Table Of ContentDETERMINANTS OF SPATIAL ORGANIZATION
The Thirty-Seventh Symposium of
The Society for Developmental Biology
Madison, Wisconsin, June 14-16, 1978
EXECUTIVE COMMITTEE:
1977-1978
IRWIN R. KÖNIGSBERG, University of Virginia, President
IAN M. SUSSEX, Yale University, Fast-President
NORMAN K. WESSELLS, Stanford University, President-Designate
WINIFRED W. DOANE, Arizona State University, Secretary
MARIE DI BERARDINO, Medical College of Pennsylvania, Treasurer
GERALD M. KIDDER, University of Western Ontario, Member-at-large
1978-1979
NORMAN K. WESSELLS, Stanford University, President
IRWIN R. KÖNIGSBERG, University of Virginia, Past-President
URSULA K. ABBOTT, University of California, President-Designate
WINIFRED W. DOANE, Arizona State University, Secretary
JOHN G. SCANDALIOS, North Carolina State University, Treasurer
GERALD M. KIDDER, University of Western Ontario, Member-at-Large
Business Manager
CLAUDIA FORET
P. O. Box 43
Eliot, Maine 03903
Determinants
of
Spatial Organization
Stephen Subtelny, Editor
Department of Biology
Rice University
Houston, Texas
Irwin R. Königsberg, Co-Editor
Biology Department
University of Virginia
Charlottesville, Virginia
ACADEMIC PRESS New York San Francisco London 1979
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Contributors and Presiding Chairpersons
Numbers in parentheses indicate the pages on which the authors'
contributions begin.
Session I Chairperson: Irwin R. Königsberg, Department of Biology,
University of Virginia, Charlottesville, Virginia
M. R. Dohmen and Ν. H. Verdonk, Zoological Laboratory, University of
Utrecht, Utrecht, The Netherlands (3)
J. R. Whittaker, The Wistar Institute of Anatomy and Biology, Philadel-
phia, Pennsylvania (29)
Gary Freeman, Department of Zoology, University of Texas, Austin,
Texas (53)
Session II Chairperson: Elizabeth Hay, Department of Anatomy, Harvard
Medical School, Boston, Massachusetts
Ralph Quatrano, Susan H. Brawley* and William E. Hogsett, Department
of Botany and Plant Pathology, Oregon State University, Corvallis,
Oregon, and *Department of Botany, University of California ,
Berkeley, California (77)
Klaus Kalthoff, Department of Zoology, University of Texas, Austin,
Texas (97)
Session III Chairperson: L. Dennis Smith, Department of Biological
Sciences, Purdue University, West Lafayette, Indiana
A. P. Mahowald, C. D. Allis, K. M. Karrer, Ε. M. Underwood, and G. L.
Waring, Department of Biology, Indiana University, Bloomington,
Indiana (127)
David Hirsh, Department of Molecular, Cellular and Developmental
Biology, University of Colorado , Boulder, Colorado (149)
Ann Janice Brothers, Department of Zoology, University of California ,
Berkeley, California (167)
vii
CONTRIBUTORS
Session IV Chairperson: David Nanney, Zoology Department, University
of Illinois, Urbana, Illinois
Christiane Nusslein-Volhard, European Molecular Biology Laboratory,
Heidelberg, Germany (185)
Joseph Frankel, Department of Zoology, University of Iowa, Iowa City,
Iowa (215)
Session V Chairperson: David Sonneborn, Department of Zoology, Uni-
versity of Wisconsin, Madison, Wisconsin
C. Peter Wölk, MSU-DOE Plant Research Laboratory, Michigan State
University, East Lansing, Michigan (247)
Richard D. Campbell, Department of Development and Cell Biology,
University of California, Irvine, California (267)
Peter J. Bryant, Center for Pathobiology, University of California, Irvine,
California (295)
Session VI Chairperson: Irwin R. Königsberg, Department of Biology,
University of Virginia, Charlottesville, Virginia
P. A. Lawrence and G. Morata*, MRC Laboratory of Molecular Biology,
Cambridge, England, and *Centro de Biologia Molecular, Universidad
Autonoma de Madrid, Madrid, Spain (317)
Preface
Developmental phenomena, whether they occur during the genesis of
a new individual or are involved in the maintenance and repair of the
adult form, are characterized by a progressive increase in complexity
that is expressed at all levels of biological organization. Not only do we
observe the emergence of a diversity of cell types, each exhibiting a
unique spectrum of macromolecules that restricts the specialized func-
tion of that cell type, but these differentiated cell types are precisely
localized within the developing embryo, bud, or regenerating part.
