Download Some Problems and Principles of Development1 University of

AM. ZOOLOGIST, 6:9-19 (1966).
Some Problems and Principles of Development1
SPRATT, J R . 2
NELSON T .
University of Minnesota
SYNOPSIS. Certain supracellular aspects of embryonic differentiation, illustrated by
studies of young chick blastoderms, are discussed. Specifically, the following principles
and guidelines seem to be involved in early development of the chick: egg organization,
relative movement, differential growth, regulation, restriction of regulative capacities,
synonomy of regulation and growth, gradients and fields1, developmental centers, dominance, integration, environmental control, and induction.
It seems appropriate to begin this "Refresher Course on Recent Advances in the
Analysis of Differentiation" by presenting
an aspect of the problem which may receive
little or no direct consideration by the
other speakers. The aspect of differentiation I have in mind may be described as
the supracellular aspect. By this is meant
the geometrical patterns in which specialized cell types appear in the cell population
constituting a developing organism. The
basic mechanisms of cellular differentiation
are probably utilized by cells whether livins; under artificial conditions such as in
cell or tissue culture or as members of a
unified cell population. However, the
methods of control of differentiation in the
intact organism would not necessarily be
evident from studies restricted to the differentiation of individual cells under experimental and artificial conditions. The
apparently greater complexity of differentiation at the organismic level does not
preclude meaningful analysis of the nature
of the over-all control mechanisms of normal development.
The orderly arrangement, of specific cell
types in tissues, organs, and the whole organism is presumably accomplished under
the control of supracellular guidelines. In
the case of embryonic development it appears, at least as a working hypothesis, that
certain of these guidelines are inherited
in the form of the cyto-architecture of the
mature egg and that other guidelines are
progressively built up during development
l Supported by grants from the National Science
Foundation.
- Most of the studies cited below were undertaken with the able assistance of my former research
associates, Dr. Hermann Haas and Dr. H. Eyal.
(9)
(Spratt, 1964). The diagram in Fig. 1 is
an elementary attempt to present the general problem of embryonic development,
to indicate its nature, and to suggest, in a
general fashion, how the ordered progression from adult to egg to adult may be controlled or guided. The diagram also indicates that formation of the egg, as well as
its development into an adult, is under
control of guidelines, both internal and
external to the system.
What is the nature of these supracellular
guidelines which presumably direct the
course of embryonic development? By
means of a summary of recent studies of
early chick development I shall attempt to
present results and observations which suggest principles and guidelines involved
perhaps not only in chick but in other patterns of development. Most of these studies
have been done on blastoderms explanted
in vitro on culture media containing egg
extract (Spratt and Haas, 1960).
EGG ORGANIZATION
Many studies of embryonic development
—discussed and reviewed by Wilson (1925),
Child (1941), Bonner (1952) and Raven
(1954)—suggest that the symmetrical cytoarchitecture of the mature egg constitutes
the primary guideline for the order of
events that are to follow. The structure of
different eggs, described by embryologists
as the "organization of the egg," is manifest
in a variety of patterns ranging from a
graded and polarized distribution of egg
constituents to a bilaterally symmetrical
localization of cytoplasmically different regions. Of special interest is the fact that
there is a general but invariable corre-
10
NELSON T. SPRATT, JR.
Progressive, Selective
Use of Instructions
ADULT
ADULT
EGG
A-
/
\
INHERITED
(genetic) ^
v'
ACQUIRED
^(epigenetic)
GUIDELINES
nuclear, cytoplasmic, environmental
FEG. 1. The diagram illustrates how both inheiitcd and acquired guidelines presumably control
the transition from adult to egg to adult.
spondence between the pattern of egg organization and the pattern of supracellular
differentiation in the embryo and adult
derived from the egg in normal development. Under abnormal conditions (most
commonly and most easily studied and
observed by experimental embryologists)
this correspondence may be modified to
varying degrees. The latter fact does not,
of course, mean that the principle of correspondence (the directive role played by
the pattern of egg organization) is any the
less important or is incorrect.
