/ . Embryol. exp. Morph. Vol. 53, pp. 15-38, 1979
Printed in Great Britain © Company of Biologists Limited 1979
\5
Notochordal-basichondrocranium relationships:
abnormalities in experimental axial skeletal
(dysraphic) disorders
By MIGUEL MARIN-PADILLA1
Professor of Pathology, Dartmouth Medical School
SUMMARY
The notochordal-basichondrocranium relationships have been investigated in cranioschisis occulta with encephalocoele (CSO-EN) and in cranioschisis aperta with exencephaly
(CSA-EX) which represent, respectively, a minimal and a severe form of experimentally
induced axial skeletal (dysraphic) disorders. Although apparently different, these two
malformations are considered to represent different degrees of the same basic abnormality
(mesodermal insufficiency) which affects the paraxial mesoderm early in embryonic development. Three different experimental models (vitamin A, sodium arsenate and clofibrate)
have been used to induce these disorders. The administration of a single dose of each of
these three agents during the primitive streak stage of embryonic development in the golden
hamster resulted in a variety of axial skeletal (dysraphic) disorders, including the two
mentioned above. Regardless of the teratogen used, the basichondrocranium in both CSOEN and in CSA-EX is shorter than normal and slightly lordotic to the vertebral axis. The
shortness of the basichondrocranium in these two disorders is caused mainly by the shortness of the basioccipital. Both the shortness of the base of the skull and its lordotic or
elevated position in relation to the vertebral axis are more pronounced in CSA-EX than in
CSO-EN. The intracranial course of the notochord in these two axial disorders is considered
to be within normal limits. However, the notochord itself is shorter than normal and depicts
terminal folds which are considered to be the result of crowding. Both the shortness of the
notochord and its terminal folding are also more pronounced in CSA-EX than in CSO-EN.
The sole difference encountered betwesn the abnormal basichondrocraniums of these two
disorders is in the severity of their common malformations (quantitative differences), but
not in their type or quality. These findings give further support to the idea that these two
axial skeletal disorders are not different types of malformations, but different degrees of the
same basic abnormality. The CNS involvement (encephalocoele or exencephaly) is also
considered to be secondary to the paraxial mesodermal insufficiency. It is believed, and
supported by the present findings, that the normal development of the paraxial mesoderm
is needed for the normal process of formation and subsequent elevation and closure of the
neural folds. A primary paraxial insufficiency could result in the partial (encephalocoele) or
the complete (exencephaly) failure of closure of the neural folds which characterizes this
type of axial skeletal (dysraphic) disorder.
In addition, the normal notochordal-basichondrocranium relationships have been investigated in a group of mammalian embryos including man, hamster, mouse, and several others
gathered from the literature. The significant developmental variations of the intracranial
course of the notochord, and of its relationships to the components of the basichondrocranium, found among different mammals have been emphasized and illustrated. The
1
Authcr's address: Department of Pathology, Dartmouth Medical School, Hanover,
New Hampshire 03755, USA.
16
M. MARIN-PADILLA
principal developmental variations encountered are those pertaining to: (a) the overall
intracranial course of the notochord; (b) the specific relationships of the notochord to the
basioccipital and the basisphenoid; and (c) the notochordal relationship (or the lack of it)
to the pharyngeal epithelium, and hence, its close association with the development of the
bursa pharyngea in some mammals (e.g. man). The segmental nature of the notochordal
enlargements of the basioccipital, found in the human and hamster embryos studied here,
as well as their relation to the somitic origin of this bone, have been analyzed and discussed.
The present study has also emphasized the need to understand the developmental variations
of the notochordal-basichondrocranium relationships among mammalian embryos for the
study and interpretation of both clinically, as well as experimentally, induced malformations
of the base of the skull.
INTRODUCTION
A variety of notochordal anomalies (bends, deviations, lack of segmentation,
focal proliferation, branching and duplications) have been described in clinical
(Budde, 1911; Feller & Stemberg, 1929; Johnston, 1932; Saunders, 1943;
Willis, 1958; Dziallas, 1962; Marin-Padilla, \965a, 1966a, 1970, 1978) as well
as in experimentally induced axial skeletal (dysraphic) disorders (Young, 1916;
Harman, 1922; Dawson, 1928; Gruenwald, 1947; Degenhart & Knoche, 1959;
Giroud & Roux, 1959; Giroud, Martinet & Lefebres-Boisselot, 1960; Murakami
& Kameyama, 1963; Marin-Padilla, 19666, 1970, 1978). The significance of
these notochordal anomalies, as well as their relationship to the various sketetal
defects which also characterize these disorders, remains poorly understood.
In addition, the difference between early and late notochordal changes described
in these disorders remains puzzling and unexplained. While only minimal
notochordal deviations (Lendon, 1975) or no apparent changes (Geelen, 1973;
Marin-Padilla, 1970, 1978) have been reported in young embryos with mesenchymal skeletal structures, prominent notochordal changes do occur in older
embryos (similarly treated) with cartilaginous or osseous skeleton (MarinPadilla, 1970, 1978). An explanation for this apparent discrepancy is also
obviously needed.
