The Emergence of
Experimental Embryology
   in the United States
.:*£&■■'
U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • National Institutes of Health 
This booklet was prepared in conjunction with an exhibit of the same title at the
   National  Library of Medicine, Bethesda, Maryland, June 1 - August 31, 1990.
Cover Illustration: Chicken embryo, from M. Malpighi,
Dissertatio epistolica de formatione pulli in ovo
(London: Joannem Martyn, 1673). 
     The  Emergence  of
Experimental Embryology
    in the United States
by S. Robert Hilfer, Ph.D.
National Library of Medicine
  Bethesda, Maryland
     1990
ITS. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • National Institutes of Health 
 This booklet is dedicated to the memory of
Professor Oscar Schotte of Amherst College.
Single copies of this publication may be obtained without
charge by writing:
                 Chief, History of Medicine Division
                 National Library of Medicine
                 8600 Rockville Pike
                 Bethesda, Maryland 20894 
BACKGROUND
F
           or many centuries embryological thought
           was dominated by  a  controversy com-
monly called the Preformation-Epigenesis argument.
In the fourth century B.C. Aristotle recognized that
development was progressive, so that organs formed
in sequence within the embryo and not all at once.
This is  the  first  statement of epigenesis. Aristotle
belonged to a Greek tradition that tried to explain
life by observation, believing that explanations for
phenomena that were not understood could be found
by studying nature rather than by invoking the super-
natural. Similar thoughts were expressed by the Greek
physician, Galen, in the second century A.D. Little
progress was made for the next 1000 years. From that
time through medieval times, the writings of earlier
workers were accepted as fact and there was little
attempt to correct inaccuracies.
  During the Middle Ages, a point of view arose that
all structures required for the production of a new
individual were preformed within the egg and that
development  required only their unfolding  and
growth in size. With the discovery that the egg and
sperm are the agents responsible for reproduction,
this argument for preformation took the form of the
egg or the sperm as containing the entire body of the
new organism. The proponents of this theory became
known  as ovists  or animiculists,  depending upon
whether they believed the egg or the sperm to con-
tain  the  intact  individual.  The preformationists
became convinced that each  new generation had to
exist preformed within the egg  or  sperm of the
preceding generation. The ridiculous nature of this
argument was pointed  out  in the 1720's by the
physicist Hartsoeker, when he calculated the size that
the fiftieth generation would have been within an egg.
This turned out to be a negative number. However,
his argument was not accepted by the preforma-
tionists for many years. Hartsoeker himself (1694, Fig.
1) had apparently accepted the preformationist view
earlier  in his career.
  With  the  emergence of  the  Renaissance, the
naturalist tradition became reestablished. The renewed
interest in  the examination of embryos provided
additional descriptions of development, for instance
by  DaVinci, Fabricius,  Harvey,  Spallanzani,  and
Malpighi (1673, Fig. 2). Most of these observations
were interpreted as supporting preformation and it
took the discoveries of cellular structure generated
by development of the microscope for real progress
to  be  made.  The  careful  observations   on the
progressive  nature of organ  formation by Wolff in
the  second half of the eighteenth  century  were
instrumental  in ending  rigid  preformationist
viewpoints.
      Figure 1. Diagram by N. Hartsoeker (1694) of how he
  imagined a sperm would look if it contained a preformed
   individual. Hartsoeker later rejected the preformationist
view, carrying out calculations of size that showed that if all
of the animals of any species had been enclosed in the first
  male or female, those animals that now inhabit the earth
  would have to be infinitely and incomprehensively small.
1 
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 Figure 2. Illustrations from M. Malpighi (1673) of developing -chicken embryos from the time of laying
to 36 hours of incubation. This plate is representative of the keen observations done on early embryos
                                                               during the 17th century.
2 
         BEGINNINGS OF
EXPERIMENTAL EMBRYOLOGY
            The early nineteenth century was marked   that the embryo developed from cell sheets. About
            by great progress in the development of   the mid-19th century, Darwin's writings on evolution
techniques to study cells and tissues as well as by the   began to appear and the  concept that developing
development of concepts important to embryological   embryos went through stages that recapitulated their
thought. The importance  of the egg for embryonic   ancestors was given an evolutionary interpretation.
development was established by von Baer (1827, Fig.   An early proponent of recapitulation was the Swiss
3), the cell theory was advanced, and it was recognized   naturalist, Louis Agassiz, who believed (Agassiz, 1859,
Figure 3. Plate from C.E. von Baer (1827) to illustrate his discovery of the human ovum. He was the first to show that the
ovum lies within the follicular structure described by de Graaf, which generally was recognized as too large to be an egg.
3 
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   Figure 4. Surface views of frog embryos from W. Roux (1888). His Figures 5 and 6 show normal embryos at early and
 late stages of formation of the neural tube. His Figures 7 to 10 show embryos in which one blastomere was killed at the
    two-cell stage. In Figure 7, the ectoderm has covered much of the surface of the dead blastomere whereas much less
repair has occurred in the other cases and it is clear that only partial embryos have formed. He interpreted these results
   as proving that development is mosaic in character, each blastomere giving rise to a restricted part of the individual.
4 
p. 174) that "the phases of development of all living
organisms corresponds to the order of succession of
their extinct representatives in past geological time."
 In the next  few decades, Haeckel popularized  the
concept and introduced several other embryological
ideas, such as the gastrea hypothesis and the theory
of germ layers. The century was also a period of
intensive  work  on  comparative   embryology  by
descriptive techniques.
  Wilhelm Roux,  the  German  embryologist, fre-
quently is given credit for founding experimental
embryology with his classical experiment published
in 1888 (Roux, 1888). Actually, experimental interven-
tion with development was tried sporadically at earlier
times, but it was not until the 1850's that experiments
were done with a clear purpose in  mind. Newport,
in  England,  began testing  the  effects of various
chemicals on fertilization of frog eggs and devised
techniques to keep the eggs in a particular position.
:/
His careful observations led to the demonstration that
the plane of the first cleavage as well as the point of
sperm entry into the egg usually mark the axis of the
embryo. Other lines of investigation in the latter half
of the century were concerned with the way embryos
inherited the characteristics of their species. Several
theories were advanced, which became the bases for
experimental work that led to modern concepts.
  Roux was a dynamic writer and speaker and his
forceful personality probably played a large part in
the reputation he received for originating the experi-
mental approach. Based upon theoretical arguments
of the time, he  used an experimental approach to
attack a fundamental problem of development. The
question Roux asked was whether the embryo con-
tains all of the information necessary for development
or if it  is influenced by the environment. He was
concerned with differentiation  of  parts of the
organism from the seemingly unspecialized egg. His
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        Figure 5. Illustrations from H. Driesch (1892) showing effects of
manipulating blastomeres of sea urchin eggs. His Figure 1 shows a normal
  16-cell stage (copied from Selenka). Figure 2 shows a half-embryo from
   an 8-cell stage and Figures 3 and 4 half-embryos from a 16-cell stage.
Figure 5 is a half-embryo at the blastula stage. These half-embryos developed
into complete individuals. Figures 7 through 9 show blastulae in the process
of dividing. These formed conjoined twins as shown in Figures 10 through 12.
