Paper - The development in vitro of young rabbit embryos (1933)
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The Development in vitro of Young Rabbit Embryos
By C. H. Waddington! and A. J. Waterman
From the Strangeways Research Laboratory and Laboratory of Experimental Zoology, Cambridge
- This work was done while I was in receipt of a part-time grant from the Medical Research Council, for which I should like to express my thanks.
- Fellow of the Commission for Relief in Belgium Educational Foundation 1931-2.
Very little experimental work has as yet been performed on the early stages of the mammalian embryo. The two main methods of experimental analysis, isolation of primordia and transplantation of fragments into different situations in the embryo, which have been applied with such success in the Amphibia, both present great technical difficulties when applied to the embryos of warm-blooded animals.
In the case of the bird the chorio-allantoic membrane of the developing embryo has been successfully used as a place for isolation of primordia (Hoadley, 1924, and others) and of portions of young blastoderms (Hoadley, 1926; Willier & Rawles, 1931). Recently Waddington (1930, 1932, 1938 a, 1933) has shown that young chick embryos may be successfully grown for several days on the surface of coagulated plasma and embryo extract. With this technique it is possible to perform various types of transplantation and deficiency experiments which are not possible with the former method. An outline of the process of determination of the embryonic axis in the bird, comparable to that worked out by Spemann and his school for the Amphibia, has been obtained in this way.
It is possible to isolate primordia of the rabbit embryo on the omental bursa of young or adult rabbits, in which environment the explant is incorporated by the tissues of the omental wall, and invaded by proliferations of host capillaries (Waterman, 1982 a): the primordia exhibit a degree of independent self-differentiation comparable to that of control embryos of the same temporal age (Waterman, 1932 b). Explants to this organ have given better results than others to various other regions of the adult.
Rabbit eggs have been grown in tissue cultures with marked success (Pincus, 1980), while Maximow (1925) has described the differentiation taking place in isolated pieces of young rabbit embryos grown in vitro. The in vitro technique has also been used by Brachet (1912, 1918) for the study of the development of entire rabbit blastocysts.
In an attempt to find a cultivation method which would make possible transplantation experiments on mammalian embryos, Waddington (unpublished data) found that young rabbit embryos of 8-9 days of development will live on the surface of plasma clots composed of adult chicken plasma and a saline extract of chick embryo and will undergo some further growth and differentiation. This was sufficiently encouraging for the authors to pursue the subject further. Various stages-of the rabbit embryo, from the late preprimitive streak stage to that of 2-4 somites, have been explanted to the surface of plasma clots. The results secured from these experiments constitute the material described in this paper.
Portions of these early stages have also been transplanted between the epiblast and endoderm of chick and duck embryos in the definite primitive streak and head process stages. Brachet’s experiments on the culture of whole rabbit blastocysts have also been repeated by one of us (Waterman) with various modifications of technique. These experiments will be described in detail in subsequent papers. It is sufficient at the present time to state that the blastocysts also will live and undergo some differentiation in this foreign medium. Development is markedly slower than the control and usually abnormal after a certain stage has been reached, depending upon the age of the blastocyst at the time of explantation.
I wish to thank Dr H. B. Fell for the courtesies extended to me at the Strangeways Research Laboratory.
Materials and Methods
The technique used in the present experiments is the well-known watchglass method of tissue culture in vitro. Equal amounts of adult chicken plasma and of a saline extract of a chick embryo of 8-9 days’ incubation are thoroughly mixed in the bottom of a watch-glass and the plasma allowed to coagulate. The watch-glass is itself enclosed in a covered Petri dish and rests upon a ring of cotton wool moistened with sterilised distilled water to prevent evaporation of the culture.
The embryos are removed from the uterus into a separate dish and then transferred together with some saline solution to the surface of the clot. After the embryo has been oriented with the dorsal side uppermost and carefully spread out flat with fine needles, the saline solution is gently removed. It is possible to explant embryos almost entirely freed from the surrounding trophoblast, but this is not usually advisable since the adjacent trophoblast, if carefully spread out, prevents the embryo itself from curling during the first few hours of culture. Even so the culture undergoes considerable shrinkage and thickening, which often results in marked distortion of the developing embryo.
