Table Of ContentCELL BIOLOGY: A Series of Monographs
EDITORS
D. E. BUETOW I. L. CAMERON
Department of Physiology Department of Anatomy
and Bio])hysics University of Texas
University of Illinois Medical School at San Antonio
Urbana, Illinois Sail Antonio, Texas
G. M. PADILLA
Department of Physiology and Pharmacology
Duke University Medical Center
Durham, North Carolina
G. M. Padilla, G. L. Whitson, and I. L. Cameron (editors). THE CELL CYCLE:
Gene-Enzyme Interactions, 1969
A. M. Zimmerman (editor). HIGH PRESSURE EFFECTS ON CELLULAR
PROCESSES, 1970
I. L. Cameron and J. D. Thrasher (editors). CELLULAR AND MOLECULAR
RENEWAL IN THE MAMMALIAN BODY, 1971
I. L. Cameron, G. M. Padilla, and A. M. Zimmerman (editors). DEVELOPMENTAL
ASPECTS OF THE CELL CYCLE, 1971
P. F. Smith. THE BIOLOGY OF MYCOPLASMAS, 1971
Gary L. Whitson (editor). CONCEPTS IN RADIATION CELL BIOLOGY, 1972
Donald L. Hill. THE BIOCHEMISTRY AND PHYSIOLOGY OF TETRA-
HYMENA, 1972
Kwang W. Jeon (editor). THE BIOLOGY OF AMOEBA, 1973
Dean F. Martin and George M. Padilla (editors). MARINE PHARMACOGNOSY:
Action of Marine Biotoxins at the Cellular Level, 1973
Joseph A. Erwin (editor). LIPIDS AND BIOMEMBRANES OF EUKARYOTIC
MICROORGANISMS, 1973
A. M. Zimmerman, G. M. Padilla, and I. L. Cameron (editors). DRUGS AND THE
CELL CYCLE, 1973
Stuart Coward (editor). DEVELOPMENTAL REGULATION: Aspects of Cell
Differentiation, 1973
In preparation
Govindjee (editor). BIOENERGETICS OF PHOTOSYNTHESIS
DEVELOPMENTAL
REGULATION
Aspects of
Cell Differentiation
Edited by STUART J. COWARD
Department of Zoology
University of Georgia
Athens, Georgia
c*
1973
ACADEMIC PRESS New York and London
A Subsidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT © 1973, BY ACADEMIC PRESS, INC.
ALL RIGHTS RESERVED.
NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR
TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC
OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY
INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT
PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC.
Ill Fifth Avenue, New York, New York 10003
United Kingdom Edition published by
ACADEMIC PRESS, INC. (LONDON) LTD.
24/28 Oval Road, London NW1
LIBRARY OF CONGRESS CATALOG CARD NUMBER: 71-187254
PRINTED IN THE UNITED STATES OF AMERICA
List of Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
W. SUE BADMAN, Society for Developmental Biology, Kalamazoo, Michi-
gan (85)
STUART BRODY, Department of Biology, University of California at San
Diego, La Jolla, California (107)
LEON S. DURE, III, Department of Biochemistry, University of Georgia,
Athens, Georgia (23)
JAMES H. GREGG, Department of Zoology, University of Florida, Gaines-
ville, Florida (85)
TOM HUMPHREYS, Kewalo Marine Laboratory, Pacific Biomedical Re-
search Center, University of Hawaii, Honolulu, Hawaii (1)
JOE L. KEY, Department of Botany, University of Georgia, Athens,
Georgia (49)
GARY KOCHERT, Department of Botany, University of Georgia, Athens,
Georgia (155)
THOMAS L. LENTZ, Department of Anatomy, Yale University School of
Medicine, New Haven, Connecticut (169)
FRANCIS J. MANASEK, Departments of Anatomy and Pediatrics, Harvard
Medical School, Boston, Massachusetts, and Departments of Cardiology
and Pathology, The Children's Hospital Medical Center, Boston, Massa-
chusetts (193)
ROBERT SEARLS, Department of Biology, Temple University, Philadelphia,
Pennsylvania (219)
LARRY N. VANDERHOEF, Department of Botany, University of Illinois,
Urbana, Illinois (49)
ix
Preface
In a field of such wide scope as developmental biology, it is impossible
to include or to do justice to each and every important facet in any one
book. I have attempted to draw together in this work some studies which
are directed toward the orderly changes in cell phenotypes which we
understand to be developmental in nature. The exciting, fundamental
advances in genetics, cell biology, and molecular biology occurring over
the last decade not only have reshaped the investigator's thinking and
reinforced his armamentarium, but have brought developmental studies
to a critical threshold point toward the elucidation of the basic mech-
anisms of differentiation. These changes, I believe, are reflected in the
nine chapters of this volume. The linear ordering of the chapters ob-
viously should not be regarded as describing the principal logical inter-
actions. This would require arrangement within a spheroid; it is for the
reader to make that juxtaposition.
