/. Embryol. exp. Morph. Vol. 36, 1, pp. 1-12, 1976
Printed in Great Britain
REVIEW ARTICLE
Mechanisms of teratogenesis*
ByLAURI SAXEN1
From the III Department of Pathology, University of Helsinki
Introduction
Site of Action
Introduction
Intracellular compartment
Cell surface
Extracellular matrix
Fetal environment
Stage of Action
Introduction
Determination
Proliferation
Organization
Migration
Morphogenetic cell death
Remarks
References
INTRODUCTION
In view of the great number of teratogenic factors known and the vast array
of congenital defects, disorders and syndromes, it would probably be a waste of
time to search for unifying mechanisms and principles in abnormal development. Instead, therefore, I shall describe a selection of teratogens and their
consequences, and try to arrange them in a certain hierarchy based on a simplified model of how they act.
The assumption underlying the model (Figs. 1 and 2) is that the result of a
teratogenic insult is determined by its site of action and the stage of development
of the target organ. This is supposed to hold for all congenital defects, whether
due to genes or caused by exogenous agents. In genetic defects the scheme
indicates the site and stage of development at which the mutant gene is expressed;
in nongenetic defects the site and stage refer to exposure to an exogenous
teratogen.
* This article is based on a paper presented in Martinique at a meeting co-sponsored by the
Institut de la Vie and the U.S. National Cancer Institute.
1
Author's address: III Department of Pathology, University of Helsinki, SF-00290,
Helsinki 29, Finland.
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Epigenetic
Fig. 1
SITE OF ACTION
The four main levels of action of a defective gene or an exogenous teratogen
are illustrated in Fig. 1. Action may primarily be exerted on (1) the intracellular
compartment, i.e. on the chain of interactions between the nucleus and the
cytoplasm leading to the specific metabolic products of the cell. The primary
action may also be expressed as (2) abnormalities in the structure and function
of the cell surface, or (3) of the extracellular matrix. Finally, detectable defects
may result from primary effects on (4) the fetal environment, i.e. from abnormalities at the organismal level or in the feto-maternal relation. Although such
a schematic classification into four main levels of teratogenic action is no doubt
an oversimplification, it may be useful to consider the mechanisms involved in
abnormal development in such a hierarchy. In what follows, examples of both
genetic and nongenetic defects of these four categories of teratogenic insult will
be presented.
Mechanisms of teratogenesis
3
Intracellular compartment
During the last two decades, the processes of nucleo-cytoplasmic interaction
have been mapped, and with this rapidly growing knowledge, information has
accumulated on the modes of action of mutant genes and of various inhibitors.
Genetic. Perhaps the best analyzed hereditary defects in the intracellular
compartment are the 'inborn errors of metabolism', a category of diseases so
named long ago by Sir Archibald Garrod. In these diseases a mutant gene leads
to deficiency of an enzyme activity, with the result that a metabolic pathway is
blocked. There are three possible consequences, (1) the metabolite before the
block may accumulate, (2) the metabolite beyond the deficient enzyme may be
lacking, and (3) the metabolite before the block may take an alternative pathway.
An example of the first type will illustrate how such an intracellular mishap may
profoundly affect the whole organism.
In the mucopolysaccharidoses, enzyme defects result in blockage of the
degradation of mucopolysaccharides and accumulation (or secretion) of their
polymers. For instance, Hurler's syndrome is due to deficiency of the lysosomal
enzyme a-L-iduronidase, and subsequent accumulation of both heparin sulfate
and dermatan sulfate in the cells. Excessive accumulation of these metabolites
in cells of various types gradually leads to the manifestation of a severe disorder,
characterized by retardation of growth, mental regression, and various skeletal
defects, accompanied by cardiac failure with hepatosplenomegaly (Leroy &
Crocker, 1966).
