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75 A FAILURE OF INDUCTION IN NORMAL DEVELOPMENT BY C. H. WADDINGTON. (Senior Student of the Royal Commissioners of the Exhibition of 1851.) (Sub-Department of Experimental Zoology, Cambridge.) (Received August 12, 1935.) (With One Text-figure.) CONTENTS. PAGE I. Introduction . . . . . . . . . . . . I I . C o m p e t e n c e o f lateral e c t o d e r m c o n t e m p o r a n e o u s w i t h i n d u c i n g c a p a c i t y o f side-plate m e s o d e r m I I I . General characteristics of a process of induction . . . . . . I V . T h e specification of p o i n t s w i t h i n t h e neural p l a t e : (a) L o n g i t u d i n a l co-ordinates . . . . . . . . . (b) T r a n s v e r s e co-ordinates . . . . . . . . . V. T h e failure of evocation outside t h e neural p l a t e : («) T h e weakness of t h e evocating powers of t h e side-plate m e s o d e r m (b) T h e w e a k e n i n g of t h e c o m p e t e n c e of t h e lateral e c t o d e r m . . VI Summary . . . . . . . . . . . . . 75 76 77 79 79 . . 80 81 84 I. INTRODUCTION. the neural plate of the Urodele Amphibia has provided the foundation on which modern experimental embryology has been built, it continues to provide us with new problems for solution. Soon after Spemann's original demonstration (1918, 1924) that the neural plate owes its inception to an inducing stimulus provided by the underlying mesoderm, Bautzmann (1926) presented evidence to show that in the young gastrula the region which is capable of inducing is more or less the same as the region which eventually comes to lie immediately under the neural plate. It therefore seemed to be true that in normal development all mesoderm which is capable of inducing actually does so. But recently facts have emerged which cast doubt on this simple hypothesis. Holtfreter (1933 a) showed that in the neurula stage the side-plate mesoderm, which does not underlie the neural plate, is also capable of inducing the formation of neural tissue in pieces of young gastrula ectoderm brought in contact with it. Now Machemer (1932), in a very careful investigation of the variation with age of capacity of gastrula ectoderm to react to an inducing stimulus, showed that this capacity to react, or competence, persists till a stage when the side-plate mesoderm is beginning to be formed. It becomes necessary to inquire whether there is any overlap of the period when the ALTHOUGH 76 C. H. WADDINGTON side-plate mesoderm can induce with that in which the ectoderm can react, and if there should be such an overlap, why no reaction actually occurs in normal development. II. COMPETENCE OF LATERAL ECTODERM CONTEMPORANEOUS WITH INDUCING CAPACITY OF SIDE-PLATE MESODERM. The question was investigated by the following experiment. In a Triton alpestris gastrula in the medium-sized yolk plug stage, a flap of ectoderm was lifted up near the ventro-lateral lip of the blastopore, the side-plate mesoderm was carefully cleaned away from underneath it, and a fragment of the dorsal lip of a young T. alpestris gastrula was substituted. The side-plate mesoderm obtained in this way was then implanted into the blastocoele of another young alpestris gastrula. Owing to lack of material and pressure of other work, comparatively few experiments were made, but the results which were obtained were perfectly clear. Both the side-plate mesoderm implanted into the young gastrulae, and the dorsal lip implanted into the old gastrulae, induced the formation of secondary neural tubes. There is no difficulty in making the grafts into the old gastrulae. In the fixed and sectioned embryos it can be seen that the graft has developed in accordance with its presumptive fate into notochord with some somitic mesoderm, as Bautzmann (1933) has described; the somites were usually very irregularly and imperfectly formed in my material. The graft mass has usually elongated in the direction of the host's gastrulation movements; no attempt was made to control the orientation of the pieces when they were grafted. In several cases the graft is accompanied throughout its whole length by an induced neural plate or tube, though sometimes the induced plate is shorter than the graft mass. The graft and induced plate may lie in the ventro-lateral region of the host, which is where the graft was originally placed, but in some specimens the graft seems to have been pulled in towards the dorsal mid-line of the host, carrying with it the induced plate which becomes united laterally with the host plate. Since the experiments did not involve heteroplastic grafting, the boundaries of the graft mesoderm cannot be precisely determined, but, while the graft notochord always remains distinct from the host notochord, it is clear that some of the graft mesoderm unites with host mesoderm to form compound, chimaerical somites. Fig. 1 shows outline drawings of the host axis and the associated induced axis in one specimen of this kind, No. D70D—2. Very similar embryos have been described by Bautzmann (1933), who exchanged dorsal and