentiation of Polysaccharides in
Duodenal Tissues Following CAM Adult Duo­
......................
denal Grafts

41

A Summary of The Results Obtained With The
Serum-agar Precipitin Test. Adult Duodenum
Antiserum was Tested With Saline Extract of
Adult and 11 Through 21-Day Chick Embryonic
Duodena. ...................... . . . . . . .

49

16

LISO? OF PLATES
Plate
77
Fig. 1*

Geometry of an Ouchterlony plate

I I ............................................. 79
Fig. 2.

Gross section through the duodenum
of a c©ntrol 18-day embryo; and

Fig* 3*

Cross section through the duodenum
of an 18-day embryo following adult
chicken duodenal grafts*

I I I ............................................. 81
Fig. 4*

Cross section through a spleen of a
control 18-day embryo; and

Fig. 3.

Cross section through a spleen of an
18-day embryo following adult duo­
denal grafts*

I V ............................................. 83
Fig* 6.

Cross section through an adult duo­
denal graft on the 18th day of
incubation; and

Fig. 7*

Cross section through a 13“day em­
bryonic duodenal graft on the 18th
day of incubation.

V ..............................
Fig. 8.

83

Photograph and diagram of an
Ouchterlony plate*

V I ............................................. 87
Fig. 9#

Photograph and diagram of an
Ouchterlony plate*

LIST OF PLATES (Cont.)
Plate
Y I I ............................................89
Fig. 10.

Photograph and diagram of an
Ouchterlony plate.

V I I I ............................................91
Fig. 11.

Photograph and diagram of an
Ouchterlony-Bjorklund in­
hibition plate.

I X ........................................... 93
Fig. 12.

Photograph and diagram of an
Ouchte rlony-Bjorklund inhibition plate.

X ....................
Fig. 13.

95

Photograph and diagram of an
Ouchterlony plate.

X I ........................................... 97
Fig. 14.

Photograph and diagram of an
Ouchterlony plate.

X I I ...........
Fig. 13♦

99
Photograph and diagram of an
Ouchterlony plate.

INTRODUCTION
The grafting of tissues to the chorioallantoic mem­
brane (CAM) of the chick embryo has been widely used for
study of organ-specific growth stimulation.

Murphy (1916)

observed that chorioallantoic (CA) grafts of adult chicken
spleen, liver, kidney and bone marrow induced enlargement
of homologous organs of host embryos, e.g., a striking en­
largement of the host spleen following grafts of adult
spleen.

Spleen hypertrophy was attributed to increased

infiltration of small lymphocytes into and confluent with
the transplanted tissue.

Further, clumps of both granular

and nongranular cells were observed adjacent to blood vessels
of the liver and kidney.

These observations were confirmed

by Danchakoff (1916) although she attributed spleen hyper­
trophy to an intense proliferation of lymphoid hemocytoblasts which ultimately differentiated into granulocytes.
In a subsequent study Danchakoff (1918) observed that trans­
formation of mesenchyme into granuloblastic cells was not
confined to the spleen but extended throughout the whole mes­
enchyme of the host.

Minoura (1921) transplanted ovarian

and testicular tissue to the CAM and observed that develop­
ment and differentiation of one sex was stimulated by se­
cretions of the gonads of the same sex and inhibited by the
gonadal secretions of the opposite sex.

2

In a study to determine the effects of thyroid tissue
on development of chick embryos, Willier (1924) observed
that CA thyroid grafts not only induced proliferation of
leucocytes but frequently induced conspicuous necrotic
nodules in the spleen and to a lesser extent in liver and
skin*

This hypertrophy of the embryonic chick spleen was

also observed following spleen, liver and thymus gland
grafts*

When using embryonic tissue (organ primordia) he

was unable to find any apparent effects on the host embryo*
Sandstrom (1932) also observed host spleen hypertrophy
following grafting of adult chicken spleen.

He attributed

hypertrophy to proliferation of granulocytes within the host
spleen*
In recent years four views have been advanced to explain
growth and differentiation, i.e., the tempi ate-antitemplate
hypothesis of Weiss (1947), the natural auto-antibody con­
cept of Tyler (1947), the hierarchy of self-limiting reaction
hypothesis of Rose (1952), and the building block hypothesis
of Ebert (1954-).
Weiss (1952, 1953 a, b) and Weiss and Kavanau (1957)
maintained that protoplasmic synthesis cf a given organ yields:
(a) molecular "templates” for further development of specific
organ molecules and (b) accessory diffusible compounds
(,,antitemplates,t) antagonistic to the former which block and
thus inhibit the reproductive activity of the corresponding

3

“templates”• This concept when first proposed, was based
on observations which demonstrated that liver and kidney
of chick embryos hypertrophied following homoplastic grafts
of corresponding organs (Weiss and Wang, 194-1)*

Weiss,

(1955) observed that addition of kidney extract to tissue
cultures of metanephros fragments reduced the frequency of
tubule differentiation*

In experiments with heart cultures

he observed that heart extract depressed differentiation
of cardiac fibrils, as evidenced by reduction in frequency
of pulsation*

Since differentiation was inversely related

to cell proliferation he concluded that substances in cell
extracts promoted homologous growth either by being incor­
porated into the corresponding cells or by neutralizing
homologous growth inhibitors of the medium*

In a subsequent

study Weiss and Andres (1952) showed that presumptive melanoblasts of dissociated embryonic cell suspensions, when
injected into the blood stream of early chick embryos,
localized in the regions characteristic of the cell type of
the donor strain.

Andres (1955) who extended the experiments,

showed that intravascular administration of mesonephros and
liver material had a specific stimulation effect on pro­
liferation in homologous organs.

The observation of Pomerat

(194*9) supported this view since he found an enormous in­
crease in the size of embryonic spleen following CA grafts
of adult spleen.

4

-

The natural auto-antibody hypothesis proposed by Tyler
(194-7) was based mainly upon the observation that both
fertilizin and antifertilizin can be found in the seaurchin egg.
The concept of a hierarchy of self-limiting reactions
was proposed by Rose (1952) and this generalization was
based on regeneration experiments made with Tubularia.
Further, in four out of twenty-six experiments in which he
cultivated frog eggs in the presence of fragments of adult
brain and heart, he observed a specific inhibition of the
developing homologous organ (Rose, 1955).

®ie experiments

of Shaver (1954-), in which he observed that a granular
fraction of adult frog brain arrested brain differentiation,
also lend support to this view.
An extensive investigation of the problem of the effect
of CA grafts of adult chicken tissue on homologous tissues
of the host embryo was carried out by Ebert (1955).

He ob­

served a very marked enlargement of spleen in host chicks
following grafts of adult chicken spleen (Ebert, 1951)*
In other experiments of this type, Ebert (1954-) observed
that nitrogen content of enlarged spleens increased and he
concluded that spleen enlargement was directly related to
increased protein content.

By the use of CA grafts labeled

with radioactive methionine Ebert (1954-, 1955) was able to
demonstrate a higher specific activity in the host spleen

5

following labeled spleen transplants and a higher specific
activity in the host kidney following labeled kidney grafts*
Grafts of labeled mouse spleen, in contrast to adult chicken spleen, did not produce any selective incorporation and
he concluded that this effect was class-specific as well
as tissue-specific*
Ebert (1954*) observed that the DNA content of enlarged
spleens differed significantly from control spleens*

He

suggested that there was no transfer of either whole cells
or "templates11 from grafts to hosts but rather a selective
incorporation of tissue-specific proteins from grafts to
homologous tissues.

A transfer of such specific protein

moieties was shown by Walter et al* (1956).
jection of either a clear supernatant of

35

Following inlabeled homo­

genized liver or heart into 9-day old embryos, a higher
specific activity was observed in the respective host
tissues of liver and heart.
Simonsen (1957) questioned the organ-specific growth
stimulation hypothesis*

He injected chick embryos incubated

for 18 days with adult chicken spleen cells and observed
that about 5 weeks post-hatching the chicks manifested
symptoms of severe haemolytic anemia and that marked hyper­
trophy occurred in the spleen, liver and thymus*

Further,

he attributed replacement of erythropoietic and myelopoietic
cells in the bone marrow by proliferating reticulo-endothelial
cells to an immunological reaction between donor cells and
host tissues.

6

An extensive review and summary of the early liter­
ature on the origin of adult antigens in the developing
chick embryo has been made by Needham (1931)» Cooper (194-6)
and Schechtman (194-7)*

Consistent results have been ob­

tained only in recent years by the use of more refined
techniques#
Burke, et_ al# (194-4-) prepared antisera against saline
extracts of adult organs (brain, testis, ovary, kidney,
liver and lens) of the chicken#

They maintained that adult

organ-specific antigens in the chick embryo appeared sub­
sequent to differentiation and development of the organ,
©•£•» ©ye lens at 14-6 hours, erythrocytes at 100 hours,
kidney at 220 hours, and brain, testis and ovary at 260
hours#

On the other hand, Schechtman (194-8) and Ebert (1930)

maintained that antigens of the brain, heart and spleen
were identifiable in the early chick blastoderm.

Further­

more, lens antigens were identified in the chick at 60 hours
of incubation by Ten Cate and Van Doorenmaalen (1930) and
Flickinger at al. (1935)*
Serum-like antigens were identified in the egg yolk
and in extracts of various ages of chick embryos#

Further

antigen activity was reduced by absorption with anti-adult
serum, anti-albumin and antiNace and Schechtman 194-8)#

-globulin (Schechtman 194-7,

Similar results were obtained

by Schechtman and Hoffman (1952) using anti-a {3- globulin
serum#

In further work Nace (1933) observed that serum of

7

chick embryos exhibited a vitelloid albumin present from
the third day, a nonvitelloid albumin by the 5th day, and
vitelloid ^globulin was detected by the 9th day and a nonvitelloid Y- globulin was not present until the 12th day
and a nonvitelloid <x|3-globulin was present from the 6th day.
He suggested that the yolk proteins may be a direct trans­
udate from the maternal serum.

Tyler (1957)» in an altern­

ative explanation for the reduction in reactivity of the
antiserum of adult chicken blood following absorption with
yolk proteins, attributed this to similar antigenic de­
terminant groups in the yolk but not necessarily to the
particular protein they were formed against.
Schechtman (1948j was the first to identify an organ
antigen in the early chick embryo, i.e., primitive streak,
early neurula and 4- to 5“Somite stage.

He prepared anti­

sera against a saline extract of perfused brain of chicks
of 19 to 20 days of incubation and observed that these anti­
sera cross-reacted with extracts of liver, heart, muscle
and blood of chicks the same age.