All operations have been performed at room temperature without any effort to maintain a constant body temperature. Embryos have sometimes been kept in the refrigerator for periods ranging from 4 to 12 hours before being transferred to the clot. If the embryo is injured by this radical lowering of temperature it is not apparent either in the appearance of the culture as a whole or in the degree of differentiation exhibited by the embryo. Except for some retardation of the onset of development, the embryos thus treated compare favourably with those which were transferred immediately to the clot. It remains to be seen whether the maintenance of a high temperature and transference to a warm clot will give still better differentiation.
A saline extract of rat embryo of approximately 14-15 days’ growth in utero has been substituted for the chick-embryo extract in the culture medium. In these experiments no effect was noticeable as regards the differentiation undergone by the embryos. Chicken embryos have also been cultivated satisfactorily on media made up with rat embryo extract.
It will be remembered that Brachet obtained similar results with both adult male and adult female rabbit plasma. Similarly, for the omental grafts of primordia (Waterman, 1932), male or female, young or old, rabbits were used as hosts with no apparent effect on the differentiation obtained. In view of these facts, it seemed probable that the embryos would show considerable tolerance for foreign plasma, and since, as is well known, the use of mammalian plasma presents certain technical difficulties, we have in these experiments used chicken plasma, which has proved so satisfactory for the cultivation of most types of tissues by the hanging-drop method. Control experiments with rabbit plasma have not yet been made, but the results obtained in the cultivation of blastocysts by Brachet’s method, using chicken plasma, compare favourably with Brachet’s own results, using rabbit plasma, and it is probable that in the watch-glass cultures, the substitution of a soft rabbit-plasma clot for the hard chicken-plasma clot would have deleterious mechanical effects which would more than counterbalance any beneficial action of the homologous plasma.
The cultures have been grown for varying lengths of time, ranging from 1 to 5 days, without change of the medium. Chick embryos develop rapidly in culture, and the liquefaction of the clot is such that it is not feasible to grow them more than 2 days. In the case of the rabbit embryos, however, very little liquefaction of the clot occurs even after 5 days of culture. Moreover, development is much slower and the culture never attains any considerable size. Efforts have been made to change the medium after 48 hours, but the resulting injury has always resulted in immediate death and degeneration. The embryos are extremely delicate, and it seems almost impossible to separate them from the old clot without injury to the lower layers of tissue. Improvements in technique will, it is hoped, minimise this difficulty.
The stages used in these experiments range from the late pre-primitive streak to the stage of 2-4 somites, approximately 6-8 days’ growth in utero. Up to approximately 7 days of growth the entire blastocyst may be removed with or without puncturing. The embryonic disc may be isolated with little difficulty from the surrounding trophoblast. Generally, however, some of this tissue remains adherent; in fact it is better for the experiment if the immediately adjacent trophoblast is not removed. It facilitates flattening the embryo and usually prevents the curling or folding which is so detrimental to differentiation.
After approximately 7 days, the blastocyst can no longer be removed entire, as implantation is initiated at this time and the blastocyst is so large that any pressure ruptures it. The embryo is therefore isolated by tearing apart the uterine wall with fine forceps along the median line of the unattached surface. The flaps are then held apart while the embryo is isolated with fine scissors.
Needless to say the strictest precautions against infection must be taken in every phase of the experiment.
The technique of transplanting portions of the primitive streak and twosomite stages between the epiblast and endoderm of the chick embryo is similar to that already described by Waddington (1932).
Cultures have been fixed in Bouin’s solution, and the whole mounts stained in Delafield’s haematoxylin.
Description of Experiments
A classification of early embryos according to their age is of little use; for instance, after exactly 7 days’ development in utero, counting for the time of copulating, the embryos from one female were in the primitive streak stages, from another in the pre-primitive streak stage and from a third in a pre-somite stage. For this reason the experimenter can never be certain of the exact stage he will find in spite of the fact that the time from the minute of copulation is known. Various factors may be postulated to account for this: possibly the length of the interval between copulation and fertilisation is important. Further, even within one uterus, not all of the embryos are of the same stage of development and this discrepancy becomes more noticeable in younger stages such as the ones used in the present experiments. After 10 or 12 days of development in utero the differences in development tend to disappear so that all of the embryos look quite alike.
The stages used in the experiments have therefore been classified according to stage of development and not according to age, as follows (fig. 1):
(a) Pre-primitive streak fig. 1, XG
(b) Primitive streak: (1) Stage of posterior thickening fig. 1, XE (2) Short . (8) Medium. . fig. 1, XL (4) Long
(c) Pre-somite fig. 1, XK
(d) Somite * fig. 1, XB
Fig. 1. Camera lucida drawings of embryonic areas at the stages of explantation (x 26).