lateral mesoderm in young gastrulae, but in his specimens the graft was originally made nearer the dorsal mid-line, and the tendency of the host mesoderm to incorporate the graft was therefore not so obvious. This tendency for the mesoderm to form a single complete system will be referred to as a function of the mesoderm individuation field (p. 78). The tendency for two contiguous neural plates to unite and even to regulate to one single plate is an even commoner and more striking phenomenon, particularly remarkable examples of which have been described in the bird embryo (Waddington and Schmidt, 1933). It would well repay a special investigation. A Failure of Induction in Normal Development 77 In grafting lateral mesoderm into young gastrulae, certain technical difficulties were met with. The amount of side-plate mesoderm which is obtained while making a graft into one mid-gastrula is hardly sufficient to make a satisfactory graft, as the cells hang together only very loosely and some are inevitably lost. It is therefore necessary to combine material from two or more gastrulae, and considerable care is required to implant the separate heaps of loosely connected cells so that they form a single mass lying against the host's ectoderm. In subsequent development, many of the grafts become entirely lost, either because the grafted cells became separated or because the mass was small enough to be completely incorporated into the host's side-plate. In other specimens the graft can be seen as a thickening Fig. I a. Fig. ib. Fig. 1. Embryo No. D70D-2. Diagrammatic sections through the combined host and induced axes (camera lucida drawing). Fig. in is further posterior than Fig. 16. Neural tissue hatched with vertical lines, host notochord widely spaced dots, graft notochord closely spaced dots. of the side-plate of the host; it never shows any trace of development into chorda or somites. In some cases the graft is accompanied by an induced neural plate or tube, which may be perfectly normally differentiated. There is a suggestion that in those specimens in which induction has occurred the graft is less well incorporated than it is in the specimens in which induction has failed to occur, but the data are not extensive enough to permit of a definite statement to this effect. III. GENERAL CHARACTERISTICS OF A PROCESS OF INDUCTION. The experiments which have been described above show that the two materials which are capable of reacting together to produce neural tissue both spread out lateral to the area in which neural plate actually forms. It is therefore impossible to suppose that the region of ectoderm which becomes the neural plate is the same 78 C. H. WADDINGTON as the area under which there lies inducing mesoderm. If we proceed then to consider how the shape of the plate is determined, we have first to remember that the shape in question is continually changing as development proceeds. The series of three-dimensional configurations assumed by a mass of neural tissue must be the result of some forces, as to whose nature we are still almost entirely ignorant. These forces produce in normal development a series of shapes which are characteristic of the well-known morphogenesis of the neural plate; and further, the same series of shapes is produced even if slight alterations are made in the size or disposition of the mass of tissue which forms the starting point of the development. In other words, the neural plate, like most other organs, has a certain regulatory power in the early stages of its development, and the normal series of shapes assumed by it in the undisturbed development of the embryo can be considered as a stable equilibrium (which is of course a function of the time, that is, a fourdimensional equilibrium), towards which the system tends to return after being altered in any way. It is convenient in such a case to refer to neural plate as an individuation field (Waddington and Schmidt, 1933), the forces concerned in the equilibrium as the field forces and the laws by which the forces are related and to which the production of the equilibrium is due as the field laws. The word "field" is already in use with a large number of meanings; it often seems to be employed merely to refer to regions whose boundaries are not sharply denned, and it therefore seems excusable to introduce the special term individuation field for regions such as we are considering, regions, that is to say, in which forces are at work moulding the tissue into a definite shape. In what follows the contraction I.F. will be used for the expression "individuation field". In considering the determination of the shape of the neural plate, we are then actually considering the agencies which define the characteristics of the neural plate I.F. In exactly the same way the whole expanse of the mesoderm is an I.F., that is to say it is the site of field forces which tend to produce a series of equilibrium shapes. We are now in a position to discuss the various processes concerned in this determination. Firstly, the mesoderm calls the