The intracranial course of the notochord and its relationships to the abnormal
base of the skull will be analysed in a selected group of experimentally induced
axial skeletal (dysraphic) disorders involving the head. The notochordalbasichondrocranium relationships will be investigated in only two basic
experimentally induced disorders: (a) cranioschisis occulta with encephalocoele
(CSO-EN) representing a minimal type of defect, and (b) cranioschisis aperta
with exencephaly (CSA-EX) representing a severe malformation. These two
axial skeletal disorders, in spite of their obvious morphological dissimilarities,
are considered to be different degrees of the same basic abnormality, caused
by an early mesodermal insufficiency (Marin-Padilla, 1978). These two fundamental defects have been selected also because they are considered to be
comparable to those involving the vertebral column (rachischisis occulta with
myelocoele and rachischisis aperta myeloschisis) in axial skeletal (dysraphic)
disorders (Marin-Padilla, 1978). In addition, these two disorders can be easily
Notochordal-basichondrocranium
relationships
17
reproduced experimentally, even in the same litter, with a variety of teratogenic
agents.
It should be pointed out that a prerequisite for this type of study should
be an understanding of the notochordal-basichondrocranium relationships
encountered in the developing mammalian embryo. It seems, therefore, appropriate to present a brief review of the notochordal relationships to the base
of the skull in a selected group of mammalian embryos prior to the presentation
of the experimental data. Such a review will emphasize the normal variations
in the intracranial course of the notochord found among different mammals.
A prior knowledge of these variations is also obviously necessary for the
interpretation of notochordal anomalies.
NOTOCHORDAL-BASICHONDROCRANIUM RELATIONSHIPS
AMONG MAMMALS
In young mammalian embryos, the head notochord runs within the cephalic
mesoderm between the ectoderm of the developing neural tube and the endoderm of the pharynx with minimal or no variations encountered among different
mammals. However, during early chondrification of the basichondrocranium,
the notochord establishes distinct relationships with its components. As the
notochord establishes relationships with the developing basioccipital and
basisphenoid, it assumes a distinct intracranial course which appears to be
characteristic for each mammalian species. In addition, it should be pointed
out that later in development, in the presence of advancing chondrification and
ossification of the base of the skull, the notochord undergoes degenerative
changes and finally disappears altogether. Therefore, in the study of the intracranial notochord, the developmental stage of the embryo is of considerable
importance, because in advancing chondrification, its course could be altered.
The intracranial course of the notochord will be described in detail in human,
hamster and mouse embryos. This will be followed by a brief analysis of the
intracranial course of the notochord in other mammalian species gathered
from the literature. The selection of the human material is due to my long
interest in the morphogenesis of developmental malformations of the base of
the skull and in the possible role played by the notochord in these disorders.
The intracranial course of the hamster's notochord has been selected because,
as far as I know, it has not been previously studied, and because the hamster
is the experimental animal utilized in the present study. The mouse has been
selected because the intracranial course of the notochord in this animal represents a unique example of this phenomenon in which the notochord remains
dorsal to and outside the entire basichondrocranium. These three examples
have been also selected because each one illustrates a very different pattern of
relationships between the head notochord and the basal plate, and is therefore
representative of the variations encountered among different mammals.
The embryos studied were processed in my laboratory and included: two
18
M. MARIN-PADILLA
human embryos obtained from hysterectomy specimens measuring 20-24 mm
in length respectively; ten 14-day-old hamster fetuses measuring between 13
and 14 mm in length; and ten 13-day-old mouse fetuses measuring between
10 and 11 mm in length. All embryos were fixed in Bouin's solution, their
heads serially sectioned (sagittally) and stained with hematoxylin and eosin.
Man
The human notochord on emerging from the vertebral axis through the
odontoid process of the second vertebra, bends slightly and forms a distinct
enlargement dorsal and posterior to the base of the skull. It then enters and
crosses diagonally the posterior region of the basioccipital, where it forms
two noticeable enlargements (Fig. \A, arrow). On leaving the basioccipital
ventrally, it establishes one or more contacts with the pharyngeal epithelium
(Fig. 1B). It then continues cephalad under the basioccipital and turns dorsally,
penetrating and terminating within the basisphenoid near the region of the
hypophysis (Fig. 1^4). The notochord terminates in a few short branches.
In essence, this description coincides with and confirms the intracranial
course of the human notochord described by early investigators (Gage, 1906;
Bardeen, 1908; Fawcett, 1910; Huber, 1912; Macklin, 1914). However, Williams
(1908) depicts the human notochord within the basal plate in its entirety
without recognizable contacts with the pharyngeal epithelium. This particular
intracranial course of the notochord could represent a normal variation,
according to some investigators (Froriep, 1882; Killian, 1888; Huber, 1912;
Snook, 1934), who pointed out that pharyngeal contacts are present in about
50 % of the embryos studied.
FIGURE 1
Composite figure (A) of a mid-sagittal section of the basichondrocranium of a
24 mm human embryo (51st day of gestation) illustrating the course of the headnotochord and its relationships to the components of the base of the skull and
other axial cephalic structures. The notochord of this embryo contacts the
epithelium of the bursa pharyngea (BP) and forms, within the posterior region of
the basioccipital (BO), two noticeable and distinct enlargements. One of these
notochordal enlargements is clearly visible in the posterior portion of the basioccipital (arrow). These enlargements are considered to represent segmental remnants of the notochord (somitic origin of the occipital bone) homologous to the
notochordal enlargements formed between the anlage of the vertebrae. The
notochord terminates within the basisphenoid (BS) forming several short branches.