STUART J. COWARD
xi
1
RNA and Protein Synthesis during
Early Animal Embryogenesis
TOM HUMPHREYS
I. Introduction 1
II. Synthesis of RNA 2
A. RNA Synthesis before Fertilization 2
B. RNA Synthesis during Development 4
C. Summary of RNA Synthesis 12
III. Protein Synthesis 13
A. Acceleration of Protein Synthesis at Fertilization 13
B. Proteins during Early Development 15
C. Summary 19
References 20
I. Introduction
Embryogenesis is an orderly progression of the organism from a rela-
tively simple fertilized egg to a much more complex functional individ-
ual. The rather similar cells resulting from cleavage must differentiate
into a host of different cell types organized into diverse tissues and
specialized for a variety of functions. Modern analysis of cellular bio-
chemistry and molecular biology has established a scheme of molecular
function an$ interdependency which leads to the general assertion that
the central event in this morphological diversification is the orderly syn-
thesis of appropriate enzymatic, structural, and regulatory proteins. It
is the proteins, with their extensive molecular diversity, which perform
most cellular functions. RNA molecules copied from the DNA encoding
the protein's amino acid sequence direct the assembly and synthesis
of the protein molecules. The activities of the DNA and RNA which
specify the structure of the proteins appear to be regulated by protein
molecules. The other molecules of cells, such as polysaccharides and
lipids, are themselves synthesized and usually organized by protein mole-
1
2 TOM HUMPHREYS
cules. Thus, the sequence of synthesis of the many specific RNA and
protein molecules required for development and the mechanisms regulat-
ing this sequence are at the crux of understanding development. Current
studies are actively pursuing this basic question. The sequence of syn-
thesis of some major classes of molecules has been worked out, and
in a few cases the sequence for defined molecules has been described.
Aspirations to penetrate beyond this gross description to reveal the
molecular regulatory mechanisms which may control synthesis have not
yet been fulfilled.
In this review I will attempt to provide a concise overview of the
studies on molecular synthesis during early development. Since an exten-
sive treatise has recently been published covering this topic (Davidson,
1969), I will not attempt to deal with the complete literature, but will
emphasize areas in which I believe new insights have been gained and
fresh viewpoints need to be expressed. I will also try to point beyond
today's results to important questions which are within the reach of
current technical capabilities. I will emphasize sea urchin embryos be-
cause they are probably the most studied and because I work on them
and know them best.
II. Synthesis of RNA
A. RNA SYNTHESIS BEFORE FERTILIZATION
RNA synthesis in the mature oocyte, or egg, before fertilization has
not been elucidated. It has been said that RNA synthesis in the unfer-
tilized egg is at a reduced, basal level (for example, Davidson, 1969).
The actual results available are fragmentary and divergent from different
organisms, probably because the storage of fully grown oocytes is
achieved differently in various species. This belief is most strongly sup-
ported by the apparent constant amount of ribosomal RNA with no
turnover of rRNA (Davidson et al., 1964) during storage of oocytes
in amphibians. However, rRNA synthesis does occur in some oocytes
including amphibian (Gould, 1969a; Smith and Ecker, 1970), but its
rate has not been measured.