Following absorption

with blood the antisera no longer reacted with yolk but did
react with the various organs and with early embryo extracts.
These results have been extended by Ebert (1950, 1951) in
which he used anti-brain, anti-heart and anti-spleen sera.
He observed that absorption of any of the three antisera
with either extracts of heart, brain or spleen removed the

8

precipitins for definitive primitive streak and early so­
mite stages, while heterologous antigen did not remove
precipitins from homologous antigens.

Therefore, it appears

that the antigen of the early blastoderm was of a more
general type than any of the tissue-specific antigens de­
scribed*
In another study Ebert (1952) was able to demonstrate
the ontogeny of tissue antigens in the developing chicken
spleen.

Antisera were prepared against saline extracts of

18-day embryonic chick spleen and tested with saline ex­
tracts of 12- and 18-day embryonic spleen.

It was observed

that the number of antigens increased from three to six
between the 12th and 18th day of incubation.

Absorption

of anti-18-day spleen sera with 12-day embryonic spleen re­
duced antigenic reactions from three to zero with 12-day
spleen antigens and decreased antigenic reactions with 18day preparations from six to three*
Immuno-embryological experiments with highly purified
cardiac contractile proteins (actin and myosin) in chick
embryos have been reported by Ebert (1955> 1955) and Ebert
et al. (1955)*

He was able to show that cardiac myosin was

not synthesized prior to the mid-primitive streak stage.
At this stage the antigen reactive groups of myosin were
not localized in any particular region*

However, at the

head fold stage it became restricted to the heart-forming
area and was sbsent at the head process*

9

A somewhat similar study of proteins in the chick was
made by Johnson and Leone (1955)*

They detected myogen in

extracts of embryos of AO hours of incubation while actomyosin could only be detected after 40 hours of incubation*
Blood proteins (serum albumins and serum globulins) were
detected in extracts of 5~ to 10-hour blastoderms and ex­
tracts of egg yolk and egg albumin.
The histochemistry and histogenesis of the duodenum
of the chick embryo has recently been studied extensively*
Moog (1950) found an accumulation of alkaline phosphomonesterase in the striated borders and in the crypts of
Lieberkuhn of the duodenum of 18-day embryos*

Moog and

Wenger (1952) identified a Schiff-positive material which
was difficult to hydrolyze , was resistent to diastase and
hyaluronidase, and was not metachromatic at sites of high
alkaline phosphatase activity*

Esterase was identified by

Richardson et al. (1955) in the connective tissues and
epithelium of the duodenum of 10-day embryos, while in later
stages it was observed only in the epithelium.

Esterase

was found to gradually increase between 9 and 17 days and
then it rapidly increased and remained at a high level
through 3 days post-hatching at which time it began de­
creasing.

Recently, Kato and Moog (1958) have demonstrated

an increase in the amount of alkaline phosphatase in the
17-day duodenum following injection of disodium phenylphosphate into chick embryos on the 14th day.

10

The histogenesis of the duodenum of the chick has- been
described by Van Alten and Fennell (1957)•

They also de­

scribed the histochemistry of the development of mucopoly­
saccharides in the connective tissues of this organ, and
observed a Schiff-positive material on the 18th day of in­
cubation which they attributed to hyaluronic acid*
The above review of literature would indicate that an
organ-specific growth stimulating material is produced
following CA grafts of adult tissue.

It would also seem

possible that by means of immunological techniques one would
be able to detect adult antigens being transferred from the
grafts to the organs.
The objectives of this investigation were to study:
(l) the effects of chorioallantoic grafts of embryonic
duodenum and various adult organs (spleen, liver, heart,
skin, brain and duodenum) upon homologous and heterologous
organs in the developing chick embryo;

(2) by means of

immunological technique the difference in antigens of adult,
embryo and embryos treated with adult spleen and duodenum
grafts; and (3) the histology of the various embryonic
organs following CA transplants.

MATERIALS AND METHODS
Hatching eggs of single-comb white leghorn chickens
were used exclusively in these studies.

The tissues used

for grafting experiments were obtained from various breeds
and ages of adult chickens.
The eggs were incubated at 37.5° C in a moistened
air, constant rotating, forced circulation incubator for
9 days prior to chorioallantoic grafting.

Subsequent to

grafting the eggs were incubated at 37#5° C in a nonro­
tating, moistened air incubator.

All weight studies were

made on chicks which were incubated 18 days, just prior
to the absorption of the yolk sac.
On the 9th day of incubation the eggs were candled
over a 75-Watt electric bulb.

An area near the bifurca­

tion of two large blood vessels was marked by swabbing with
tincture of merthiolate 1:1000 which was also used for
sterilization of the shell.

Prior to candling, the air sac

end of the egg was also swabbed with tincture of merthio­
late.

Subsequently, a small hole was drilled by means of

a motor-driven burr into the air sac and a second was care­
fully drilled through the shell to expose the shell mem­
brane above the bifurcated vessels.

A small sterile nee­

dle was used to puncture the shell membrane.

Care was

taken not to puncture or tear the underlying vascular

12

chorioallantoic membrane * Simultaneously decreasing the
pressure at the air sac caused the CAM to drop away from
the shell membrane thus displacing the air sac,

The hole

over the CAM was enlarged by very carefully removing the
shell with a pair of sharply pointed forceps.
Chickens of both sexes, of different breeds and of
ages ranging from 3 months to about one year post-hatch­
ing were used as donors of tissues for grafting.

The chick­

en was killed by removing the head and various tissues (duo­
denum, spleen, liver, heart, skin and brain) were quickly
removed and cut into very small pieces in chick Ringer’s
solution.

In order to sterilize the duodenum it was placed

in 200 ml of chick Ringer's solution containing 1000 mg of
Chloromycetin for 10 minutes*

One of the prepared pieces

of tissue, 2-3 mm, was then placed at the bifurcation
point of the blood vessels on the CAM.

The hole through

which tissues were inserted were sealed with Scotch tape
and melted paraffin and the hole leading into the former
air sac was sealed with just the melted paraffin.

The

egg was then placed in the incubator with the window faced
upward.

The eggs continued to incubate until the 18th

day when embryos and various vital organs were weighed and
some were preserved for further study.

The above procedure

was also used for duodenal grafts of 15“ * 16-, 17-, 18and 20-day embryos and for adult rat duodenum.

Controls

13

of two types were used, either a drop of Ringer's solution
or a piece of 2% agar were placed on the GAM.
In order to ascertain the effect of soluble cell free
material of the duodenum, liver and heart of the adult
chicken on the chick embryo the following procedure was
carried out.

The adult chicken was killed by decapitation

and the above tissues were removed under sterile conditions
and put into cold sterile saline.

The duodenum was open­

ed, thoroughly washed and then treated in a solution of
1000 mg of Chloromycetin in 200 ml of 0.15 M saline.

(The

adult tissue was homogenized in a Waring blender with a 1
to 5 ratio of 0.15 M saline (buffered to pH 7*4- with 0.005
M phosphate buffer).

It was homogenized for 20 minutes at

4° C and then centrifuged at 2000 RPM for 50 minutes at
4° C.
sac.

The supernatant was used for injection into the yolk
Eggs which had been incubated for 96 hours were

candled and the shell was swabbed with tincture of merthiolate just below the equator.

At this site a small hole

was drilled through the shell but not through the shell mem­
brane by means of a motor-driven burr, so that a 23“gauge
needle could be thrust into the yolk sac.

Eggs were in­

jected by means of a lee tuberculin syringe and O.lcc of
adult tissue homogenate or 0.15 M saline was used.

The

hole was closed with melted paraffin and the eggs were re­
turned to the incubator but were candled every day and dead

14

ones were removed#

Various dilutions of the stock antigens

were prepared and injected by the same method#
The agar diffusion technique of Ouchterlony (1949)
(which has recently been reviewed by Oudin, 1952) was used
in the study of adult antigen formation in the duodenum
(11 through 21 days of incubation) and for comparison of
17-day spleens and duodena following CA grafts of adult duo­
denum and adult spleen#

All tests were made in 90 mm Petri

dishes which were prepared in the following manner#

A 2%

agar solution in 0#85% saline buffered with 0.005 M phos­
phate buffer at pH 7*4 was prepared, filtered and measured
out into 30 ml quantities#

Eight 1 5/8 inches x 1/2 inch

strips of filter paper (Whatman #1) were arranged equi­
distant around the lip of the male half Petri dish with a
small portion lying on the bottom of the dish#

A stainless

steel wire was placed inside the male half Petri dish se­
curing the paper strips against the wall and bottom of the
dish#

The dish was then sterilized by dry heat at 120° C

for 12 hours.

Pollowing sterilization 30 ml of sterile agar

wer epoured into the male half of the dish and the agar was
allowed to harden.

After hardening, a template was placed

over the plate, the wells were cut and the pieces of agar
left in the wells were removed (fig. 1)#

A drop of agar

was then placed in each well to seal the bottom#
the above wells held 0#15 ml of test solution.

Each of
Due to the

15

difficulty in analyzing complex systems containing num­
erous antigenic components the Bjorklund (1952) specific
inhibition technique was employed.

In this case the anti­

gen which was used for inhibition was placed in the antiserum well (fig* 1) in doses of 0*15 ml until the desired
number of inhibiting doses were absorbed*

Subsequent to

the last dose the plates were set aside for 48 hours be­
fore addition of the test antigen and anti serum.
Adult chicken duodenum (from which the pancreas had
been removed), liver, heart, spleen and brain were used
for preparing antigens*

These organs were removed from sev­

eral adult chickens ranging in age from 3 months to 1
year.

The organs were drained on several thicknesses of

filter paper, weighed, mixed with 3 ml of cold 0.15 M
saline (buffered to pH 7*4 with 0.005 M phosphate buffer)
per gram of tissue, homogenized in a Waring blender for 10
minutes and further homogenized with a glass homogenizer
for 30 minutes at 4° C.

The homogenate was centrifuged

at 4° C for 30 minutes at 2000 STM and frozen until ready
to use.

Antigens of embryonic chick duodena

of 11 to 21

days inclusive (from which the pancreas had been removed)
and spleens of 17-day embryos were prepared in a similar
manner except that all homogenizing was done with only a
glass homogenizer.

16

Antisera were prepared against adult chicken duodenum
and spleen by injecting the pooled antigen into three male
rabbits*

Five intravenous injections were given during a

10-day period, one every other day*

The first two in­

jections were 0*5 ml of antigen each; the third was 1 ml,
the fourth 1*5 ml and the fifth was 2 ml*

The rabbits

were rested for two weeks following the injections and then
given an anamnestic injection of 2 ml of antigen intra­
venously.

Seven days later the rabbits were bled and a

precipitin titer was run.

