/ . Embryol. exp. Morph., Vol. 15, 3, pp. 261-269, June 1966
With 2 plates
Printed in Great Britain
261
Mesodermal alterations induced by
hypervitaminosis A
By MIGUEL MARIN-PADILLA1
From the Department of Pathology, Dartmouth Medical School
INTRODUCTION
In a recent experimental study (Marin-Padilla & Ferm, 1965) of the teratogenic effects of vitamin A, a distinct cellular necrosis in the somites of young
hamster embryos was found. This necrosis appeared at 12 h and the maximum
degree of damage was reached some 24 h after the administration of the vitamin.
The somite damage was considered then to be the earliest alteration induced
by vitamin A which, in turn, causes disturbances in the axial mesoderm.
Disturbances in the axial mesoderm could explain the vitamin A induced
cranioschisis and sacral rhachischisis. The study of human cranioschisis (MarinPadilla, 1965 b) and craniorhachischisis (Marin-Padilla, 1966) has also shown
that unknown mesodermal conditions, affecting the cephalic and the axial
mesoderm respectively, are the most likely underlying causes of these two
malformations. Similar axial mesodermal conditions may also explain the
notochordal alteration encountered in human cranioschisis (Marin-Padilla,
1966). ('Cranioschisis' ('cranium' and 'schisis') is another term for anencephaly, and' craniorachischisis' (' cranium' and' rachis-schisis') is a term for the
combination of anencephaly and the total failure of closure of the spinal canal
(vertebral column). These terms express better the basic developmental defect
in these malformations, namely, failure of closure of the neural tube.) Furthermore, a study (Marin-Padilla, 1965 a) of the sphenoid bones of unequally affected
human cases of cranioschisis and craniorhachischisis has shown that the
sphenoid bones are similarly malformed. This was considered to be suggestive
of the existence of a common, segmental disturbance of the cephalic mesoderm
in these malformations. It was suggested, in view of the experimental observations, that somite alterations could explain the common, segmental condition
affecting the cephalic mesoderm of these malformations.
However, the possibility of a condition affecting primarily the presomite
cephalic and the axial non-segmented mesoderm (embryonic body) and thus
secondarily involving the somites has not yet been investigated. This possibility,
on the other hand, could better explain the embryogenesis of cranioschisis and
1
Author's address: Department of Pathology, Dartmouth Medical School, Hanover,
New Hampshire, U.S.A.
17
JEEM 15
262
M. MARIN-PADILLA
craniorhachischisis. Primary presomite disturbances with secondary somite
damage have been previously suggested by Gallera (1951), Stephan & Sutter
(1960), Griineberg (1963) and Jurand (1963).
Both the investigation of this possibility and the necessity for a sequential
study of the earliest possible teratogenic effects of vitamin A to clarify the nature
of the previously reported somite necrosis were indicated. This double objective
was carried out using vitamin A as the teratogenic agent and with the golden
hamster as the experimental animal. Vitamin A was selected for this study not
only for its known teratogenic effects but also because it is known to affect
mesodermal tissues (Chung & Houck, 1964; Weissmann, 1964; Nelken et al.
1965; Harrison, 1965; Marin-Padilla & Ferm, 1965). The observations made in
this investigation are presented and discussed.
MATERIALS AND METHODS
Twenty female golden hamsters were Used in this investigation. The animals
were separated in five groups of four animals. The four animals in each group
were bred on the same day, at the same time, and kept under identical general
conditions. Two animals in each group received 20000 u of vitamin A by gavage
on the 8th day of gestation at 8 a.m. The other two animals in each group were
used as controls and received nothing. The use of a control animal for each
experimental animal was an absolute requirement in this investigation since
the observations of early changes must be based on a continuous comparative
analysis of treated and control animals.
The two treated and the two control animals in each group were sacrificed
as follows. The first group at 3 h; the second at 6; the third at 8; the fourth at
10; and the fifth at 12 h after the administration of vitamin A to their pregnant
mothers.