Nongenetic. The various steps of the nucleocytoplasmic interactions may be
blocked more or less specifically by appropriate inhibitors. As, by definition,
these inhibitors prevent the process of the reading of genetic information, they
should all cause defects mimicking hereditary disorders (phenocopies). However, this may be difficult to demonstrate for two reasons. If the function inhibited is essential to life, the effect is usually lethal and so can never give rise to
a detectable malformation (Wilson, 1973). Secondly, the effect may be reversible,
and the result therefore transient. The first difficulty has been overcome by
treating isolated organs in vitro followed by retransplantation. By this method,
actinomycin D has been shown to be teratogenic to early brain anlagen,
where it produces severe eye defects (Diethelm & Schowing, 1974). Concerning
reversible inhibition, tissue culture methods have proved useful, because normal
and disturbed development can be followed constantly. Cycloheximidine, when
added to cultures of kidney rudiments, stops both protein synthesis and morphogenesis. But after the drug is withdrawn, both processes continue normally, and
the short lag period is masked by the subsequent normal development (Saxen,
unpublished observations).
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Cell surface
Experiments of various types have repeatedly shown the importance of the
cell surface in many developmental processes such as cell recognition and
aggregation, morphogenetic cell interactions, and cell migrations (see Moscona, 1974a). Consequently, it may be expected that both genetic defects and
experimentally induced alterations of the cell surface will lead to impaired
development.
Genetic. Extensive studies on mice with mutant genes at the T locus have
shown that the homozygotes have various defects and are usually inviable. One
of these lethal alleles, t9, seems to exert its effect via a primary abnormality of
the cell surface. Development seems to proceed normally until gastrulation.
Then, migration of the mesodermal cells between the ecto- and entoderm is
severely impaired, and ectodermal-mesenchymal relations are disrupted.
Electron microscopy shows that at the gastrula stage embryos homozygous for
the /9 gene have mesodermal cells of abnormal shape and with abnormal intercellular relations. Genes at the T locus also determine surface components on
spermatozoa carrying the various mutants. The surface antigens of the four t
alleles so far tested are different, and no cross-reactions have been detected.
Thus the t9 allele seems to act primarily upon the cell surface components,
thereby leading to impaired cell-to-cell interactions and altered cell behavior
and ultimately to a total block of development (Bennett, 1975).
Nongenetic. The formation of the secondary palate terminates with a fusion
of the palatal shelves, a process which can occur only after the epithelium lining
the mesenchymal shelves has disappeared. If this 'morphogenetic cell death'
were prevented, fusion would be incomplete, and the result would probably
be a cleft palate. Death of the epithelial cells and fusion of the shelves were
preceded by rapid synthesis of mucopolysaccharides on the epithelial surface.
This observation led Pratt and his collaborators (Pratt & Hassell, 1975;
Greene & Pratt, 1975) to study the effect of inhibiting polysaccharide synthesis.
The antagonist of glutamine, 6-diazo-5-oxo-norleucine (DON), was used to
block the synthesis of glycosaminoglycans, and the success of the treatment was
confirmed by various techniques of light and electron microscopy. In the presence of DON, epithelial necrosis was prevented, and subsequent fusion of the
shelves was incomplete. Excess of glutamine restored the synthesis of the surfaceassociated glycosaminoglycans and allowed programmed death of the epithelial
cells and complete fusion.
Extracellular matrix
In various tissues, structure and function are defined by an extracellular
matrix. Each matrix has a composition unique to its particular tissue. Hence a
teratogen may be expected to affect the function of a tissue by interfering with
the production or maturation of this extracellular material.
Mechanisms of teratogenesis
5
Genetic. Dermatosparaxis is a hereditary disease recently detected in cattle,
and characterized by a fragile skin with an inelastic dermal tissue. The basic
defect seems to be abnormal or insufficient production of collagen in the dermal
connective tissue. The mutant cells are devoid of the enzyme procollagen
peptidase. Consequently, the procollagen accumulated is abnormal, containing
N-terminal extensions of noncollagen protein and its capacity to form fibrils is
defective. This, in turn, leads to a severe disturbance in the architecture of the
collagen fibrils and in their cross-linkages (Lenaers, Ansay, Nusgens & Lapiere,
1971; Hanset & Lapiere, 1974). The same enzyme defect was subsequently
found in patients suffering from one form of the hereditary Ehlers-Danlos
syndrome (Lichtenstein et al. 1973).