If the titer was at least 1:64

the rabbit was bled on the following day, sera collected,
1:5000 merthiolate added, complement inactivated by heat­
ing at 56° C for 50 minutes and the serum was frozen un­
til used.

If the titer was not 1:64, the rabbit was given

another 2cc injection of antigen on the following day and
a titer was run again in another 8 days.

This procedure

was carried out until a sufficient titer was obtained.
The statistics used in comparing the weight of the
chick, spleen, liver, heart, intestine and duodenum follow­
ing various treatments to the CAM, were an analysis of
variance (Dixon and Massey, 1951) and the multiple range
test for heteroscedastic means (Duncan, 1957).

The former

test, analysis of variance, is based upon the fact that if
means of groups are greatly different the variance of the
combined groups is much larger than the variance of the

17

separate groups.

However, an F test alone, when it rejects

the homogeneity hypothesis, gives no decisions as to which
differences among the treatments may be considered signif­
icant and which are not.

The purpose of using the multiple

range test was to designate which differences among the
treatment means might be considered significant and which
were not.
Duncan (1957) proposed the following basic rule for
an cx-level multiple range test.

"Any subset of P means is

homogeneous if the largest adjusted difference in the sub­
set fails to exceed the critical value R'P.

Any two means

not both contained in the same homogeneous subset are sig­
nificantly different.

Any two means both contained in the

same homogeneous subset are not significantly different;{t
To illustrate the procedure used, Table I shows the pro­
cessing of some data obtained from one of the experiments.
Following various CA grafts, tissues of spleen, liver,
heart, duodenum and CAM were fixed in Bouin*s fixative for
24 hours.

Tissues were then transferred to 70% alcohol

(5 changes of 24 hours each), dehydrated and embedded in
paraffin by routine methods of tissue preparation.

Tissue

sections were cut at 5 microns and affixed to clean glass
slides.

All tissues were routinely stained with hema­

toxylin and eosin, Gomori's trichrome stain (Gomori, 1950),
the triple stain for DHA polysaccharides and proteins of

TABLE 1
MULTIPLE RANGE TEST
Analysis of Varience
Source
Total
Between
Within

d.f.
1Q7
3
104

Sum Squares
501.04107.20
394.84

Mean Squares
35.73 F * 9.4-1
3.797 S = VMT s T * 1.9486

Ranked Treatment Means and Replication Numbers
A
D
B
C
Mean
16.25
17.60
18.13
18.86
n
(33)
(21)
(29)
(25)
Critical Values: R'p = S*Zp
P:,
Zp-1R ’P

(2)
2.80
5.4561

(3)
2.95
5.7484

(4)
3.05
5.9432

Arithmetical Details for Calculating Adjusted Differences
(C-A)' = (C-A)AAC = 2.61 V2(33)(25)/’5S = 13.92096
Similarly:
(C-D)’ »-1.26(4.7777) = 6.01990 (B-A)' - 1.88(5.5562) - 10.4457
(C-B)' =0.73(5.1819) = 3.78279 (D-A)' * 1.4-0(5.0662) * 7.0927
(B-D)* *0.53(4-.9356) * 2.61587
Test Sequence
(C—A) '>R'4-,
(B-A) '>R'3»
(D-A)’>R'2

( O D ) i>R,3t
(B-D)'>R'2

(OB)'3>R'2

Results
(BC)
(BD)

Re suit
(B0)

(BD)

Any two means appearing together within the same par­
enthesis are not significantly different at the 5% level.
Any two means not appearing together within the same par­
enthesis are significantly different.
^Values for Zp are obtained from tables in Duncan (1955).

19

Himes and Moriber (1955) and the Azure II eosin procedure
(Lillie* 1954-)*

elastic tissue Weigert's resorcin

fuchsin was used and Van Giesen's picro-acid fuchsin was
used for collagenous tissue.

RESULTS
1.

THE EFFECT OF CHORIOALLANTOIC TRANSPLANTS OF ADULT

AND EMBRYONIC CHICKEN TISSUES ON THE WEIGHT OF HOMOLOGOUS
AND HETEROLOGOUS TISSUES OF THE HOST EMBRYO.
It is evident from Table 2 that following adult duo­
denal grafts there was a significant reduction in weight
of the whole embryo, a marked enlargement of the spleen,
liver and heart and that there was no significant weight
difference in the intestine or duodenum.

It can also be

seen that subsequent to liver grafts the liver and heart
showed a significant enlargement over those of controls
(sham operation).

However, the liver and heart were sig­

nificantly larger subsequent to grafts of duodenum than
those following liver grafts.

When alcohol inactivated

adult chicken duodenum was transplanted to the CAM it was
observed that there was a decrease in weight of both the
whole embryo and the duodenum.
It is evident from the multiple range test in Table
3 that the weight of the whole chick significantly decreased
while the spleen, liver, heart and duodenum all exhibited
a significant increase in weight following grafts ofadult
duodenum.

It was further observed that following heart

grafts there was a significant increase in weight of both

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23

the intestine and duodenum*

A comparison of the obser­

vations in Table b wxuh those in Table 3 shows that in the
former the weights of the intestine and duodenum differ
from those of the latter and are not always significantly
larger.

Transplantation of alcohol inactivated chicken

duodenum induced a significant decrease in the weight of
the whole embryo and the duodenum (Table 3)*
From the multiple range test as given in Table 4 it
is seen that subsequent to duodenal transplants the spleen,
liver, heart, and duodenum showed a significant enlargement.
When adult spleen was grafted the host spleens were sig­
nificantly larger than control spleens but were still sig­
nificantly smaller than the spleens subsequent to duodenal
grafts.
In Table 5 it is evident that the spleen and heart
were significantly larger when brain was grafted to the CAM
than when control procedures were used.

The duodenum was

larger but other significant growth changes were not ob­
served following grafts of rat duodenum.
It is evident on the basis of the multiple range test
(Table 6)that following duodenal grafts the weight of the
whole embryo was decreased significantly and the weights
of the spleen, liver and heart were significantly increased
in contrast to decreased weights of these organs in controls
and following lyophilized duodenal grafts.

On the other

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27

hand, when lyophilized duodenum was placed on the CAM
the weight of the host heart was significantly higher
while the duodenum was significantly lower*
On the basis of evidence presented in Table 7 it can
be seen that when adult chicken duodenum was placed on the
CAM there was a significant decrease in the weight of the
whole chick and a significant increase in the weight of
individual organs, i.e., spleen, liver and heart.

Follow­

ing grafts of either heated or alcohol extracted chicken
duodenum there were no significant weight changes observed
in either the weight of the whole embryo or the individual
organs •
Table 8 shows that on the basis of the multiple range
test there was no significant differences observed in the
weights of whole chicks or of individual organs follow­
ing heart grafts.

Further, following the grafting of skin

the multiple range test shows that spleen, liver and duo­
denum had significantly increased in weight.
The question naturally arises as to whether enlarge­
ment of host embryonic organs following grafts of adult
tissue was dependent on an increase in protoplasm or was
merely a manifestation of edema.

The following analysis

indicates that the enlargement was due to an increase in
protoplasm.

It is evident in Table 9 that the fresh and

dry weights of the spleen and heart were significantly

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31

larger following spleen and duodenal grafts and it also
shows that following duodenal grafts the fresh and dry
weights of the liver were also significantly increased*
The dry weight of the duodenum was not significantly dif­
ferent from the controls following either duodenal or
spleen grafts*

Of the weight increases induced by the

grafting of duodenum or spleen, the former produced the
greatest weight increase of host organs.
It is evident in Table 2 that the weight of the whole
chick following duodenal grafts was lower than that of con­
trol embryos.

It is also evident that on the basis of the

multiple range test the weight of the duodenum in both the
control and experimental (duodenal) grafts was not sig­
nificantly different.

The question arises as to whether

the relative weights of duodena following duodenal grafts
are significantly higher than those of the controls.
The covariance test was used to ascertain whether the
weight

of the whole chick had any effect on the weight of

the duodenum.

The

value given in Table 10 measures the

difference between the two sample regression coefficients*
This value is 6.6 with a df. * 1, 51 and at the 95% level
this is significant.

This clearly indicates that the weight

of the duodenum following duodenal grafts is statistically
larger than following control procedures.

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33

Since transplantation of adult tissue to the CAM induces
profound changes in various organs of the embryo, the
question arises as to whether or not grafts of embryonic
f
tissue would have the same effect as adult tissues on host
organs*

Since the adult duodenum was the most effective

stimulant for host tissues, duodena from various ages of
embryos were transplanted to the CAM.
The multiple range test in Table 11 shows that the
weight of the whole chick following grafts of 16-day em­
bryonic duodenum is significantly higher than the weight
of embryos following grafts of 18- and 20-day duodena but
is not significantly different from control experiments*
However, the weight of the whole chick following 18-day
embryonic duodenal grafts is significantly less than the
controls*

[Further, the weight of the spleen, liver, heart

and intestine are not significantly higher following grafts
of either 16-, 18- or 20-day embryonic duodenum from that
of the control.

However, it is evident that following

grafting of 20-day chick embryonic duodenum the weight of
the duodenum is significantly decreased while weights of
duodena following grafting of 18- and 16-day duodenum were
not significantly different from controls.
It is evident in Table 12 that the multiple range test
shows that the weight of the whole chick is not signifi­
cantly different from that of the control following grafts
of 15“ , 16- and 17-day chick embryonic duodena.

Further,

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36

the weights of liver, heart and intestine in each ex­
perimental procedure were not significantly different from
controls*

On the other hand, the weight of the spleen of

embryos receiving 15-day chick embryonic duodenum was sig­
nificantly lower than the controls.

The duodenum follow­

ing 16-day embryonic duodenal grafts was significantly
greater than the weight of duodena obtained with 17-day
embryonic duodenal grafts.
During the course of this investigation it was ob­
served that subjection of adult chicken duodenum prior to
transplantation to either 95% alcohol at -20° C for 24hours, lyophilization, a temperature of 80° C for 20 min­
utes or immersion into 1:10,000 merthiolate in 0.15 M
sodium chloride for 30 minutes, host tissues did not re­
spond to the stimulus.
2.

THE EFFECT OF SOLUBLE ANTIGENS OF ADULT AND EMBRYONIC

CHICKEN ORGANS ON THE HOST EMBRYO WHEN INJECTED INTO THE
YOLK SAC
Soluble antigens of 0.1 ml of adult chicken duo­
denum, liver and heart were injected into the yolk sac of
96-hour embryos while controls were injected with 0.1 ml
of 0.15 M sadine.

A mortality rate of 100% was observed

following the injection of supernatant of homogenized duo­
denum.

