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/ . Embryol exp. Morph. Vol. 53, pp. 237-243, 1979
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237
The use of avian double monsters in studies on
induction of the nervous system
By R. J. ULSHAFER 1 AND A. CLAVERT 1
From the Laboratoire d'Embryologie, Faculte de Medecine,
Universite Louis Pasteur, Strasbourg, France
SUMMARY
Ninety-eight duck double monsters (siamese twins) were studied between 48 and 72 h of
incubation in whole-mount or serial-sectioned preparations. Each case was classified into
one of four categories depending on the orientations of the two embryonic axes. In all
classes, defects in primary neural induction of various parts of the embryonic nervous
system were observed which depended to a large extent on the distance separating the anterior
tips of the two notochords or on the relative positions of the notochords. Our observations
suggest that: when the same inducing field of one embryo overlaps to a critical extent with
that of the twin, the two inducers act as one; induction of forebrain and optic vesicles is purely
prechordal; the notochord exerts an inhibition on induction of forebrain by prechordal
mesoderm; and the presence of rhombencephalic structures depends on both prechordal and
chordamesodermal interactions.
INTRODUCTION
The study of naturally occurring or mechanically induced double monsters
(siamese twins) offers excellent model systems for problems concerning organizing phenomena, symmetry of body plan, teratogenesis/?er se and also induction
and regulation. The phenomenon occurs spontaneously quite frequently in
birds, especially in ducks where about 2 % of all fertilized eggs form double
monsters; the effect is believed to be caused by orientation changes of the egg
within the uterus during the critical period of symmetrization which results in
the formation of two (or more) organizing centres (Vintemberger & Clavert,
1956; Clavert, 1962). The phenomenon can also be produced experimentally
with up to 80 % success by removing eggs from the uterus prior to the critical
period and subjecting them to series of rotations in a mechanical apparatus
(Vintemberger & Clavert, 1960).
From an experimental point of view, this anomaly corresponds to a heterotopic graft of the primary organizer. The relative positions of the two. organizing
centres determine the orientations of the embryonic axes and location of
inducing tissue which may therefore have consequences on induction of
embryonic structures. The purpose of the present study was to determine if
1
Authors' address: Laboratoire d'Embryologie, Faculte de Medecine, Universite Louis
Pasteur, 11 Rue Humann, 67085 Strasbourg Cedex, France.
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R. J. ULSHAFER A N D A. CLAVERT
differences exist in brain structure between the twins as a result of interference
with induction caused by orientation and location of the embryonic axes of
the double monsters. Since, in our study, the effect occurs spontaneously in ovo
without the intervention of microsurgery, it presents the advantage of eliminating artifacts that may be introduced during experimental manipulation.
MATERIALS AND METHODS
Ninety-eight spontaneous double monsters were obtained from about 5000
duck eggs between 48 and 72 h incubation, used in previous or concurrent
studies. The blastoderms were excised, photographed using reflected light and
then fixed in Bouin's fixative. In about half the cases permanent whole-mount
preparations were made, while in the others the embryos were embedded in
paraffin, sectioned at 6 jtcm and stained with haematoxylin and eosin. Sections
were analysed by counting the number of forebrains, optic vesicles or cups,
hypophyses, hindbrains and otic vesicles. In relevant cases the distance separating
the anterior extremities of the notochords of the twins was measured using a
micrometer having 5 /*m per division under a Leitz optical microscope.
RESULTS
We observed four different groups of double monsters, as distinguished by
the relative positions of the embryonic axes and the number of differences in
brain structures noted between the two embryos.
Group I. 13 cases. The notochords approached each other medially. The
twins shared a common cephalic extremity, having one well-formed prosencephalon with one set of optic rudiments and one or two hypophyses (Fig. 1).
Group II. 41 cases. The notochords approached each other medially. Each
embryo had its own forebrain which had either both or only one lateral optic
rudiment (Fig. 2).
F I G U R E S 1-4
Fig. 1. Group I double monster at about 72 h incubation. The twins share a common
cephalic extremity. Serial sections of this embryo revealed one forebrain, one set of
optic cups, one hypophysis, two hindbrains and two sets of otic vesicles.
Fig. 2. Group II double monster at about 60 h incubation. Two separate heads are
visible, each having a pair of optic cups, one hypophysis, a normal hindbrain and
a pair of otic vesicles.
Fig. 3. Group ITI double monster at about 60 h incubation. The twins approach each
other frontally, neither one having forebrain or optic rudiments. Serial sections of
this monster revealed two hindbrains, but only the one on the left had otic rudiments.