XG. Late pre-primitive streak. XE. Stage of posterior thickening. XL. Medium primitive streak. XK. Pre-somite. XB. Three somite.
p.st. primitive streak; p.pl. prochordal plate;
c.pl. chorda plate; p.m.8. pre-mesodermal somite;
. p.kt. primitive knot; 8. somites,
The results secured from the culture of each stage will be discussed separately. In general, the younger the stage explanted the less has been the development attained.
Table I includes the results obtained in the experiments. Unless so indicated all the cultures were grown upon clots composed of equal parts of adult chicken plasma and chick-embryo extract.
(a) Late pre-primitive streak stage (fig. 1, XG).
Cultures of this stage have been grown on the surface of plasma clots for as long as 5 days, during which time no visible differentiation could be distinguished either in the living culture or after fixation and staining. During the first 24 hours of culture the whole explant contracts or shrinks and becomes markedly thicker. The embryonic area becomes smaller, greatly thickened and rather irregular in outline. No outwandering of cells from the explant can be seen, nor has there occurred any noticeable liquefaction of the plasma.
In the succeeding days it becomes increasingly more difficult to distinguish the embryonic area from the trophoblast. After the third day the two cannot be distinguished and the whole explant appears as a very thick, dark mass of tissue of irregular outline. Outwandering of cells occurs very seldom. In several instances when the fixing fluid was poured into the watch-glass, the explant floated from the clot. This would indicate that the explant only survived, but made no use of the medium either as a source of food or as a supporting medium. Maximow considers that the failure of cultures of 6 days of age to differentiate was due to the destruction of the embryonic area by the invasion and phagocytic activity of the cells of the trophoblast. Whether this may be considered to be the case in the present experiment cannot be determined, as no sections have as yet been made of embryos explanted at this stage.
(b) The stage of posterior thickening and elongation of the embryonic disc (fig. 1, XE).
For the first 8 days the appearance of the explant is similar to that described above for the late pre-primitive streak stage, with the exception that outwandering of cells occurs about the second day. The embryonic area is still distinct on the third day, but has undergone considerable thickening, and its outline has become markedly irregular. Very little liquefaction of the plasma has occurred. This last is true of practically all the stages explanted. The outwandering of the trophoblastic cells indicates that the plasma is being used as a supporting medium. Moreover the food materials present in the embryo extract must be used, since the explant increases slightly in size. Some differentiation is apparent by the fourth or fifth day, when one or two pulsating vesicles may appear. Fig. 2 is a camera lucida outline drawing of a culture of 5 days’ growth in which are seen two pulsating tubes widely separated by an irregular, very dark body of undetermined differentiation. Immediately above The Development in vitro of Young Rabbit Embryos 361
the dark body is a clear, vesicular cavity, while below it the explant is thinner than around the periphery. These thin areas probably represent the former embryonic disc, and the thicker peripheral 7 margin the trophoblast. The protocol of the experiment states that the embryonic area was more or less distinct on the fourth day, while on the second day there was a very short primitive streak.
In another example (Case XE6) the explant on the fourth day seemed everywhere of uniform thickness and condition except at one end where a single pulsating dumbbell-like body could be seen in association with a clear vesicle. As no further differentiation had occurred by the fifth day and as the heart was feebly beating, the culture was fixed.
Thus this stage possesses greater capacity than that of the late pre-primitive streak stage pig 2, Dorsal view of culture XEI. to maintain itself and to undergo differentiation, 5 days’ cultivation. Camera lucida but its development is still markedly influenced utline drawing (50). 4. pulby the very foreign environment. The chief sating tubule. increase in the differentiation exhibited is the occurrence of one or more pulsating vesicles or tubes.
(c) The short primitive streak stage.
A slight advance in the activity of the embryonic area is shown by this stage. In one example in particular the embryonic area, as well as the primitive streak, was markedly elongated at the end of the first day. The whole explan* ‘became increasingly more dense during the subsequent days until all distinction between embryonic area and trophoblast disappeared. After 48 hours in culture two widely spaced pulsating tubes were present at the extreme edges of the explant while, after 72 hours, these were increased to three. The third appeared also at the extreme edge, but almost between the other two. In no other culture of this or older stages have more than two pulsating tubes differentiated. No other differentiation occurred anywhere in the explant by the fifth day so the culture was killed. Other than the three “hearts” the culture appeared as a homogeneously thick, dark, flattened mass. It had practically doubled its size.
