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/ . Embryo/, exp. Morph. Vol. 25, 2, pp. 189-201, 1971
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189
The mechanism of axial rotation in the rat embryo:
an experimental study in vitro
ByE. M. DEUCHAR 1
From the Department of Anatomy, The Medical School,
University of Bristol
SUMMARY
Axial rotation has been studied in 9- to 11-day rat embryos grown in culture by New's
watch-glass technique.
Unlike the mouse, the rat embryo rotates towards its right side and rotation starts with
the head end only. The twist then passes caudaiwards until the whole axis has reversed its
dorsoventral orientation and curvature. Contractions in cervical and cardiac regions appear
to initiate the rotation.
Posterior parts of 9- and 10-day embryos, isolated by transections at mid-trunk or cervical
levels, show much less ability to rotate than unoperated controls: the frequencies of fully
turned, partially turned and unturned embryos have been compared between control and
experimental groups and show significant differences. There is more marked inhibition of
rotation when the operation is performed at 9 days than at 10 days, and more with cervical
than with mid-trunk transections. In all, 67 % of embryos transected at the mid-trunk level
and 98 % transected at the cervical level were unable to rotate the posterior parts. Extrusion
of embryos from the amniotic cavity also resulted in abnormal or incomplete axial rotation.
The role of the membranes in facilitating rotation is discussed briefly.
INTRODUCTION
Considerable attention has been paid by descriptive embryologists to the
so-called 'inversion of the yolk sac' which occurs in rat and mouse embryos,
the best-known of laboratory rodent material. Surprisingly little attention has
been given, however, to the inversion and reversion of the embryo itself which
is consequent upon the original layout of the yolk sac. The changing topography
may be summarized as follows: the embryonic disc is from its initial formation
convex ventrally and concave dorsally with the yolk sac reflected upwards on
all sides of it. The axis of the embryo therefore develops in a U shape, the
outer margin of the U being ventral and the inner margin dorsal. This orientation
persists until 10£ days' gestation in the rat (8-| days in the mouse: see New.
19666; Snell, 1941). Then, in the rat from 10-| to \\\ days and in the mouse
from 8 | to 9\ days, the embryo rotates round its longitudinal axis and at the
same time the amniotic cavity expands, with the result that the dorsoventral
orientation and the curvatures of the embryo are completely reversed, and it
1
Author's address: Department of Anatomy, The Medical School, University Walk,
Bristol BS8 1TD, U.K.
13-2
190
E. M. DEUCHAR
is now convex dorsally and concave ventrally for the rest of its development.
This rotation process has been only very briefly described in the literature and
only for the mouse, not the rat (Snell, 1941; Snell & Stevens, 1966). The earlier
descriptions were based mainly on serial sections of preserved material and
not on any continuous in vivo observations, though they included photographs
of intact mouse embryos.
More recently, New & Stein (1964) have shown that rat embryos will rotate
successfully in culture. The watch-glass culture method of New (1966 a) makes
it possible to grow rat embryos with their membranes attached, during the
period from 9 to 11 days' gestation, and to observe the rotation process under
normal and experimental conditions. Using this method, a preliminary investigation of the mechanism of rotation has been undertaken in the work to be
reported below. Observations on controls (Series 1) indicated that rotation was
initiated in the head and neck regions and that the rest of the axis was perhaps
dragged round passively. Some transection and wounding experiments were
therefore carried out (Series 2-4) to isolate posterior regions and to see if, in
these and other circumstances, the posterior axis had any ability to rotate by
itself. In a previous series of experiments (Deuchar, 1969) it had been shown
that posterior parts could survive and differentiate normally when severed
from anterior parts.