Other structures illustrated include: the hypophysis (H); the Rathke's pouch (R)
clearly separated and distinguishable from the bursa pharyngea; the tongue (T);
the pharynx with the entrance to the larynx (L) and the esophagus (E); and the
basilar artery (BA) under the developing nervous system (N). H & E, x 50. B
represents a higher magnification of the rectangular area outlined in A, illustrating
the notochordal relationships to the epithelium of the bursa pharyngea. The notochord runs ventral to the cartilage of the basioccipital (large arrows) and sends
short branches (small arrows) which seem to make contacts with the epithelium
forming the bursa. H & E, x 400.
Notochordal-basichondrocranium relationships
20
M. MARIN-PADILLA
The distinct contact between the human notochord and the epithelium of
the bursa pharyngea illustrated in this study (Fig. 1B) has also been described
by early (Froriep, 1882; Killian, 1888; Gage, 1906; Huber, 1912) as well as
by recent (Snook, 1934; Slipka, 1972) investigators. The bursa pharyngea
appears to be a peculiar human developmental structure seldom described
in other mammals (Tourneux, 1912). Practically nothing is known about the
significance or function of this structure of which only a mere mention can
be found in leading books on human embryology.
In the human embryos, as well as in the hamster embryos studied (see later),
the notochord penetrates the posterior region of the basioccipital, where it
forms noticeable focal enlargements (Figs. 1, 2, 5, 7, 10) which are similar to
those described by Slipka (1974) in pig embryos. I fully agree with Slipka's
interpretation concerning the segmental nature of these occipital notochordal
enlargements. He considers them to be homologous to the notochordal enlargements formed between the anlage of the developing vertebrae. The
occipital bone (basioccipital, lateral occipital, and the planum nuchale of the
squama) develops, as do the vertebrae, from three pairs of somites which
constitute rudimentary occipital vertebrae (De Beer, 1937; Hamilton, Boyd &
Mossman, 1972). The two occipital notochordal enlargements described in
human, hamster and pig embryos could represent the intermediate segments
between the three rudimentary occipital vertebrae. The notochordal enlargement
formed between the vertebral column and the basioccipital, which is recognizable
in most of the embryos studied herein, could well represent the third segment.
The segmented (somitic) nature of the occipital bone disappears early in
development when its various elements fuse together to form a single structure
FIGURES 2 AND 3
Fig. 2. Mid-sagittal section of the basichondrocranium of a 14 mm hamster embryo
(14th day of gestation) illustrating the intracranial course of the notochord and its
relationships to other axial cephalic structures. The somewhat advancing chondrification of the base of the skull of this embryo has obscured the notochordal
course, making it unclear at this magnification. In view of this, the notochord
has been outlined with black ink, making it more clearly visible in the figure. A
typical notochordal enlargement within the posterior region of the basioccipital
is marked by an arrow. Other axial cephalic structures illustrated include: the
hypophysis (H); the basioccipital (BO) and basisphenoid (BS); the basilar artery
(BA); the palate (P), tongue (T), larynx (L) and esophagus (E) and the Rathke's
pouch (R). H & E, x 40.
Fig. 3. Mid-sagittal section of the basichondrocranium of an 11 mm mouse
embryo (12th day of gestation) illustrating the intracranial course of the notochord
and its relationships to other axial cephalic structures. The notochordal borders
have been lightly outlined with white ink to make the chorda more clearly visible
at this magnification. The notochord of the mouse remains dorsal to the entire
basichondrocranium, thus representing an interesting variation of this developmental phenomenon. The captions of this figure are self-explanatory (see also
Figs. 1 and 2). H & E, x 70.
Notochordal-basichondrocranium relationships
21
22
M. MARIN-PADILLA
which then becomes incorporated into the base of the skull. The presence of
more than one hypoglossal nerve canal in the lateral occipitals is considered
to be an indication of the segmental origin of this bone (De Beer, 1937).
Furthermore, in some pathological conditions known to affect the vertebrae
specifically, such as some chondrodystrophies, the occipital bone is also affected,
behaving in these cases more as a vertebra than as a component of the base
of the skull (Marin-Padilla & Marin-Padilla, 1977).
Hamster
The intracranial course of the notochord in the golden hamster (Fig. 2) also
represents a unique example of this developmental phenomenon. The notochord, on emerging from the vertebral axis, bends slightly and forms a distinct
enlargement dorsal and posterior to the basioccipital. It then penetrates and
crosses diagonally the posterior region of the basioccipital. On leaving the
basioccipital, the notochord runs a short distance ventrally and outside the
cartilage. It then turns dorsally and crosses again, diagonally, the basioccipital
to emerge at its dorsal surface. From this point it continues cephalad to terminate near the region of the hypophysis, remaining dorsal to the basisphenoid
without penetrating it.
In some embryos the notochord forms noticeable enlargements within the
posterior region of the basioccipital (Fig. 2, arrow) similar to those described
in the human embryo. It should be pointed out that at the developmental
stage studied here, the notochord does not establish recognizable contacts with
the pharyngeal epithelium. However, such a possibility could occur in younger
embryos because the head notochord in the hamster runs for a short distance
under the basal plate and therefore close to the pharyngeal epithelium. Further
investigation is needed to clarify this point.