Another example, XA2, showed a marked elongation of the embryonic area and primitive streak. After 48 hours the latter was comparable to that of a long primitive streak stage. It was somewhat smaller than the control, while the embryonic area and trophoblast were very thick. Unfortunately degeneration occurred on the third day.
In comparison with the stage of posterior thickening a greater resistance to environmental influences is shown by this stage. Noticeable are the elongation of the embryonic area and primitive streak after 24 hours of culture, and the appearance of pulsating vesicles on the second day. In explants of the stage of posterior thickening, pulsating vesicles appeared only after 4 or 5 days of culture.
(d) The medium primitive streak stage (fig. 1, XL).
The stage of medium primitive streak is the earliest stage from which these developed any embryonic primordia other than pulsating tubes.
Reference to Table I will show that a relatively small percentage of embryos develop and that these never develop very far. In most of the examples the phenomena occurring in the cultures are similar to those already described for the earlier stages. Thus there is a shrinkage and thickening of the explant, early elongation of the primitive streak and embryonic area, subsequent loss of individuality of embryonic area and trophoblast, and finally the appearance of one or two pulsating tubes in an otherwise thickened, more or less homogeneous mass of tissue.
Exceptions to this occurred in cultures XL2—-XL4. Case XL2 gave rise after 48 hours’ culture to a somewhat distorted but otherwise complete embryo with four pairs of somites. After 24 hours of culture the embryonic area as well as the primitive streak were markedly elongated and the stage was comparable to the control pre-somite stage. By the second day there had occurred a considerable advance in the degree of development; a typical three-part brain composed of prosencephalon, mesencephalon and myelencephalon was visible, as well as a short neural tube bordered by four pairs of somites, and there was a considerable thickening of tissue in the region of the tail. The initial appearance of the head fold separated the anterior tip of the head from the remainder of the embryonic area. A single elongated pulsating heart extended anteriorly to the head. The optic vesicles were indicated, and there was a short rod lying beneath the anterior end of the neural tube which was interpreted as the notochord. After 72 hours of culture the heart was no longer beating, and as visible necrosis was present in certain parts of the explant, it was killed.
Case XL2 was by far the best example obtained from explants of this stage. Case XL8 failed to show any differentiation other than a localised pulsation after 24 hours of culture. Cases XL and XL4 developed typical three-part brains, and short neural tubes, but only XL4 formed two hearts. Cases XAI and XC1—XC4 gave no differentiation whatsoever, and appeared as thickened masses of tissue. In case XC5 two pulsating vesicles were visible on the third day of culture.
Cases XL2 and XL3 are especially interesting, as they were grown upon a medium composed of adult chicken plasma and rat-embryo extract. It cannot be said at this time that the high degree of differentiation present in case XL2 was the result of the rat-embryo extract. Reference to cases XK1 and XK8 below, in which the pre-somite stage was explanted to the same medium, show Table I. Summary of culture experiments. (Unless so indicated all cultures were grown in chicken plasma and chick-embryo extract.)
Days cultured Cases Comments , A ~ 1 2 3 4 5 Late pre-primitive streak:
XF6 Shrinkage Shri e Thick mass No differenand thicken- and thicken- tiation ing ing
XF7 ” Elongated *”
XF '8 ” ” ”
XG6 ” ” ”
X@Q7 ” ” ”
XGs » Elongated ”
mass Posterior thickening and initial elongation:
XE2 Disc dis- Shri e No differentinct, and thicken- tiation shrinkage ing
XE5 - ” Infected *
XE6 » ” 1 heart No further differentiation
XEI1 ” » Elongation 2 hearts,
(Fig. 2) and thicken- neural ing of disc tissue? Short primitive streak:
XLl Elongation Thickening, 3 hearts, No further No further of primitive 2 hearts culture very differentia- differentiastreak, thick- dense tion tion ening
XA2 Elongation Pre-somite Necrosis of disc stage
Medium primitive streak:
XAl No differen- No differen- Necrosis
tiation tiation, changed to fresh medium
XCl Shrunken Thickening No differen- No differen tiation tiation
XC2 Shrunken Medullary » ” and thick- plate? ened
XCc3-— ” Thickening ” ”
XC5 Curled mass Thick mass 2 hearts, No further
medullary differenplate? tiation
XC6_ In refrige- Thickened No differen- ”
rator12hr. mass tiation
XC7 ” ” No differen- 1 heart »
XL2 Rat-embryo Elongation 4-somite Fixed
extract of embryonic embryo, disc an 1 heart, primitive brain, etc. streak XL Used as host » Embryo with Fixed for graft of | Also brain, neural portion of shrunken tube, no pr. st. beating
hearts Cases Comments XL3 Rat embryo extract
Long primitive streak:
XB4 Embryo very short
XK4 = Rat-embryo extract
32-198 (Fig. 4)
32-199 (Fig. 5)
XK1 = Rat-embryo extract
XK3_ Rat-embryo extract 32-231 (Figs. 7, 8)
Two somite: XBl
XB2 (Fig. 6)
XB3 Cut transversely between somites A, anterior piece.