  These results were interpreted as demonstrating regulative development.
                     (Courtesy of the Marine Biological Laboratory.)
5 
(1874) had earlier proposed that different regions of
the cytoplasm of a fertilized egg contain  different
materials needed to  make  specific  parts  of the
developing embryo. Roux tested this hypothesis by
killing one cell of a two-celled  frog embryo (Fig. 4).
He reasoned  that if different  regions  of the egg
formed different parts of the embryo, the organism
resulting from his manipulation should be defective,
whereas it should be normal  if controlled by the
environment.  The result  he obtained was a partial
embryo and gave rise to a point of view  that can be
considered a modified expression of preformationism.
   It was shown in subsequent experiments that Roux
misinterpreted his results. At the time, however, his
experiments renewed  interest  in  the  idea   that
formation of an embryo results from  mechanical
partitioning of a specialized egg. This modified view
of preformation  became known as determinate
development and Roux compared the structure of the
egg to a mosaic in which small regions  of the
cytoplasm constructed specific parts of the adult, just
as small parts of the mosaic construct a picture. In
contrast, experiments by Driesch (1892, 1902) on sea
urchin  embryos gave the opposite result  (Fig. 5).
Driesch  isolated blastomeres of two-cell stages by
shaking the embryos in sea water. Each individual
blastomere was able to develop into a complete  indi-
vidual, thus showing that  the embryo could regulate
to replace the missing parts that normally would have
been formed  by the  missing  cytoplasm.  If the
blastomeres did not separate  completely,  double
embryos were formed.  This developmental pattern
was  called  indeterminate  and corresponds  to
epigenetic development. Thus, the theoretical argu-
ment had shifted from preformation vs epigenesis to
mosaic vs regulative development. On the one hand,
the search for preformed individuals within egg or
sperm was replaced by attempts to recognize specialized
cytoplasmic domains within the fertilized  egg. On the
other  hand,  the epigenetic  approach gradually
became  shaped into  studies on the structure of
cytoplasm and the establishment of pattern by interac-
tions among cells of the embryo or by external forces
as development proceeds.
     Figure 6. Photograph of Louis Agassiz giving a lecture.
Agassiz was a dynamic speaker and attracted large audiences
    at popular lectures in the United States. He married an
  American and settled at Harvard University. He did much
to popularize biology and was responsible for training a number
   of embryologists in summer classes as well as at Harvard.
           (Courtesy of the Marine Biological Laboratory.)
6 
ORIGINS OF EXPERIMENTAL EMBRYOLOGY       THE ROLE OF MARINE STATIONS
          LN THE UNITED STATES                 IN EXPERIMENTAL EMBRYOLOGY
B
           y the mid 1800's, a naturalist tradition
           1 was well established in the United States.
Most  biologists were  teachers and there  was little
opportunity for graduate training. The first signifi-
cant American  training centers were established by
Agassiz, who had come to Harvard from Switzerland,
and by W. K. Brooks, who established the Darwinian
tradition at Johns Hopkins. Interest in the experimen-
tal approach was generated by contact with European
laboratories and writings. C. O. Whitman broke with
American  tradition  and  went  to Germany for
graduate study. He returned to assume a professor-
ship at the University of Chicago, bringing the new
interest in experimental embryology with him. Four
students of Brooks - E. G. Conklin, E. B. Wilson, T.
H. Morgan, and R. G. Harrison - also joined the ranks
of the new experimental biologists and made major
contributions to the establishment of experimental
embryology in  this country. All worked at times in
European laboratories; for instance, Conklin, Wilson
and Morgan were introduced to  marine organisms
at the recently established Naples  Zoological Station.
T
            he embryos of marine organisms became
            favored  material  for  embryological
research because they were easier to observe and
manipulate than amphibian or bird embryos. Euro-
pean marine stations were established as centers of
experimental research and many European workers
such as the Hertwigs, Boveri, Driesch, and Herbst used
their facilities. In the United States, the first marine
stations were established as teaching facilities. Agassiz
opened a small summer school for teachers on the
island of Penikese off Woods Hole, Massachusetts in
1873 (Fig. 6). Brooks at Johns Hopkins established the
Chesapeake Zoological Laboratory in Virginia in 1878
and one of Agassiz's students, Hyatt, founded a school
at Annisquam, north of Boston, in 1880.
  The Marine Biological Laboratory in Woods Hole,
Massachusetts grew out of the Annisquam school. It
was founded with a research mission as well as that
of teaching and consequently played a significant role
in the development of experimental embryology in
this country. Another of the students in Agassiz's first
summer class, Whitman, was appointed director and
the first building was constructed in 1888 for classes
that summer (Fig.  7).  The faculty were involved in
Figure 7. Photograph of a laboratory class at the Marine Biological Laboratory, circa 1900. Leonard W.
   Williams (standing, center) was the instructor. From its inception, courses at the Marine Biological
 Laboratory have played an important role in training teachers and scientists in modern experimental
            techniques, a role that continues today. (Courtesy of the Marine Biological Laboratory.)
7 
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Figure 8. Diagrams of early cleavage stages of the tunicate, Cynthia partita, from Conklin (1905). The four cells at
the bottom of the upper left figure are traced through succeeding cleavages into a series of blastomeres that give
 rise to  segmental muscles and other mesodermal derivatives. This is an example of the painstaking  care that was
                                        needed to trace regionalization of egg cytoplasm through development.
8 
CELL LINEAGE AND THE ATTACK ON
  HAECKEL'S GERM LAYER THEORY
research programs as well as in teaching and tended
to return year after year to do research. The teaching
and research missions overlapped and researchers
and students were exposed to the latest ideas in
embryological theory, as well as other subjects, in the
series of evening lectures that were held each summer.
The   speakers  ranged  from Whitman  discussing
preformation vs epigenesis, through  Wilson's and
Morgan's  arguments  on heredity, to lectures  on
geology, evolution, and  other branches of science.
Visitors came from Europe, including well establish-
ed embryologists as well as younger ones.
  Some embryologists, such as Harrison,  did not
participate in  research  on marine organisms; the
questions they formulated were better studied  on
vertebrates. They also found it disruptive to work on
material that was available during only a part of the
year and did not want to move their laboratories to
a summer station. This did not diminish their contri-
butions  to  experimental  embryology, as will  be
described below.
   Figure 9. Diagrams of cleavage stages of a polyclad worm
(A and C), a mollusc (D) and annelid worms (B and E) from
 Wilson (1892). Wilson was impressed with the similarity in
   the formation of mesoderm by these different groups of
        organisms. The shaded cells are the precursors of
    mesoderm. Wilson interpreted studies of cell lineage as
                   showing that development is mosaic.
            Haeckel's theory of the germ layers pro-
            posed that organization of the embryo
occurred only after the three germ layers of ectoderm,
mesoderm, and endoderm became established. This
represented an epigenetic position that was attack-
ed by the proponents of determinate development.
The first approach that was taken to discover if early
cell divisions could be related to developmental pat-
tern, was the tracing of cell lineages, which became
known as mapping cell fates. This procedure allowed
the descendants of early blastomeres to be traced to
the organs they formed during normal development.
Organisms were chosen for study that had distinctive
cleavage patterns or  pigments to provide natural
markers which could be followed during successive
cleavages.
  The Woods  Hole workers were active in this field.
An early study was done by Whitman (1878) in Europe