The time of death varied with the strength of the in­

jection. The results of this experiment have been summar­
ized in Table 13*

37

TABLE 13
THE EFFECT OF SOLUBLE ANTIGENS ON THE HOST EMBRYO
WHEN INJECTED INTO THE YOLK SAC

Amount mg wet wt*
20
10
7.7
6.7

mg
mg
mg
mg

Time death occurred
24 hours
36-48 hours
48-60 hours
56-72 hours

Similar results* i.e., all embryos died, were observed
following the injection of adult chicken liver and heart
and 20-day embryonic duodenal soluble proteins*

Death of

the embryo subsequent to injection with these soluble ma­
terials could not be attributed to a pressure effect be­
cause the control embryos survived until the 20 th day of
incubation at which time they were harvested*
3*

MORPHOLOGY OF CHICK EMBRYOS AND VARIOUS HOMOLOGOUS AND

HETEROLOGOUS ORGANS SUBSEQUENT TO CHORIOALLANTOIC TRANS­
PLANTS OF ADULT AND EMBRYONIC CHICKEN TISSUES
Whole embryo. It was observed that when embryos were
harvested on the 18th day of incubation subsequent to graft
ing of adult chicken duodenum there was a marked decrease
in the size of the chick and it also appeared pale, ashen
and anemic.

When adult chicken spleen was transplanted to

the CAM there were a few chicks which appeared pale but
none as marked as in the case of the duodenal transplants.

38

All embryos appeared normal following CA grafts of adult
chicken liver, heart, brain, skin, the various treated duo­
dena, embryonic duodenum and controls*
Duodenum* 'The gross morphology of the duodenum appear­
ed essentially the same following CA transplants of adult
and embryonic chick tissue and control procedures (fig. 2).
On the other hand the differentiation of the micro­
scopic structure of the duodenum was somewhat altered follow­
ing grafting of adult tissues to the CAM.

Following graft­

ing of adult duodenum the connective tissue within the villi
consisted of a compact mesenchymal layer until the 15th day
and this was succeeded by the lamina propria mucosae on the
16th day.

Van Alten and Fennell (1957) under normal con­

ditions observed the lamina propria mucosae on the 17th day.
Further, Van Alten (1955) identified goblet cells on the
17th day of incubation in untreated embryos, however, follow­
ing adult duodenal grafts goblet cells were identified in
great abundance on the 16th day of incubation.

These ob­

servations indicate that duodenal grafts accelerated differ­
entiation of the duodenal tissues of the host embryo.
It appeared that following grafting of adult duodenum
there were more goblet cells in the host duodenum than in
the control duodenum.

Table 14 shows the number of goblet

cells found in host duodena following CA grafts of various
tissues and control procedure® (all counts were made by

TABLE 14
THE EFFECT OF CHORIOALLANTOIC GRAFTS ON THE DEVELOPMENT
OF GOBLET CELLS IN THE CHICK DUODENUM
Treatment to CAM

Mean no. Goblet Cells/6 Fields

A. Adult chicken duodenum

167.00

B. Adult chicken liver

113.83

C. Control

95.50

D. Adult chicken heart

108.50

E. Adult chicken duodenum

120.67

F. Adult chicken spleen

143.00

G. Control

104.33

H. 16-day embryonic duodenum

160.00

I. 20-day embryonic duodenum

83.50

J. 17~day embryonic duodenum

111.83

Shatis'bical F Test; = 10.36*
Multiple Range Test:
(ABDEFGHJ), (BCDEFGHJ), (BCDEFGIJ)

40

averaging three counts per field and counting six different
fields of six different sections from the same duodenum).
It is evident in Table 14 that the mean number of goblet
cells following adult duodenal grafts is 167 and 120.67*
On the basis of the multiple range test the higher number
of cells, i.e., 167* in the host duodenum is significantly
higher than control C but not higher than control G.

On

the other hand, the number of cells following grafts of
adult chicken duodenum E was not significantly higher than
controls C and G.

These observations indicate that the

number of goblet cells following CA transplants of adult
chicken duodenum was not consistently greater than those
following grafts of other adult or embryonic tissues and
control procedures*
During the course of this study other observations
show that following grafting of either adult chicken duo­
denum or spleen there was an infiltration of lymphocytes
into the duodenal tissue but this was not observed follow­
ing liver, heart, brain, skin or embryonic duodenal
grafts (fig* 3)*
It is evident from the results summarized in Table 13
that transplantation of adult duodenum to the CAM accel­
erates the histochemical differentiation of both connect­
ive tissues and goblet cells in the embryonic duodenum.
The muscle connective tissues differentiated on the 13th

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42

day following duodenal grafts while in controls differen­
tiation was not observed until the 15th day.

Further, the

development of the lamina propria mucosae, submucosa and
goblet cells developed 24 hours earlier than in controls.
It is evident from the observations presented in the
preceding paragraphs that both morphological and histochemical differentiation were accelerated following graft­
ing of adult chicken duodenum.
Spleen. Spleens of host organs following duodenal
grafts were markedly enlarged with a diameter of about 10
mm while the maximum in control embryos was a diameter of
2 mm.

Ihe surface of enlarged spleens exhibited numerous

white tumor-like protuberances.

Essentially the same type

of reaction occurred following spleen and skin grafts.
On the other hand, Control spleens and host spleens follow­
ing grafts of heart, liver, inactivated duodena and embry­
onic duodena (15~* 16-, 17-» 18- and 20-day embryos) were
spherical and dark reddish-brown in color.
In prepared sections of enlarged spleens of 18-day
embryos, which had received OA grafts of either adult chick­
en duodenum, spleen or skin, marked changes were observed
(fig. 5).

Ihere were fewer venous sinuses than in the con­

trol spleens (fig. 4).

Within the pulpa there were few

reticular cells, i.e., mesenchymal cells, and many hemocytoblasts were observed.

Large noduxar areas (as many as

43

six in a single section) filled with granulocytes were
walled off by reticulo-endothelial cells and multinucleated
giant cells were found to be quite numerous and uniform­
ly distributed throughout these enlarged spleens.

In

contrast to control spleens, which contained many erythro­
cytes within the venous sinuses, very few erythrocytes
were observed in enlarged spleens.
It is evident from the above observations that follow­
ing grafts of adult chicken duodenum, spleen or skin the
host embryonic spleen is transformed into a granuloblastic
center.
Liver. On the 18th day of incubation the liver con­
sisted of right and left lobes and exhibited what Kingsbury
et al. (1956) described as a deep sulfur-yellow color.

This

was essentially the way the liver also appeared following
CA transplants of adult chicken spleen, liver, heart, brain,
inactivated duodenum (by heat, lyophilization or alcohol)
and embryonic duodenum.

Following grafts of adult skin the

liver of host embryos (18th day of incubation) was a buff
color with small red spots of about 2 mm in diameter uni­
formly distributed over the entire liver surface.

(The

most marked color change was observed following GA grafts
of adult chicken duodenum.

The host livers were bile green

in color, friable and usually exhibited several relatively
large gray areas.

44

Kingsbury et al*

(1956) have described the micro­

scopic structure of the liver parenchyma of the chick em­
bryo as a continuous network of anastomosing tubules de­
limited by slit-like sinusoids*

By the 18th day of in­

cubation in both control and treated embryos lymphocytes
existed as small nodules in the connective tissues.
Microscopic examination of the enlarged green livers re­
vealed that from a histological point of view they were
essentially similar to those of control embryos.
Heart* The heart of 18-day embryos following OA
grafts of adult duodenum was pale pinkish-gray in color
and appeared to have a length in excess of 10 mm.

Hearts

of control embryos of the same age had a maximum length
of approximately 10 mm and had a bright pink color*
'The wall of the heart of the 18-day embryo consisted
of three poorly differentiated layers, i.e., the endocardium,
myocardium and epicardium.

The myocardium, which is the

thickest , consisted of a spongy network of muscular bun­
dles*

The spaces separating individual bundles of muscle

fibers exhibited sparse amounts of tissue fibers.

Elastic

tissue in the myocardium was confined to the internal sur­
face of the arterioles.

Following duodenal grafts the

various layers of tissue in the heart of host embryos appear­
ed to be essentially similar to those found in control em­
bryos.

Further, other tissues (adult spleen, liver, heart,

4-5

skin, brain and embryonic duodenum) when grafted to the
GAM did not induce gross or microscopic differences in
heart tissues.
Reaction of the Chorioallantoic Membrane. The graft­
ing of small pieces of either adult duodenum or spleen to
the CAM induced the development of numerous small tumor­
like masses which were sufficiently extensive to cover the
major portion of the CAM*

Following the grafting of heart

tissues to the CAM hemorrhagic regions were frequently ob­
served adjacent to or in the vicinity of the grafted tissue
of host embryos on the 18th day of incubation*
A microscopic examination of the CAM adjacent to the
graft site* i.e., grafts of adult duodenum, spleen, liver,
heart and skin, exhibited an abundance of granuloblasts and
granulocytes.

The cytoplasm of these cells exhibited an

abundance of eosinophilic granules.

Many basophilic cells

were also observed in the graft region and these cells
were identified as hemocytoblasts.

In almost every in­

stance there were loci of grafted tissue which had not sur­
vived and these appeared as an acidophilic mass with numer­
ous basophilic granules (fig. 6).
When embryonic duodenum, i.e., duodenum from 15” ? 16-,
17-, 18- and 20-day chicks was transplanted to the CAM the
integrity of the original tissue was not destroyed.

The

46

GAM around the graft contained numerous vessels.

Within

the grafted tissue the mucosa, lamina propria, submucosa
and tunica muscularis were identifiable.

The epithelium

of the mucosa overlying the villi consisted of simple colum­
nar epithelium with numerous functional goblet cells.

The

lamina propria, which forms the core of the villi, was
structurally well differentiated.

The muscularis mucosae

was in most instances confluent with the circular layer of
the tunica muscularis except in areas where the submucosa
was definite.

The tunica muscularis was seen to consist of

two layers, i.e., the inner circular layer and the outer
longitudinal layer (fig. 7)*
The preceding observations demonstrated that follow­
ing grafts of adult chicken tissue to the CAM there was a
loss of morphological integrity of the original tissue trans­
planted and in the graft region a myeloid metaplasia occurred.
On the other hand, when embryonic duodenum was placed on
the CAM the tissue retained its morphological integrity and
continued to differentiate.
4.

THE APPEARANCE OF ADULT DUODENAL ANTIGENS IN THE DE­

VELOPING EMBRYONIC DUODENUM OF THE CHICK
Antisera against saline extracts of adult chicken duo­
denum were tested with saline extracts of adult and 11through 21-day embryonic chick duodena by a modification of
the methods of Ouchterlony (1949) and Bjorklung (1952).