Although studies of the organization of
the bird egg are scant and incomplete, these
studies do suggest that egg organization
constitutes an important guideline for the
pattern of supracellular differentiation
which begins with cleavage of the egg
(Harper, 1902; Bartelmez, 1912, 1918; Olsen, 1942; Clavert, 1960). The diagrams of
Fig. 2 indicate how the polar (dorso-ventral)
EGG ORGANIZATION
Ec
FIG. 2. Correspondence between the cyto-architectural pattern of an egg and the pattern ot supiacellular differentiation which appears during early
cleavage at the animal (top) pole of the egg. D =
dorsal, V = ventral, A = anterior, P =r posterior:
lie =: ectoderm, End = endoderm.
PRINCIPLES OF DEVELOPMENT
axis and the axis of bilateral symmetry
(embryonic axis) of the fertilized egg correspond with the pattern of early cellular
differentiation. Thus, during cleavage the
cytoplasmic pattern of the egg becomes parcelled out to the increasing number of cells
but in an orderly way such that the cell
population as a whole possesses the same
pattern as that present in the animal pole
region of the uncleaved egg. As a consequence, cells of the population contain different fractions of the egg cyto-architecture
(e.g., different quantities and/or kinds of
yolk and other ultrastructural components).
The two recognizably different cell types
(ectoderm and endoderm or epiblast and
hypoblast) present in the unincubated
chick blastoderm appear to have been
formed in this way. Although the upper
(outer) and lower (inner) groups of cells
differ initially because of their cytoplasmic
inheritance, this does not mean, as we shall
see below, that further specialization of the
cells is independent of influences external
to them.
RELATIVE MOVEMENT
A rather characteristic feature of the
early development of animal embryos is
the movement of one layer of cells relative
to another. Such movements, mostly translocations of sheets of cells (tissues), usually
described as morphogenetic movements, are
particularly evident during gastrulation.
The movement of layers of cells not only
brings together different types of cells for
heterotypic interactions but also results in
local increases in density of the cell population (number of cells per unit volume)
with all the consequences relating to microenvironmental influences on cellular differentiation.
Studies of unincubated chick blastoderms
(Spratt and Haas, 1960; 1961a, b; 1962;
1965) indicate that the primary morphogenetic movement is the translocation of
the lower and lower middle layers across
the underside of the relatively, immobile
upper layer of the blastoderm. For simplicity the term "lower layer" will be used
to describe the combined lower and middle
11
layers (Spratt and Haas, 1965). The lower
layer of cells moves anteriorly in a fountainlike pattern, as a coherent sheet from a
source (growth center) near the posterior
margin of the pellucid area (Spratt and
Haas, 1960, Fig. 15). This pattern of movement has been revealed by marking the
blastoderms with carbon, carmine, or vital
dyes. By experimental manipulation of the
movement pattern in the lower layer (diverting it, opposing at various angles the
axis of symmetry of two or more movement
patterns, partial splitting of the moving
sheet, initiation of a new movement pattern, mechanical blocking of all lower layer
movement, etc.) it has been shown that
there is a one-to-one correspondence between the presence, position, and number
of movement patterns and the presence,
position, and number of primitive streaks
and eventually embryo bodies formed by
the blastoderm (Spratt and Haas, op. cit.).
These experiments demonstrate that movement of the lower layer plays a decisive
role in polarizing and bilateralizing the
potentially radially symmetrical upper
layer. Since no embryo body develops
when formation and movement of the
lower layer are prevented, it appears that
organized cellular differentiation within
the upper layer depends upon interaction
of the upper layer cells with the moving
lower layer cells. It also seems probable
that the orderly pattern of arrangement of
specialized cell types in the upper layer is
controlled by the direction of movement
of the lower layer. No preformed, stable
pattern of bilateral symmetry can be detected in either the upper or lower layers;
formation of the lower layer can be experimentally initiated from any point in the
circumference of the marginal zone (germ
wall or junction zone of the pellucidopaque area). Formation of the pattern
of supracellular differentiation of the embryonic body axis is thus primarily an
epigenetic process. Movement of the lower
layer across the underside of the non-motile
upper layer results in a geometrically organized and time sequential pattern of lengths
of exposure of the upper to the lower cells.