on the leech Clepsine (now Glossiphonia). He showed
that  the mesoderm, nervous system, and excretory
organs arise at distinct cleavages from pairs of cells
that he called teloblasts. This type of ordered develop-
ment was shown for the worm, Nereis, by Wilson
(1892), the mollusc, Crepidula, by Conklin (1897) and
the ascidian, Cynthia (now Styela), also by Conklin
(1905, Fig.  8). All of these organisms were similar in
that entire organ systems could be traced back to one
or a pair of blastomeres at an early developmental
stage. Furthermore, blastomeres having similar posi-
tions in different organisms were  responsible for
similar organs  (Fig. 9). Thus, development of different
organisms  showed homology  that was assumed to
relate to their evolutionary origins. These studies did
much  to  discredit  the  germ  layer  theory and
strengthened  the ideas that specific regions of the
zygote (fertilized egg) are set aside (or determined)
early in development to form specific organs.
   Fate mapping continues to be a useful technique
to the  present time. Techniques have changed to
match  the organism being studied. The  German
embryologist W. Vogt (1925) introduced the use of
vital  dyes  to  follow  cleavages and  movements of
amphibian blastomeres. Dyes were not appropriate
for the small cells of bird embryos and Spratt (1946)
introduced the technique of marking surfaces with
fine carbon particles. In more recent times bird and
mammalian fate maps have been prepared by using
new fluorescently labeled dyes that can be injected
into single cells, by transplanting groups of cells
labeled with radioactive precursors of DNA synthesis,
or by  making chimeras between  species having
distinctive nuclear structure. These studies have all
been important in tracing the movements of cells
during normal development.
9 
    Figure 10. Experiments by J. Loeb, as in this paper from 1893, gave additional support to the argument
  that sea urchin eggs do not contain specialized areas of cytoplasmic determinants. Treatment of fertilized
   eggs with hypotonic sea water resulted in the formation of cytoplasmic blebs (upper left) that remained
attached to the main embryo during cleavage (upper right) and resulted in doubled embryos at the blastula
          stage (lower left). These embryos developed into Siamese twins (lower right). The egg cytoplasm
                                   was able to form most of the parts of more than one individual.
     STUDIES ON EPIGENESIS -
THE PHYSICOCHEMICAL NATURE
           OF CYTOPLASM
             The alternate concept to that of deter-
             minate development was that blasto-
 meres become specialized as a result of the position
 they come to occupy in the embryo. This represents
 the  extreme regulative position  as  opposed  to
 mosaicism. Proponents of this viewpoint believed that
 answers to how blastomeres become specialized lie
 in the physicochemical characteristics of cytoplasm,
 which  appears  homogeneous  in   the  zygote.
 Experiments had been done to distort the organiza-
 tion of egg cytoplasm  as a way of testing for mosaic
 development. In England, Jenkinson (1909) subjected
 eggs to  pressure, while other investigators placed
 zygotes or early embryos in centrifugal fields. In spite
 of these insults,  the embryos  developed normally.
Jaques Loeb was a strong proponent of this viewpoint
 and as a result of his studies much was learned about
 the physical properties of cytoplasm. One of his early
 experiments was to cause rupture of the cell mem-
brane by placing fertilized  eggs  in  hypotonic sea
water. Blebs formed that either separated completely
from the cleaving cytoplasm or remained partially
attached. The result was duplication, either as com-
plete embryos or as Siamese twins or triplets (Fig. 10).
Loeb concluded that "the number of embryos which
come from one ovum is not determined by prefor-
mation of germ regions in the protoplasm, or nucleus,
but  by the geometric shape  of the  ovum and the
molecular condition of the protoplasm" (Loeb, 1893,
p. 58). Loeb's main contributions were on the physical
chemistry of proteins. His interest in the chemistry
of cytoplasm led him to work with fertilization and
as a result he discovered the  cause of artificial
parthenogenesis.
   Other proponents of a physicochemical interpreta-
tion of development included Matthews, Lillie, and
Just. All  three had serious disagreements with Loeb
and held to old interpretations of colloid chemistry 
Figure 11. Illustrations from E. B. Wilson (1904), showing the effect of removing the polar lobe in the mollusc, Dentalium.
         Figures 4 to  9 show normal early cleavages. Regions of cytoplasm have different densities and shadings; a special
      cytoplasmic region becomes segregated in the polar lobe  (h and 12). When the polar lobe was removed, major parts
             of the larval body were missing (Figures 32 to 37)  as compared with normal development  (Figures 29 to 31).
                                  These results suggested that the polar lobe contains special cytoplasmic determinants.
11 
 EXPERIMENTAL ANALYSIS OF
CYTOPLASMIC DETERMINANTS
while Loeb's  ideas  were continuously  changing.
Matthews  worked on  the effects of  salt  on  the
properties of cytoplasm, using some  embryological
material, but  in  the latter  part  of  his  career he
concentrated on nerve cells. Lillie was  a student of
Whitman, whose primary interest was  in fertilization;
he is best known for his fertilizin-antifertilizin theory
of fertilization. He served as the second director of
the Marine Biological Laboratory and was instrumen-
tal  in its expansion.
  Just was a student of Lillie's and also was greatly
influenced by Matthews. As a black biologist, he was
confronted by the prejudices of the time  and his work
was accomplished under the serious disadvantages of
not gaining an appointment at a  research oriented
university; thus he  had no graduate students, had
access to colleagues with similar interests only during
the summer,  and had an excessive  teaching load
during the rest of the year. He has been described
as a brilliant observer and a perfectionist. He became
the outspoken proponent of Lillie  and Matthews
against Loeb and was subjected to personal as well
as scientific attacks by Loeb because of his stand. Just's
major contribution  to the analysis of cytoplasm was
in establishing the role of the egg cortex during fertili-
zation. He also provided some of the early experimen-
tal support for Lillie's theory. The later work of Just
included analyses of all of the chemical  components
of the egg cell, including  nucleic  acids. In his later
unpublished writings he may have been  on the verge
of discovering the role of nucleic acids as the genetic
material, although   to the  end of  his  career he
remained  a  strong  proponent   of regulative
development.
   Investigations on  the properties of cytoplasm also
led to discoveries on cell motility and relationships
of  cells to  their substrate. Ross Harrison (1910)
originated procedures for growing cells in isolation
from the animal body, thus establishing the tech-
niques of tissue culture. His early work  was directed
at solving problems of nerve development. He showed
that nerve cells produce axons by outgrowth and not
by fusion of several cells. S. Ramon y Cajal in  Spain
discovered the growth cone and Paul Weiss showed
that nerve fibers become oriented by the substratum.
At the same time, Warren and Margaret Lewis were
successful in growing chick and mammalian cells in
culture and studied their migrations, while Holtfreter
isolated amphibian cells and studied their behavior.
       A position intermediate between indeter-
             minate and determinate development
was expressed by Wilhelm His (1874). He believed that
the cytoplasm of the egg contains germinal regions
that are responsible for forming organs as a result
of physiological interactions during the course  of
development. His believed that development was not
truly mosaic nor was it totally regulative. The fate
mapping  experiments of Whitman, Wilson,  and
Conklin supported His's argument. It appeared that
organ-forming regions could be related to specific
regions of cytoplasm in the zygote. Constituents  of
these cytological germ regions of His later became
known as cytoplasmic determinants.
  The  idea of cytoplasmic determinants was tested
by several experimental approaches. Separation  of
blastomeres of  Amphioxus by Wilson (1893), of a
ctenophore  by Driesch and Morgan (1895), and  of
tunicates  by Conklin (1905) suggested  that some
regulation could occur but that eventually during
development  defects  were   produced  when