MATERIAL AND METHODS
Wistar rat embryos of 9 and 10 days' gestation were used (day 0 = the
morning on which vaginal plugs were observed, after mating the previous
night, with females in oestrus). Embryos were removed from the uterus with
fine forceps in warm Tyrode's saline and cultured by the watch-glass method
of New (1966«), in rat serum obtained by heart puncture of the same females
that were used as sources of embryos. The cultures were incubated in 95%
O2/5 % CO2 at 37 °C for 24-48 h. The operations were carried out with glass
needles when the embryo was in the watch-glass of serum. At the end of the
culture period embryos were fixed in Bouin's fluid and stained in to to with
1 % aqueous eosin so as to be easily visible through later procedures. After
dehydration in alcohols and clearing in methyl benzoate they were photographed
whole, then embedded in paraffin wax. Sections 8 /i thick were stained with
Weigert's haematoxylin and eosin.
RESULTS
Series 1: normal rotation in the rat embryo
Some fifty unoperated embryos that had been cultured as controls in the
various experimental groups, as well as others removed from the uterus at
stages between 10-| and 1 1 | days, were observed both in toto and in serial
sections. Surprisingly, it was found that their rotation was in the opposite
direction to that described in the mouse by Snell (1941). Whereas Snell describes
Axial rotation in the rat embryo
191
the mouse embryo as rotating its dorsal surface towards its left side, it was
clear that the opposite occurs in the rat: it turns its dorsal surface towards its
right side. The sequence of turning in the rat is illustrated in Fig. 1 and in
Fig. 2 A-C. Another point of difference from Snell's description of the mouse
was that in the rat the tail end showed no sign of twisting until the head and
Fig. 1. Diagrams to illustrate axial rotation in the rat embryo. (A-C) Intact embryo
within its membranes: A, 9\ days; B, 10| days; C, \\\ days. (D-F) Transverse
sections, in plane indicated by straight arrows in A-C. Curved arrows show
direction of rotation, all, Allantois; am, amnion.
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E. M. DEUCHAR
anterior parts had turned through at least 90°. Rotation always began in the
head and cervical regions, then proceeded caudalwards from there, looking as
if the posterior regions were dragged round passively by the initial twist of
B
D
all ys
Axial rotation in the rat embryo
193
anterior parts. Observations on twenty-one 10-day embryos while the heart
was beating suggested that the earliest tendency to twist was at the cardiac
level, helped both by the heart-beat which caused rhythmic dipping movements
of the head towards the right, and by the left-over-right twist of the cardiac
tube during its morphogenesis.
Fig. 3. Diagrams to illustrate types of operation performed. (A, C) Nine-day
embryo; (B, D) 10-day embryo. Lines labelled 2 and 3 represent transection levels
in experimental Series 2 and 3 respectively, C and D show the lines along which the
amnion was cut in experiments of Series 4. tr, Trophoblast; all, allantois; am, amnion.
FIGURE 2
(A) U-shaped embryo in which axial rotation has not commenced, photographed
within its membranes, x 30.
(B) Dorsal view of semi-rotated embryo. Anterior axis lies on its right side. Tail
end (unrotated) and allantois project upwards towards camera. Inner membrane,
close to embryo, is the amnion: outer membrane is yolk sac. x 30.
(C) Fully rotated embryo within amnion. x 30.
(D) Photomicrograph of transverse section through embryo at U-shaped stage.
Note 'back-to-back' orientation of the head and tail portions. Neural folds not yet
closed in brain region. x45.
Abbreviations: all, allantois; am, amnion; ht, heart; R, Reichert's membrane;
ys, yolk sac.
194
E. M. DEUCHAR
Other observations indicated that the cervical region had special contractile
activity. When 9-|-day embryos, still in the U-shaped stage, were dissected
from their membranes, they were found to contract in the cervical region in
such a way as to narrow the U shape. Later stages in mid-twist, however
(cf. Figs. IB, 2B), tended to slip round into completion of the twist as they
were removed from their membranes; especially when the membranes were
ruptured at cervical levels. The posterior trunk was apparently pulled round
to complete rotation, as soon as the cervical region was released from its
attachments to amnion and yolk sac.
One of the advantages of New's culture method is that, provided there is
sufficient depth of serum, the amniotic cavity can expand to the same extent
as in utero, giving ample three-dimensional space for normal axial rotation to
occur. The healthy embryos in this series were in every way comparable to
embryos removed from the uterus at equivalent gestation ages. The amnion
and yolk sac were well expanded and the latter was highly vascularized.