The pregnant uteri of treated and control animals were submitted to the
following procedure. Dissection of the uterus in its individual gestational areas
was followed by a superficial coronal incision of the uterine musculature at each
gestational area. By this method the decidual casts with the embryonic cavities
were released from the uterine cavity either spontaneously or after gentle
manipulation. These decidual casts are egg-shaped with a superior pointed area
containing the embryonic cavity and an inferior rounded one composed of
decidua. The decidual casts were then divided into two halves. The superior
one with the embryonic cavity was fixed immediately in Bouin's solution; the
inferior one was discarded. After 24 h of fixation, nine to twelve embryonic
cavities thus obtained can be mounted as a single group in paraffin. The paraffin
blocks were serially sectioned. Approximately one-third of these sections contain
embryonic tissue. All sections were stained with hematoxylin and eosin. A
total of 64 000 sections from both treated and control animals were examined.
Mesodermal alterations
p.
.
263
RESULTS
First group
No appreciable changes or alterations were noted in the embryos recovered
3 h after the administration of vitamin A when compared with the control
embryos.
Second group
Alterations become apparent in embryos recovered 6 h after the administration of vitamin A. These alterations are minimal and subtle, and only appreciated after extensive comparative analysis with control embryos. The alterations
are only present in the mesodermal tissue of the cephalic region. They consist of
a slight shrinkage of the cytoplasm of the mesodermal cells and an increase in
the size of the intercellular spaces. No changes are seen in the nuclei of the
mesodermal cells. The enlargement of the intercellular spaces appears to be
the result of accumulation of fluid. Dilatation of the marginal vascular spaces
(sinusoids) directly under the cephalic neuro-ectoderm is encountered in some
embryos. No appreciable changes are seen in the axial non-segmented mesoderm
of the embryonic body or in the somites of the embryos of this group.
Third group
Similar but more marked alterations in the cephalic mesoderm are found in
embryos recovered 8 h after the administration of vitamin A. Also, shrinkage
of the cytoplasm and an enlargement of the intercellular spaces become apparent
in the axial non-segmented mesoderm of the embryonic body in the embryos
of this group. No distinct alterations in the somites are found in these embryos.
Fourth group
The mesodermal damage induced by vitamin A reaches a maximum 10 h
after the administration of the vitamin to the pregnant hamsters. In view of this,
the mesodermal alterations and changes observed in this group of embryos will
be presented in more detail. All mesodermal elements are affected.
The cephalic mesoderm is the more severely affected region of these embryos.
The alterations observed in this region remain the same as in the previous
groups of embryos, namely shrinkage of the cell cytoplasm and enlargement of the
intercellular spaces due to accumulation of fluid (compare Plate 1,figs.A-B and
C-D). The shrinkage of the cytoplasm of the mesodermal cells is so severe that
some of them appear to be totally deprived of it. In the cephalic mesoderm the
number of mesodermal cells (compare figs. A-B and C-D) and the number of
mitoses appeared to be reduced in the treated embryos when compared with the
controls. The enlargement of the intercellular spaces has reached its maximum
in these embryos (compare figs. A-B and C-D), and the vascular spaces are
also greatly dilated, especially branches of the dorsal aortae and the marginal
vascular sinusoids under the cephalic neuro-ectoderm. The neuro-ectodermal
17-2
264
M. MARIN-PADILLA
tissue covering the altered cephalic mesoderm does not appear to be affected
and shows a normal number of mitoses (compare figs. C-D). However, a slight
reduction in the thickness of the cephalic neuro-ectoderm is observed in some
embryos.
The axial non-segmented mesoderm of the embryonic body of the treated
embryos show similar but less marked changes to those observed in the cephalic
mesoderm. The mesodermal cells show shrinkage of their cytoplasm and the
intercellular spaces are enlarged (compare figs. E-F and G-H). The number of
cells of the axial non-segmented mesoderm appears to be reduced as well as the
number of mitoses in the treated embryos when compared with the controls.
Some treated embryos appear slightly retarded in their development and the
neural tube has not yet closed (compare figs. G-H). The neuro-ectoderm of
these embryos appears to be within normal limits.