Mouse
The intracranial course of the notochord in the mouse is quite different from
that found in man or hamster (Fig. 3). It represents an interesting variation
of this phenomenon because the notochord does not penetrate the basioccipital.
The mouse head notochord, on emerging from the vertebral axis, bends slightly
and forms a noticeable enlargement posterior to the basioccipital. It then
remains dorsal to the basioccipital and basisphenoid in its entirety without
penetrating them. It terminates dorsal to the basisphenoid near the region of
the hypophysis. Although the notochord appears to be very close to the
cartilage of the basal plate (Fig. 3), it is distinctly separated from it. The mouse
head notochord does not come near the pharyngeal epithelium at any point.
The intracranial course of the notochord in the mouse described here confirms
the observations of Tourneux & Tourneux (1912).
The intracranial course of the notochord and its relationships to the basi-
Notochordal-basichondrocranium relationships
23
chondrocranium have been investigated in few other mammalian embryos.
Some examples have been gathered from the literature and tabulated (Table 1)
to facilitate their analysis and to illustrate the variations encountered among
different mammals.
MATERIALS AND METHODS
Pregnant golden hamsters treated with one of three experimental compounds
-vitamin A, sodium arsenate and clofibrate (an antilipidemic agent)-have
been utilized in this study. The administration of a single dose of one of these
three teratogenic agents to a pregnant hamster early in gestation, during the
primitive streak stage of development, resulted in a variety of axial skeletal
(dysraphic) disorders, some of which involve specifically the cephalic region.
Only embryos of the same litter with small encephalocoele (cranioschisis
occulta) and with exencephaly (cranioschisis aperta) were selected for study.
The embryos were obtained between the 13th and the 14th day of gestation,
during the cartilaginous stage of skeletal development. The embryos were
fixed in Bouin's solution. After fixation, their heads were serially sectioned
(sagittally) and stained with hematoxylin and eosin. Complete reconstructions,
using serial sections of the entire basichondrocranium, were done in a few
selected cases.
The doses used were: a single oral dose of 20000 i.u. vitamin A; an intravenous injection of 20 mg/kg sodium arsenate; or an intravenous injection of
lOOmg/kg clofibrate. The administration of these agents was done early in
the morning of the eighth day of gestation in the hamster. Some of the teratogenic effects of these agents have been previously investigated (Marin-Padilla
& Ferm, 1965; Marin-Padilla, 19666; Carpenter & Ferm, 1977; Ferm, Saxon
& Smith, 1971; Ferm, 1978).
OBSERVATIONS
The present study has emphasized the significant variations in the intracranial course of the notochord encountered among different mammals.
Although some of these variations were pointed out by early investigators
(Gage, 1906; Bardeen, 1908; Williams, 1908; Fawcett, 1910; Huber, 1912;
Rand, 1917), they have remained practically ignored [excepting De Beer's
(1937) monumental work] and are not generally recognized. Attention is called
in this report to concerned investigators about these developmental variations,
because such knowledge should be a prerequisite in any teratological study.
Once the intracranial course of the notochord in the hamster has been
established and compared with that of other mammals, it is possible to undertake the study of its developmental abnormalities. The hamster's intracranial
notochordal course represents a unique example in which the chorda crosses
the basioccipital twice and terminates dorsal to the basisphenoid without
penetrating it.
Cat
Armadillo (Dasipus)
Mole {Taipei)
Armadillo (Tatusia)
Rat
Mouse
Hamster
Rabbit
Mammal
Posterior
basioccipital
Dorsal + inside
Dorsal + inside
Dorsal + inside
Dorsal + inside
Dorsal + inside
Dorsal
Dorsal
Dorsal
Dorsal
Dorsal
Dorsal
Ventral
Dorsal + inside
Dorsal + inside
Dorsal + inside
Dorsal + inside
C-R
length
(mm)
12-5
12-5
17
45
13-14
10-11
12
9
9-15
9-13
10-12
17-5
7-5
19-27
15
23-32
Ventral
Ventral
Inside
Inside
Inside
Dorsal
Dorsal
Dorsal
Dorsal
Dorsal
Dorsal + inside
Ventral
Ventral
Ventral
Ventral
Ventral + inside
Anterior
basioccipital
Dorsal
Dorsal
Dorsal
Dorsal
Dorsal
Inside +
ventral
Ventral
—
Inside
Inside
Inside
Inside
Inside
Inside
Inside
Dorsal
Basisphenoid
—
—
—
—
—
—
—
—
—
One or two
enlargements
—
—
—
—
—
—
Occipital chordal
enlargement
De Burlet, 1913
De Beer, 1937
De Beer, 1937
Williams, 1908
De Beer, 1937
Marin-Padilla, 1979
Tourneux & Tourneux, 1912
Huber, 1912
Tourneux & Tourneux, 1912
Geelen, 1973
De Beer, 1937
De Beer & Woodger, 1930
Voit, 1909, 1911
Huber, 1912
De Beer, 1937
Marin-Padilla, 1979
Authors
The intracranial course of the notochord in several mammalian species gathered from the literature, including those described
in this report. The course of the head notochord is illustrated by determining its relationship to three basic areas of the
basichondrocranium, namely: the anterior and posterior regions of the basioccipital and the basisphenoid. The position of the
notochord is recorded as dorsal, ventral or inside each of the three selected areas of the basichondrocranium. The table illustrates
the significant developmental variations encountered among different mammals, and the occipital notochordal enlargements.