Table 1. Results of mid-trunk severance
Age
at operation
(days)
Total
examined
Not
rotated
Partially
rotated
Fully
rotated
9
10
22
88
11
38
9
14
2
36
Series 2: mid-trunk severances
In this series of experiments, 9- or 10-day embryos were severed with a glass
needle at the mid-point of the axis, i.e. the apex of its U shape (see Fig. 3). This
operation necessarily damaged the amnion and yolk sac in this region and
resulted in a little loss of amniotic fluid, but never more than one-fifth of its
total volume. The amnion, however, healed quickly and was later also covered
by yolk-sac tissue. So any loss of anchorage that the embryo suffered immediately
after the operation was quickly restored. In addition, as reported in a previous
paper (Deuchar, 1969), 63 % of 9-day embryos and 39 % of 10-day embryos
showed some healing of the trunk severance by the end of the culture period.
It had also been established in the previous work that the isolated posterior
axis was able to differentiate normally in the majority of cases.
Table 1 summarizes the effects of mid-trunk severance at either 9 or 10 days,
on rotation of the embryonic axis. Results in both groups were judged at
11 days, i.e. after 48 h culture of embryos operated on at 9 days and after 24 h
culture of embryos operated at 10 days. It can be seen from the Table that
only two of the 9-day embryos were able to rotate completely after the operation.
Half of these embryos had not even begun to twist; their axes remained
U-shaped, with the dorsal side concave and in transverse section the head and
Axial rotation in the rat embryo
195
tail ends were still exactly back-to-back as in the normal 9-day embryo
(Figs. ID, 2D). In these the membranes were somewhat shrunken and the
amniotic cavity small. Embryonic differentiation had, however, advanced
considerably and was comparable to that at 11-days' gestation.
The nine embryos of this group which were classed as 'partially rotated'
showed varying degrees of twisting of the anterior axis, while regions posterior
to the original severance seemed not to have moved from their original
orientation. The resulting appearance of head and tail ends in transverse
section varied from oblique back-to-back orientation (seven cases, cf. Figs. 1E,
4A) to side-by-side orientation (two cases, cf. Fig. 4B). In two cases the anterior
end had bent downwards into the amniotic cavity, as well as rotating 180°, and
so finally lay in the same orientation as the rotated tail when viewed in transverse
section (Fig. 4C).
In contrast to the 9-day embryos, a considerable number of the embryos
severed at 10 days were able to rotate completely (36/88 = 41 %). Rotation
did not occur at all in 38 cases (43 %), however, and the remaining 14 embryos
had only partly rotated, at the anterior but not the posterior end. These showed
the same range of orientations in transverse section as described above for the
9-day embryos.
A remarkable feature of all the embryos in this series was their ability to
survive the operation in good condition and to continue differentiating normally.
By the 11-day stage a beating heart and circulatory system, eye rudiments and
up to twenty pairs of somites were usually visible. So it was clear that the
failures in rotation were not secondary to any general deleterious effects of the
operation. They may, however, have resulted partly from failure of the amnion
to expand normally after healing (see remarks above).
Series 3: severance at the cervical level
This operation was carried out on both 9-day and 10-day embryos, with
glass needles, as in Series 2. The cut was made immediately caudal to the heart
rudiment (see Fig. 3). Nine-day embryos were then cultured for a further 48 h
and 10-day embryos for a further 24 h as in the previous series. Probably
owing to haemorrhage, ten embryos of this series did not survive the operation:
these have not been included in the summary of results in Table 2.
As Table 2 shows, no embryo was able to rotate completely after severance
at the cervical level. More than half of the embryos (27/49) did not rotate at
all, and the remainder showed partial rotations, of the anterior end only, as
described in Series 2. All of them had rather distorted membranes and a much
reduced amniotic cavity.
The posterior axis in embryos of this series showed stunting and partial
necrosis in seven cases. There was also some evidence of delayed differentiation:
for instance, six embryos still showed an open neural plate at the 11-day stage.