P, posterior piece
XD4 XD3_ Cut transversely just anterior to somites.
A, anterior; P, posterior XD12 In refrige(Fig. 3) rator at 5° C.
for 6 hours
Table I (continued)
1 2 Differentia- No hearts tion obscure, thick mass, beating Thickened 2 hearts, mass neural tube,
Embryo of 5 Fixed somites,
Embryo of 6 Fixed somites, brain,
tube, 2 hearts Brain, neural Fixed tube, ear
Embryo of 5 Fixed somites
4 somite No change embryo, 2 hearts, brain 8 somites, No heart brain, no beat, fixed heart beat 6 somites, Heart beatbrain ing, fixed 5 somites, Fixed brain, 2 hearts 5 somites, brain, fixed 9 somite Appearance embryo of membranes 5 somite Fixed embryo 8 somite Covering by embryo, 2 membranes hearts, brain 9 somite ” embryo A. 2 hearts, brain. B. Neural tube, somites 6 somite embryo. fixed
Fixed, no further differentiation
No further development
2 hearts, brain
A. Brain, hearts, neural tube, somites. B. Neural tube, somites
that the resulting embryos were certainly no better, and perhaps even worse, than others grown upon chick-embryo extract, and the same is true of case XK4, an explant of a long primitive streak stage. These experiments were tried for the purpose of finding a better growth-promoting medium. The most that can be said at the present time is that rat-embryo extract does not furnish obviously better growth-promoting factors for the rabbit embryo than does chick-embryo extract.
The effect of a lowering of temperature upon the development of these early stages is a question which is still unsettled. As was stated in the section on methods, all experiments were performed at room temperature and explantation made to the surface of a cold clot. In the case of the chick embryo, similar methods appear to have no effect upon the development of the explant, but it is well knowh that chick embryos can undergo lowering of incubation temperature, as, for instance, at the gastrula stage, with no apparent ill-effects. If for some reason the hen fails to deposit a fertilised egg and it remains in the uterus overnight, development continues, and at the time of laying the embryo usually is in the gastrula stage. If not incubated at once, the embryo may remain at this stage for a relatively long time, development continuing when the egg is incubated. The mammalian embryo is never subjected to such a change of temperature at any point in the course of its development, consequently at first sight it might be expected that such a change of temperature as occurred during these experiments would greatly influence subsequent development. To test this question further, portions of the uterus containing embryos in the medium primitive streak stage were placed in a refrigerator at 5° C. for 12 hours, after which the embryos were explanted. In case XC6 no differentiation occurred, and the culture resembled others of the same stage not similarly treated, in which no differentiation occurred. Case XC7 contained one pulsating tube after 72 hours of culture. Portions of a similar stage transplanted between the epiblast and endoderm of a chick blastoderm lived and became incorporated as grafts. A two-somite embryo (XD12), which had been in the refrigerator for 6 hours, developed after 24 hours into an embryo with six pairs of somites, brain, neural tube and notochord, which was quite similar to other explants which had not been treated in this manner (fig. 3). Moreover portions of two-somite stages subjected to this same lowering of temperature for 5-6 hours and subsequently transplanted to the chick blastoderm became incorporated as grafts and differentiated into somites and neural tube.
These experiments would seem to indicate that the mammalian embryo, at least in its earlier stages, may be subjected to considerable lowering of temperature without markedly affecting its development in vitro otherwise than by retarding it. It remains to be seen whether the maintenance of a constant body temperature will give more complete and further differentiation in vitro.
Fig. 3. Dorsal view of culture XD12. 243 hours’ cultivation. Camera lucida outline drawing ( x 33).
(e) The long primitive streak, pre-somite, and two or three somite stages (fig. 1, XK, XB).