neural plate into being; without the presence of the mesoderm the neural plate and its I.F. would not be there, as Holtfreter (1933 b) has finally demonstrated. This process of calling into being, originally referred to as "induction as such" (Waddington and Schmidt, 1933) and later as evocation (Needham, Waddington and Needham, 1934), is due to the diffusion of a chemical substance from the mesoderm into the overlying ectoderm, as recent research has shown. In the normal development of the chick, this diffusion does not seem to be regulated by the mesoderm field laws in the dorso-ventral direction, that is, it is not controlled so as to build up any unit shape, since the axial mesoderm can evocate a neural tube against its ventral surface even when it is already in contact with a neural plate lying against its dorsal surface, and it can thus evocate more neural tissue than is required for the normal equilibrium shape (Waddington and Schmidt, 1933). The experiment does not seem to have been performed in the Amphibia, but it is likely that the same conclusion holds there A Failure of Induction in Normal Development 79 also. We shall have to discuss later whether the mesoderm I.F. controls the area of mesoderm out of which this diffusion can occur. IV. THE SPECIFICATION OF POINTS WITHIN THE NEURAL PLATE. (a) Longitudinal co-ordinates. The neural plate must also be "individuated" (Waddington and Schmidt), that is, two co-ordinates must be specified for each point, giving its position on the anterior-posterior and transverse axes (neglecting for the present purposes its position on the dorso-ventral axis). A process of induction, as opposed to a process of evocation, must involve the specification of these two sets of co-ordinates. There are two possibilities; either the co-ordinates are specified by the mesoderm I.F., or they are previously fixed in the ectoderm, which would then have certain field laws before the specifically neural plate field laws were evocated in it. Spemann (1931) has discussed both possibilities with reference to the longitudinal set of co-ordinates, and decided rather in favour of the second. Waddington and Schmidt (1933), on the basis of work performed on the bird embryo, criticised Spemann's arguments and favoured the first alternative, and more recently Holtfreter (1934) has supported the same view in connection with the Amphibia. Possibly the two alternatives are not mutually incompatible, but the experiments reported in the present communication shed no further light on the determination of the longitudinal set of co-ordinates. (b) Transverse co-ordinates. Our data concern more closely the specification of the transverse co-ordinates. Two facts emerge from previous work. In the first place, a complete set of transverse co-ordinates can be produced by the ectoderm without the co-operation of the mesoderm I.F. This is demonstrated by those cases in which a transversely asymmetrical part of the mesoderm I.F., which moreover remains asymmetrical during the process, induces a symmetrical neural plate: examples are found in the work of Holtfreter (1933 a) on induction by side-plate mesoderm in the neurula stage. The same principle is exemplified in those comparatively few cases in which a dead organiser, in which the field is presumably destroyed, has evocated a symmetrical neural plate of normal shape in ectoderm which is not connected to the main axis of an embryo. Even those cases in which a "fieldless" (dead or dissolved) organiser evocates a symmetrical neural tube in ectoderm which is still part of a complete embryo may probably be taken to support the same conclusion, since although the host's mesoderm I.F. might well affect the longitudinal co-ordinates of the evocated field, it is more difficult to see how it could influence the transverse co-ordinates. Although the ectoderm can, as we have just seen, produce a field completely specified in the transverse direction, other evidence makes it probable that this specification can also be affected by an underlying mesoderm I.F. The most conclusive evidence comes from the observation of compound neural plates underlain 80 C. H. WADDINGTON and induced by compound, unequilibrated mesoderm fields. If a mass of neural tissue is induced by two masses of mesoderm which for some reason do not completely attain the unitary equilibrium shape, the neural plate which is formed shows on the one hand a tendency to form a single unit, which tendency might be attributed to the self-sufficiency of the transverse component of the ectoderm I.F., and on the other hand a definite doubleness which can only be attributed to the doubleness of the mesoderm I.F. Examples of this are very frequent; one has been described and figured above and many other examples will be found in the literature; some particularly striking examples have been described in birds (Waddington and Schmidt). Other evidence which demonstrates the effect of the mesoderm field in this respect is derived from those experiments in which the size or specific nature of the