It was therefore evident that the operation had other deleterious effects than
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E. M. DEUCHAR
simply an inhibition of rotation. The closeness of the cut to the heart rudiment
may have been one adverse factor. It was also noted that in two unrotated
embryos the paired heart rudiments had failed to fuse mid-ventrally and a
ht
D
Axial rotation in the rat embryo
197
double heart had resulted: the significance of this finding is discussed later.
The possible effects of distortion of the membranes have also to be considered,
in view of the results below (Series 4).
Table 2. Results of cervical severance
9
10
14
35
Not
rotated
oo
Total
examined
ON
Age
at operation
(days)
Partially
rotated
Fully
rotated
5
17
0
0
Table 3. Results of extrusion from amniotic cavity
Age
at operation
(days)
Total
examined
Not
rotated
Partially
rotated
Fully
rotated
9
10
29
36
16
10
13
24
0
2
Series 4: extrusion of the embryo from the amniotic cavity
In thirty-six 10-day embryos a split was made in the amnion at some distance
from the embryonic axis, and the embryo was then manipulated through this
opening so that it lay outside all its membranes but still attached to them
mid-ventrally (Fig. 3D). This procedure could not be carried out in the same
way with 9-day stages, since these had not yet developed head- and tail-folds.
Instead, cuts were made so as to split the amnion away from the head and
lateral regions of the embryo and it was left attached to the membranes at the
tail-end only (Fig. 3C). The main object of both operations was to sever those
FIGURE 4
(A) Transverse section of embryo in which anterior parts have begun rotation, and
lie at an oblique angle to posterior parts. Section passes through hindbrain, pharynx
and bulbus regions in the anterior axis, x 75.
(B) Transverse section of embryo in which rotation has achieved an almost
'side-by-side' orientation of the two parts of the axis. Anterior parts, with heart,
shown on right-hand side of photograph, x 30.
(C) Transverse section of embryo in which anterior axis (shown on right) has
rotated fully but tail end has not, so that the two dorsal sides now point in the same
direction. x45.
(D) ' Buckled' embryo from Series 4. Lower vesicle contains heart and head region;
rest of axis, bent into Z shape, can be made out with difficulty in upper vesicle.
x30.
(E) Embryo from Series 4 with multiple amniotic vesicles. Axis is not visible, x 20.
Abbreviations: all, Allantois; am, amnion; hb, hindbrain; ht, heart;/?//, pharynx;
vv, amniotic vesicles.
198
E. M. DEUCHAR
regions in which rotation should be initiated from their anchorage to the
amnion.
A summary of the results in this series is given in Table 3. The majority of
embryos operated on at 10 days were afterwards capable of turning partially,
and two achieved complete rotation. The degree of rotation in these embryos
could be assessed with reference to their residual point of attachment to the
membranes: for example, in those fully rotated, the ventral surface was turned
towards the membranes, instead of the dorsal surface as in Fig. 3 C, D. Among
the embryos operated on at 9 days, however, a lower proportion (13/29) were
able to turn partially and none achieved complete rotation. Ten of the 10-day
and sixteen of the 9-day embryos did not rotate at all after the operation.
In this series, the category 'partially turned' includes four 9-day and nine
10-day embryos which had undergone abnormal contortions as a consequence
of their extra-amniotic position. Fig. 4D shows an example of this. The embryo
is buckled: its head has apparently begun a normal twist, but the rest of the
body has formed irregular bends without rotating on its longitudinal axis.
There were other cases, less easy to see in toto, in which parts of the axis had
become re-enclosed in irregular vesicles of healed amnion. As has been noted
in previous work, there is very rapid healing of wounds in the amnion (Deuchar,
1969). In the present series, several small accessory amniotic vesicles, partly
covered by yolk sac too, were formed as a result of this healing. One or more
of the vesicles might enclose parts of the embryonic axis. Fig. 4E shows one
of these 'multi-vesicle' embryos. It should be stressed that, despite their partial
enclosure sometimes within such vesicles, none of these embryos was able to
undergo a complete rotation: they were never more than partially rotated.
The two 10-day embryos which were able to rotate completely may perhaps
have already begun to twist at the time of operation: they were recorded at
that time as already having a heart-beat and appearing unusually advanced.