Fig. 4. Group IV double monster at about 60 h incubation. The embryonic axes
intersect each other almost perpendicularly at the level of the hindbrain. The
embryo on the left lacked forebrain and optic vesicles but had normal hindbrain
and otic rudiments. The embryo on the right had a normal nervous system.
Induction in avian double monsters
239
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R. J. ULSHAFER AND A. CLAVERT
Table 1. Distance separating the anterior extremities of the notochords and the
resulting number of forebrain structures in double monsters which approached
each other medially
Number of
cases
Distance
separating the
Number
anterior notochord
of
(mm)
forebrains
6
3
9
0-20-0-40
0-40-0-50
0-45-0-80
Number
of optic
vesicles
Number of
hypophyses
Class
2
2
4
1 or 2
2
2
I
II
II
1
2
2
Table 2. Distance separating the anterior extremities of the notochords and the
resulting number of hindbrain structures in the frontal approach of the notochords
(Group III)
Number of
cases
4
1
1
9
Distance separating
the notochords
(mm)
Number of
hindbrains
Number of
otic rudiments
0
1
2
2
0
1
2
4
000 (fused)
0-30
0-40
0-48-0-70
Table 3. Level where secondary embryo intersects the first and the resulting
presence of neural structures in the secondary embryo of Group 111 monsters
Level where secondary
Number of
embryo meets
first
cases
Fore-/midbrain
Hindbrain
Spinal cord
6
5
4
Presence of
forebrain
Presence of
hindbrain
Presence of
spinal cord
+
0
0
+
+
+
+
+
+
Group III. 27 cases. The notochords approached each other frontally. Both
embryos lacked all forebrain structures. In some cases rhombencephalic
structures of both embryos were normal while frequently one or both twins
lacked otic rudiments and/or hindbrain entirely (Fig. 3).
Group IV. 17 cases. The secondary embryo approached the first somewhat
perpendicularly. The primary embryo was normal while the twin lacked optic
vesicles and frequently other brain structures (Fig. 4).
Within groups I and II a correlation existed between the distance separating
the anterior tips of the notochords and the lack of forebrain. structures (Table 1).
When the two notochords were separated by a distance less than 0-40 mm
(mean 0-30 mm), only one forebrain was induced. When a mean distance of
0-45 mm separated the anterior notochords, two forebrains resulted but only
Induction in avion double monsters
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one optic rudiment per head formed, and always on the lateral side. Only when
a distance of at least 0-50 mm (with one exception at 0-45 mm) separated the
two notochords, did two normal forebrains occur, each with its own pair of
optic vesicles.
In group III the presence of hindbrain structures also depended on a critical
distance separating the anterior tips of the notochords, which in this case
approached each other head-on (Table 2). When the two notochords were
separated by a distance of at least 0-48 mm, both embryos developed normal
rhombencephalic structures. When the two notochords were fused, no hindbrains or otic rudiments were induced. Two intermediate cases were seen, one
having one hindbrain and one otic vesicle and the other having two hindbrains
and two otic vesicles. Prosencephalons and optic rudiments never formed in
this group.
In group IV the axis of one embryo intersected that of the twin almost
perpendicularly at the level of the brain or spinal cord. In this group, the embryo
whose anterior notochord was located near the trunk of the second developed
little or no forebrain and no optic rudiment at all. When the embryos met near
the anterior neural tube somewhat more development of the brain occurred
than when they met more posteriorly, although optic vesicles never developed
in this group (Table 3).
DISCUSSION
Symmetrization in birds is related to direction of rotation of the egg within
the uterus. Eggs of species which have unusually large and elastic uteri, such as
the duck, frequently undergo rolling movements which reverse the orientation
of the egg in the uterus and thus change the direction of rotation. If this reversal
occurs during the critical period of symmetrization (within a span of a few hours)
a new centre of symmetrization and therefore a new embryonic axis forms — the
result being a double monster. That one of the resulting embryos is always
slightly older than the other also indicates that symmetrization, and subsequently
gastrulation, of the two embryos do not coincide in time. Due ultimately to the
telolecithal nature of the bird egg, a certain degree of continuity of germ layers
exists between the two embryos. Even though the neural tubes are frequently
discrete, the twins almost always share structures derived from all germ layers.
Consequently, identical twins are very rare in birds while Siamese twins are
relatively more common.