Nongenetic. The antibiotics of the tetracycline group will serve to illustrate
how teratogens may affect development by acting on extracellular compounds,
and not directly on the cells producing these molecules. The tetracyclines are
known to be incorporated in the mineralizing tissues, where they may retard
growth and cause hypoplasia. Their mode of action has been thoroughly
analyzed in vitro, with fetal mouse bones as target tissue. At concentrations that
inhibited bone growth, tetracycline hydrochloride did not reduce the rate of
proliferation of chondroblasts. Nor did these concentrations affect the synthesis
of collagen and mucopolysaccharides, which are the main components of the
cartilaginous matrix. The primary action of the drug was finally shown to be
exerted on the bone mineral crystals; in the presence of low concentrations of
tetracycline their formation and growth were definitely retarded. The drug
apparently acts by competing with the inorganic ions building the bone mineral
(Kaitila, 1971; Saxen & Kaitila, 1972).
Fetal environment
The development of a mammalian embryo is controlled by a complex of
factors, maternal, placental, and autogenous; these factors include hormones,
protective mechanisms (immune system), and nutritional factors. Changes in
these might be expected to lead to developmental abnormalities.
Genetic. Numerous hereditary defects give rise to changes in the internal
environment that secondarily affect various organs. Many endocrine disorders
can be placed in this category. For example, the hereditary Pendred's syndrome
is characterized by congenital deafness and hypothyroidism. To study the
association between these two defects, Deol (1973) mimicked the syndrome in
mice by treating pregnant females with propylthiouracil. This treatment led to
severe lesions in the inner ear, especially in the tectorial membrane, which
caused loss of hearing. Addition of thyroxine to the mother's drinking water
resulted in offspring with normal morphology of the inner ear and normal
hearing. These experimental results suggest that in Pendred's syndrome, deafness is a secondary consequence of the endocrine disorder.
A peculiar hereditary syndrome in mice is caused by the l tail-short' mutant
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gene. This mutant is characterized by a short, kinky tail associated with various
other skeletal defects and retarded growth. Deol (1961) showed that in the
early stages of development affected embryos suffer from transient anemia. This
can be traced back to the formation of blood in the yolk-sac; at this stage the
mutants have smaller blood islets than the wild-type embryos. This led Deol to
suggest that anemia, by leading to differential retardation of various organs,
might be the primary cause of the syndrome.
Nongenetic. The embryonic 'macromilieu' is susceptible to many external
influences: nutritional deficiencies, placental insufficiency, altered maternal
endocrine status and immunization following feto-maternal incompatibility
may have deleterious effects on the developing organism. As an example, I shall
cite the work of Brent (summarized in 1971). He showed that rabbit antisera
against certain rat tissues, when injected into pregnant rats, caused congenital
defects in the offspring. Yet the antibodies never crossed the placenta. They
accumulated in the yolk-sac epithelium, a structure peculiar to the placenta in
rodents and rabbits. There, apparently, the immunoglobulins impaired placental
function by impeding the transport of nutritional and other vital factors. A
similar mode of action has been suggested for certain azo dyes that cause
specific defects in embryos without actually entering the fetal organism (Beck,
1967).
STAGE OF ACTION
Introduction
The development of various organs during embryogenesis is best considered
not as a continuous process of 'maturation', but as a chain of distinguishable
steps strictly controlled in time and space. The fate of cells, which were originally
identical with the same genome, diverges step by step, and this determination
is followed by various events that lead to an orderly arrangement of tissue
components of specific shape, size and function. There is good reason to believe
that these basic steps of development are highly sensitive to both hereditary and
exogenous factors, and that insults at such stages will have profound teratological consequences. Thus, development involves a series of tight corners at
which normal embryogenesis is determined, but vulnerability is increased.
Fig. 2 illustrates the development of a hypothetical organ, in which the
following events can be distinguished: determination, proliferation, cellular
organization, migration, and morphogenetic cell death. Several congenital defects
and syndromes, both genetic and nongenetic, can be traced back to failures in
these events.
Determination
The determination of embryonic cells with the same genetic information and
their subsequent morphogenesis are processes guided by the position of the cell
and its relation to other cells. Determination thus depends on interaction
between cells. Numerous examples of such 'inductive cell interactions' have
Mechanisms of teratogenesis
Morphogcnctic
cell death
Organization
Proliferation
Determination
Progressive development
Fig. 2
been described, but the underlying molecular mechanisms are virtually unknown
(see Kratochwil, 1972; Saxen et al. 1976). These events are likewise sensitive to
both genetic and exogenous factors.