The mechanisms involved in the generation of this high degree of spa-
tial organization have continued to intrigue investigators since the
emergence of the discipline of developmental biology. In examining
these phenomena during the formative period of this science, several
major observations were made suggesting the operation of mechanisms,
unique to early development, that regulate subsequent gene expression
in the various subgroups of the expanding cell population. The first of
these was the establishment of the fact that developing zygotes of a wide
variety of organisms contain morphogenetic determinants localized to
discrete regions of the egg cytoplasm and that the blastomeres formed in
these areas give rise to specific differentiated cell types.
These early-segregating cells were also shown to exhibit both gradient
properties within a single morphogenetic area (for example, the amphib-
ian gray crescent area) or interacting gradients between the blastomere
lineages, which arise from each of two spatially separated cytoplasmic
areas (such as in the developing sea urchin). Similarly, at later stages of
development and during the regeneration of ablated structures, the exis-
tence of field or gradient properties also suggests that some system of
cell communication represents a second-order mechanism for establish-
ing spatial organization in developing systems.
It was my purpose in organizing this symposium to bring together a
diverse group of investigators who are analyzing these problems from
different vantage points, employing a variety of experimental systems in
innovative ways. The development of a program that would adequately
treat the topic within the constraints of time involved many hard choices
as well as a number of seemingly trivial chores. One has to decide, for
ix
PREFACE
example, who goes first, who follows, and how to keep the unavoidable
breaks for lunch and other physiological needs from interrupting the
continuity. The scheduling became simpler when at some point during
these deliberations, I became convinced that, indeed, the research in-
terests of these symposium speakers were closely interrelated. I imagine
other organizers of symposia have clutched at the same straw under
similar circumstances. As Clifford Grobstein observed, however, in his
preface to the twenty-first symposium of this Society, speakers fre-
quently do not share the organizer's view of the unifying theme of a
symposium. If such a division of opinion occurred in Madison, it was
not evident, and I have every reason to hope that this volume, which
reports the proceedings, reflects a synthesis of interests of investigators
who are (1) probing the mechanism of localization and the nature of
morphogenetic determinants in the developing zygote, (2) employing
maternal-effect mutants to study the roles of cytoplasmic determinants
and the expression of gradient properties in early development, and (3)
using genetic, micromanipulative, and biochemical tools to study pat-
tern formation in simple and more complex forms.
During the first session, which dealt with cytoplasmic localization of
determinants, Gary Freeman introduced his presentation by projecting
the frontispieces of two volumes: one, The Cell in Development and Hered-
ity, published by Edmond Β. Wilson in 1928, and the other, the second
edition of Eric Davidson's Gene Activity in Early Development published in
1977. Freeman's appraisal was that very little had been added between
the two publication dates. The five papers presented during the first day
of the symposium (including Freeman's) indicate that the appraisal, al-
though a good opening gambit, should be taken with a grain of jovial
salt.
Within the past decade, at least, our knowledge of the cytoplasmic
localization of developmental information has been extended by the ap-
plication of new approaches to this old problem. Dohmen and Verdonk,
for example, have studied the fine structure of cytoplasmic specializa-
tions of the polar lobe of molluscan embryos and observed a unique
aggregate of vesicular structures in Bithynia. By the more classical cen-
trifugation and deletion experiments this "vegetal body" appears to
carry the lobe-associated determinants. Similarly, scanning electron
microscopy reveals striking differences in the surface architecture in the
prospective polar lobe region and the possible role of these surface
specializations is discussed.