Although the orientation of the embryonic axes of the two embryos ultimately
results from the location of the symmetrization centre as a function of direction
of rotation and orientation of the egg within the uterus, the morphological
groups of double monsters that we described are also due to interactions between
tissues of the twins during gastrulation and induction,
The monsters in group I arise because of complete overlapping of two
prechordal plates, which has the effect of a single inducing field, thus producing
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R. J. ULSHAFER AND A. CLAVERT
one set of forebrain structures of normal dimensions. As the distance separating
the anterior tips of the notochords increases, the inducing effect of each prechordal plate becomes more and more distinct, producing the monsters of
group IT. It appears that the size of the induced organ therefore does not depend
on the mass of inducing tissue but rather on its own intrinsic capacity to respond
to the message within a given area. It should be emphasized, however, that the
relative distances noted here do not correspond to the actual distances which
separated the tissues during primary induction, due to growth of embryonic
organs and interstices. In group III the notochords were always so close as to
obliterate totally or inhibit the action of the prechordal plate mesoderm on
overlying ectoderm, and therefore no forebrain developed. Prechordal plate
mesoderm, however, appears necessary for normal induction of hindbrain
structures since, in the monsters in which the two notochords were fused no
hindbrains were induced, while in those cases where the two notochords were
separated by a space containing prechordal plate, two normal hindbrains
formed. In group IV one embryo was always normal while the one approaching
it from the side always lacked brain structures when it met the first at the level
of the trunk but not when they met more anteriorly. Here, again, the prechordal
plate of the approaching embryo overlapped the inducing field of the chorda
mesoderm of the primary embryo which apparently inhibited prechordal plate
from inducing forebrain. Since prechordal plate was present, however, hindbrain structures in the approaching embryo were normal. The more caudal the
location of the approaching embryo, the more drastic was the reduction in brain
structures. These observations suggest that induction of forebrain and optic
vesicles is purely prechordal, that the notochord exerts an inhibitory effect on
induction of forebrain by prechordal plate, and that this effect is stronger in
the more caudal regions of the notochord.
Naturally occurring multiple monsters resemble those produced experimentally by heterotopic grafting of Hensen's node, as reported by Gallera & Ivanov
(1964) and Gallera (1971). All of their monsters would belong to our groups III
and IV. That none was induced resembling our groups I and II is probably due
to their precise orientations of the grafts with respect to the embryonic axis of
the host (anterior end of the graft was always turned toward the centre of the
host). Although they did not report any inhibiting effect of chorda mesoderm on
forebrain, it appears in their figures that the same phenomenon occurred during
their experiments. In amphibians, however, only monsters corresponding to our
groups I and II are possible (after Spemann, 1962), primarily due to the ovoid
nature of the amphibian egg.
Our results therefore correspond to most of the generally accepted theories
on induction of the nervous system (reviewed by Saxen & Toivonen, 1962) in
that the more anterior parts of the nervous system are induced predominantly
by prechordal plate, spinal cord is induced purely by chorda and somitic
mesoderm, and intervening brain structures by an interplay of the two.
Induction in avian double monsters
243
This work was supported by Grant no. ATP 68.78-100 from the INSERM. Dr Ulshafer
is supported by the CNRS/NSF Exchange of Scientists Program.
REFERENCES
CLAVERT, J. (1962). Symmetrization of the egg of vertebrates. Adv. Morphogenesis 2, 27-60.
GALLERA, J. (1971). Primary induction in birds. Adv. Morphogenesis 9, 149-180.
GALLERA, J. & IVANOV, L. (1964). La competence neurogene du feuillet externe du blastoderme
de Poulet en fonction du facteur 'temps'. J. Embryol. exp. Morph. 12, 693-711.
SAXEN, L. & TOIVONEN, S. (1962). Primary Embryonic Induction. London: Logos Press
limited.
SPEMANN, H. (1962). Embryonic Development and Induction. New York: Hafner Publishing
Company.
VJNTEMBERGER, P. & CLAVERT, J. (1956). Sur la frequence de la monstruosite double et le
degre d'instabilite de I'orientation de l'oeuf dans I'uterus chez diflferents Oiseaux. C. r. hebd.
Seanc. Acad. Sci., Paris 243, 2149-2151.
VINTEMBERGER, P. & CLAVERT, J. (1960). Sur le determinisme de la symetrisation bilaterale
chez les Oiseaux: les facteurs de I'orientation de l'embryon par rapport a l'axe de l'oeuf
et la regie de Von Baer a la lumiere de nos experiences d'orientation dirigee sur l'oeuf de
Poule extrait de I'uterus. C. r. Seanc. Soc. Bio/. 154, 1072-1076.
(Received 8 January 1979, revised 27 April 1979)