Genetic. A mutant strain of mice in which the homozygotes are born eyeless
or with severe microphthalmia was analyzed by Chase & Chase (1941). In
homozygous embryos the early development of the optic vesicle was completely
normal and progressed as in the wild type. Before the stage at which the optic
cup makes contact with the overlying presumptive lens epidermis, growth
seemed to slow down and the two tissue components either failed to meet or
established contact over a restricted area only. Consequently, the 'inducing
stimulus' which determines the lens and requires close apposition of the tissues
was not passed from the optic bud and no lens developed (or the lens formed was
very small). Subsequent studies on this mutant strain have suggested that the
primary cause of incomplete interaction might be not the retarded growth of
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LAURISAXEN
the optic bud, but failure of some intervening mesenchymal cells to undergo
normal 'morphogenetic cell death' (see below) (Silver & Hughes, 1974).
Nongenetic. Inductive cell interactions can be blocked by various means, both
chemical and physical. The effects of various chemicals are difficult to interpret,
because our knowledge of the molecular basis of these interactions is so fragmentary. Like various mutant genes, an inhibitor may act on the inductor
tissues, on the transmission of inductive signals, or directly on the responding
tissue (see Saxen, 1973). Some direct physical effects are easier to explain. The
example deals with one of the best studied interactive systems, that occurring
between the amphibian gastrula ectoderm and the invaginating mesoderm of
the archenteron roof. In his classic experiment, Mangold (1931) interfered with
this process by a microsurgical manipulation. He cut a flap in the responding
ectoderm, the presumptive neural plate, lifted it, removed a piece from the
midline of the underlying archenteron roof, and replaced the ectoderm in its
original position. The wounds healed. The ectoderm was now exposed to an
abnormally narrow inductive 'template' of mesoderm. As a result, the neural
plate was narrowed and the lateral edges approached each other, the outcome
frequently being fused optic cups (synophthalmia). The extreme form was
complete fusion of the eyes (cyclopia).
Proliferation
As spatially and temporally controlled proliferation of cells is vital for normal
embryogenesis, disturbances in proliferation rates should result in a variety of
defects. General inhibition would result in abnormally small embryos and more
localized effects in organ hypoplasia or - when differential proliferation rates
determine the shape of the organ - to misshapen or defective organs.
Genetic. The anomaly known as dextrocardia, which is frequently associated
with situs inversus, appears to result from the action of an autosomal recessive
gene. During early embryonic life, the primitive cardiac loop, instead of bulging
to the left, bulges to the right. Normal curvature of the cardiac loop and regional
differentiation and shaping of the heart have been shown to result from differential proliferation of the embryonic heart cells. The heart cells display this
intrinsic capacity in vitro in the absence of any other tissues, thus showing that
they are somehow programmed for differential proliferation. Consequently,
dextrocardia is the result of a failure or rather a reversal of the normal pattern
of proliferation in the embryonic heart (DeHaan, 1967).
Nongenetic. Irradiation and radiomimetic drugs acting primarily on dividing
cells have a typical stage-dependent spectrum of consequences as the tissues
usually affected are those that proliferate most rapidly. This can be shown in
the developing eye. During early development, mitoses occur throughout the
outer layer of the neural retina, but as cytodifferentiation of the central part
advances, proliferation is restricted to a narrow peripheral zone. Consequently,
triethylene-melamine (TEM), a radiomimetic drug, has different effects at
Mechanisms of teratogenesis
9
different stages. When applied in early development, it produces cell necrosis
and blocks mitoses all over the retina, the result being anophthalmia. At a later
stage, treatment affects only the peripheral zone of proliferation and the endresult is less drastic, mainly microphthalmia (see Saxen & Rapola, 1969).
Organization
Determination and differentiation of cells lead to surface specialization, with
the result that like cells can recognize each other. They then tend to adhere to
each other and form aggregates. These are the anlagen for various organs and
components of organ.