blastomeres were deleted. Similarly, removal of polar
lobes from eggs of spirally  cleaving molluscs  and
worms  resulted in defective larvae (Fig. 11). Eventually
Wilson evolved an explanation which  stated  that
development starts as regulative and that different
organisms became  mosaic  at  different points
during embryogenesis.
  The  interest in and search for cytoplasmic deter-
minants continues to the present time. This field has
seen a merging of ideas from the other lines  of
embryological research. As advances were made  in
the newly emerging discipline of genetics, it became
clear that the composition of cytoplasm is controlled
by active genes and that all genes need not be active
in all cells. It also was established that genetic infor-
mation is stored in the egg during maturation and
is used during early embryogenesis. Identification of
stored proteins and messenger RNA that are differen-
tially distributed within the zygote's cytoplasm and
in emerging embryos gives promise of leading to an
understanding of developmental control mechanisms
at a genetic level. The recent molecular biological
approaches are confirming the theories proposed  by
His, Wilson and their contemporaries.
12 
     ESTABLISHMENT OF
DEVELOPMENTAL GENETICS
            The modified preformationist argument
            of the late 1800's implied that embryo-
genesis occurs during cleavage through distribution
of inherited information to specialized regions of the
body. His's argument was that inheritance occurs
through distribution of cytoplasmic factors. In con-
trast, August Weismann suggested that inheritance
occurs through the cell nucleus and that the develop-
ing organism is a mosaic of nuclei having specialized
potentials.  This was part of the Germ Plasm Theory,
which postulated that hereditary determinants were
carried only in the cell lineage giving rise to gametes.
Roux set out to test the hypothesis that nuclei become
specialized with his experiments in which blasomeres
of frog embryos were killed; he interpreted the results
as support for Weismann. However, it soon became
apparent that Roux's original experiment was flawed.
The presence of the killed blastomere influenced the
attached living one, whereas the first two blastomeres
when separated from each other both developed
normally (McClendon, 1910). Nevertheless, as in other
so-called regulative organisms, blastomeres of later
stages were not able to form complete embryos.
  In the early twentieth century, Spemann tested the
potential of nuclei at later developmental stages by
a clever procedure. He reasoned that if nuclei become
specialized during cleavage, they should not be able
to support complete development if introduced at a
later time into  uncleaved cytoplasm. He used a fine
hair to constrict frog eggs so that  nuclei could not
Figure 12. When lecturing, O.E. Schotte jokingly il
                      and an embryologist (
                               (Drawing b
enter part of the cytoplasm. After the fourth cleavage
when 16 nuclei were present, he released the constric-
tion and  a  nucleus  entered  the uncleaved zygote
cytoplasm. In spite of the delay in arrival of nuclei
until a stage when isolation experiments gave partial
embryos, these embryos were normal. It was not until
much later that it became possible to transplant nuclei
of older embryos into the cytoplasm of an uncleaved
egg  (Briggs and  King, 1952).  By this method it
was shown that nuclei from blastula through early
neurula stages are able to support development of
a complete embryo. More  recently, it  has been
demonstrated that nuclei from even mature tissues
when transplanted to an  egg can support normal
development of a  fertile adult organism. These
experiments have proved that specialization of nuclei
during development does not involve loss of genetic
information and support the alternate  hypotheses of
Weismann's contemporaries that inheritance occurs
through differential  nuclear activity rather than by
loss of genetic information.
  The rediscovery of Mendel's writings did much to
stimulate interest in  inheritance  in the early 1900's.
Wilson became interested in the  cytological basis of
inheritance  after  performing his experiments on
cytoplasmic determinants. He  came to  view  the
nucleus as  playing  a  crucial role in controlling
development and devoted much of his later career
to these problems. His book on "The Cell in Develop-
ment and Heredity"  (1925) is a classic which is  still
lustrated with this diagram the way a geneticist (left)
ight) viewed the cell. The nucleus is shown hatched.
' S. R. Hilfer, from memory of an embryology class.)
13 
worth reading because it is so modern in its approach.
  T.H. Morgan was the most influential scientist in
establishing genetics as a separate field of biology. In
doing so, he changed from a strong supporter of
totally regulative development to a proponent of
nuclear control of development. He also gave up at
least temporarily his interests in embryology to pur-
sue the  establishment of the genetic basis of in-
heritance.  He was  instrumental in causing a split
between embryology and genetics that lasted for many
years. In fact,  O.E. Schotte pictured the way an
embryologist and  geneticist looked upon the cell
during this time as either mostly cytoplasm or mostly
nucleus (Fig. 12).
  Morgan was concerned primarily with establishing
the gene as the basis of genetics.  He  did this by
examining mutants and established Drosophila as the
organism of choice for these studies. He was respon-
sible for describing many characteristics and showing
that they could be related to sequential arrays on
specific chromosomes. Nevertheless, Morgan retained
a concern for with the problem of how genetic control
might be exercised during development. In his Nobel
Prize address, delivered in 1934, Morgan (1935, p. 15)
remarked that "It  is conceivable that different bat-
teries of genes come into action one after the other,
as the embryo passes  through its stages  of develop-
ment. This sequence might be  assumed  to be an
automatic property of the chain of genes." In discuss-
ing the genetic  control of development, he stated
(Morgan, 1935, p.  16):
       We have also  come  to realize that the
     problem of development is not as  simple
     as I have so far assumed to be the case, for
     it depends, not only on independent cell
     differentiation of individual cells, but also
     on interactions between cells, both in the
     early  stages of development and on the
     action of hormones on the adult organ
     systems. At the end of the last century, when
     experimental  embryology greatly flourish-
     ed, some of the most thoughtful students
     of embryology laid emphasis on the impor-
     tance of the interaction of the parts on each
     other, in contrast to the theories of Roux
     and Weismann that attempted to explain
     development  as  a progressive series of
     events that: are  the  outcome  of  self-
     differentiating processes or, as we would say
     today, by the  sorting out of genes during
     the cleavage of the egg. At that time there
     was almost no experimental evidence as to
     the nature  of the postulated interaction of
     the  cells. The idea  was a generalization
     rather than an experimentally determined
     conclusion, and,  unfortunately, took  a
     metaphysical turn.
       Today this has changed, and owing mainly
     to  the  extensive experiments of the
     Spemann school of Germany, and to the
     brilliant results of Horstadius of Stockholm,
     we have positive  evidence of  the  far-
     reaching importance of interactions bet-
     ween  cells  of  different  regions of the
     developing  egg. This implies that original
     differences are already present, either in the
     undivided egg or in the early formed cells
     of different regions. From the point of view
     under consideration results of this kind are
     of interest  because they bring up  once
     more, in a slightly different form, the pro-
     blem as to whether the organizer acts first
     on the  protoplasm of  the neighboring
     region with which it comes in contact, and
     through the protoplasm of the cells on the
     genes; or whether the influence is more
     directly on the genes. In either case the pro-
     blem  under discussion remains exactly
     where it was before. The conception of an
     organizer has not as yet helped to solve the
     more fundamental relation between genes
     and differentiation, although  it certainly
     marks an important step forward in our
     understanding of embryonic development.