With a similar goal in mind, Mahowald characterized the fine struc-
ture of the polar granules of Drosophila eggs. Having an unequivocal
marker for the germ cell determinants, he has recently been successful in
PREFACE xi
first obtaining a granule-enriched fraction and then identifying a unique
polar granule protein in 2-D gels. Following the synthesis of this protein
during oogenesis, he is currently testing a scheme of the continuity of
the polar granule in the germ cell lineage, which he postulated earlier on
the basis of fine-structure studies. Polar granule morphology was also
used earlier to confirm cytoplasmic transfer of germ plasm (with Illmen-
see). Such studies provided the first demonstration that the transfer of a
specific cytoplasmic determinant to an "ectopic" region of the egg di-
rects the development of the blastomeres that form in that region into
the lineage specified by that determinant. Similarly, in studies of As-
cidian development, Whittaker has now shown, by altering the cleavage
pattern rather than by microinjection, that determinants of muscle cell
proteins will alter the fate of blastomeres normally destined to form ec-
toderm.
Freeman has also employed techniques to delay cleavage and to alter
the cleavage pattern, in both Cerebratulus and Mnemiopsis, to pose sig-
nificant questions by-passed in the classical period. Using inhibitors of
karyokinesis he has been able to uncouple develomental time from
cleavage and to determine the respective roles played by cell division
and cleavage planes in the localization of cytoplasmic determinants.
These studies indicate that, contrary to the classical view, that determi-
nants in the zygotes in some species are not definitively localized during
the postfertilization cytoplasmic streaming but become progressively
segregated during cleavage processes linked to the cell division cycle.
One might anticipate that knowing how these determinants are translo-
cated might provide, as well, an approach to their identity.
Quatrano's research on the polarity of rhizoid formation in Fucus has,
in fact, followed such a trail and might provide a paradigm for the locali-
zation of specific macromolecules in embryonic anlagen. Starting with a
consideration of how polar gradients are established and fixed, Qua-
trano and his colleagues moved on to the identification and characteriza-
tion of rhizoid specific polysaccharides and of the localization of such
molecules to preformed sites in the zygote.
The data presented by Kalthoff at this symposium also trace a line of
research leading from initial phenomenological observations to the es-
tablishment of a body of evidence that suggests that a localized deter-
minant of the cephalic region of the embryo of the choronimid Smittia
may be one of a limited number of species of RNA. It was observed
initially that UV microbeam irradiation of a precise region of the anterior
pole at early stages leads to the induction, in high frequency, of bicaudal
embryos. Action spectra suggested the presence of both a protein and
nucleic acid moiety in the target area. The photoreversibility of the mor-
PREFACE
phological effect again suggests the involvement of nucleic acid, which
is further supported by Kalthoff's observation of the formation and
decay of pyrimidine dimers in RNA after UV irradiation followed by
exposure to visible light. Since either RNAase or UV irradiation applied
to the same site results in double abdomen formation, the simplest
hypothesis, Kalthoff suggests, is that both inactivate a single type of
cytoplasmic determinant.
The strategy of the "new look" at cytoplasmic localization that seems
to be emerging is first to define the phenomenon in more precise terms.
This redefinition frequently involves a clearer characterization of the cell
properties specified by a given determinant such as Quatrano's rhizoid-
specific polysaccharides, Freeman's use of light emission by the photo-
cyte, or Whittaker's exploitation of cell-type-specific histochemically
demonstrable enzymes. Other studies have focused on a fine-structural
identification of the cytoplasmic inclusion with which the determinant is
associated (the "ventral body" of Dohmen and Verdonk and
Mahowald's polar granule) or on the experimental lability of the deter-
minant property. How these "handles" are used varies considerably
from Mahowald's use of ultrastructural criteria to obtain granule-
enriched fractions and examine subunit composition to Quatrano's
analysis of the role of sulfation in the localization of fucoidin. What is
most important, however, is that the more precise characterizations lead
to more readily resolved questions, and if there is any doubt that such
seemingly prosaic beginnings can lead to highly significant findings I
suggest that the reader carefully think through Whittaker's paper, not
only where he has been, but where he is going and how he intends to
get there.