Table 1. Variations of the notochordal-basichondrocranium relationships among mammals
f
***
O
'
Dorsal + inside
Dorsal + inside
Dorsal + inside
Dorsal + inside
Dorsal + inside
Ventral + inside
20
30
36-40
19
27
92
Horse
Bull
Seal (Weddell's)
Whale (Humpback)
Man*
17-88
6-70
20-32
43
20-24
40
40
20
Dorsal + inside
Dorsal + inside
18-21
19-25
19-21
Dorsal + inside
Dorsal + inside
27
17
Dog
Pig
Mammal
Posterior
basioccipital
C-R
length
(mm)
Inside
Ventral, ±
pharyngeal
contact
Inside
Inside
Inside
Inside
Dorsal
Inside
Inside
Inside
Huber, 1912
De Beer, 1937
Tourneux, 1912
Slipka, 1974
K)
a-
*••+.
5'
a
o
a
r
o
Tourneux & Tourneux, 1912
Rand, 1917
"—4.
o
Authors
DeBeer, 1937
Fawcett, 1918
De Beer, 1937
Gage, 1906
Fawcett, 1910
Bardeen, 1908
Two enlargements Marin-Padilla, 1979
Link, 1911
—
De Beer, 1937
—
Macklin, 1914
—
Froriep, 1882
—
Huber, 1912
—
Williams, 1908
—
—
—
—
—
—
—
—
—
enlargements
Two
—
Inside
Inside
Inside
Occipital chordal
enlargement
Basisphenoid
Ventral + inside
Inside or ventral+
pharyngeal contact
Inside or ventral +
pharyngeal contact
Inside
Inside
Inside or ventral +
pharyngeal contact
Ventral
Inside
Dorsal
>
Anterior
basioccipital
Table 1 (cont.)
26
M. MARIN-PADILLA
The external morphological features of experimentally induced cranioschisis
aperta with exencephaly (CSA-EX) are well known, and therefore there is
no need to describe them again. On the other hand, the cranioschisis occulta
with encephalocoele (CSO-EN) induced by these three teratogenic agents is
analyzed and described here for the first time in detail. The encephalocoeles
are necessarily associated with cranioschisis occulta for obvious reasons. The
membranous neurocranium is absent at the level of the encephalocoele (Fig. 4).
The encephalocoeles with cranioschisis occulta (CSO-EN) induced by these
three teratogens are all similar types of defect. All of them consist of a small
saccular distension of the top of the head located in the midline which is
clearly visible to the naked eye (Fig. 4, insert). All of them are small saccular
dilatations of the dorsal wall of the cerebral aqueduct, and are located posterior
to the pineal invagination (Fig. 4, arrow). Some may be more anterior than
others. The wall of the encephalocoele at its central region is very thin, rhomboidal in shape, and somewhat tense due to the pressure of the cerebrospinal
fluid (Fig. 4). The lateral edges of the thin central rhomboidal region of the
encephalocoele fuse with the thick neuroectoderm in which nerve elements
are recognized.
The wall of the encephalocoele at its central region is composed of three
different layers of flattened cells which are clearly recognized by light microscopy. The outer layer is recognized as atrophic and very thin surface ectoderm
(epidermis); the mid-layer is composed of mesodermal cells and a few capillaries
(dermis); and the inner layer is composed of a single layer of flattened cells,
FIGURES 4 AND 5
Fig. 4. Mid-sagittal section of the head of a sodium arsenate-treated embryo
(14 days old) with cranioschisis occulta and small encephalocoele (arrow). Insert.
Head of another treated embryo of same age, illustrating the anatomical features
of the induced encephalocoele. The encephalocoeles consist of a saccular dilatation
of the dorsal wall of the cerebral aqueduct posterior to the pineal invagination.
The wall of the encephalocoele is very thin and composed of three layers of flattened
cells. The outer layer is atrophic surface ectoderm (epidermis), the mid-layer is
mesodermal tissue with few capillaries (dermis), and the inner layer is often discontinuous or fenestrated, and its cells appear to be of ependymal origin. The
membranous neurocranium is missing at the level of the encephalocoele. Vertical
bar = 1 mm.
Fig. 5. Mid-sagittal section of the basichondrocranium of a sodium arsenate-treated
embryo (14 days old) with cranioschisis occulta and encephalocoele, illustrating
the intracranial course of the notochord and its relationships to the basioccipital
(BO) and the basisphenoid (BS). The basichondrocranium is shorter than normal
and slightly lordotic to the vertebral axis. The notochordal enlargement of the
posterior region of the basioccipital (arrow) and its terminal folding are clearly
illustrated. The notochord is also shorter than normal. The upper border of the
notochord has been outlined with white ink to make it more clearly visible at this
magnification. Other cephalic structures illustrated are: the hypophysis (H); the
palate (P); the tongue (T); the larynx (L); and the entrance of the esophagus (E).
Vertical bar = 0-25 mm.