Answers to the questions raised above are becoming
available only today with  the advent of molecular
genetics  and a  renewed  merging of interest  in
developmental biology and genetics.
  Another line of research that contributed basic in-
formation for the eventual merging of embryology
and genetics was the physiological approach espoused
by Goldschmidt. Goldschmidt came to this country
to escape  Nazism and found it difficult to  gain
acceptance in the American scientific community.
Part of this came from the unpopular point of view
he took of attempting to unite embryology and
genetics  into  a  common field of developmental
genetics. He stimulated work on genetic analysis of
development through  use of mutants rather  than
through surgical manipulation. Embryology to him
was  subservient  to  genetics and simply a way  of
examining the time that mutations had their effect
on development. His point  of view  was  expressed
most forcefully in his book, "Physiological Genetics"
(1938). Goldschmidt's position that the genes in the
nucleus are the most important factor in development
gained support from Hammerling's experiments  on
the alga, Acetabularia. In the 1930's, Hammerling
showed that the structure of the spore-forming body
is controlled by  the nucleus. The cap-like structure
14 
 EMBRYONIC INDUCTION AND
CONTROL OF DIFFERENTIATION
differs in different species and upon transplanting
the nucleus of one species  into the cytoplasm of
another, the structure was changed to that of the
species from which the nucleus came. Goldschmidt's
work eventually  resulted  in renewed interest  by
geneticists in the embryo. Current work on pattern
forming genes is an outgrowth of a long line of studies
in developmental genetics, primarily on Drosophila,
but on mammals as well. The renewal of a combined
interest  in  genetics and  development, however,
depended upon the writings of other workers. One
of the most influential was Waddington in England,
who in  "Organizers and Genes" (1940)  played  an
important role in  the  process of  reuniting  the
two disciplines.
I
          n the early 1900's embryologists increasing-
         .ly turned away from the mosaic-regulative
argument and became more concerned with  the
problems of  differentiation of organs. One of the
prime movers in this new interest was the German,
Hans Spemann.  By using hairloops to constrict
embryos, he  showed that duplications of the head
region could be produced by pinching along the axis
of the egg. As a result, he questioned how different
organs arose  and what controlled the order with
which they formed. The first step was to discover what
part  of  the embryo controlled  duplications;
presumably this event occurred before gastrulation
was completed because duplications were not ob-
tained from older embryos. Furthermore, the dorsal
edge of the blastopore, where cells initially move from
the surface to the interior of the embryo during
gastrulation, seemed to be most important. Only con-
strictions that went through the dorsal "lip" caused
duplications.  Testing the role of the dorsal lip was
accomplished by transplanting this region  of the
embryo to different sites in  a host embryo. Hilde
Mangold (nee Proscholdt), a Ph. D. student to whom
he assigned this problem, discovered that the dorsal
lip of the blastopore, when placed into the future
abdominal region, produced duplications (Spemann &
Mangold, 1924). These always occurred as a duplication
Figure 13. The classical experiments of Spemann and H. Mangold (1924) demonstrated induction in
   amphibian embryos. A graft of the dorsal lip of the blastopore was made from an unpigmented
  species to a pigmented species. The graft was placed in the future ventral region of the body. At
the neurula stage, the normal neural plate is found on the dorsal surface (left) and a second neural
      plate, containing the graft on the ventral surface (center). The result was the formation of a
                         secondary embryonic axis which produced Siamese twinning (right).
15 
of the body axis (Fig. 13), and Spemann coined the
term "organizer" to describe the role and "induction"
to describe the action  of the dorsal lip. The extra
tissue resulted in the formation of a "secondary axis".
Even before  this publication Spemann, as well as
W. Lewis in  the United States, had demonstrated
interactions among different parts of the embryo as
necessary for normal development, the best known
being the requirement of the eye primordium for
development of a lens.
   Spemann had a number of talented students who
in the next few decades examined the properties of
what became known as embryonic induction. The
organizer was shown to correspond with the presump-
tive notochord region and to have a differential effect
at  different  levels along  the axis of the  embryo
(O. Mangold, 1933). Although cells could be induc-
ed to form new structures, these were limited to the
genetic information in the responding cells and not
to the inducing tissue. A prime example was the work
done on induction of mouth parts in amphibians
(Spemann & Schotte, 1932). Transplantation of ecto-
derm from the region that was fated to become belly
skin in  a frog to the mouth region of a salamander
produced  mouth parts but had  the  organization
belonging to structures of the frog mouth region. The
reverse experiment  produced  salamander  mouth
parts in the  frog mouth region.  Thus, the mouth
region  was able to induce  belly ectoderm to make
mouth structures, but they conformed to the genetic
information provided by the organism from which
the donor ectoderm came.
   Finally, it was shown by Holtfreter, another student
of Spemann's, that exogastrulation, in which the cells
move outside instead of into the embryo, results in
an absence  of  induction.  The  endodermal and
mesodermal mass formed some internal organs, but
there was  a failure of axial organs, including the
nervous system  and somites. Holtfreter  also was
instrumental in  providing a new method of testing
induction. He realized that transplants  to a host
embryo, or insertion into the blastocoel cavity as was
used in later Spemann experiments, did not isolate
the responding ectoderm from organismal influences.
As a result he searched for a way to test the respon-
ding tissue in isolation. This was  accomplished by
devising a  medium of physiological salt solution in
which isolated amphibian cell layers remained healthy
and responsive to inductive influences. He showed
that combinations such as ventral ectoderm and
chordamesoderm would form neural structures just
as in the embryo. This system also served to study
other sources of induction,  such as chemicals.
  Within a decade, the role of induction came into
question because of the discovery that various killed
tissues could cause induction of limited neural struc-
tures. The response to these findings took two forms.
Holtfreter stimulated a line of research that showed
these "evocations," as he called them, to result from
limited proteolysis of the responding cells. He  con-
cluded that potential in the cells was released, or as
Needham  and Waddington  in England proposed,
evoked by limited damage caused by the killed tissue
or chemical agents. This line of research has resulted
currently in a better understanding of the capabilities
for  self differentiation of groups of  cells  within
different regions of the developing embryo. The other
line of research concerned attempts to elucidate the
chemical nature  of the organizer, especially of the
neural "inducer"  after evocation was discovered.
  There was great interest in the chemical control of
development at the time. It had been demonstrated,
for instance, that different regions of the embryo  have
different respiratory rates. This was incorporated by
Child into a theory that development can be explain-
ed by gradients existing within different planes of the
embryo. For  the amphibian, he proposed that one
gradient exists from animal to vegetal pole and that
a second dominant gradient is established at the
dorsal lip at  the onset of gastrulation.  Attempts to
document gradients within the embryo as well as to
isolate active inducing substances led to confusing
results. The work was done in England, Sweden, and
Germany as well as by Barth and his coworkers in the
United States. It appeared that these experiments sup-
ported the notion that a variety of substances could
release the ability of competent ectoderm to undergo
only limited development. It  is only in the  last few
years that progress has again begun to be made in
understanding the way the  cells  of the dorsal lip
establish the  axis of the developing embryo.
16 
*, CD

