ABSTRACT
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 caudalwards 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 days’ gestation in the rat (
days in the mouse: see New, 1966b; Snell, 1941). Then, in the rat from
to
days and in the mouse from
to
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 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 8μ 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 and
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 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. 2A-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 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 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.
Diagrams to illustrate axial rotation in the rat embryo. (A-C) Intact embryo within its membranes: A, days; B, 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.
(A) U-shaped embryo in which axial rotation has not commenced, photographed within its membranes, × 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. × 30.
(C) Fully rotated embryo within amnion, × 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. ×45.
(A) U-shaped embryo in which axial rotation has not commenced, photographed within its membranes, × 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. × 30.
(C) Fully rotated embryo within amnion, × 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. ×45.
Other observations indicated that the cervical region had special contractile activity. When -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.
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.
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.
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.
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 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).
(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, × 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, × 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. × 45.
(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. × 30.
(E) Embryo from Series 4 with multiple amniotic vesicles. Axis is not visible, × 20.
(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, × 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, × 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. × 45.
(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. × 30.
(E) Embryo from Series 4 with multiple amniotic vesicles. Axis is not visible, × 20.
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 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 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).
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 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).
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 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.
RÉSUMÉ
Le mécanisme de la rotation axiale dans Vembryon du rat: une étude expérimentale in vitro
La rotation axiale a été étudiée chez des embryons de rat cultivés selon la technique du verre de montre de New.
A la différence de la souris, l’embryon de rat effectue une rotation vers son côté droit et la rotation commence à l’extrémité céphalique seulement. La torsion passe alors vers la partie caudale jusqu’à ce que l’axe entier ait renversé son orientation dorso-ventrale et sa courbure. Des contractions dans les régions cervicales et cardiaques semblent amorcer la rotation.
Des parties postérieures d’embryons de 9 et 10 jours isolées par des sections transversales au milieu du tronc ou au niveau cervical, montrent moins d’aptitude à effecteur une rotation que les témoins non opérés: les fréquences de rotation totale, partielle et nulle ont été comparées chez les embryons témoins et les embryons opérés; elles présentent des différences significatives. Quand les opérations sont effectuées le 9ème jour, l’inhibition de la rotation est plus marquée que lorsque les opérations sont effectuées le lOème jour, l’inhibition est plus importante pour des sections cervicales que pour des sections au milieu du tronc.
En tout, 67% des embryons sectionnés au milieu du tronc et 98% de ceux sectionnés dans la région cervicale sont incapables d’effectuer la rotation des parties postérieures. L’expulsion des embryons de la cavité amniotique donne lieu aussi à des rotations axiales incomplètes. L’aptitude que des membranes pourraient jouer à faciliter la rotation est discutée brièvement.
Acknowledgements
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.