The somites of some embryos of this group appear also affected, they are not
as clearly established and well delineated as in the control embryos (compare
EXPLANATION OF PLATES
NOTE. All the sections depicted in thefiguresbelong to embryos recovered 10 h after the
administration of vitamin A unless otherwise stated.
PLATE 1
Fig. A. Cross-section of the superior portion of the cephalic region of a control embryo
showing the neuro-ectoderm in the superior and inferior edges and the endodermal tube in
the center. Notice the richness and abundance of cells of the cephalic mesoderm. (H and E.,
x 160.)
Fig. B. Cross-section of the superior portion of the cephalic region of a treated embryo,
showing the neuro-ectoderm in the superior and inferior edges and a central endodermal
tube. Notice the shrinkage of mesodermal cells, the enlargement of intercellular spaces and
the dilatation of the vascular and of the marginal sinusoids. (H. and E., x 160.)
Fig. C. Cross-sections of the head region of a control embryo showing the anterior (left) and
the posterior (right) areas of the neuro-ectoderm and the central endodermal tube. Notice the
richness and abundance of cells in the mesodermal tissue and in the maxillary processes
(arrows). The neuro-ectoderm shows abundant superficial mitosis. (H. and E., x 160.)
Fig. D. Cross-section of the head region of a treated embryo showing the anterior (left) and
the posterior (right) areas of the neuro-ectoderm and a central endodermal tube. Notice the
mesodermal changes, the dilatation of the intercellular and vascular spaces and the reduction
of the number of mesodermal cells elsewhere and in the maxillary process (arrow). The
neuro-ectoderm shows abundant superficial mitosis. (H. and E., x 150.)
Fig. E. Cross-section of the middle thoracic region of a control embryo, showing the neural
tube and the somite (left) and the pericardial cavity with the heart (right). Notice the richness
and abundance of cells in both the non-segmented and in the somitic mesoderm and the
superficial mitosis of the neural tube. (H. and E., x 140.)
Fig. F. Cross-section of the middle thoracic region of a treated embryo showing the neural
tube and somite (above) and the pericardial cavity with the heart (below). Notice the scanty
cellularity in both the non-segmented and the somite mesoderms and the superficial mitosis
of the neural tube. The somites are poorly formed and cannot be easily identified. (H. and
E., xl40.)
J. Embryo/, exp. Morph., Vol. 15, Part 3
PLATE
M. MAR IN-PADILLA
facing p. 264
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PLATE 2
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facing p. 265
Mesodermal alterations
265
figs. I-J). The affected somites have also a smaller number of cells (compare
figs. E-F and I-J). The somites most frequently affected are those of the cephalic
and cervical regions. The dorsal, lumbar and sacral somites are little affected
or not at all. The mesodermal concentrations in the cephalic region such as
the maxillary processes, although not affected themselves, are surrounded by an
altered mesoderm. In some treated embryos the maxillary processes give the
impression of a loose arrangement of their cellular component when compared
with controls (compare figs. C-D).
In addition to the alterations described, the mesoderm of the treated embryos
shows generalized changes. These changes appear to be the direct consequence
of the mesodermal alterations. The changes consist of a mesodermal derangement and, more importantly, of a mesodermal collapse. The first consists of a
disorganization of the mesodermal and somitic cells. The second, as its name
indicates, is literally a collapse of the mesodermal tissue, or, in other words,
a breakdown of the mesoderm as a whole. The term collapse, however, describes
better the phenomenon observed in the mesoderm of these embryos (figs. K, L).
It is most frequently encountered in the cephalic region of the embryo. The
cephalic mesoderm collapse causes an exterioration, an exposure and a flattening
of the cephalic neuro-ectoderm (fig. K). The exposed and flat neuro-ectoderm of
the cephalic region does not appear to be affected. It gives the false impression
of an overgrowth over the cephalic mesoderm (figs. K, L). However, the
PLATE 2
Fig. G. Cross-sections of the thoracic and lumbar regions of a control embryo, showing the
richness and cellularity of the non-segmented and somitic mesoderms. (H. and E., x 80.)