In sea urchins, where the egg is stored after the meiotic divisions,
the egg is impermeable to radioactive RNA precursors (Piatigorsky and
Whiteley, 1965). I have attempted to set an upper limit for RNA synthe-
sis in sea urchin eggs by measuring the specific activity of the precursor
pool during labeling experiments as we have during embryogenesis
(Emerson and Humphreys, 1971; Brandhorst and Humphreys, 1971).
1. UNA AND PROTEIN SYNTHESIS IN EMBRYOGENESIS 3
Very high concentrations of radioactive precursor (50 μθ'ι [3H]adeno-
sine/ml) were used in these experiments without achieving significant
radioactivity in RNA. Sometimes, small amounts of radioactive ribo-
somal RNA were obtained but this may have been due to immature
oocytes even though all preparations used contained more than 99.5%
normal eggs. When the specific activities of the ATP pools were measured
during these incubations, they were so low that RNA synthesis at the
rate observed in eggs just fertilized would have gone undetected. The rate
and kinds of RNA synthesized in unfertilized eggs could be identical
to those of the eggs just fertilized. Since, as I will show below, RNA
synthesis on a per nucleus basis is very active immediately after fertiliza-
tion, failure to label RNA is no support for the idea that synthesis
is reduced or at a basal level.
In the mature amphibian oocyte, heterogenous RNA synthesis ap-
pears to be rather rapid. Brown and Littna (1966) showed that a very
large amount, considering the egg has one nucleus, of heterogenous RNA
was radioactive in Xenopus eggs being shed by females injected with
32P. The amount of RNA was constant through cleavage, and they be-
lieved that it was synthesized during ovulation and persisted. Recently
Smith and Ecker (1970) have shown that RNA can be labeled at all
stages of egg maturation. This finding suggests that the radioactive RNA
observed by Brown and Littna is a steady state level of unstable RNA
which is turning over and continually resynthesized and can become
radioactive whenever precursor is added. Since the amount of this RNA,
10"9 gm per egg, is above two orders of magnitude more than is accumu-
lated by a single nucleus in most animal cells (for example, see Brand-
horst and Humphreys, 1971), I suggest that it might be mitochondrial
RNA synthesized on the very large excess of mitochondrial DNA in
the amphibian egg.
The activity of the ribosomal RNA genes is thought to be reduced
in mature oocytes and eggs (Davidson, 196|8|) but again quantitative
data is lacking. Although an absolute rate has not been measured, Smith
and Ecker (1970) show labeled nucleoli in mature oocytes suggesting
some rRNA synthesis. Calculations I have done suggest that the large
amount of heterogenous RNA synthesized in mature oocytes could mask
the activity of the large number of amplified ribosomal genes present
at this stage even if they were transcribed at the rate observed in gas-
trulae (Brown and Littna, 1966). The full implications of not shutting
down the ribosomal genes at the end of oogenesis are not clear to me,
but in many amphibians such as Rana pipiens, it would make little
difference since synthesis would stop as the temperature dropped in
the fall after the eggs are fully grown. The same argument does not
4 TOM HUMPHREYS
hold for Xenopus, but this problem has not been examined in enough
detail to answer the question either way.
The best documented case of rRNA synthesis in full-grown oocytes
is in Urechis capo (Gould, 1969a). All stages of these eggs are permeable
and ribosomal RNA synthesis is easily detected. The data do not permit
an estimate of the actual rates of synthesis, and the synthesis observed
may be a residual, repressed level. It also might as easily be equal
to synthesis in the young, growing oocytes. The evidence suggests that
the ribosomal RNA synthesized in the mature oocytes of Urechis is
not fully processed and does not enter ribosomes. It, therefore, might
not contribute to the store of egg ribosomes. It would seem most logical
that RNA synthesis, similar to some other metabolism including protein
synthesis, is reduced in the storage oocyte or egg and is reactivated
at ovulation, fertilization, or later. However, sound evidence to justify
this conclusion is lacking and the details of the regulation of RNA syn-
thesis at the end of oogenesis and at fertilization are yet to be worked
out.