Genetic. The recognition mechanism and adhesive properties of embryonic
cells may be genetically defective. This was shown by Ede and his collaborators,
who studied the talpid3 mutant chick characterized by a variety of skeletal
defects (Ede & Agerback, 1968; Ede, Bellairs & Bancroft, 1974). The authors
used the technique of enzymatic disaggregation and subsequent reaggregation
to study the mesenchymal cells of these embryos. Comparison with a wild-type
embryo at the same stage showed that reaggregation of the mutant cells was
enhanced and the aggregates formed were larger. Ultrastructural studies showed
changes in the surface of these cells and their intercellular relations. In all probability, the many defects in this mutant strain are due to altered surface properties
of the mesenchymal cells resulting in increased adhesiveness and abnormal
intercellular relations.
Nongenetic. The cells of the embryonic neural retina synthesize glutamine
synthetase (GS), an enzyme which can be induced with hydrocortisone (Moscona,
1972). This induction depends on homotypic contacts between the retinal cells
and their retinotypic organization. In vitro, dispersed cells on monolayers do
not respond to hydrocortisone, but when freshly dissociated cells are allowed to
reassemble, the clusters can be induced to synthesize GS (Morris & Moscona,
1971). In vivo, the dependence can be demonstrated by treating the embryos
with 5-bromodeoxyuridine (BrdU). This thymidine analog is incorporated into
DNA and interferes with cell proliferation. When applied to early embryos with
mitoses still occurring throughout the neural retina, it disorganizes the tissue
and the chaotic cell population that results does not respond to hydrocortisone.
When given somewhat later, similar BrdU treatment does not markedly harm
the architecture of the neural retina, and, consequently synthesis of GS can be
induced with hydrocortisone (Moscona, 19746).
Migration
During morphogenesis, in addition to moving and aggregating at random,
cells and cell clusters undergo oriented movements. Such migrating cells are
restricted to certain paths, apparently provided by neighboring cells or their
products. Interference with this guiding principle would stop migration or lead
to formation of abnormal patterns.
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Genetic. The postnatal development of the weaver mutant mouse (wv) is
characterized by gradual regression of the cerebellar cortex and consequent
motoric dysfunction. Here electron microscopy has revealed a primary failure
in the migration pattern of the granular cells. Normally, these cells proliferate
in the external granular layer and migrate from there to their adult position in
the granular layer. This migration, a prerequisite for the maturation of the cells,
is guided by the fibers of the Bergman glial cells, which extend all the way from
the external granular layer to the granular layer. In the mutant mice, Bergman
cells are mostly lacking, and the granular cells, deprived of their guiding effect,
never reach their final position and ultimately degenerate. Similar changes,
though less marked, have been demonstrated in the +fwv heterozygote mice
(Rakic & Sidman, 1973; Sidman, 1974).
Nongenetic. The vertebrate heart develops from two lateral populations of
precardiac mesenchymal cells. These migrate towards their final anterior and
ventral position, and again, this migration of cells and cell clusters seems to be
guided. The filamentous processes of the mesenchymal cells sense a path provided by the underlying entoderm. If the binding Ca 2+ ions are removed from
the milieu experimentally, contact between the filaments and their substances
is broken, migration stops, and the cells form a paired heart anlage (cardia
bifida). The key role of the Ca2+ ions was further demonstrated in experiments
in which the chelating agent added to the medium was accompanied by an
equimolar amount of Ca2+. Then migration and heart formation proceeded
normally (DeHaan, 1958).
Morphogenetic cell death
Various embryonic organs and tissue components are eliminated selectively
during embryogenesis, and this programmed 'morphogenetic cell death' can be
regarded as one important event in morphogenesis, shaping and carving different organs. Both excessive and inhibited cell death should therefore result in
abnormal development and congenital anomalies.
Genetic. Various sites and stages of limb development are characterized by
cell death. This is strictly timed and localized. At the borders of the limb-bud,
necrotic zones determine the size and shape of the limb, and necrosis between
the anlagen separates the digits. In talpid3 mutant chick embryos, these necrotic
processes are impaired. Absence of necrotic zones at the limb-bud borders leads
to abnormally large limb-buds with supernumerary digits, and incomplete
interdigital necrosis results in partial soft-part syndactyly, in which the digits are
webbed (Hinchliffe & Ede, 1967; Hinchliffe & Thorogood, 1974).