Fig. H. Cross-sections of the thoracic and lumbar regions of a treated embryo, showing the
alteration in the non-segmented and somitic mesoderms. Also, this embryo shows a retardation in the closure of the neuro-ectoderm which may be caused by the altered underlying
mesoderm. (H. and E., x 80.)
Fig. I. Longitudinal section of the body of a control embryo to show the somites. Notice the
richness and abundance of cells in the somites and the clear demarcation of the individual
somite. (H. and E., x 140.)
Fig. J. Longitudinal section of the body of a treated embryo to show the affected somites.
With the exception of the large somite (left) the other somites (arrows) are not clearly established and they are composed by a smaller number of cells. (H. and E., x 140.)
Fig. K. Oblique section of the cephalic and thoracic region of a treated embryo to show the
typical mesodermal collapse with the exposure and flattening of the cephalic neuro-ectoderm.
The cephalic mesoderm also depicts the typical alterations. The neuro-ectoderm gives the
false impression of overgrowth above the collapsed mesoderm. This embryo represents the
earliest example of vitamin A induced cranioschisis (10 h after administration). (H. and E.,
xlOO.)
Fig. L. Oblique section of the cephalic and thoracic regions of a treated embryo recovered
12 h after the administration of vitamin A to show the persistence of the typical mesodermal
collapse. This embryo represents another example of the earliest form of experimentally
induced cranioschisis (12 h after administration). (H. and E., x 100.)
266
M. MARIN-PADILLA
reason for this false impression is actually the collapse of the underlying cephalic
mesoderm.
Occasionally an abnormal focus of cellular necrosis is encountered in the
cephalic mesoderm of treated embryos. No necrosis in the somites is present at
this time.
Fifth group
The mesodermal alterations, shrinkage of cell cytoplasm and enlargement of
intercellular spaces are also present in treated embryos of this group. However,
these alterations are less accentuated than in the previous group as if these
embryos were already recovering from the teratogenic insult. The mesodermal
collapse of the cephalic region with flattening and exposure of the neuroectoderm is still present and prominent in these embryos (fig. L). Cellular
necrosis of mesodermal cells of the cephalic region and of the cephalic neuroectoderm are observed in the treated embryos. An occasional somite in the
cephalic and in the cervical region may depict focal cellular necrosis.
DISCUSSION
The administration of vitamin A to the pregnant hamster is followed almost
immediately (6 h) by mesodermal alterations in the embryos which reach their
maximum severity after 10 h.
The mesodermal regions of the embryo are affected in the following order:
the cephalic mesoderm, the axial non-segmented mesoderm and, finally, the
segmented mesoderm, especially the somites, The nature of these mesodermal
alterations is not known. From other sources it is known that vitamin A plays
an important role in the permeability of the cellular membrane and that its
excess causes instability of the membranes (Lucy & Dingle, 1964). Also, it has
been shown that vitamin A in excess inhibits the formation of new blood vessels
(Nelken et ah 1965) and the growth of a rabbit's papilloma by apparently
affecting its dermal (mesoderm) support (Harrison, 1965). It is possible then
that biochemical alterations of the cell metabolism and disturbance of the cell
membrane may explain the shrinkage of the cell cytoplasm and the accumulation of fluid in the extracellular space of the vitamin A induced mesodermal
alteration in young embryos.