As a hypothesis it has been suggested that
12
NELSON T. SPRATT, JR.
this polarized movement of the lower layer
is the mechanism for establishing bilateral
and patterned cellular differentiation in
the upper (and also lower) layers.
DIFFERENTIAL GROWTH
Evidence that differential growth (cellular multiplication) patterns exist in the
early chick blastoderm has been summarized in previous reports (Spratt and Haas,
1960; 1961b; 1965; and Spratt, 1963). This
evidence consists of several kinds of observations including that of differential
growth of isolated parts of the blastoderm
(Fig. 3), the pattern of incorporation of H 3
thymidine, differential rates of separation
of groups of cells marked with carbon or
carmine, and mitotic index patterns. Two
examples of the mitotic index pattern in
the uppermost layer of a prestreak and an
early streak blastoderm are illustrated in
Fig. 4. The percents shown denote the percent of cells in metaphase and anaphase in
various areas of the blastoderms. In general, the pattern of mitotic frequency agrees
with the other observations noted above
in indicating the presence of a center of
cellular proliferation in the posterior part
FIG. 3. Diagrams illustrating differential growth ot isolated parts of unincubated chick blastoderms and the pattern of cell population density (density of stippling).
PRINCIPLES OF DEVELOPMENT
13
FIG. 4. Mitotic index patterns in a prestreak (left) and early streak (right) blastoderm.
of the uppermost layer of the pellucid area
and the presence of a ring of proliferating
cells in the uppermost layer of the marginal
zone. The density of stippling in Fig. 3
denotes the geometry and gradient features
of the differential growth pattern of an
unincubated blastoderm. Presumably, the
pattern of cellular density in the blastoderm (also indicated by stippling in Fig. 3)
is a consequence of the growth pattern.
Of special interest in relation to guidelines for early development is the probability that the pattern of differential
growth is one of the earliest detectable
patterns of supracellular differentiation.
Differential growth seems to be basic to
many of the activities and properties exhibited by the early blastoderm: formation
of germ layers; morphogenetic movements,
formation of the primitive streak, regulation, dominance, etc.3
REGULATION
A well-known property of many k,inds of
embryos, particularly at early stages of development, is the capacity of a separated
part of the embryo to reconstitute a complete embryo, identical with the original
whole except in initial size. Few kinds of
embryos exhibit regulative capacities to a
greater degree than the chick (Spratt and
Haas, 1960) and duck (Lutz, 1949) blastoderms. Of particular interest in relation to
supracellular differentiation is the demonstration that the symmetrical patterns of
differential growth, density of cellular
population, and morphogenetic movement
are labile. Furthermore, only those separated parts of the chick blastoderm which
re-establish the supracellular patterns of
symmetry, growth, movement of lower
layer, etc., are capable of reconstituting the
whole blastoderm and embryonic body.
The necessity of these patterns as guide3 The mitotic index pattern in the upper layer
of the older, definitive streak and head-process
blastoderm is characterized in part by a particularly
high frequency of. cells in metaphase and anaphase
stages in and around the primitive streak. The
index in the anterior one-third of the streak ranges
from about ]4%-17%. Furthermore, within the
anterior third of the streak 43%-81% of the mitotic
spindles are oriented vertically to the surface of
the blastoderm. In all other areas of the blastoderm
at these stages the percent of vertically-oriented
spindles ranges from about 0%-7%. Obliquelyoriented spindles seem to be quite rare. The impression provided by these observations of the
orientation and distribution of the mitotic spindles
in the upper surface layer is that many cells are
being added to the middle germ layer of the streak
by the division of upper layer cells—a process
which presumably begins during early stages of
cleavage (cf. Fig. 2).