47

Figure 8 shows a photograph and diagram of an
Ouchterlony plate obtained with anti-adult duodenum serum*
This plate consisted of agar with four wells; the lower
well contained the antiserum while the other three wells
contained saline extracts of 12-, 13- and 14-day embryonic
duodenum*

In the plate a series of lines of precipitate

have developed where there was optimum concentration of
antigens and antiserum and these have been numbered 1, 2,
and 3 in the diagram.
The results of these experiments show that there were
changes in the number of antigens present in the duodenum
of the chick embryo between 11 and 21 days of incubation.
From the 11th through the 13th day there were only two
lines formed.

On the 14th day a third line was present,

i.e., line 3 in figure 8.

Line 4 appeared on the 15th day

and was also found on the 16th and 17th days (fig* 9).
However, on the 18th day only lines 1, 2 and 3 were present
and line 4 was absent (fig. 10).

Lines 1, 2 and 3 remained

through the 21st day and a fourth line wat present at this
stage, but it is not possible from the data to ascertain if
this line was identical to line 4 in 15-day duodena.
The distribution of line 4 has been confirmed by rep­
etition of experiments and also by means of the Bjorklund
inhibition technique.

When the latter technique was used

results were obtained which are shown in figure 11.

In

48

this plate six doses of 0*15 M saline extract of 18-day
embryonic duodenum were put into the antiserum well prior
to putting the antigens and anti serum into their respec­
tive wells.

It was shown in figure 10 that 18-day duodenal

antigen reacted with antiserum to form lines 1, 2 and 5*
Following the inhibition procedure lines 1, 2 and 3 were
absent but line 4 developed adjacent to the 15-day well
(fig, 11),

The presence of line 4 and also the line ad­

jacent to the 21-day well suggests that they may be qual­
itatively distinct from those found in the 18-day duo­
denum,
The results presented in figure 12 were obtained in
essentially the same manner as in figure 11 with the ex­
ception that adult chicken serum was used as the inhibiting
antigen.

The position of lines 1, 2 and 3 adjacent to

20- and 21-day wells is comparable to those in figure 10,
This suggests that lines 1, 2 and 3 are not serum anti­
gens,

The absence of the line adjacent to the 21-day well,

as illustrated in figure 11, suggests that this antigen has
reactive groupings in common with serum.
Table 16 summarizes the distribution of duodenal anti­
gens which was observed in various ages of embryos.

The

inhibition analyses are also summarized in this table.
From the evidence at hand, it has been demonstrated
that: (1) the pattern of adult duodenal antigens found in

TABLE 16
A SUMMARY OP THE RESULTS OBTAINED WITH THE SBRUMAGAH PRECIPITIN TEST. ADULT DUODENUM ANTISBRUM
WAS TESTED WITH SALINE EXTRACT OP ADULT AND
11 THROUGH 21—DAT CHICK EMBRYONIC DUODENA
Antiserum

Antigen

No. Lines

Anti-Adult
duodenum

11-day embryo
duodenum

2

12d4ggeg$F^°
13-day embryo
duodenum
14-day embryo
duodenum
15-day embryo
duodenum
16-day embryo
duodenum
17-day embryo
duodenum
18-day embryo
duodenum
19-day embryo
duodenum
20-day embryo
duodenum
21-day embryo
duodenum
21-day embryo
duodenum
Adult duodenum
Adult Serum

2

t?

»t

n

n

w

rt

it

ft

n

M

tt

n
n

Inhibition

No. Lines

2

4

18-day embryo
duodenum

1

4
4
18-day embryo
duodenum

0

3

Adult Serum

3

4

Adult Serum

3

4

18-day embryo
duodenum

3
3

9
3

Adult Serum

1

0

50

the developing duodenum changes during development and
(2) at least three of the antigens found in the embryonic
duodenum do not appear to be present in ddult serum*
5.

THE EFFECT OF CHORIOALLANTOIC TRANSPLANTS OF ADULT

CHICEEN DUODENUM AND SPLEEN ON THE DEVELOPMENT OF ADULT
DUODENAL AND SPLEEN ANTIGENS IN THE DUODENUM AND SPLEEN
OF 17-DAY EMBRYOS
Antisera against saline extracts of pooled adult chick­
en duodenum or against adult chicken spleen were tested
with saline extracts of: (1) normal 17-day embryonic chick
duodenum or spleen; (2) 17-day embryonic duodenum or spleen
which was stimulated by CA grafts of adult chicken duo­
denum; (3) 17-day embryonic duodenum or spleen which was
stimulated by CA grafts of adult chicken spleen.

Methods

similar to those mentioned in the preceding section were
used*
The antigenic pattern of the developing duodenum
following CA grafts of adult chicken duodenum or spleen
is shown in figure 13*

In this figure the antiserum well

contained anti—adult chicken duodenum and the other wells
contained adult spleen stimulated 17-day embryonic duodenum
(Spleen Duo), adult duodenum stimulated 17-day embryonic
duodenum (Duo-Duo) and normal control 17-day embryonic duo­
denum (17-Duo).

It may be seen that opposite wells 17-Duo,

Duo-Duo and Spleen Duo, lines 1 through 4 are present*

5 1

This demonstrates the presence of an antigenic component
of identical specificity in all three preparations.

It

will he further noted that line 5 is opposite only wells
Duo-Duo and Spleen Duo.

On the other hand, lines 6 and 7

are present only opposite well Duo—Duo*
figure 14 shows an Ouchterlony plate obtained with
anti—adult chicken duodenum serum and tested with antigens
of adult duodenum (Adult Duo), duodenum stimulated 17-day
embryonic duodenum (Duo-Duo) and untreated control 17-day
embryonic duodenum.

It was evident in figures 13 and 14

that grafting of adult duodenum to the CAM of 9-day chick
embryos caused an increase in antigenic components from
4 to 7 in the 17-day embryonic chick duodenum.

It was al­

so observed that if adult chicken spleen was transplanted
to the CAM there was one extra antigenic component found
but this one was also found if duodenum was used on the
CAM*
It has been shown above that the spleen of the embryo
undergoes a marked increase in size subsequent to grafting
adult duodenum or adult spleen to the CAM.

Figure 15 shows

an Ouchterlony plate obtained with anti-adult dhicken spleen
in the antiserum well and saline soluble antigens obtained
from normal control 17-day embryonic spleen (17-day Spleen),
adult spleen stimulated embryonic spleen (Spleen Spleen)
and adult duodenum stimulated spleen (Duo-Spleen).

Lines

52

1, 2 and 4 can "be seen opposite well 17-day Spleen and
from other plates it has been confirmed that line 4 con­
sists of two antigenic components which are seen as lines
3 and 4 opposite the Duo-Spleen well.

Only two lines can

be seen opposite well Spleen Spleen but these also have
been shown to each consist of two lines, therefore, it can
be concluded that lines 1, 2, 3 and. 4 are all present.
Lines 3 and 4 are clearly seen opposite well Duo-Spleen.
However, lines 1 and 2 form one line as they did opposite
well Spleen Spleen.

It is clear then that lines 1 through

4 are opposite each of the antigen wells and are continuous
and therefore demonstrate antigenic components of identical
specificity.

However, because the lines are together oppo­

site some of the wells and divided when opposite others
this shows that there are quantitative differences in the
various spleens.

Line

oa the other hand, is present

only opposite well Duo-Spleen so that this demonstrates a
clearly qualitative difference.
From the above results it can be seen that grafting
of adult chicken duodenum to the CAM of 9-day chick embryos
causes both quantitative and qualitative antigenic differ­
ences in the duodenum and spleen of 17-day embryos.

Adult

spleen, on the other hand, when grafted to the CAM pro­
duces both qualitative and quantitative changes In the anti­
genic picture of 17-day embryonic duodenum but in 17-day

53

embryonic spleen only quantitative changes were ob
served*

DISCUSSION
1.

WEIGHT AND MORPHOLOGICAL CHANGES ON HOST ORGANS FOLLOW­

ING CA GRAFTS
Observations presented in the preceding pages demon­
strated that profound changes occurred in the chick embryo
subsequent to grafting of adult chicken tissues to the CAM*
The host spleen was enlarged following grafts of adult
spleen, duodenum, brain and skin.

The host liver was en­

larged following transplants of adult liver, spleen and duo
denum, while the heart was enlarged following either spleen
liver or duodenal grafts.

In all cases the adult duodenum

produced the most pronounced enlargement of the embryonic
organs studied, i.e., spleen, liver and heart.

Enlarge­

ment of the host duodenum following grafts of adult duo­
denum could be demonstrated only on a relative weight basis
i.e., the weight of the whole embryo decreased while the
weight of the duodenum remained about the same as that of
controls.
Histological studies of the enlarged spleen showed
that the enlargement was primarily caused by granulopoi­
esis regardless of the adult organ (spleen, duodenum, skin
or brain) used to stimulate it.

Although the gross mor­

phology of the host liver (bile green color) and the heart

55

(enlarged and pale color) was altered following CA grafts
of adult chicken duodenum the microscopic anatomy was
similar to that of the controls*
The hypothesis of organ-specific growth stimulation
was proposed by Weiss (194-7) and Ebert (1955) and has been
based largely on the observations of Murphy (1916),
Danchakoff (1916), Willier (1924), Weiss (194-7) and Ebert
(1951)*

These workers found that when a small fragment of

adult chicken organ was transplanted to the vascular bed
of an embryo (either CAM or vascular area of the blasto­
derm) it greatly stimulated growth of the homologous em­
bryonic organs*

Weiss and Andres (1952) observed an in­

crease in the mitotic rate of the embryonic kidney follow­
ing injections of kidney brei into the CA blood vessels
and also in the mitotic rate of the kidney and liver follow­
ing injections of mesonephric brei (Andres, 1955)*

Some­

what similar results were reported by Teir (1952) for the
orbital gland in rats following intraperitoneal inject­
ions of orbital gland.

Sundell and Teir (1954-) also found

a specific factor in the digestive tract (including the
duodenum) of rats which had a mitotic-stimulating effect
on the epithelium of the digestive tract.
study Teir et al.

In a recent

(1957) presume that this mitotic-stim­

ulating effect takes place through the nucleic acids (RNA and
DNA).

56

Kohn (1958) has demonstrated an inhibitory factor
for liver regeneration in the serum of normal rats.

This

supports the views of Rose (1952) who maintained that
organ-specific growth control substances of homologous
tissues inhibited rather than stimulated growth.
Observations made during the course of this study did
not support the hypothesis of organ-specific growth stim­
ulation.

Danchakoff (1918) observed that enlargement of

embryonic spleen after CA transplants of adult spleen was
not confined to the spleen but extended throughout the
whole mesenchyme of the host.