y-
     Figure 14. Summary diagram of experiments by S. Horstadius (1928) on regulative development in
  sea urchins. These experiments, combining animal hemispheres, (closed semicircles) with macromeres
(large circles) and micromeres (small circles), showed that normal development results from a balance of
     animal and vegetal blastomeres. Too few macromeres and/or micromeres reduces the completeness
  of endodermal and mesodermal differentiation. Note also that skeleton (rods), normally formed from
  micromeres, can form in the absence of micromeres if the macromere population is sufficiently large.
  Another  line  of  investigation  that provided
evidence that cells of one part of an organism exert
an influence upon another part concerned the sea
urchin. This work was an  outgrowth  of the ex-
periments of Driesch, followed by the work  of the
American embryologists. It  became apparent that
even  sea  urchin  embryos  are  not  completely
regulative. It was recognized that the vegetal pole cells,
the micromeres formed at the fourth cleavage, are dif-
ferent from the rest of the blastomeres in other ways
besides their smaller size. In a series  of brilliant
experiments the Swedish embryologist,  Horstadius,
demonstrated that the micromeres play an important
role in  the control of morphogenesis. It had been
shown earlier that a variety of chemical treatments
produced two classes  of embryos, those in which the
formation of either dorsal or ventral structures was
inhibited. Horstadius  (1928) was able to separate and
combine layers of blastomeres into hybrid embryos.
He showed that either the central layers or the animal
and vegetal ends, when combined, could regulate to
produce a  normal  larva  (Fig.  14). Furthermore,
addition of micromeres to a layer of animal tier
blastomeres was enough to permit normal develop-
ment. As a result, Horstadius proposed that develop-
ment of the sea urchin is regulated  by gradients of
an inductor released by  the animal pole  and an
opposing inductor released by the vegetal pole. This
scheme was similar to that proposed for amphibian
induction and the parallel between the two organisms
was heightened by the ability of extra  micromeres
implanted  in the animal region to induce a second
embryonic axis consisting of a duplicated digestive
tract. As with amphibian embryos, the search for
chemical inducers has resulted in marginal success.
17 
TISSUE INTERACTIONS
AND DIFFERENTIATION
          Interest  in  tissue  interactions  during
          embryogenies were pursued along lines
other than studies on induction. One of the leaders
in this field was Ross Harrison, already mentioned
above,  who  occupies  as  prestigious  a  niche  in
American  scientific history as Spemann does  in
Germany.  As early as 1904  Harrison showed an
interest that was to occupy most of his later career.
He tested the role of nerves in muscle development
by removing the neural tube from an early embryo
and showed that limb development still was normal.
From 1920 on, Harrison devoted most of his efforts
to  explore the origin of axial organization and
symmetry  in the embryo. He was particularly con-
cerned with the establishment of symmetry in the limb
and used the method of transplantation to ask what
controls the establishment of the limb axes (Harrison,
1921, Fig.  15).
  Holtfreter also was interested in tissue interactions
other than induction. He combined different  layers
of the embryo  in his cultures and showed  that they
behaved in isolation the same way as they did  in the
embryo, endoderm taking an internal position when
combined with mesoderm and ectoderm becoming
external. The study of cell interactions as opposed
to tissue interactions became a reality when Holtfreter
discovered (as  often occurs in science, based upon
preliminary  experiments by others)  that lowered
calcium concentration in the culture medium resulted
in  the  cells of a tissue becoming  separated,  or
dissociated. He then was able to reassemble masses
of cells from different tissue origins and to test their
interactions. His discovery that the cells separated into
groups based upon their origin was a revolutionary
finding. It had  been anticipated in earlier studies by
H. V. Wilson and  Galtsoff on sponges, who showed
that dissociated  cells  from different  species will
segregate or "sort out" when they are intermixed.
Holtfreter proceeded to catalogue the positions that
cells took in different combinations, and concluded
that they conformed to the normal relationships in
the embryo (Townes & Holtfreter, 1955, Fig. 16). This
work led to current investigations on cell adhesion
molecules, cell communication through specialized
junctions,  and  interest in the role of  extracellular
matrix in morphogenetic movements.
  Holtfreter's work was done on amphibian embryos.
It took many more years for methods to be devised
that allowed the cells of birds and mammals to  be
separated  while  retaining  their  viability.  Two
important contributions were made by Moscona, who
showed that trypsin, and Zwilling, who showed that
a calcium chelating agent, would effect dissociation
in bird embryos. Moscona's experimental work has
contributed significantly to the understanding of cell
     Figure 15. Illustrations from a paper by R. G. Harrison
(1921) on the establishment of symmetry within the embryo.
  Limb primordia were transplanted in various orientations
  to the flank region or to the normal position of the limb.
 This series of illustrations shows an embryo in which a left
 limb bud was implanted into the same location after being
rotated 180°. Figures 49 & 50 are dorsal and lateral views at
    4 days, 51 & 52 at 7 days, 53 & 54 at 10 days, and 55 a
  dorsal view at 17 days after the operation. Duplication of
                           distal limb parts occurred.
18 
     IMMIGRATION AND
EMBRYOLOGICAL RESEARCH
interactions in a variety of organs, while Zwilling con-
centrated on interactions occurring among cells of
the limb.
  Finally, an area of cell interaction that has had a
tremendous  influence  on  current  embryological
research is of recent origin. Clifford Grobstein (1953)
demonstrated that the mesenchyme surrounding an
epithelial organ plays an important role in controlling
its shape. He used  trypsin to separate  the mesen-
chymal capsule from the epithelial surface of organ
primordia and combined mesenchyme from  one
organ with epithelium of another. He showed that the
initiation of shape changes in some organs  depends
upon a highly specific association with their own
mesenchyme and that the mesenchymal influence can
be  transmitted through a semiporous  filter. This
research  also contributed  to  the  current  active
field of investigations  on the roles of extracellular
matrix,  membrane receptors,  and  cytoplasmic
filaments in changes in cell shape that occur during
organ  formation.
            The scientific well  being of the United
            States has been enhanced by continued
immigration to this country. Agassiz came from
Switzerland to settle at  Harvard in 1847 and Loeb
came from Germany to  teach first at Bryn Mawr in
1891 and later at the University of Chicago and
University of California. Although some immigration
occurred during World War I, the greatest influx of
scientists occurred with  the rise of Nazism. Schotte,
who was stranded as a student in Switzerland at the
time of the Russian revolution,  left  Spemann's
laboratory to  be  a Silliman Fellow at Yale under
Harrison. In 1933 he obtained  a  teaching post at
Amherst College. Other embryologists who came from
Germany included D. Bodenstein, V. Hamburger, J.
Holtfreter, R. Goldschmidt, P. Weiss,  and Salome
Waelsch. Migrations also occurred from Asia.
  Figure 16. Illustration from Townes and Holtfreter (1955) demonstrating
sorting of dissociated cells from different embryonic layers in amphibians.
 Cells from different germ layers sorted into clumps and tended to take a
           position resembling that of normal embryonic  development.
19 
   DISSEMINATION OF INFORMATION
     - ESTABLISHMENT OF SOCIETIES
               AND JOURNALS