mesoderm I.F. determines the size of the induced neural plate, for instance in the experiments of Ruud and Spemann (1923) on half-gastrulae and of Holtfreter (1935) on Anural-Urodele chimaeras. It seems clear then that the transverse component of the neural plate I.F. is determined by a collaboration of the mesoderm I.F. with a tendency which is already present in the ectoderm and can be released by the mere process of evocation. V. THE FAILURE OF EVOCATION OUTSIDE THE NEURAL PLATE. The final aim must be to discover what is the material basis underlying the specification of a co-ordinate. We are still far from this goal. The next immediate step is the investigation of the consequences of any particular specification. Within the limits of the neural plate itself these consequences are summed up in the results of morphological and histological, and eventually chemical, examination. But for the regions lateral to the neural plate we have to consider the further fact, which is the subject of this paper, namely that there is a failure of evocation. This failure of evocation cannot be ascribed only to the weakness of the evocating powers of the lateral mesoderm, since this mesoderm, as has been shown above, can evocate in young gastrula ectoderm, and should therefore evocate in the lateral ectoderm which it normally underlies if that ectoderm persists unchanged from the young gastrula stage. On the other hand, it is equally clear that it is not only the ectoderm which is at fault, since that same ectoderm can react to a stimulus as powerful as that of the young dorsal lip. We must assume both that the sideplate mesoderm evocates less strongly than does the young dorsal lip, and that the lateral ectoderm reacts less readily than young gastrula ectoderm. (a) The weakness of the evocating powers of the side-plate mesoderm. There are two main possibilities as regards the evocating power of the sideplate mesoderm; the mesoderm at this stage may be able to exert only a weak evocating stimulus even under the best conditions, either because only a weak evocating power has as yet developed or because the evocating power has been irreversibly affected by the remaining parts of the general mesoderm I.F. ; or the side-plate mesoderm may be potentially able to exert a strong stimulus which is A Failure of Induction in Normal Development 81 normally controlled and weakened by a reversible action of the general mesoderm I.F. We have not as yet sufficient data on the evocating powers of the side-plate mesoderm after isolation from the mesoderm I.F. to give an accurate evaluation of these two possibilities. The evidence which is available suggests that both may be to some extent realised. On the one hand, the inductions performed by the sideplate mesoderm, transplanted from the yolk-plug gastrula, have never been as extensive as the normal inductions by young dorsal lip, which suggests that they are reactions to a weaker stimulus. It is quite possible that the intrinsic evocating capacity of the side-plate mesoderm increases rapidly from the young gastrula, in which Bautzmann (1926) found an almost complete inability to evocate, through the fairly weak phase of the mid-gastrula, to the strongly acting condition described by Holtfreter (1933 a) in the neurula. On the other hand, the influence of the mesoderm I.F. in suppressing evocation is suggested by the observation that evocation is nearly always absent in those cases in which the grafted side-plate mesoderm has been completely incorporated into the side-plate of the host. A similar suppression by the mesoderm I.F. of a possible evocation certainly occurs in the chick embryo (Waddington, 1932; Waddington and Schmidt, 1933). (b) The weakening of the competence of the lateral ectoderm. For the weakening of the reaction of the ectoderm there are three factors to be considered. Each individual cell of the ectoderm may become less reactive through an inherent process, or the reactivity of the lateral ectoderm may be suppressed by the underlying part of the mesoderm I.F., that is, by the side-plate mesoderm, or the reactivity may be suppressed by the neural plate field which is gradually arising; or finally the suppression may be due to some combination of these factors. It is easy to make a direct test of the first possibility, and this has been done. Ectoderm was removed from several young gastrulae (Triton alpestris) and was then cultivated in Holtfreter solution along with other gastrulae of the same age until such time as these gastrulae developed open neural plates. Evocating tissue (archenteron roof from the neural plate embryos) was then implanted into the isolated ectoderm fragments. In some cases, though not in all, neural tissue was induced, although it is well known that the ectoderm of the neural plate stage is normally quite incapable of reacting to the stimulus which evocates neural differentiation. One must conclude that the rapid loss of competence of the ectoderm between the gastrula and neurula stages is not due to an inherent process, though there can