Weiss (1955a) in re-eval-

uating his experiments with organ injection, found that the
increase in the weight of the homologous organs could be
attributed to trapping of debris and hemorrhages.

Andres'

(1955) observations are much less convincing when one con­
siders the work of Wilson and Leduc (194-7) and Levey (1956).
©he former authors question the validity of mitotic rate
counts in unstandardized material while the latter author
did not observe changes in the kidney, i.e., the retro­
gression of the mesonephros following CA grafts of 18day embryonic mesonephros or metanephros.

The observations

of Saetren (1956) raise additional questions concerning
the interpretation of Andres* (1955) work.

He found that

following the injection of macerated homologous tissue in­
to the peritoneal cavity of rats following partial nephrectomy

57

or removal of a portion of the liver, there was a marked
inhibition of mitoses in regenerating portions of kid­
ney and liver*
The tracer studies of Ebert (1954) showed that when
labeled tissues were placed on the CAM, homologous organs
had a higher specific activity than heterologous organs.
Waddington and Sirlin (1955J* w^° nsed autoradiographic
procedures, were unable to demonstrate organ-specific
transfer from grafts to homologous tissues in early stages
of amphibia.

Further, Horn and House (1955) observed that

when they injected tagged homogenates of liver, kidney,
spleen and thymus into young mice the uptake value of the
spleen was consistently higher than other organs,

fhey

suggested that the spleen was the most effective organ
of the reticulo-endothelial system for removing foreign
protein from the circulation.

CDhe observations of

Levi-Montalcini (1952) also cast doubt on the validity of
the organ-specific growth hypothesis.

She found that the

spleen and liver of the chick were enlarged subsequent to
transplantation of mouse sarcoma 37 or 180 to the allantoic
vesicle of the chick.
An alternative hypothesis for the enlargement of em­
bryonic spleen following spleen grafts was proposed by
Simonsen (1957).

He believed that there was a direct trans­

fer of cells by way of the blood vascular system from the

58

graft to homologous tissue where they colonized.

'These

cells were thought to multiply and form antibodies against
the host.

However, this hypothesis does not explain why

there is an increase in weight of heterologous tissues
following duodenal, brain and skin grafts.
It was shown in the preceding paragraphs that valid
objections have been raised to both organ-specific growth
stimulation and direct cell transfer hypotheses for in­
creased rates in homologous tissues following GA grafts.
Observations made during the course of this study do not
completely support either view.

For this reason it is

interesting to speculate on the factors responsible for
the growth of embryonic organs following CA grafts.

Q?he

observations made in this study demonstrated that the mes­
enchyme of the embryonic spleen exhibited a marked increase
in mononucleated stem cells and granulocytes.

On the other

hand, enlargement of the liver following grafts of adult
duodenum, liver and skin was attributed to an increase in
parenchyma tissue.

Both fresh and dry weights of stim­

ulated liver were higher than those of controls.

Further,

the secretion of bile was accelerated as evidenced by the
complete discoloration of the liver with bile.

It is well

established that the reticular tissue of the spleen and
the reticulo-endothelial cells of the liver, i.e., stellate
cells of von Kupffer, are concerned with removal of foreign

59

material from the "blood vascular system.

Horn and House

(1955) maintain that following injections of various
tagged tissues into young mice, the spleen consistently
gave higher uptake values than other organs.

These authors

suggest that the spleen is the most effective organ for
removing foreign protein from the circulation.
Antigenic studies made during the course of this in­
vestigation demonstrated that antigens made with stim­
ulated spleens exhibited a different antigenic pattern
from control spleens.

This would indicate that grafted

tissue releases materials (proteins) into the circulation
which stimulate the reticulo-endothelial system of the
spleen.

Further, certain organs, viz., heart, liver and

embryonic duodenum, probably do not contain this reticulo­
endothelial stimulating material, because the spleen re­
mained essentially normal subsequent to grafting of these
organs.

It also has been demonstrated in this study that

the embryonic duodena contain fewer antigenic components
than do adult duodena.

Additional evidence in support of

this view, viz., that enlargement is dependent upon an
imbalance of proteins in stimulated organs, is found in
the antigens made with stimulated duodena.

In the latter,

seven lines were present while in controls only four lines
could be identified.

In the spleen there was an increase

in mononucleated cells, granulocytes, and multinucleated

60

giant cells , while in the duodenum an abundance of mononucleated cells was observed,

(Phis indicates that en­

largement of organs probably was induced by a reaction to
a fairly specific protein,

The mechanism in this reaction

is inherent in the tissues themselves, i,e., the reticulo­
endothelial cells have the ability to remove not only par­
ticulate matter but also dissolved materials.

Lewis (1931)

demonstrated that monocytes and macrophages would not only
ingest foreign particulate materials but would "drink”
various liquefied proteins, globulins and other complex
substances.

However, it is a well known fact that young

animals are less reactive to foreign agents than older ones.
It would seem from the antigenic differences observed in
stimulated duodena and spleens that this may accelerate
differentiation of the phagocytic cells following GA grafts
of organs which contain these antigens.
It appears then, that removal of non-specific pro­
teins from the blood in the embryo produces a noninfectious
tpye of inflammation.

The response to this stimulant is

an increase in the number of specific cell types, especial­
ly granulocytes.

Menkin (19^5) has observed that associated

with the inflammatory processes, there is concomitant
hyperplasia of granulocytes in the bone marrow which may
spread to other lymphatic organs such as the spleen and
thymus.

Thus, it would appear that enlargement of the

61

embryonic spleen and liver of the chick after grafting of
adult tissues may be due to a noninfectious inflammatory
reaction caused by an imbalance of proteins in the embryo.
Heart enlargement was probably due to compensatory
reaction which was related to an anemic condition of the
chicks.

This anemic condition was most likely due to the

fact that the spleen was almost totally given over to the
production of granulocytes rather than erythrocytes.

The

grafts were made on the 9th day of incubation at which time
Fennell (194*7) observed that the definitive erythrocytes
had replaced the primitive erythrocytes to become the most
numerous type in peripheral blood.

Thus, spleen and liver

enlargement may be due in part to removal of primitive gen­
eration of blood calls and foreign substances from the
blood vascular system.

On the other hand, heart enlargement

may be due in part to compensation.
It was also observed in the course of this study that
when either normal duodenum, liver or heart were injected
into the yolk sac on the 4-th day of incubation they caused
all the embryos to die within 72 hours.

This observation

is in keeping with that of Fennell (194-7) who using a much
more dilute inoculum of minced normal liver observed that
67% of the embryos died within 5 days after inoculation.
He further observed that normal liver mince injections pro­
duced blood changes which met the requirements for

62

hemocytoblastbsis.

This reaction on the part of the host

embryo also can be attribued to a reaction caused by im­
balance of proteins in the embryo.
2.

IMMUNOLOGICAL STUDIES
It was observed in this study that adult chicken duo­

denum antigens can be identified in the 11-day embryonic
duodenum.

However, from the 11th through the 21st day of

incubation there is an increase in the number of antigens
in the developing duodenum, e.g., two antigens were pre­
sent in 11-, 12- and 13-day embryos; three antigens in 14day embryos; four antigens in 15-* 16- and 17-day embryos;
three antigens in 18-, 19- and 20-day embryos; and four
antigens in 21-day embryos.

Ebert (1952) identified an in­

crease in the number of antigens in the development of the
chick spleen.

He observed three antigens in spleens of

12-day embryos and six in spleens of 18-day embryos when
he used 18-day embryonic spleen antiserum.
The appearance of an antigen on the 15th day and its
disappearance on the 18th day is rather striking.

A some­

what similar situation has been reported by Telfer and
Williams (1955) in the Cecropia silk worm.

They identi­

fied five pupal blood antigens present throughout meta­
morphosis with a sixth appearing at the fifth instar.

This

persisted during the pupal stage and disappeared in the
adult.

In the case reported in this study the antigen

63

observed on the 15th day was also found in the adult be­
cause anti serum was prepared against adult duodenum.

It

must be kept in mind that at the present time it is not
possible to prove the complete absence of an antigen on
the basis of its absence in agar plates.
Several explanations for the appearance and dis­
appearance of an antigen during the course of development
have been advanced,

Tyler (1937) suggested that many of

the large molecular substances of an organism may have sim­
ilar determinant groups and therefore may react with the
same anti serum.

Observations made during the course of

this study lead one to believe that this view does not ad­
equately explain the disappearance of antigens in the
course of development.
During morphogenesis there may be a structural change
in antigenic macromolecules.

Fox (1958) proposed that an

antigen may be transformed from a complete antigen into
an incomplete antigen.

An incomplete antigen is one which

has the specificity for antibody formation but it will not
react in the precipitin test.

A somewhat different change

in antigenic structure could occur, i.e., a change from a
globular to a fibrous protein.

Mazia and Dan (1932) ob­

served that during spindle fiber formation there was a
change from globular to fibrous protein.

Heidelberger

(1936) suggests that antigenic specificity may be due to

64

multiple reactive areas on the surface of antigens.

He

believes that changes in specificity of native proteins
can be accounted for by the unfolding of polypeptide
chains which leads to their separation from adjoining
chains and as a consequence the specificity of the anti­
gen is altered.
If Heidelberger's concept correctly explains antigen
specificity it is reasonable to assume that during the
course of development of an antigen its polypeptides may
undergo foldings and separations.

Under these conditions

the reactive surface of the molecule is altered and cannot
be detected by immunological methods.

On the basis of the

evidence on hand and the wide divergence of views of anti­
gen reactivity, a final explanation cannot be given why
line 4 appears in 15-day duodena and disappears in 18-day
duodena.
It has been further observed in the course of this
study that transplantation of adult chicken organs (duodenum
and spleen) to the GAM caused changes in the antigenic
pattern of the duodenum and spleen.

This change was most

pronounced following grafting of adult duodenum.

Adult

duodenal grafts increased the number of antigens from four
to seven in the 17-day embryonic duodenum and from four to
five in the spleen.

The grafting of adult spleen altered

the antigenic pattern of the 17-day duodenum (from four

65

to five) "but was without effect in the spleen*

It must be

kept in mind, however, that even these changes in the anti­
genic picture are the end products of differentiation and
not the causal factors.
Ebert (1955) maintained that growth of host tissues
following homologous grafts was due to a "building block”
rather than a "template" or catalytic mechanism (Weiss, 194-7)*
The findings in this investigation tend to support this
"building block" hypothesis, i.e., selective incorporation
of tissue-specific antigens from grafts into homologous
host tissues.

However, this hypothesis does not adequate­

ly explain the generalized effects, i.e., growth of heter­
ologous organs.