           The  expansion  of biological research at
           various institutions in the United States
beginning in  the 1880's resulted in desires to com-
municate orally and in writing. At this  time, the
American  Association  for  the  Advancement  of
Science (AAAS)  was the only national scientific society
and was considered too broad by the biologists. Those
interested in biology became served by a loose federa-
tion,  the American Society of  Naturalists, that
separated from  the AAAS. The Society of Naturalists
of the Eastern United States was founded in 1883 and
changed  its  name to  the American Society  of
Naturalists in 1886. The motivating force was pro-
vided by A. Hyatt, who played a role in the founding
of the Marine Biological Laboratory, and  S.F. Clark
of Williams  College, who  was among the  first to
receive a degree in the new Ph. D. program at Johns
Hopkins. The  society  served as a  source  for the
establishment of smaller  societies that tended to
develop along narrow disciplinary lines; those which
had some interest in embryology or were founded by
the new  experimental embryologists included the
Association of American Anatomists (later changed
to the American Association of Anatomists)  in 1888
and  the American  Morphological Society (later
changed  to the American Society of Zoologists) in
1890. The Association of American Anatomists was
founded as an offshoot of the American Medical
Association and at first included primarily teachers
of anatomy;  only around 1900  did  membership
become limited to trained investigators.  Whitman
provided the initiative for founding the  American
Morphological  Society. These  societies  were  not
limited  to  embryology and it  was not  for many
years that  a society  dedicated  to the  discipline
was established.
  The Society for the Study of Growth and Develop-
ment, now named the  Society  for  Developmental
Biology, was founded in 1940 after a symposium on
Growth and Development was held at Truro on Cape
Cod in August,  1939. It was organized by the editors
of Growth, a journal  founded  by the Lankenau
Hospital Research Institute's Marine Research Station
in Truro. The meeting generated enthusiasm for the
establishment of a continuing symposium series spon-
sored by  the newly organized society. The symposia
were designed to allow much time for discussion in
small, informal  groups in addition to the discussion
periods after  formal lectures. This format was main-
tained for  many years  and  the  program  was
deliberately diverse, with  speakers recruited from
fields other than embryology where the  work was
related to development and growth. Speakers at the
first symposium included Lewis, Schott, Glaser, and
Sinnott from the United  States and Waddington,
Needham, and Stern from overseas. The second sym-
posium was concerned  entirely with physical  and
chemical aspects of growth and development.  The
topics of the society continued to be at the forefront
of embryological knowledge. This, plus the inclusion
of speakers at the periphery of embryology and the
encouragement of informal  discussion, made the
meetings an exciting time. The  relatively small size
and informal atmosphere  also encouraged interac-
tions between students and established investigators,
adding to its value as a stimulator of research.  The
society celebrated its 50th anniversary in 1989.
  A number of journals were founded in the same
time  period. Whitman founded the Journal of
Morphology in  1887,  which became unofficially
affiliated  with  the  Morphological  Society.  The
American Journal of Anatomy (1901) and Anatomical
Record (1906), both started with private funds, served
the Anatomists. The Journal of Experimental Zoology
was founded in 1904 by Harrison at Yale, and the
Marine Biological Laboratory  launched  its  own
journal, the Biological Bulletin, in 1898. Prior to that,
summer lectures at the laboratory were published in
bound volumes, beginning  in  1893. All of these
journals  exist  today  and  publish papers  on
developmental biology.
20 
SOURCES OF RESEARCH FUNDING
           Funding  of research  by  government
           agencies is a relatively recent develop-
ment in American science. Europe has a much longer
tradition of institutes established and funded by the
State. In the 1800's most embryologists paid for their
own research materials and attendance at research
stations in  the summer. Those who  were lucky re-
ceived  support from their institutions. The first
substantial  grants were provided by private founda-
tions,  such  as the Rockefeller Foundation and the
Carnegie Foundation. The larger grants were made
to institutions but some small grants also were award-
ed to individuals.
  Support targeted for embryology through Federal
grants began in 1952, with the establishment of the
National Science Foundation (NSF). An average of
11 awards per year were made in the  first four years,
but the numbers climbed to average 100 in the period
from 1961 to 1964. During that same time period the
budget climbed from an  average of approximately
$98,200 in the first four years  to  an average of
approximately  $3,450,000 in  the  early  1960's.
Currently the budget for  Developmental Biology is
approximately $18,000,000 for all  programs with
approximately 60 new grants funded each year at a
cost of between $2.0 and 2.5 million. New grants
represent  approximately  one third  of the  total
number of research grants that are  supported.
  Funding  of grants specifically in  developmental
biology started at the National Institutes of Health
in 1966. In  that year, the National Institute of Child
Health and Human Development awarded approx-
imately $3,400,000 to approximately  150 research
grantees. A decade later,  246 research awards were
made for approximately $13,400,000 while 371 awards
were made in 1989 for $50,200,000. These  figures
represent new and continuing awards and are not
comparable to the NSF figures. Over the years, the
number of awards as well as the dollar amount of
individual  grants has increased dramatically. The
increase in  funding in more recent years reflects not
only the rate of inflation but also the increased costs
for the more sophisticated equipment that is used.
21 
 