The somite alterations induced by vitamin A occur later than the mesodermal
alterations and consist of the formation of abnormal somites, and later in
somite necrosis. The cause of these somite alterations is not known. Two possibilities may be considered. They may be the result of a direct effect of vitamin A
upon the somite cells as previously suggested (Marin-Padilla & Ferm, 1965), or
they may be the result of an indirect effect secondary to the primary alterations
in the axial non-segmented mesoderm. This last possibility appeared more
likely in view of the present observations. It is also supported by the observations
Mesodermal alterations
267
of other investigators. Gallera (1951) suggested that early mesodermal (chordomesodermal) disturbances may cause somite alterations and platyneuria
(craniorhachischisis) in chicks. Stephan & Sutter (1960) described blockage of
cellular proliferation in the undifferentiated axial mesoderm and in the somitic
centers and also the formation of edema fluid in chick embryos treated with
trypan blue. Jurand (1963) observed early mesodermal disorganization, lack
of segmentation and, later, somite necrosis (in severe cases) in the mouse after
the administration of a nitrogen mustard derivative. Griineberg (1963), discussing some malformations of the axial skeleton, states that disturbances of
segmentation may secondarily cause somite alterations. Hicks (1954) induced
severe skeletal malformations (anencephaly) after X-irradiation of young
mammalian embryos before the formation of somites, or during one or two
somite stages. Whatever the nature of the mesodermal alterations may be, they
are of great significance for the understanding of vitamin A induced malformations. They are the first appreciable alterations induced by vitamin A and
the cause of significant changes in the mesodermal tissue.
In view of the observations presented here, those made from the previous
experimental studies described above, and those made from the study of human
axial skeletal malformations, the embryogenesis of these malformations can
now be better understood. Axial skeletal malformations such as cranioschisis,
craniorhachischisis and rachischisis are the result of the same fundamental
disturbances of the mesoderm. This disturbance affects primarily the presomite
cephalic and axial non-segmental mesoderm of the body of the embryo and
secondarily the somitic mesoderm. Direct consequences of these alterations are
a derangement of mesodermal cells and, more significantly, a mesodermal
collapse. If the mesodermal collapse occurs before the closure of the neural
tube it will cause exposure, exterioration and flattening of the neuro-ectoderm.
The neuro-ectoderm thus affected and exposed obviously will fail to close, not
as a primary neuro-ectodermal disturbance but as a failure of the collapsed
mesoderm to enclose it. The exposed neuro-ectoderm which is not affected at
first continues its development causing the false impression of an overgrowth
over the collapsed mesoderm. Later the neuro-ectoderm starts to fold, causing
the typical appearance of exencephaly. This phenomenon of apparent overgrowth and folding of an exposed nervous tissue has been beautifully shown in
the experimentally induced exencephaly which follows the opening of the roof
of the rhombencephalon in the chick (Jelinek, 1960; Klika & Jelinek, 1961).
Even later in development, a degeneration of the exposed neuro-ectoderm begins,
resulting in its destruction (anencephaly). Thus, the embryos depicted in figs.
K and L may be regarded as the youngest known examples of vitamin A
induced cranioschisis. If the mesodermal alterations occur after the closure of
the neural tube only skeletal malformations will occur. The secondary alterations of the somites (abnormal somites and somite necrosis) and of the notochord (duplication and deviations) appear to influence the final morphology of
268
M. MARIN-PADILLA
the skeletal malformations (Marin-Padilla, 1965 a; Marin-Padilla, 1966). The
teratogenic effects of vitamin A in the golden hamster in relation to the embryogenesis of the skeletal malformations are graphically expressed in Table 1.
Table 1. Tetratogenic effects of vitamin A on the hamster embryo
Injury to the somites1
/
Biochemical injury to the /
presomite (cephalic and (
non-segmented) mesoderm\
Failure to enclose the1
developing notochord
Skeletal malformations (cranioschisis, craniorhachischisis and
rhachischisis) with degeneration
of exposed areas of neural tissue
Cephalic and axial mesodermal
derangement and collapse
Failure to enclose the
developing neural tissue
1
Interaction of these factors influence the final morphology of the skeletal malformations.
SUMMARY
The administration of a single dose (20000 u) of vitamin A by gavage to the
pregnant hamster is followed almost immediately by changes in the mesoderm
of the embryos. These changes appear 6 h after the administration and after
10 h they have reached their maximum severity. The alterations appear first in
the cephalic mesoderm, later in the axial non-segmented mesoderm of the body
of the embryo and finally in the somitic mesoderm. They consist of shrinkage
of the cell cytoplasm, enlargement of the intercellular spaces (fluid accumulation)
and reduction of the mitotic rate of mesodermal cells. These alterations precede
the cellular necrosis which appeared later in the somites and is considered a
secondary change. An analysis of the embryogenesis of the vitamin A induced
skeletal malformation based on the changes described is discussed.