14
NELSON T. SPRATT, JR.
PROGRESSIVE RESTRICTION OF CAPACITY
YOUNG
6HRS.
WINTER
UNINCUBATED
12 HRS.
SUMMER
15 HRS.+
FIG. 5. Gradual restriction in the capacity to form
embryonic body. The positions oC the arrows denote
the positions of embryonic bodies formed in right,
left, anterior, or posterior halves of explanted
blastoderms. The head of an arrow denotes the
head of the embryo. The length of an arrow denotes the relative frequency of embryos formed at
a particular stage. The longer the arrow the greater
the frequency.
lines for all further development is thereby
indicated.
derms studied seems to be equally shared
by all regions of the blastoderm (cf. studies
by Lutz, 1949, and Lutz, el al., 1963, of duck
blastoderms). The capacity then becomes
restricted, but equally, to all parts in the
circumference of the marginal zone. With
further increase in developmental age, a
bilaterally symmetrical gradient in embryo
body-forming capacity is present in the
marginal zone. Finally, the capacity becomes restricted to increasingly more posterior parts of the marginal zone. Eventually, the only isolated region that can develop an embryonic body is the primitive
streak. It may be significant that the changing regional pattern of regulative capacity
coincides spatially and temporally with an
increase in steepness of the radial and bilaterally symmetrical gradient patterns in
cell density (cf. Fig. 3).
PROGRESSIVE RESTRICTION OF CAPACITIES
A progressive restrictidn in regulative
capacities seems to be a universal characteristic of embryonic development. In
other words, the labile pattern of differentiation characteristic of early stages of egg
development is gradually replaced by an
increasingly stable pattern. The regulative
capacities of separated areas of the chick
blastoderm become restricted in a temporally and geometrically orderly pattern
(Fig. 5). The results summarized in the
figure are based on numerous studies
(Spratt and Haas, I960, and Eyal and
Spratt, 1965). Embryo body-forming capacity in the youngest unincubated blasto-
PRINCIPLES OF DEVELOPMENT
15
GROWTH AND REGULATIVE CAPACITY
YTS
FIG. 6. Diagrams illustrating the relationship between growth and legulative capacity and the dependence of both on the nutritional composition o£
the culture medium. The arrows within the outlines of the blastoderms denote the positions oC the
embryonic bodies. The dashes along the sides of
the arrows denote the rows of somits. R = right
half, L = left half ol a single unincubated blastoderm. A = anterior half, P = postciior half of a
single unincubated blastoderm. Alb. Extr. = egg
albumen culture medium, Egg Extr. = whole egg
(yolk plus albumen) culture medium.
REGULATION AND GROWTH
half on the nutritionally poor egg albumen
medium, and only half embryos develop in
right and left halves on this medium. However, a posterior halt (greatest cell number
and population density of any half) explanted on an albumen medium may form
a small, complete embryonic body. By contrast, any half on the superior, growthpromoting, egg-extract medium may develop a complete embryonic body. Results
such as those illustrated in Fig. 6 seem to
warrant the conclusion that regulative potentiality is essentially synonymous with
growth or synthetic capacity.
Unpublished studies (Haas and Spratt,
1962, and Spratt and Haas, 1965) indicate
that the ability of an explanted part of an
unincubated blastoderm to form a complete, bilaterally symmetrical embryonic
body depends not only on the region of the
blastoderm present in the part, on its intrinsic growth potentiality, and on its cell
number and population density, but also
on the growth-promoting properties of the
culture medium. Fig. 6 illustrates in summary form how realization of the regulative capacity of isolated right and left, and
anterior and posterior, halves of the blastoderm depends upon the nutritional properties of the culture medium. For example,
no embryonic body forms in an anterior
GRADIENTS AND FIELDS
The pattern of early supracellular differentiation in the chick blastoderm, and in
16
NELSON T. SPRATT, JR.
many other embryonic systems, is of the
gradient and/or embryonic field type
(Spratt and Haas, I960, 1965). The term
gradient is used to denote the giadual and
quantitative differences between different
parts of the pattern of organization of the
early blastoderm. The term, field, is used
to denote the set of presumably graded
environmental conditions under which the
blastoderm develops. The observed gradient patterns of cell population density
and differential growth in the unincubated
blastoderm probably constitute primary but
labile guidelines for the control of supracellular differentiation. It seems notable
that the gradient pattern of embryo-forming capacity, exhibited by isolated parts
of the blastoderm, is congruent with the
cell population density and growth pattern.