Series 5: extra controls to check for variability of rotation
under culture conditions
Since in earlier series it had not always been recorded whether or not the
unoperated controls had rotated completely at 11 days, an extra 37 were
cultured without operation. The results (Table 4) show that 25 of these, i.e.
68 %, rotated completely and 9 partially. The 3 which did not rotate had been
smaller than normal at the start of the culture period and were not healthy
at the end of it.
Comparing the percentages of embryos (a) fully turned, (b) partially turned
and (c) unturned in these controls with each of the experimental groups 2-5,
it is clear that all experimental procedures significantly inhibited rotation and
that the results were not due simply to the vagaries of culture conditions
(Table 4).
Axial rotation in the rat embryo
199
Table 4. Comparisons of rotation in controls with totals
of experimental groups
Controls
Series 2 (mid-trunk cut)
Series 3 (cervical cut)
Series 4 (ex. amnion)
Total
examined
No
rotation
Partially
rotated
Fully
rotated
37 (100)
110 (100)
49 (100)
65 (100)
3 (8-D
49 (44-5)
27 (55-1)
26 (400)
9 (24-3)
23 (20-9)
22 (44-9)
37 (56-9)
25 (67-6)
38 (34-5)
0
(0)
2
(3-D
DISCUSSION
From these limited types of operation that could be performed successfully
on rat embryos in vitro, some preliminary conclusions can be drawn about the
mechanism of axial rotation which serve as a pointer to further problems worth
investigating.
The operations in Series 2 and 3 were designed to isolate the posterior part
of the axis from those anterior regions in which rotation appeared to be
initiated, and to see if in these circumstances the posterior axis had any independent powers of rotation, or whether it was dependent on the anterior axis
to 'drag it round'. From the results of Series 2 it is clear that if the transection
is carried out early enough, at 9 days, posterior parts are never able to rotate
on their own. This is all the more striking in view of the fact that, as noted in
an earlier paper (Deuchar, 1969), the two cut ends often heal and the axis
then partially turns. As has been described above, however, in these cases of
partial rotation it is only the regions anterior to the transection that turn, and
not posterior regions.
The fact that 41 % of the 10-day embryos in Series 2 were able to rotate
completely after transection could be explained by assuming that some brief
initial stimulus is required from the anterior end, rather than a continuous
traction. It could be argued that, in the embryos which turned successfully,
this stimulus had already passed from anterior to posterior parts before the
time of the operation. Once this stimulus had been received, the posterior axis
could continue turning on its own, independently of whether or not it healed
on to anterior parts later. There are difficulties in this interpretation, however,
when the results of Series 3 are also considered. If the ability of posterior parts
to rotate depends on a stimulus spreading progressively caudalwards, one
would expect greater success in turning of posterior parts in Series 3 than in
Series 2, since the Series 3 isolates included regions ahead of the mid-axial
flexure, which might already have received this stimulus at a time when parts
caudal to the flexure had not yet received it. But in fact Series 3 showed less
rotation ability than Series 2 (compare Tables 1 and 2). It seems clear that
200
E. M. DEUCHAR
transections at the cervical level are more effective in inhibiting posterior
rotation than are cuts at mid-axial level.
It was noted in series 1 that contractility could be observed in the cervical
region of living control embryos; also that the normal twisting of the cardiac
tube, together with the rhythmic rightward and downward movements of the
head that resulted from the heart-beat, might play some part in initiating axial
rotation. It therefore fits with expectation that the transections of Series 3,
which were near the heart, in the cervical region, had the most marked effect
on rotation. It has already been pointed out that in this series there was a risk
of damage to the heart. It was also of particular interest that in two of the
embryos which had not rotated at all, the heart rudiments had failed to fuse
ventrally.
All rotation movements, in either cardiac, cervical or mid-trunk regions,
encounter some resistance from the extra-embryonic membranes. Unless these
have expanded so as to give plenty of free space (amniotic cavity) round the
embryonic axis, rotation is not possible (cf. the unrotated embryos of Series 2
and 3). At the same time, the membranes provide anchorage and a fixed point
around which pivoting can take place. So, when the membranes are extensively
damaged as in Series 4, rotation may be inhibited.