No tochordal-basichondrocranium relationships
27
28
M. MARIN-PADILLA
and appears in many areas to be discontinuous or fenestrated. Tn the fenestrated
areas, only the mid-layer and the superficial layer are recognized by light
microscopy.
The cells of the inner layer of the encephalocoele, at the edges of the defect,
seem to fuse or become continuous with either the ependymal cells (neuroectodermal origin) or the pia-arachnoidal cells (neural crest origin). Light
microscopy alone has proven to be inadequate to solve the embryonic origin
(nature) of some of the cells of the inner layer of the encephalocoeles. Electron
microscopic studies of the wall of these experimental encephalocoeles are
being carried out at present, and will be reported elsewhere.
BASICHONDROCRANIUM IN CRANIOSCHISIS OCCULTA
WITH ENCEPHALOCOELE
The basichondrocranium of all treated embryos, regardless of the teratogen
used (vitamin A, sodium arsenate or clofibrate), is shorter than normal (Figs.
5, 8, 10) and appears to be similarly affected in all cases [see also Marin-Padilla
(1978) for examples of vitamin-A-induced defects]. The longitudinal reduction
of the basichondrocranium in CSO-EN is of the order of 7-9 % of the normal
control.
The reduction of the longitudinal diameter of the base of the skull in embryos
with encephalocoeles is caused primarily by the shortness of the basioccipital
(Figs. 5, 8, 10). The basichondrocranium of these embryos is also abnormally
elevated or lordotic in relation to the vertebral axis (Fig. 10). In addition, an
analysis of the complete reconstruction of the base of the skull in these embryos
FIGURES 6 AND 7
Fig. 6. Mid-sagittal section of the head of a sodium arsenate-treated embryo
(14 days old) with cranioschisis aperta and exencephaly. The base of the skull
is considerably shorter than normal and lordotic to the vertebral axis. The cranial
cavity of these affected embryos is small and obviously inadequate in size to
lodge and to accommodate the developing brain, which is forced out of it (exencephaly). The exposure of the developing brain to the deleterious effects of the
amniotic fluid causes a progressive degeneration which may result in its total
destruction (anencephaly). Vertical bar = 1 mm.
Fig. 7. Mid-sagittal section of the basichondrocranium of a sodium arsenate-treated
embryo (14 days old) with cranioschisis aperta and exencephaly, illustrating the
intracranial course of the notochord and its relationships to the basioccipital (BO)
and the basisphenoid (BS). The base of the skull, especially the basioccipital, is
considerably shorter than normal and lordotic to the vertebral axis. The notochordal enlargement of the posterior region of the basioccipital (arrow) and the
prominent terminal folds are also clearly illustrated. The notochord is also shorter
than normal. Serial reconstruction of the terminal folds has demonstrated that they
are caused by the crowding of the notochord, forced to grow into a reduced space.
The borders of the notochord have been outlined with white ink to make them
more clearly visible at this magnification. Vertical bar = 0-25 mm.
Notochordal-basichondrocranium relationships
29
EMB 53
30
M. MARIN-PADILLA
has demonstrated a slight reduction of its transverse diameter. Therefore the
overall growth of the basichondrocranium in these induced encephalocoeles is
insufficient, inasmuch as its longitudinal growth is more retarded than its
transversal one.
The notochord in both control and embryos with encephalocoeles (cranioschisis occulta) depicts a similar intracranial course (Figs. 5, 10) which is
considered to be within normal limits. As the notochord emerges from the
odontoid process of the second vertebra, it bends and penetrates the basioccipital and crosses it. It then runs cephalad for a short distance under the
cartilage before bending and crossing the basioccipital again. It terminates near
the region of the hypophysis, remaining dorsal to the basisphenoid and without
penetrating it (Figs. 5, 7, 9, 10). The typical notochordal enlargement of the
posterior region of the basioccipital is recognizable in all embryos with
encephalocoeles (Figs. 5, 10). In some treated embryos, the notochordal
enlargement of the basioccipital appears to be slightly larger and more prominent than in control embryos (Fig. 5, arrow).
Although the intracranial course of the notochord in induced encephalocoeles
is within normal limits, the notochord itself is shorter than normal (Fig. 10).
In addition, the notochord depicts terminal bends or angulations which are
not found in control embryos (Figs. 5, 10). These types of terminal notochordal
bends are quite prominent in embryos with cranioschisis aperta and exencephaly
(Figs. 7, 9, 10) of the same litter. Only the reconstruction of the entire basichondrocranium in these treated embryos permits the visualization of the
complete notochord. The study of these reconstructions has demonstrated that
the terminal bends are indeed a continuous folding of the notochord in an
accordion-like fashion. This type of folding suggests a crowding of the developing notochord, which must grow in a reduced space.
BASICHONDROCRANIUM IN CRANIOSCHISIS APERTA
WITH EXENCEPHALY
The basiochondrocranium of embryos in induced cranioschisis aperta with
exencephaly (CSA-EX), regardless of the teratogenic agent used, is shorter
FIGURES 8 AND 9
Fig. 8. Mid-sagittal section of the basichondrocranium of a clofibrate-treated
embryo (14 days old) with cranioschisis occulta and encephalocoele. The basichondrocranium is shorter than normal and slightly lordotic to the vertebral
axis. Compare with Fig. 5. Vertical bar = 0-25 mm.