RESUME
Alterations du mesoderme produites par une hypervitaminose A
L'administration d'une dose unique (20.000 ju) de vitamine A par le gavage
d'une hamster gravide est suivie presque immediatement par des changements
dans le mesoderme de l'embryon. Ces alterations apparaissent 6 heures apres
radministration et atteignent leur maximum apres 10 heures. Elles se manifestent d'abord dans le mesoderme cephalique, ensuite dans le mesoderme axial
non segmente et finalement dans le mesoderme somitique. Elles consistent en
une retraction du cytoplasme des cellules, agrandissement des espaces inter-
Mesodermal alterations
269
cellulaires (accumulation de liquide) et une reduction du taux des mitoses des
cellules mesodermiques. Ces alterations precedent les necroses cellulaires qui
sont apparues ulterieurement dans les somites et qui sont considerees comme
secondaires. Une analyse de Pembryogenese des malformations squelettiques
induites par la vitamine A, et basees sur les changements qui ont ete decrits, est
discutee.
This investigation was supported by USPHS Grant GM 10210.
REFERENCES
A. C. & HOUCK, J. C. (1964). Connective tissue. IX. Effects of hypervitaminosis A
upon connective tissue chemistry. Proc. Soc. exp. Biol. Med. 115, 631-3.
GALLERA, T. (1951). Influence de l'atmosphere artificiellement modifiee sur le developpement
embryonnaire du poulet. Acta Anat. 11, 549-85.
GRUNEBERG, H. (1963). The Pathology of Development. Disorder of Segmentation. Pp. 170.
Oxford: Blackwell.
HARRISON, M. (1965). Inhibition of growth of Shope rabbit papilloma by hypervitaminosis A.
Cancer Res. 25, 947-55.
HICKS, S. (1954). Mechanism of radiation anencephaly, anophthalmia and pituitary anomalies. Repair in the mammalian embryo. A.M.A. Archs Path. 57, 363-78.
JELINEK, R. (1960). Development of the experimental exencephalia in the chick. Cslkd Morf.
8, 368-78.
JURAND, A. (1963). Anti-mesodermal activity of a nitrogen mustard derivative. /. Embryol.
exp. Morph. 11, 689-96.
KLIKA, E. & jELfNEK, R. (1961). Histogenesis of the experimental exencephalia in the chick.
Cslkd Morf. 9, 162-73.
LUCY, J. A. & DINGLE, J. T. (1964). Fat soluble vitamins and biological membranes. Nature,
Lond., 204 (1), 156-60.
MARIN-PADILLA, M. (1965 a). Study of the sphenoid bone in human cranioschisis and craniorhachischisis. Virchows Arch. path. Anat. Physiol. 339, 245-53.
MARIN-PADILLA, M. (19656). Study of the skull in human cranioschisis. Ada Anat. 62,
1-20.
MARIN-PADILLA, M. (1966). Study of the vertebral column in human craniorhachischisis.
The significance of the notochordal alteration. Acta Anat. (in the Press).
MARIN-PADILLA, M. & FERM, V. (1965). Somite necrosis and developmental malformation
induced by vitamin A in the golden hamster. /. Embryol. exp. Morph. 13, 1-8.
NELKEN, D., PICK, E., GABRIEL, M., BITTERMAN, W. & Boss, J. H. (1965). Vitamin A induced
rejection of autographs and nomographs. Nature, Lond., 205 (2), 1022.
STEPHAN, F. & SUTTER, B. (1960). Action du blue trypan sur la differentiation du mesoderm
axial chez l'embryon de Poulet. C. r. Seanc. Soc. Biol. 154, 1620-2.
WEISSMANN, G. (1964). Labilization and stabilization of lysosomes. Fedn. Proc. Fedn. Am.
Socs. exp. Biol. 23, 1038-44.
CHUNG,
(Manuscript received 8 November 1965)