The experiments of Series 4 are difficult to interpret because of the frequent
distortions of the embryonic axes and the irregular amniotic vesicles which
in some cases partly enclosed them. But from a comparison of the data of
Table 3 with the frequency of normal rotation in the Series 5 controls (see
Table 4), it is clear that detachment or extrusion of the embryos from their
membranes significantly reduces their ability to rotate. This result again fits
with expectation, since the cardiac and cervical regions, where rotation seems
to be initiated, lie immediately adjacent to the roots of the extra-embryonic
membranes and would normally derive anchorage from them which could
contribute to the control of normal rotation. The experiments show that
when this anchorage was removed there was a tendency for exaggerated bends
and buckling, instead of twisting, in posterior axial regions.
To discover more precisely the mechanism by which contractions of cervical
and cardiac levels may initiate axial rotation in the rat embryo, other types
of experimental approach are required. Crude surgery can only provide the
indications that have already been discussed. Cinematographic observations
of rotation are now being started, and it is hoped also to undertake a more
detailed study of the structure and contractility of cervical somites and cardiac
tissues in 9 to 11-day rat embryos, which may throw further light on axial
rotation mechanisms.
Axial rotation in the rat embryo
201
RESUME
Le mecamsme de la rotation axiale dans Vembryon du rat:
une etude experimentale in vitro
La rotation axiale a ete etudiee chez des embryons de rat cultives selon la technique du
verre de montre de New.
A la difference de la souris, l'embryon de rat effectue une rotation vers son cote droit
et la rotation commence a l'extremite cephalique seulement. La torsion passe alors vers la
partie caudale jusqu'a ce que l'axe entier ait renverse son orientation dorso-ventrale et sa
courbure. Des contractions dans les regions cervicales et cardiaques semblent amorcer
la rotation.
Des parties posterieures d'embryons de 9 et 10 jours isolees par des sections transversales
au milieu du tronc ou au niveau cervical, montrent moins d'aptitude a effecteur une rotation
que les temoins non operes: les frequences de rotation totale, partielle et nulle ont ete
comparees chez les embryons temoins et les embryons operes; elles presentent des differences
significatives. Quand les operations sont effectuees le 9eme jour, 1'inhibition de la rotation
est plus marquee que lorsque les operations sont effectuees le lOeme jour, 1'inhibition est
plus importante pour des sections cervicales que pour des sections au milieu du tronc.
En tout, 67% des embryons sectionnes au milieu du tronc et 98% de ceux sectionnes
dans la region cervicale sont incapables d'effectuer la rotation des parties posterieures.
L'expulsion des embryons de la cavite amniotique donne lieu aussi a des rotations axiales
incompletes. L'aptitude que des membranes pourraient jouer a faciliter la rotation est
discutee brievement.
I am grateful to Mrs F. M. Parker for technical assistance and to Mrs P. Walton for help
with photography. Some of the costs of the research were covered by a grant from the
Agricultural Research Council.
REFERENCES
E. M. (1969). Effects of transecting early rat embryos on axial movements and
differentiation in culture. Acta Embryo I. Morph. exp. 157-167.
NEW, D. A. T. (1966O). Development of rat embryos cultured in blood sera. /. Reprod. Fert.
12, 509-524.
NEW, D. A. T. (19666). The Culture of Vertebrate Embryos. London: Logos Press.
NEW, D. A. T. & STEIN, K. F. (1964). The cultivation of post-implantation rat and mouse
embryos on plasma clots. 7. Embryol. exp. Morph. 12, 101-111.
SNELL, G. D. (1941). The early embryology of the mouse. In Biology of the Laboratory
Mouse, pp. 1-54. London: Constable.
SNELL, G. D. & STEVENS, L. C. (1966). Early embryology. In Biology of the Laboratory
Mouse, 2nd. edn. (ed. E. L. Green), pp. 205-246. London: McGraw-Hill.
DEUCHAR,
{Manuscript received 23 July 1970)