Fig. 9. Mid-sagittal section of the basichondrocranium of a clofibrate-treated embryo
(14 days old) with cranioschisis aperta and exencephaly, illustrating the intracranial course of the notochord and its relationships to the basioccipital (BO)
and the basisphenoid (BS). Compare with Fig. 7. The borders of the notochord
have been outlined with white ink to make them more clearly visible at this
magnification. Vertical bar = 0-25 mm.
Notochordal-basichondrocranium relationships
31
3-2
32
M. M A R I N - P A D I L L A
Fig. 10. Composite figure of camera lucida drawings of the basichondrocraniums of
normal (N) and treated embryos with cranioschisis occulta with encephalocoele (E)
and cranioschisis aperta with exencephaly (A), illustrating the intracranial course
of the notochord. The shortness and the lordotic position of the basichondrocraniums of the treated embryos are also illustrated to facilitate their comparative
analysis. Other structures illustrated include: the notochord (N); the basioccipital
(BO); the basisphenoid (BS); the hypophysis (P); the Rathke's pouch (R); the
odontoid process of the second vertebra, the third vertebra and the intervertebral
notochordal segmental enlargements. These segmental notochordal enlargements
are considered to be homologous to those found within the posterior region of
the basioccipital.
Notochordal-basichondrocranium relationships
33
than normal and appears to be similarly affected in all cases (Figs. 6, 7, 9, 10).
in addition, the basichondrocranium in all treated embryos is abnormally
elevated or lordotic to the vertebral axis. It should be emphasized that the
degree of shortness, and that of the lordosis of the basichondrocranium, is
greater, or more accentuated, in cranioschisis aperta (exencephaly) than in
occulta (encephalocoele), but the nature of the defects appears to be identical
(compare Figs. 5,7,9,10). The longitudinal reduction of the basichondrocranium
in CSA-EX is more pronounced than that of CSO-EN, and it is of the order
of 12—15 % of the normal control.
Although the intracranial course of the notochord has been found in induced
CSA-EX to be within normal limits, the notochord itself is considerably shorter
than normal and depicts prominent terminal notochordal folds (Figs. 7, 9, 10).
The degree of shortness of the notochord appears to be related to that of the
basichondrocranium, and hence is more severe in cranioschisis aperta than in
occulta (compare Figs. 5, 7, 8, 9, 10). Similarly, the terminal notochordal
foldings are more accentuated in CSA-EX than in CSO-EN, although they
seem to be of a similar nature. The notochordal enlargement formed in the
posterior region of the basioccipital is slightly larger than normal and clearly
recognizable in cranioschisis aperta with exencephaly (Fig. 7, arrow). It should
be noted that the transverse diameter of the basichondrocranium in these
induced disorders is also slightly reduced.
DISCUSSION
The basichondrocranium in both CSA-EX and CSO-EN has been found
to be similarly affected and to be shorter (and slightly narrower) than normal
following exposure of pregnant hamsters to three different teratogens. In both
disorders the shortness of the basichondrocranium is considered to be caused
primarily by the reduced or insufficient longitudinal growth of the basioccipital
(Fig. 10).
The sole difference encountered between the abnormal basichondrocraniums
of these two disorders has been in the degree of severity of their common basic
abnormalities (quantitative differences), but not in their nature or quality. The
similarity of the abnormal basichondrocranium of these two apparently different
disorders gives further support to the idea that they indeed represent different
degrees of the same basic defect involving the axial skeleton (Marin-Padilla,
1978). The deficient growth of the base of the skull, especially that of the
basioccipital, has been considered to be caused by a primary mesodermal
insufficiency (Marin-Padilla, 19666, 1970, 1978).
In this respect, it should be pointed out that no apparent differences have
been found among the axial skeletal defects studied here, induced by vitamin
A, sodium arsenate or clofibrate. This observation suggests the possibility of
a common mechanism of action for the three teratogens. The three teratogens
34
M. MARIN-PADILLA
are administered, in a single dose, at the primitive streak stage of embryonic
development when the paraxial mesoderm and the notochord are actively
growing, forming together the first axial skeleton of the embryo. In addition,
there are experimental observations suggesting that the three teratogens used
here affect, in one way or another, the embryonic mesoderm (Marin-Padilla,
19666; Morris, 1972, 1973; Carpenter & Ferm, 1977; Ferm, 1978).
The mammalian axial skeleton is almost entirely of mesodermal origin.
Cells, probably of ectodermal origin, begin to proliferate at the primitive knot
region of the embryo, and wedge themselves between the ectoderm and the
endoderm (Jurand, 1974). This growing mass of cells constitutes the primordium
of the embryonic mesoderm, from which the paraxial mesoderm is derived.