Nongenetic. Exogenous factors will also prevent interdigital necrosis in
normal, wild-type embryos. Chick embryos develop soft-part syndactyly when
treated with Janus green before interdigital necrosis is visible (Menkes &
Delanu, 1964; Saunders & Fallon, 1966). The experiment has been repeated in
rabbit embryos with the same result (Saxen, unpublished observations).
Mechanisms of teratogenesis
11
REMARKS
The schemes of Figs. 1 and 2 postulate that the effect of a teratogen and the
pathogenesis of the resulting maldevelopment are determined by the site of
action of the teratogen and the stage of development of the target tissue.
Various examples from genetics, human pathology and experimental teratology
seem to support this classification, but do not necessarily establish its validity.
Like all classifications, the scheme should be regarded mainly as an attempt to
produce order out of chaos. It is intended as an operational guide until a deeper
knowledge of teratogenesis yields a simpler, and, perhaps, more unifying theory
of abnormal development.
REFERENCES
BECK, S. L. (1967). Effects of position in the uterus on foetal mortality and on response to
trypan blue. /. Embryol. exp. Morphol. 17, 617-624.
BENNETT, D. (1975). T-locus mutants: Suggestions for the control of early embryonic organization through cell surface components. In The Early Development of Mammals (ed. M.
Balls & A. E. Wild), pp. 207-218. Cambridge University Press.
BRENT, R. L. (1971). Antibodies and malformations. In Malformations Congenitales des
Mammiferes (ed. H. Tuchmann-Duplessis), pp. 187-220. Paris: Masson.
CHASE, H. B. & CHASE, E. B. (1941). Studies on an anopthalmic strain of mice. I. Embryology
of the eye region. J. Morphol. 68, 279-302.
DEHAAN, R. L. (1958). Cell migration and morphogenetic movements. In The Chemical
Basis of Development (ed. W. D. McElroy & B. Glass), pp. 339-377. Baltimore: The Johns
Hopkins Press.
DEHAAN, R. L. (1967). Development of form in the embryonic heart. An experimental
approach. Circulation 35, 821-833.
DEOL, M. S. (1961). Genetical studies on the skeleton of the mouse XXVIII. Tail-short.
Proc. Roy. Soc. B 155, 78-95.
DEOL, M. S. (1973). An experimental approach to the understanding and treatment of
hereditary syndromes with congenital deafness and hypothyroidism. /. med. Genet. 10,
235-242.
DIETHELM, F. & SCHOWING, J. (1974). Action directe de l'actinomycine D sur l'encephale
embryonnaire du poulet. Obtention experimentale de la cyclopie. Wilhelm. Roux Arch.
EntwMech Org. 175, 163-172.
EDE, D. A. & AGERBAK, G. S. (1968). Cell adhesion and movement in relation to the developing limb pattern in normal and talpid3 mutant chick embryos. /. Embryol. exp. Morph. 20,
81-100.
EDE, D. A., BELLAIRS, R. & BANCROFT, M. (1974). A scanning electron microscope study of
the early limb-bud in normal and talpid3 mutant chick embryos. /. Embryol. exp. Morph.
31, 761-785.
GREENE, R. M. & PRATT, R. M. (1975). Inhibition by diazo-oxo-norleucine (DON) of rat
palatal glycoprotein synthesis and epithelial cell adhesion in vitro. J. CellBiol. (In the Press).
HANSET, R. & LAPIERE, C. M. (1974). Inheritance of dermato-sparaxis in the calf. /. Hered.
65, 356-358.
3
HINCHLIFFE, J. R. & EDE, D. A. (1967). Limb development in the polydactylous talpid
mutant of the fowl. /. Embryol. exp Morph. 17, 385-404.
HINCHLIFFE, J. R. & THOROGOOD, P. V. (1974). Genetic inhibition of mesenchymal cell death
and the development of form and skeletal pattern in the limbs of talpid3 (ta3) mutant chick
embryos. /. Embryol. exp. Morph. 31, 747-760.
KAITILA, I. (1971). The mechanism by which tetracycline hydrochloride inhibits mineralization in vitro. Biochim. biophys. Acta 244, 584-594.
12
LAURISAXEN
RRATOCHWIL, K. (1972). Tissue interaction during embryonic development: General properties. In Tissue Interactions in Carcinogenesis (ed. D. Tarin), pp. 1-47. London: Academic
Press.