There is evidence that a growing organ will

remove from the blood vascular system metabolites which
play an important part in growth.

Kato and Moog (1958)

found that after injection of disodium phenylphosphate in­
to the chick on the 14-th day of incubation there was an
increased alkaline phosphatase activity in the duodenum,
liver, mesonephros and metanephros on the 17th day of
incubation.
It was shown in this study that the spleen, duodenum,
skin, brain and liver stimulated growth of embryonic organs.
It is interesting to speculate on the factors responsible
for the growth of these organs.

If it is assumed that a

possible increase in substrate concentration in the embryonic

66

circulatory system could ensue following grafting of adult
tissues, then growth may he attributed to specificity of
substrate for certain organs.

Under certain of these con­

ditions these enzyme systems would be accelerated and
growth would be enhanced.

SUMMARY
1.

It was observed in this study that following CA

grafts of adult chicken duodenum there was a marked de­
crease in the absolute weight of the host, a marked in­
crease in the weight of the spleen, liver and heart and a
relative weight increase in the duodenum.

Further, follow­

ing grafts of adult skin and brain the spleen and liver
were significantly heavier.

Following liver grafts the

liver and heart showed a significant increase in weight.
Adult chicken spleen grafts caused a marked increase in
the weight of the spleen and heart.

Further, it was ob­

served that regardless of what tissue was used for graft­
ing, nine days later the morphological integrity of the
graft was destroyed and the area was replaced by a myeloid
metaplastic center.

On the other hand, embryonic duodena

retained their integrity and continued to differentiate.
2.

Grafting of adult duodenum caused acceleration of

tissue differentiation of the host duodenum.

Following

grafting of duodenum, spleen and skin the host spleen ex­
hibited a marked increase in granuloblasts and granulo­
cytes.

The heart and liver following grafts were essen-

ially like those in control embryos.
3.

Observations made following treatment of the adult

duodenum prior to grafting with either 95% alcohol for 24

68

hours at —20° C, lyophilization, heating at 80° C for 20
minutes or treatment with 1:10,000 merthiolate in 0.15M
sodium chloride for 30 minutes, showed that the grafts
were inactivated and the host was not effected.
4.

When duodena of 15“ * 16-, 17” * 18- and 20-day em­

bryos were grafted to the CAM no changes in the weight of
host spleen, liver or heart were found.

Following 20-day

duodenal grafts the weight of the duodenum was signifi­
cantly decreased.
5#

All embryos receiving grossly normal adult duo­

denum, liver and heart supernatant injections into the
yolk sac at four days of incubation died within 72 hours
after inoculation.
6.

The polysaccharides in the connective tissue and

goblet cells of the duodenum following CA grafts of adult
duodenum, differentiated at least 24 hours earlier than
in control chicks.
7.

The development of adult duodenal antigens in the

embryonic duodenum was studied by means of the Ouchterlony
agar diffusion technique.

In 11-, 12— and 13-day embryonic

duodena there were two antigens; by the 14th day three
antigens; in the 15-* 16- and 17-day duodena four antigens;
while in 18-, 19- and 20-day duodena there were only three
antigens with a fourth antigen again present in the 21day duodenum.

The disappearance of the fourth antigen

69

found, in 15-> 16- and 17-day duodena was attributed to a
change in molecular configuration in the process of differ­
entiation*
8*

Following grafts of adult duodenum there was an

increase of three antigens observed in the host duodenum
and an increase of one antigen in the host spleen.

When

adult spleen was grafted there were no extra antigens in
the host spleen but there was one extra antigen in the
duodenum.
9*

The results are discussed in the light of the organ-

specific growth stimulation hypothesis and this hypothesis
is rejected.

It has instead been postulated that the adult

grafts may provide substrate for enzyme systems in host
organs and this upsets the balance and causes the more gen­
eralized results reported in this study.

LITERATURE CITED
Andres, G. 1955 Growth reactions of mesonephros and
liver to intravascular injections of embryonic liver
and kidney suspensions in the chick embryo. J. Exp.
Zool., 150:221-249.
Bjorklund, B. 1952 Specific inhibition of precipitation
as an aid in antigen analysis with gel diffusion method.
Proc* Soc. Exp. Biol. & Med., 79:319-524.
Burke, V., N.P. Sullivan, H. Petersen and R. Weed 1944
Ontogenetic change in antigenic specificity of the
organs of the chick. J. Inf. Dis., 74:225-233.
Cooper, R. S. 194-6 Adult antigens (or specific combining
groups) in the egg, embryo and larva of the frog.
J. Exp. Zool-., 101:143-172.
Danchakoff, V. 1916 Equivalence of different hematopoietic
anlages. (By method of stimulation of their stem cells).
Am. J. Anat., 20:255-327.
Danchakoff, V. 1918 Equivalence of different hematopoietic
anlages (by method of stimulation of their stem cells).
II. Graphs of adult spleen on the allantois and response
of the allantoic tissues. Am. J. Anat., 24:127-189.
Dixon, W. J. and P. J. Massey, Jr. 1951 Introduction to
Statistical Analysis McGraw-Hill Book Company,
i^iew York 370 pages.
Duncan, D. B. 1955 Multiple range and multiple F tests.
Biometrics, 11:1-42 *
Duncan, D. B. 1957 Multiple range tests for correlated
and heteroscedastic means. Biometrics, 13:164-176.
Ebert, J. D. 1950 An analysis of the effects of anti-organ
sera on the development, in vitro. of the early chick
blastoderm. J. Exp. Zool., 115:351-378.
Ebert, J. D. 1951 Ontogenetic change in the antigenic
specificity of the chick spleen. Physiol. Zool.,
24:20-41.

71

Ebert, J. D. 1952 Appearance of tissue-specific proteins
during development. Ann. N.Y. Acad. Sci., 55:67-84.
Ebert, J. D. 1953 An analysis of the synthesis and dis­
tribution of the contractile protein, myosin, in the
development of the heart. Proc. Nat. Acad. Sci.,
22.:333-344.
Ebert, J. D. 1954 Ihe effects of chorioallantoic trans­
plants of adult chicken tissues on homologous tissues
of the host chick embryo. Proc. Nat. Acad. Sci.,
40:337-347.
Ebert, J. D. 1955 Some aspects of protein biosynthesis
in development. In Aspects of Synthesis and Order in
Growth. D. Rudnick editor. "Princeton University
Press, Princeton, N.J. 69-112.
Ebert, J. D., R. A. Tolman, A. M. Mun and J. P. Albright
1955 ®b.e molecular basis of the first heart beats.
Ann. N.Y. Acad. Sci., 60;968-985.
Fennell, R. A. 1947 Ihe relation between age, number,
and types of cells in the peripheral circulation of
chicken embryos under normal and experimental con­
ditions. J. Ag. Research, 74:217-239.
Flickinger, R. A., E. Levi, and A. E. Smith 1955 Some
serological experiments relating to the embryonic de­
velopment of the lens. Physiol* Zool., 28:79-85.
Fox, A. S.

1958

Personal communication.

Gomori, G.
1950 A rapid one-step trichrome stain.
Am. J. Clin. Path., 20:661-664♦
Heidelberger, M. 1956 Lectures in Immunochemistry.
Academic Press, New York. 150 Pages.
Himes, M. and L. Moriber 1956 A triple stain for des­
oxyribonucleic acid, polysaccharides and proteins.
Stain Technol., 51:67-70.
Horn, E. C. and M. E. House 1955 A test of specific up­
take from organ homogenates by homologous organsin
the young mouse. J. Elisha Mitchell Sci. Soc.,
Zl:171.

72

Johnson, X* S. and C. A. Leone 1955 ^he ontogeny of pro­
teins of adult chicken heart as revealed hy serolog­
ical techniques. J. Exp. Zool., 130:515-534.
Kato, Y. and F. Moog 1958 Differences in response of
phosphatases in chick embryo to injection of sub­
strate. Science, 127:812-813.
Kingsbury, J. W., M. Alexanderson and E. A. Kornstein 1956
The development of the liver in the chick. Anat. Rec.,
124;165-188.
Kohn, R. 1958 Effect of administration of rat serum
on rat liver regeneration. Exptl. Cell Research,
14:228-230.
Levi-Montaicini, R. 1952 Effects of mouse tumor trans­
plantation on the nervous system. Ann. N.Y. Acad. Sci.,
55.:330-343.
Levy, C. K. 1956 The role of extrinsic factors in the
retrogression of the mesonephros of the embryo chick.
University of North Carolina PhD. Thesis.
Lewis, W. H.
42.:17-28.

1931

Pinocytosis.

Bull. John Hopkins Hosp.,

Lillie, R. D. 1954 Histopathological Technic and Practical
Histochemistry. Blakiston Co., New York. 501 Pages.
Mazia, D. and K. Dan 1952 The isolation and biochemical
characterization of the mitotic apparatus of dividing
cells. Proc. Nat. Acad. Sci., 38:826-838.
Menkin, V. 1945 Chemical basis of fever with inflammation.
Arch. Path., 39:28-36.
Minoura, T. 1921 A study of testis and ovary grafts on
the hen's egg and their effect on the embryo.
J. Exp. Zool., 33 sl-61.
Moog, F. 1950 The functional differentiation of the small
intestine. I. The accumulation of alkaline phosphomonesterase in the duodenum of the chick. J. Exp. Zool.,
115:109-129.
Moog, F. and E. L. Wenger 1952 The occurrence of a neutral
mucopolysaccharide at sites of high alkaline phosphatase
activity. Am. J. Anat., 90:339-378.

73

Murphy, J. B. 1916 The effect of adult chicken organ
grafts on the chick embryo, J. Exp, Med,, 24:1-6,
Nace, G. W. 1933 Serological studies of the blood of the
developing chick, J. Exp, Zool,, 122:425-448,
Nace, G, W, and A, M, Schechtman 1948 Development of
nonvitelloid substances in the blood of the chick
embryo. J. Exp. Zool,, 108:217-254.
Needham, J. 1931 Chemical Embryology.
ity Press 5 vol., 2021 Pages.