In other words, the gradient nature of the
micro-environmental pattern of the early
blastoderm makes regulation possible since
any part of the environmental field pattern, within limits, has a pattern smaller
but otherwise identical with that of the
whole field. The unincubated blastoderm
is probably best described as a developmental field out of which one or more
embryonic body fields may arise, depending
upon local changes in cellular environments.
cells of the population—are their differentially greater mitotic activity, metabolic
activity, and cell population density. In
respect to the formation of a spatiallyordered (symmetrical) pattern of cellular
specialization, a developmental center possesses an autonomy not shared with groups
of cells outside the center.
DOMINANCE
Demonstration of the multiple embryoforming capacity of the unincubated blastoderm (Fig. 5) raises an important problem:
"What control mechanisms prevent more
than one embryonic body from forming in
a single whole blastoderm in the course
of normal development?" Many observations (Spratt and Haas 1960; 1961a, b;
1962) indicate that the answer to this question resides in the fact that one region of
the blastoderm (the developmental center)
acquires dominance over all other regions.
In the unincubated blastoderm the dominant region is the posterior marginal zone
and later the growth center and primitive
streak derived from this part of the marginal zone. It seems that the greater cell
production and cell population density of
the center give it its qualities of dominance.
It has been suggested that the sheet of
lower cells moving radially from the growth
center mechanically inhibits the tendencies
of the lower surface of the remainder of
DEVELOPMENTAL CENTERS
the marginal zone to move inward (cenA common, if not universal, feature of tripetally). Thus, the initiation of the
the pattern of supracellular differentiation primitive streak and embryonic body is inis its progressive formation from a center. hibited at all points of the marginal zone
The dorsal lip of the blastopore of an ring but one, namely the prospective
amphibian gastrula, the node of the avian embryo-forming point.
primitive streak, the shoot apex of a plant,
etc., are familiar examples of developmenINTEGRATION
tal centers (Spratt, 1955). More recent
studies indicate that germ layers, primitive
Studies of the regulative capacities of
streak, and formation of the embryonic unincubated chick blastoderms suggest
body proceed from a center of cellular ac- that the movement of the lower layer of
tivity (proliferation and movement) lo- cells over the underside of the upper layer
cated in the posterior half of the pellucid is a basic integrative mechanism. Only
area of the unincubated blastoderm (Spratt those isolated pieces of the blastoderm in
and Haas, I960; 1965). It is suggested that which the lower cells spread below the
the primary properties of the group of cells upper in a fountain-like pattern become
constituting a developmental center—prop- reconstituted as whole blastoderms capable
erties which distinguish them from other of forming a single embryonic body. Fur-
PRINCIPLES OF DEVELOPMENT
17
ENVIRONMENTAL CONTROL
FOLDED BLASTODERM
ANTERIOR
FIG. 7. Diagrams illustrating the transformation
of the anterior ectoderm of a folded unincubated
blastoderm into endoderm. a = anterior; p — posterior. Ec = prospective anterior ectoderm, E =r
thermore, unification of disarranged or
synthetic blastoderms (made by fusing parts
of different blastoderms, or by fusing two,
three, or four whole blastoderms) occurs
only when one of the two or more growth
centers present is dominant. The dominant
center is usually the older center with
greater cell population density or a whole
rather than a half center. The dominant
center initiates a pattern of lower cell movement which becomes the single pattern of
movement of lower cells across the underside of the pellucid area. When two or
more growth centers are evenly balanced
in respect to cell population density, or
when the area of the synthetic system is
more than three times that of a single
blastoderm, unification of the system by a
single center does not occur.