This paraxial mesoderm, together with the notochord, begins to grow in a
cephalo-caudal direction (longitudinal growth) and constitutes the first axial
skeleton of the embryo. From this paraxial mesoderm, cells start to migrate
laterally, forming progressively the somites from which the sclerotomes, and
hence the vertebrae, and also the occipital bone, will eventually develop (De
Beer, 1937). An injury to this paraxial mesoderm early in embryonic development could result in a primary mesodermal insufficiency which, in itself, may
be the cause of a variety of developmental abnormalities involving the various
axial structures. A primary insufficiency of the paraxial mesoderm might well
be the mechanism for the following developmental abnormalities: (a) longitudinal growth impairment of the axial skeleton, resulting in shortness of the
region affected; (b) growth impairment in the formation and progressive
elevation of the neural folds, resulting in a variety of dysraphic (partial or
complete failure of closure of the neural folds) disorders; (c) folding (crowding)
of the developing notochord (itself probably unaffected) underlying the affected
and shortened region of the axial skeleton; and (d) folding (crowding) of the
developing neuroectoderm (probably primarily unaffected) overlying the affected
and shortened region of the axial skeleton, giving the impression of a focal
overgrowth of the neural tube. Needless to say, all of these developmental abnormalities have been recognized in the two types of experimental axial skeletal
(dysraphic) disorders studied here, as well as in many others (Marin-Padilla,
1965a, b, 1970, 1978; Geelen, 1973; Fields, Metzner, Garol & Kokich, 1978).
In both CSA-EX and CSO-EN studied here, the shortness of the basichondrocranium, especially that of the basioccipital (Fig. 10), is considered to
be the most basic abnormality. This shortness is considered to be the direct
consequence of the primary mesodermal insufficiency believed to be the cause
of these axial skeletal disorders. On the other hand, the abnormal position
(lordosis) of the basichondrocranium, the terminal notochordal folds, and the
dysraphic defects (encephalocoele or exencephaly) are all considered to be
secondary abnormalities indirectly related to the primary mesodermal insufficiency and probably caused, in part, by the shortness of the affected region
of the axial skeleton.
Notochordal-basichondrocranium relationships
35
In the course of embryonic development, the insufficient longitudinal growth
of the basichondrocranium on the one hand, and the apparently normal
longitudinal growth of other axial cephalic structures (known to be unaffected
in these axial skeletal disorders) on the other hand, resulted in the abnormal
lordotic position of the base of the skull, a condition which characterizes
these disorders. In addition, the presence of a partial or a complete dorsal
dysraphic defect in these disorders probably contributes also to the abnormal
lordotic position of the base of the skull.
The terminal notochordal folds (crowdings) encountered in both CSA-EX
and CSO-EN are also considered to be secondary abnormalities caused by the
shortness of the basichondrocranium. The developing notochord, which is
probably primarily unaffected in these disorders, is forced to grow into a
reduced or short space, causing it to fold as it grows. The degree of folding
will reflect the degree of shortness of the base of the skull. This explains the
more prominent notochordal folds found in cranioschisis aperta (shorter basichondrocranium) as compared to those found in occulta (Fig. 10). Furthermore,
this interpretation will also explain the difference between the early or minimal
notochordal changes and the more prominent changes found in later embryonic
developmental stages. Obviously the notochordal folding should increase in
the course of embryonic development, becoming more prominent in later
stages.
It should be emphasized that the study of a single histological section of
these folds could be erroneously interpreted as either notochordal deviation,
displacement, duplication or focal proliferation, depending on the angle of
sectioning. Needless to say, all of these types of notochordal anomaly have
been described in these disorders, and all of them can be the result of the
type of folding described herein. The need to study complete reconstructions
of the entire basichondrocranium of these disorders is essential to understand
the notochordal changes.
A similar interpretation is suggested by these findings to explain the apparent
local overgrowth of the neural tube (Patten, 1953) which is so often described
in axial skeletal (dysraphic) disorders. The developing neural tube growing
over a primarily shortened area of the axial skeleton will be forced to fold on
itself (crowding), which could give the impression, in single histological sections,
of a localized overgrowth. In addition, the shortened cranial or spinal cavities
(Marin-Padilla, 1965a, b, 1966a, 1970, 1978) become inadequate to lodge
and accommodate the developing neural tube which is being progressively
forced out of these spaces, thus becoming everted to extroverted (exencephaly,
myeloschisis).
A primary insufficiency of the paraxial mesoderm is also considered to be
the underlying cause of the dysraphic (exencephaly and encephalocoele) defects
which characterize these malformations (Marin-Padilla, 1978). A mesodermal
insufficiency will affect the normal process of formation and progressive
36
M. MARIN-PADILLA
elevation of the neural folds which could result in their complete (exencephaly)
or partial (encephalocoele) failure of closure. A recent analysis (Marin-Padilla,
1978) of the morphogenesis of the various types of axial skeletal (dysraphic)
disorders emphasizes the participation of the neural folds as a whole, and of
each of its components (surface ectoderm, mesoderm, neural crest and neuroectoderm), rather than the neural tube (neuroepithelium) alone, as has been
often erroneously considered.
Axial skeletal (dysraphic) disorders are complex developmental malformations
characterized by multiple abnormalities and the involvement of many axial
structures. In the study of these disorders, it is absolutely necessary to differentiate between those anomalies which are more directly related to the basic
defect (primary anomalies) and those which are indirectly related (secondary
anomalies), but which represent nonetheless important components of the
whole malformation. Because of the obvious clinical implications, a considerable emphasis on the anomalies involving the CNS has permeated the entire
literature on these malformations, often ignoring totally the underlying axial
skeletal defects which are important, perhaps fundamental, defects in these
malformations.
This work has been supported by a grant - HD-09274 - from the National Institute of
Child Health and Human Development from NIH, USA.
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