Cambridge Univers­

Ouchterlony, 0. 1949 Antigen-antibody reactions in gels.
Arkiv. Eemi, Mineral, Geol., 26B, Nr pp. 1-9*
Oudin, J. 1952 Specific precipitation in gels andits
application to immunochemical analysis. In Methods in
Medical Research, 5.edited by A. C. Corcoran! The Year
Book Publishers, Inc., Chicago, p. 335*
Fomerat, C. M. 19^9 Morphogenetic effects of spleen
antigen and antibody administrations to chick embryos.
Exptl. Cell Research, suppl. 1:578-581.
Richardson, D., S. D. Berkowitz and P. Moog 1935 2?he
functional differentiation of the small intestine. V.
The accumulation of non-specific esterase in the duo­
denum of chick embryos and hatched chicks. J. Exp. Zool.,
130:57-70*
Rose, S. M. 1952 A hierarchy of self-limiting reactions
as the basis of cellular differentiation and growth
control. Am. Nat., 86:537-354.
Rose, S. M. 1955 Specific inhibition during differentiation.
Ann. N. Y. Acad. Sci., 60:1156-1159*
Saetren, H. 1956 A principle of auto-regulation of growth.
Production of organ specific mitose-inhibitors in kidney
and liver. Exptl. Cell Research, 11:229-254.
Sandstrom, C. J. 1932 Heteroplastic transplants of duck
kidney tissue on the chorio-allantois of the chick in
conjunction with adult chicken spleen grafts. Anat. Rec.,
22:69-81.

74-

Schechtman, A. M. 194-7 Antigens of early developmental
stages of tlie chick. J. Exp. Zool., 105:329-348.
Schechtman,
emhryo.

A. M. 1948 Organ antigen in the early chick
Proc. Soc. Exp. Biol. Med., 68:263-266.

Schechtman, A. M. and H. Hoffman 1952 Serological studies
on the origin of globulins in the serum of the chick
embryo. J. Exp. Zool., 120:375-390*
Shaver, J. R. 1954- Inhibition of nervous system develop­
ment in the frog produced by tissue fractions of adult
homologous organ. Anat. Rec. ,118:64-6-64-7.
Simonsen, M. 1957 The impact on the developing embryo
and newborn animal of adult homologous cells. Acta.
Path. et. Microbiol. Scand., 4-0:4-80-500*
Sundell, B. and H. Teir 1954- Growth factors in the di­
gestion apparatus of rat. Exptl. Cell Research,
6 :321-326.
Teir, H. 1952 Experimental alteration of cell size and
mitotic activity in the outer orbital gland of the white
rat. IV. Influence of parenterally applied extracts
of outer orbital gland. Acta. Path. Microbiol. Scand.,
20:158-183.
Teir, H., A. Larmo, A. Alho and K. Blomqvist 1957 In­
fluence of intraperitoneally injected homogenates of
the outer orbital gland and the liver on ERA and DNA
in these organs in rats. Exptl. Cell Research,
12.:14-7-157.
Telfer, W. H. and C. M. Williams 1953 Immunological
studies of insect metamorphosis. I. Quantitative and
qualitative description of the blood antigens of the
Cecropia silkworm. J. Gen. Physiol., 36:389-4-13*
Ten Cate, G., and W. J. van Doorenmallen 1950 Analysis of
the development of the eye lens in chicken and frog
embryos by means of the precipitin reaction. Proc.
Koninkl. Ned. Akad. Wetenschap., 53:894— 909*
Tyler, A. 194-7 An auto-antibody concept of cell structure,
gfcowth and differentiation. Growth, 10 (suppl.):7-19.

75

Tyler, A. 1957 Immunological studies of early develop­
ment. In The Beginnings of Embryonic Development
Edited by A. Tyler, R. C. von Borstel and C. B. Metz
Publication no. 48 of A A A S. p. 341-382.
Van Alten, P. J. 1955 Development and histogenesis of
the avian digestive tract with special reference to the
histochemistry of the connective tissue. Michigan
State University Unpublished M. S. Thesis.
Van Alten, P.J. and R. A. Pennell 1957 Histogenesis and
histochemistry of the digestive tract of chick embryos.
Anat. Re c ., 127 s677-696.

Waddinfeton, C. H. and J. L. Sirlin 1955
labelled grafts in amphibian embryos.
Soc. Edinburgh, 24:28.

Experiments with
Proc. Poy.

Walter, H., D. W. Allmann and H. R. Mahler 1956 In­
fluence of adult tissue homogenates on formation of
similar embryonic proteins. Science, 124;1251-1252•
Weiss, P. 1947
development.
Weiss, P. 1952
own products.

The problem of specificity in growth and
Yale J. Biol. Med., 19:235-278.
Self-regulation of organ growth by its
Science, 115:487-488.

Weiss, P. 1953a
Remarks made at a symposium on the Bio­
chemical and Structural Basis of Morphogenesis.
Arch. Neerland. Zool., 10 (suppl. 1), 159-161.
Weiss, P. 1953b
Some introductory remarks on the cellular
basis of differentiation. J. Embryol. exp. Morph.,
1:181-211.
Weiss, P. 1955Specificity in growth control. In
Biological Specificity and Growth. Edited by E. G.
Butler, Princeton University Press. (12th Growth
Symposium 1953)*
Weiss, P., and G. Andres 1952 Experiments on the fate of
embryonic cells (chick) disseminated by the vascular
route. J. Exp. Zool., 121:449-488.
Weiss, P., and J. L. Kavanau 1957 A model of growth and
growth control in mathematical term. J. Gen. Physiol.,
41:1-47.

76

Weiss, P., and H. Wang 194*1 Growth response of the liver
of embryonic chick hosts to the incorporation in the
area vasculosa of liver and other organ fragments,
Anat, Rec,, 22. (Suppl.):62,
Willier, B, H, 1924- The endocrine glands and the develop­
ment of the chick. I, The effects of thyroid grafts.
Am, J. Anat., 55:67-103*
Wilson, J, W., and E, H. Leduc 194-7 Mitotic rate in mouse
liver following intraperitoneal injection of liver,
kidney and egg yolk. Anat. Rec., 97:4-71-4-94-.

77

PLATE I
Fig* 1.

Geometry of an Ouchterlony plate.
An. Antiserum well;
1. Antigen well;
2. Antigen well; and
5• Antigen well.

An

©

79
PLATE II
Fig. 2.

Gross section through, the duodenum of a control
18-day embryo.
A.
B.
G.
D.
E.

Fig. 3.

Epithelium;
Goblet cell;
Lamina propria;
Tunica muscularis;
Blood vessel.

Gross section through the duodenum of an 18-day
embryo following adult chicken duodenal grafts.
A. Epithelium;
B. Goblet cell;
G . Lamina pr opri a;
D. Tunica muscularis;
E. Blood vessel;
F. Lymphocytes.
Micrometer scale insert:! space® 0.01 mm.
All sections were stained with Himes and Moriber
triple stain.

81
PLATE III
Fig. 4-.

Cross section through a spleen of a control 18
day embryo.
G.
H.
I.

Fig. 5.

Vein;
Artery;
Reticular structure

Cross section through a spleen of an 18-day embryo
following adult duodenal grafts.
G.
H.
J.
K.
L.

Vein;
Artery;
Hodule;
Multinucleated giant cells;
Lymphoid hemocytoblast.

Micrometer scale insert; 1 space = 0.01 mm.
All sections were stained with Himes and Moriber triple
stain.

83
PLATE IV
Pig* 6*

Cross section through an adult duodenal graft on the
18th day of incubation*
F.
M.

Fig. 7*

Lymphocytes and granulocytes;
Foci of degenerating adult duodenal tissue.

Cross section through a 15“day embryonic duodenal
graft on the 18th day of incubation*
A.
B.
C*
D.
E.
N.

Epithelium;
Goblet cell;
Lamina propria;
Tunica muscularis;
Blood vessel;
Chorioallantoic membrane.

Micrometer scale insert: 1 space = 0.01 mm.
All sections were stained with Himes and Moriber
triple stain.

85

PLATE V
Pig. 8.

Photograph, and. diagram of an Ouchterlony plate.
An, Anti-adult chicken duodenum;
12-Duo,
Antigenof 12-day embryonic duodenum;
13-Duo,
Antigenof 13-day embryonic duodenum;
14-Duo.
Antigenof 14-day embryonic duodenum.

I

13-DUO

14 -DUO

AN

©

87

PLATE VI
Fig* 9*

Photograph. and diagram of an Ouchterlony plate*
An. Anti-adult chicken duodenum;
15-Duo.
Antigenof 15-day embryonic duodenum;
16-Duo*
Antigenof 16-day embryonic duodenum;
17-Duo*
Antigenof 17-day embryonic duodenum*

89

PLATE VII
Fig. 10.

Photograph and diagram of an Ouchterlony plate.
An. Anti-adult chicken duodenum;
18-Duo.
Antigenof 1 8 -d a y embryonic duodenum;
19-Duo.
Antigenof 19-day embryonic duodenum;
20-Duo.
Antigenof 20-day embryonic duodenum.

19-DUO

2 0 -D U O

18-DUO

AN

(5)

9 1

PLATE VIII
Pig* 11.

Photograph and diagram of an Ouchterlony-B^orklund
inhibition plate*
An.

Six
gen
15-Duo.
21-Duo*
18-Duo*

doses of 18-day embryonic duodenum anti­
prior to anti-adult duodenum;
Antigen of 15-day embryonic duodenum;
Antigen of 21-day embryonic duodenum;
Antigen of 18-day embryonic duodenum.

1 8 -D U O
15-DUO

AN

93

PLATE IX
Pig.

12.

Photograph and diagram of an Ouchterlony-Bjorklund
inhibition plate.
An.

Four doses of adult chicken serum prior to
anti-adult duodenum;
20-Duo. Antigen of 20-day embryonic duodenum;
21-Duo. Antigen of 21-day embryonic duodenum;
Serum. Blood serum of adult chickens.

SERUM

95
PLATE X
Pig* 13.

Photograph and diagram of an Ouchterlony plate*

An. Anti-adult duodenum;
17-Duo. Antigen of control 17-day embryonic duo­
denum;
Duo-Duo• Antigen of 17-day embryonic duodenum
following CA grafts of adult duodenum;
Spleen Duo. Antigen of 17-day embryonic duodenum
following GA grafts of adult spleen.

DUODUO

17DUO

SPLEEN

DUO

AN

©

97

PLATE XI
Pig* 14.

Photograph and diagram of an Ouchterlony plate.
An. Anti-adult chicken duodenum;
17-Duo. Antigen of control 17-day embryonic duo­
denum;
Duo-Duo. Antigen of 17-day embryonic duodenum
following CA grafts of adult duodenum;
Adult Duo. Antigen of pooled adult duodena.

DUO
DUO

DUO

AN

99

PLATE XII
Fig, 15.

Photograph and diagram of an Ouchterlony plate.
An. Anti-adult chicken spleen;
Luo-Spleen. Antigen of 17-day embryonic spleen
following CA grafts of adult duodenum;
Spleen Spleen. Antigen of 17-day embryonic spleen
following CA grafts of adult spleen;
17-Lay Spleen. Antigen of control 17-day em­
bryonic spleen.

SPLEEN
SPLEEN

17- DAY
SPLEEN

DUOSPLEEN

AN