ENVIRONMENTAL CONTROL
In a highly regulative system such as the
chick blastoderm it is not surprising that
specialization of the individual cells is not
a consequence of their origin from a particular part of the egg cytoplasm but primarily the result of their response to the
particular micro-environment they happen
to occupy. In other words, the position of
a cell in the cell population controls its
behavior and eventually its specific fate.
ECTODERM
ENDODERM
endoderm. The arrow in the diagram on the right
denotes the position of the single embryo with
foregut inpocketing from the upper surface (originally prospective ectoderm).
Much of the history of experimental embryology attests to the truth of this principle. For example, the properties which
distinguish cells of the developmental center (growth and embryo-forming center) of
an unincubated chick blastoderm seem to
be solely a consequence of the position of
these cells. Thus, transplantation of the
cells constituting the developmental center
to the geometrical center of the blastoderm
results in the loss of the properties originally peculiar to the developmental center
(Spratt and Haas, i960). Other cells which
move into the position formerly occupied
by the developmental center acquire the
distinctive properties of that center. Other
examples of local environmental influence
on cellular differentiation include the
transformation of cells at and near the
cut edge of the pellucid area of an unincubated half-blastoderm into cells typical
of the opaque area. A further example is
the inside-outside (pellucid-opaque area)
pattern of cellular differentiation in fragments isolated exclusively from either the
pellucid or opaque areas of an unincubated
blastoderm.
A more recent demonstration of the influence of the cellular environment is illustrated by the diagrams of Fig. 7. When
an unincubated blastoderm is explanted
with the ectoderm down against the agar
NELSON1 T. SPRATT, JR.
18
surface of the culture medium and the anterior half of the blastoderm is folded over
the posterior, the prospective anterior ectoderm cells may differentiate in the endodermal direction. A single embryonic body
develops, with foregut inpocketing from the
upper (prospective anterior ectoderm) surface, (Eyal and Spratt, 1964). It should be
noted that contact of prospective ectodermal cells with a solid substratum (the
agar medium surface in vitro; the vitelline
membrane in ovo) presumably favors differentiations of the ectodermal type. Conversely, differentiation of endodermal type
seems to be favored by contact of this cell
layer with fluid (the fluid film covering
explants in vitro; the subgerminal fluid
in ovo).
In general, it appears that the pattern of
diverse, probably graded, micro-environments occupied by different cells of the
early blastoderm constitutes an important
guideline for the pattern of supracellular
differentiation—particularly in those developmental patterns which are primarily
regulative.
INDUCTION
Descriptive and analytical studies of embryonic induction are legion. The importance of interactions between cells as a
method of differentiation has been demonstrated repeatedly. Yet we know little
about the supracellular aspects of inductive processes, i.e., inductions contributing
to the establishment of the orderly pattern
of arrangement of cell types. In the brief
description above of the principle of relative movement it was suggested that the
progressive formation and movement of
the lower cell layer over the underside of
the upper layer constitutes a mechanism
for symmetrical and patterned induction
in the upper layer.
The results of recent studies of the development of folded blastoderms (Eyal and
Spratt, 1964) indicate that the movement
of the lower layer may simultaneously induce the formation of two embryonic axial
systems, one in the prospective posterior
half, the other in the prospective anterior
half of the blastoderm. Such a result is
obtained when the posterior half of an
early streak blastoderm, explanted ectoderm down, is folded over the anterior
half. The two embryonic bodies are more
or less congruent, i.e., corresponding structures along the head-tail axis lie one above
the other, and are in a belly-to-belly
position.
SUMMARY
The observations and experiments described above were undertaken in the belief
that some aspects of the problem of cellular differentiation can be analyzed even
though the detailed mechanism of differentiation at the cellular and subcellular
levels is unknown. Thus, studies of the
properties of a developmental system like
the chick blastoderm may reveal some of
the guidelines for the orderly patterns of
cellular specialization appearing during
development. By manipulating one or
more of the guidelines, a measure of control over the differentiation not only of
individual cells but also of organized populations of cells has been achieved. The
next step would be an analysis of the
mechanism of operation of the demonstrated guidelines (primarily, properties of
the cellular environment). This would
bring us close to the level of the individual cell.