LENAERS, A., ANSAY, M., NUSGENS, B. V. & LAPIERE, C. M. (1971). Collagen made of extended a-chains, procollagen, in genetically-defective dermatosparaxic calves. Eur. J.
Biochem. 23, 533-543.
LEROY, J. G. & CROCKER, A. C. (1966). Clinical definition of the Hurler-Hunter phenotypes.
A review of 50 patients. Am. J. Dis. Child. 112, 518.
LICHTENSTEIN, J. R., MARTIN, G. R., KOHN, L. D., BYERS, P. H. & MCKUSICK, V. A. (1973).
Defect in conversion of procollagen to collagen in a form of Ehlers-Danlos syndrome.
Science, N.Y. 182, 298-300.
MANGOLD, O. (1931). Das Determinationsproblem. III. Das Wirbeltierauge in der Entwicklung und Regeneration. Ergebn. Biol. 7, 193-403.
MENKES, B. & DELANU, M. (1964). Leg differentiation and experimental syndactyly in the
chick embryo. II. Experimental syndactyly in chick embryo. Revue Roum. Embryol.
CytoL, Ser. Embryol. 1, 69-77.
MORRIS, J. E. & MOSCONA, A. A. (1971). The induction of glutamine synthetase in aggregates
of embryonic neural retina cells: correlation with differentiation and multicellular organization. Devi Biol. 25, 420-444.
MOSCONA, A. A. (1972). Induction of glutamine synthetase in embryonic neural retina: A
model for the regulation of specific gene expression in embryonic cells. In Symposium on
Biochemistry of Cell Differentiation, vol. 24 (ed. A. Monroy), pp. 1-23. London: Academic
Press.
MOSCONA, A. A. (1974a). Surface specification of embryonic cells: Lectin receptors, cell
recognition, and specific cell ligands. In The Cell Surface in Development (ed. A. A.
Moscona), pp. 67-99. New York: John Wiley and Sons.
MOSCONA, A. A. (ed.) (19746). The Cell Surface in Development. New York: John Wiley and
Sons.
PRATT, R. M. & HASSELL, J. R. (1975). Appearance and distribution of carbohydrate-rich
macromolecules on the epithelial surface of the developing rat palatal shelf. Devi Biol. 45,
192-198.
RAKIC, P. & SIDMAN, R. L. (1973). Weaver mutant mouse cerebellum: Defective neuronal
migration secondary to adnormality of Bergmann glia. Proc. natn. Acad. Sci. U.S.A. 70,
240-244.
SAUNDERS, J. W., JR. & FALLON, J. F (1966). Cell death in morphogenesis. In Major Problems in Developmental Biology (ed. M. Locke), pp. 289-314. New York: Academic Press
SAXEN, L. (1973). Tissue interactions and teratogenesis. In Pathobiology of Development (ed.
E. V. Perrin & M. J. Finegold), pp. 31-51. Baltimore: Williams and Wilkins Co.
SAXEN, L., KARKINEN-JAASKELAINEN, M., LEHTONEN, E., NORDLING, S. & WARTIOVAARA, J.
(1976). Inductive tissue interactions. In Cell Surface Interactions in Embryogenesis (ed. G.
Poste & G. L. Nicolson). Amsterdam: North-Holland Division of ASP Biological and
Medical Press. (In the Press).
SAXEN, L. & KAITILA, I. (1972). The effect and mode of action of tetracycline on bone
development in vitro. In Adv. exp. Biol. Med. 27, 205-218. New York: Plenum Publ. Co.
SAXEN, L. & RAPOLA, J. (1969). Congenital Defects. New York: Holt, Rinehart and Winston.
SIDMAN, R. L. (1974). Contact interaction among developing mammalian brain cells. In
The Cell Surface in Development (ed. A. A. Moscona), pp. 221-253. New York: John
Wiley and Sons.
SILVER, J. & HUGHES, A. F. W. (1974). The relationship between morphogenetic cell death
and the development of congenital anophthalmia. /. comp. Neurol. 157, 281-302.
WILSON, J, G. (1973). Environment and Birth Defects. New York: Academic Press.
{Received 17 February 1976)