B. RNA SYNTHESIS DURING DEVELOPMENT
Many early embryos exhibit unique patterns of RNA synthesis which
are not observed in other kinds of cells. With development, the patterns
pass through a series of changes and finally revert to the pattern typical
of most animal cells with a rapid synthesis of large nuclear RNA
and a predominate accumulation of ribosomal and transfer RNA. These
changes have been described best in sea urchin embryos (Emerson and
Humphreys, 1970). Right after fertilization the RNA labeled during
an incubation with radioactive RNA precursors is small heterogenous
RNA which sediments from 5 to 25 S. Toward the end of exponential
cell division the predominate RNA becomes larger and ranges up to
50 S or more. At about gastrulation, when the rapid cell divisions of
early development are complete, ribosomal and transfer RNA become
the predominate RNA's accumulating. Although the data are fragmen-
tary, this pattern appears to occur in amphibians, teleosts, Urechis, in-
sects, ascidians, and snails (Brown and Littna, 1964; Belitsina et ah,
1964; Gould, 1969b; Harris and Forrest, 1967; Markert and Cowden,
1965; Collier, 1966).
These changing patterns have been recognized for a decade but have
only recently been understood. At present we may determine four sepa-
rate stages of RNA synthesis. The initial period of small heterogenous
RNA consists of two separate phases with different kinds of RNA pre-
dominating. In the first phase most of the RNA is synthesized from
1. RNA AND PROTEIN SYNTHESIS IN EMBRYOGENESIS 5
the mitochondrial DNA, which, because of the large cytoplasm to nucleus
ratio, is the most abundant DNA in many eggs (Dawid, 1966; Piko
et al. 1967). As nuclei increase in number during cleavage, their RNA
r
synthesis predominates. However, during cleavage they appear to syn-
thesize mostly small molecular weight heterogenous RNA (Emerson and
Humphreys, 1970). Presumably this RNA codes for rapid nuclear protein
synthesis which occurs during cleavage (Nemer and Lindsay, 1969;
Kedes and Gross, 1969). As cleavage slows, the more typical large mo-
lecular weight RNA characteristic of nuclear RNA begins to predominate
(Emerson and Humphreys, 1970). Accumulation of detectable, newly
synthesized ribosomal RNA begins at about gastrulation when the syn-
thesis of heterogenous RNA declines. In the following sections, I will
summarize the data which establish the existence of these four periods
of RNA synthesis.
Mouse and Ascaris embryos show little or no indication of these pe-
riods when heterogenous RNA predominates (Ellen and Gwatkin, 1968;
Woodland and Graham, 1969; Piko, 1970). In these embryos ribosomal
RNA predominates from very early stages. The reasons for the differ-
ences among early embryos is unknown, but the embryos which show
the changes in patterns of RNA synthesis have rapid cleavage stages
while those which do not show the changes cleave rather slowly.
1. Mitochondnal RNA Synthesis
Eggs contain tens to thousands of times more cytoplasm than the aver-
age diploid somatic cell. This cytoplasm contains mitochondria and thus
much more mitochondrial DNA than is usual for a single cell. In sea
urchin eggs at least 80% of the DNA is mitochondrial (Piko et al.,
1967). The amphibian egg DNA likewise contains a very large per-
centage of mitochondrial DNA (Dawid, 1966). In sea urchins this DNA
appears to serve as a template for much of the RNA synthesized right
after fertilization. The first data suggesting this came from experiments
with artificially activated, enucleate half eggs. RNA synthesized in the
enucleate halves was identical in quantity and kind to that synthesized
in nucleate halves and whole eggs (Chamberlain, 1967, 1970). The nature
of this RNA synthesized in the egg cytoplasm was established by showing
that it hybridized well with purified sea urchin mitochondrial DNA
(Craig, 1970) but not with DNA from sea urchin nuclei. The RNA
synthesized during this time is heterogenous with a sharp peak at 15
to 17 S. It may be mitochondrial messenger RNA (Craig, 1970), but
its nature and its role in mitochondrial physiology is essentially unex-
plored despite the fact that these early zygotes provide an ideal system
for analyzing the details of mitochondrial RNA synthesis.