REFERENCES
Barlelme*, G. W. 1912. The bilaterali'Ly of the
pigeons egg. J. Morphol. 23:269-328.
Bartelmez, G. W. 1918. The relation of the embryo
to the principal axis of symmetry in the bird's
egg. Biol. Bull. 35:319-361.
Bonner, J. T. 1952. Morphogenesis. Princeton
Univ. Press.
Child, G. M. 1941. Patterns and problems of development. Univ. Chicago Press.
Clavert, J. 1960. Determinisme de la symetrie bilaterale chez les oiseaux. IV. Existence d'une
phase critique pour la symetrisation de l'oeuf.
Arch. d'Anat. Micr. 49:345-361.
Eyal, H., and N. T. Spratt, Jr. 1964. Embryo formation in folded chick blastoderms. Amer. Zool.
4:428.
Eyal, H., and N. T. Spratt, Jr. 1965. Unpublished
studies.
Haas, H., and N. T. Spratt, Jr. 1962. Development
PRINCIPLES OF DEVELOPMENT
in vitro of the unincubated chick blastoderm on
synthetic culture media. Anat. Rec. 142:338-339.
Harper, E. H. 1902. The fertilization and early
development of the pigeon's egg. Diss. Univ.
Chicago.
Lutz, H. 1949. Sur la production experimentale
de la polyembryonie et de la monstruosite double
chez les oiseaux. Arch. d'Anat. Micr. 38:79-144.
Lutz, H., M. Departout, J. Hubert, and C. Pieau.
1963. Contribution a l'etude de la potentialite du
blastoderme non incube chez les oiseaux. Devel.
Biol. 6:23-44.
Olsen, M. W. 1942. Maturation, fertilization, and
early cleavage in the hen's egg. J. Morphol. 70:
513-534.
Raven, C. P. 1954. An outline of developmental
physiology. McGraw-Hill, New York.
Spratt, N. T., Jr. 1955. Studies on the organizer
center of the early chick embryo, p. 209-231. In
D. Rudnick (ed.), Aspects of synthesis and order
in growth. Princeton Univ. Press.
Spratt, N. T., Jr. 1963. Role of the substratum,
supracellular continuity, and differential growth
in morphogenetic cell movements. Devel. Biol.
7:51-63.
Spratt, N. T., Jr. 1964. Introduction to cell differentiation. Reinhold, New York.
Spratt, N. T., Jr., and H. Haas. 1960. Integrative
19
mechanisms in development of the early chick
blastoderm. I. Regulative potentiality of separated parts. J. Exptl. Zool. 145:97-137.
Spratt, N. T., Jr., and H. Haas. 1961a. Integrative
mechanisms in development of the early chick
blastoderm. II. Role of morphogenetic movements and regeneiative growth in synthetic and
topographically disarranged blastoderms. J.
Exptl. Zool. 147:57-94.
Spratt, N. T., Jr., and H. Haas. 1961b. Integrative
mechanisms in development of the early chick
blastoderm. III. Role of cell population size and
growth potentiality in synthetic systems larger
than normal. J. Exptl. Zool. 147:271-293.
Spratt, N. T., Jr., and H. Haas. 1962. Integrative
mechanisms in development of the early chick
blastoderm. IV. Synthetic systems composed of
parts of different developmental age. Synchronization of developmental rates. ]. Exptl. Zool.
149:75-102.
Spratt, N. T., Jr., and H. Haas. 1965. Germ layer
formation and the role of the primitive streak
in the chick. 1. Basic architecture and morphogenetic tissue movements. J. Exptl. Zool. 158:
9-38.
Wilson, E. B. 1925. The cell in development and
heredity. Macmillan, New York.