Since it is clear that cytoplasmic determinants must be synthesized
and stored during oogenesis, maternal-effect mutants of developmental
processes offer promising tools to investigate the time of synthesis, na-
ture, and mechanism of expression of cytoplasmic determinants. In this
class of mutants the developmental defect is an expression of the mater-
nal genome and not that of the zygote. Whether the defect represents
the absence of a particular gene product, the production of an altered
gene product, or distortions of egg organization, the primary event must
occur prior to fertilization.
This unique type of gene expression was first recognized in the pair of
alleles that control dextral and sinistral cleavage in the egg (and sub-
sequent coiling of the shell) in the fresh water snail Lynnea stagnalis. First
described by Boycott and Diver (1923-1938), the mode of inheritance of
these traits was subsequently analyzed by Sturtevant in 1923. The most
extensive developmental study of a mutant of this type has been per-
PREFACE
formed in the 0 mutant (ova deficient) of the Mexican axolotl. The mu-
tant phenotype observed in the progeny of homozygous females is arrest
of development at the gastrula stage. Brothers has reviewed the evi-
dence that gastrular arrest is due to a deficiency in the synthesis of 0+
factor normally synthesized during the germinal vessicle stages and re-
leased into the cytoplasm at the 1st meiotic division. The defect can be
corrected by injecting either germinal vessicle nucleoplasm or egg cyto-
plasm from eggs of wild-type females into fertilized, uncleaved eggs of
homozygous mutant females. Rescue of a blastula nucleus of an embryo
fated for gastrula arrest can be effected by transplantation into an enu-
cleated egg containing 0 + substance provided the nuclear transfer is per-
formed before the stage (late blastula) at which normal nuclei are acti-
vated by the factor. Conversely, late blastula nuclei of normal embryos,
having been activated by 0 + factor, support normal development when
transplanted into enucleated eggs of homozygous mutant females. Acti-
vation in such nuclei is stable and heritable through at least 30 mitotic
divisions in clonal serial transplants in enucleated eggs of homozygous
mutant females. The basis of the maternal effect in the 0 mutant is the
inability to synthesize and store a soluble gene product that at late blas-
tula activates, in stable fashion, one or more gene functions required to
carry the embryo through normal gastrulation and neurulation.
In theory, a variety of maternal-effect mutations should occur affect-
ing not only cleavage and early morphogenetic and inductive processes
but the establishment of embryonic symmetry and polarity as well. One
mutation of the latter type, bicaudal, was described in Drosophila by Bull
in 1966. Unfortunately the low penetrance and expressivity of the mu-
tant gene precluded more extensive analysis. The most extreme expres-
sion of this mutant phenotype, however, was remarkably similar to the
double abdomen embryos of Smittia, which Kalthoff has since experi-
mentally produced.
Recently Nüsslein-Volhard has been able to increase the frequency of
mutant expression by constructing hemizygous mutant females in
which the single point bic mutation is balanced against a homolog carry-
ing a deletion in the bicaudal region. One would assume, therefore, that
bicaudal is a hypomorphic mutation producing a smaller amount of the
normal gene product.
The mutant phenotype spectrum varies continuously from an embryo
lacking only a head to completely symmetrical double abdomens with a
distribution frequency, which suggests that the mutation shifts the pat-
tern between the two more stable extremes. A number of observations
suggest that the mutation affects the pattern of segments in both an-
terior and posterior halves of the embryo, thereby suggesting that the