                                         BIBLIOGRAPHY


General References

Goldschmidt, R.B. 1938 Physiological Genetics. McGraw-Hill, New York.

Horder, T.J., J.A. Witkowski, and C.C. Wylie, eds. 1986 A History of Embryology. Cambridge University Press,
       Cambridge. Contains papers by the editors and others on Harrison, Spemann, and Morgan as well
       as  other interesting topics.

Just, E.E. 1939 The Biology of the Cell Surface. Blakiston, Philadelphia.

Maienschein,J. 1989  100 Years Exploring Life, 1888-1988, The Marine Biological Laboratory at Woods Hole.
       Jones and Bartlett, Boston.

Moore, J.A.  1987 "Science As A Way Of Knowing. IV. Developmental Biology." Am. Zool. 27:415-573.

Rainger, R., K.R.  Benson, and J.Maienschein, eds. 1988 The American Development of Biology. University
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Spemann, H. 1938 Embryonic Development and Induction. Yale University Press, New Haven. Reprinted by
       Hafner, New  York,  1962.

Willier, B.F. and J. M. Oppenheimer 1974 Foundations of Experimental Embryology. Hafner, New York. Con-
       tains translated reprints of papers by Roux, Driesch, Child,  Spemann & Mangold, Holtfreter, and others.

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Baer, C.E. von 1827  De ovi mammalium et hominis genesi. L. Vossius, Lipsiae.

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Briggs, R.  & T.J. King 1952  "Transplantation of living nuclei from blastula cells into  enucleated frog's eggs."
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Child, CM.  1929 "The physiological gradients." Protoplasma 5:447-476.

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Conklin, E.G.  1905  "The  organization and cell lineage of the ascidian egg." J. Acad. Nat.  Sci. Phila.
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Driesch, H.  1892  "Entwicklungsmechanische Studien. I."  Zeit. Wissenschaft. Zool. 53:160-178.

Driesch, H. 1902 "Neue Erganzungen zur Entwickelungsphysiologie des Echinidenkeimes." Arch. Entwick. Organ.
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Driesch, H. & T.H. Morgan 1895 "Zur Analyse der ersten Entwicklungsstadien der Ctenophoreneies. I. Von
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Grobstein, C. 1953 "Epithelio-mesenchymal specificity in the morphogenesis of mouse sub-mandibular rudiments
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Harrison,  R.G. 1910 "The outgrowth of the nerve fiber as a mode of protoplasmic movement." J. Exp. Zool. 
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Hartsoeker, N. 1694 Essay de Dioptrique. Jean Anisson,  Paris.

His, W. 1874 Unscr Korperform und das Physiologische Problem ihrer Entstehung. Vogel Verlag, Leipzig.

Holtfreter, J. 1934 "Der Einfluss thermischer mechanischer und chemischer Eingriffe auf die Induzierfahigkeit
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Horstadius, S. 1928 "Uber die Determination des Keimes bei Echinodermen." Acta Zool. 9:1-191.

Jenkinson, J.W. 1909 Experimental Embryology. Clarendon, Oxford.

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