In every case explants of the long primitive streak stage develop embryos with brain, neural tube, somites, one or two hearts, and possible notochord. Several of the better differentiated embryos are included in Table I. Maximum development is attained after 2448 hours of culture and the embryos have usually been killed on the second day. No further differentiation occurs after this interval as far as can be seen from the observation of living cultures. The hearts cease to beat and centres of necrosis appear which gradually spread throughout the culture. Similar phenomena occur in cultures of the older stages. In fact explants of these three stages cannot be successfully cultivated longer than 48 hours.
Cases 198 and 199 (figs. 4, 5) are camera lucida outline drawings of explants of the long primitive streak stage which have been cultivated for 48 hours: In both instances a recognisable young embryo may be clearly seen; that in fig. 5 shows better differentiation than the other as regards the somites. A normal three-part brain, auditory vesicles, optic vesicles, five pairs of somites, neural tube and head fold are present in fig. 5. In the living culture two beating heart primordia lay on either side of the embryo in the region of the mesencephalon. Considerable outwandering of cells was visible around the periphery of the culture. Efforts to subculture such stages after 48 hours have resulted in the death of the embryo. The embryonic tissues, especially the endoderm, adheres so firmly to the plasma that the embryo cannot be removed without injury. If greater technical skill will permit this, it is possible that the embryo may be kept alive for longer periods of time.
Another explant of the same stage (Case XK4) was grown upon a medium composed of adult chicken plasma and rat embryo extract. The degree of . differentiation was similar to that just described above with the exception that six pairs of somites were visible. This difference is not great enough to be significant.
Fig. 4. Dorsal view of culture 32-198. 48 hours’ cultivation. Camera lucida outline drawing (x 33). 6., brain; 7.t., neural tube; a.v., auditory vesicle; c., trophoblast.
Fig. 5. Dorsal view of culture 32-199. 48 hours’ cultivation. Camera lucida outline drawing (x 33). 8., somite; other lettering as before.
It is interesting to note that explants of the pre-somite and two-somite stages fail to develop beyond that of the embryo just described. It might be expected that, considering the advanced degree of differentiation present at the time of explantation, more normal embryos might result. But this is not the case (cf. figs. 6, 7, 8). In some instances eight or even nine pairs of somites developed, but otherwise no difference could be seen in comparison with the embryos from the long primitive streak stage. The use of rat embryo extract likewise did not apparently affect development. In one example (XK1) the embryo contained eight pairs of somites, in another five pairs.
Apparently the development of the explants beyond that described above is prohibited by the mechanical restrictions of the environment. The embryo develops in the form of a flattened sheet without the differentiation of body form, the feature of which in turn checks organ differentiation.
Fig. 6. Dorsal view of culture X B2, explanted in pre-somite stage. 29 hours cultivation. Camera lucida outline drawing ( x 33). h., heart, other lettering as before.
- The results of the present experiments show that young rabbit embryos may be grown in vitro upon a medium of adult chicken plasma plus a saline extract of chick-embryo extract and that considerable differentiation may occur,
- A previous lowering of the temperature has no effect other than a slight slowing up upon this differentiation. Moreover rat-embryo extract may be substituted for that of the chick without affecting the results.
- The older and more differentiated the embryo at the time of explantation, the greater is the differentiation exhibited by the culture.
- The stage of late pre-primitive streak gives no apparent differentiation as seen in whole mount preparations. Localised thickenings only occur.
- The stage of posterior thickening and initial elongation of the embryonic disc develops one or two beating hearts, and localised thickenings after 4-5 days’ growth in vitro.
- The stage of short primitive streak undergoes marked elongation of the primitive streak and embryonic dise on the 2nd day; two, and in one case three, beating hearts appeared after 2-3 days of culture.
- The stage of medium primitive streak gives results comparable to (c). In several instances brain, hearts, neural tube and somites appear. Fig. 7. Dorsal view of culture 32-231, at time of explanting: late pre-somite stage. (Approx. x 30.) Photograph.
- The stage of long primitive streak gave rise to embryos with as many as six pairs of somites after 1 day of culture, and the pre-somite and two-somite stages give only slightly, if at all, better development.
- Pulsating muscular tissue (? heart tissue) differentiates very easily from the earliest stages where no differentiation of any other primordia is noticeable.
- The distortion present in most of the cultures is the result of the mechanical conditions, which greatly hinder the development of folds.
Fig. 8. Dorsal view of culture 32-231, after 24 hours’ oultivation. Note optic vesicles, somites, folding of embryonic area and contraction of trophoblastic portions. (Approx. x 25.) Photograph. .
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