be little doubt that this competence is eventually lost even in isolated ectoderm, and the processes which cause this slow loss may have begun to play some role even before gastrulation is complete. Returning to the other factors mentioned above, it is easy to see that the mere presence of side-plate mesoderm cannot alone be the condition responsible for the suppression of the competence of the lateral ectoderm, since if it were the sideplate mesoderm should not be able to perform evocations in young gastrula ectoderm, as it actually can. We can also exclude the possibility that the loss of JBB-XIIli 6 82 C. H. W A D D I N G T O N competence is dependent solely on continued connection with the neural plate I.F., since if mid-gastrula lateral ectoderm together with the underlying side-plate mesoderm is cultivated in isolation from the presumptive neural plate, no neural evocation occurs. The isolations which were made to test this point were not cultivated long enough for a complete development of the mesoderm, but there was every indication that the mesoderm had self-differentiated into its presumptive fate. In any case, the failure of evocation in this experiment shows that there is neither an acquirement by the ectoderm of a reactivity as sensitive as that of young gastrula ectoderm nor an acquirement by the mesoderm of a power of evocation equal to that of the young dorsal lip. Therefore we can neither attribute the whole of the loss of competence of the ectoderm to the continued presence of the neural plate, nor, to return to a subject we have discussed earlier, can we hold the mesoderm I.F. entirely responsible for the weakness of the evocating capacity of the side-plate mesoderm, though as we have seen it may have some influence of this kind. We are left with the alternatives that the side-plate mesoderm is able to suppress the competence of the lateral ectoderm only while it is still in its place in the whole mesoderm field, or that in the period between the young and middle gastrula the ectoderm has been brought, either by virtue of its connection with the developing neural plate or through inherent change, into a state in which it can react with the side-plate in such a way as to lose its competence. We can test the last two of these possibilities by isolating ectoderm and presumptive side-plate mesoderm before the neural plate I.F. has begun to develop. If it is the neural plate I.F. which brings the ectoderm into a state in which its competence is suppressed by the side-plate, then the suppression should fail to occur in the isolates and neural tissue should be developed; while if the preparation of the ectoderm is done by an inherent process, we would expect to find no neural tissue. In fact, the ventral half of a young gastrula develops into a " Bauchstuck", in which the included ventral and lateral mesoderm develops into its presumptive fate and in doing so does not induce neural tissue. This evidence suggests that the preparation of the ectoderm is mainly due to an inherent process. But the evidence from a Bauchstuck cannot be regarded as conclusive, since Mangold and Seidel (1927) have described the presence of neural tissue in such systems, though they themselves expressed a doubt as to the validity of their result. No experiments have yet been made which allow one to evaluate the probability of the first of the three alternatives mentioned above, namely that the competence of the lateral ectoderm is suppressed by the side-plate mesoderm only if the mesoderm remains part of the whole mesoderm I.F. We are left, then, with three factors which may be invoked to explain the weakness of the competence of the lateral ectoderm. The first is that the ectoderm changes, through some inherent process, in such a way that at the mid-gastrula stage it is in a state in which it reacts with the side-plate mesoderm with a rapid loss of competence. This inherent process of change might perhaps be connected with the process which causes the slow loss of competence in isolated ectoderm fragments. The second possibility can only be entertained if we accept the presence A Failure of Induction in Normal Development 83 of neural tissue in a Bauchstuck; it also supposes that the ectoderm is changed between the young and mid-gastrula stages, but it attributes this change to the developing neural plate I.F. We have already seen that the developing neural plate I.F. can specify the transverse set of co-ordinates within the neural plate itself, and this second hypothesis merely supposes that it can also do so in the regions lateral to the plate; it supposes, in fact, that the neural plate field is not confined to the neural plate but extends throughout the whole ectoderm. The third possibility is that the loss of competence is dependent on the mesoderm I.F., the suppression actually being carried out by the side-plate mesoderm which forms part of the field. Again, we have seen that the mesoderm field can specify the set of transverse co-ordinates of points within the plate, and there seems no theoretical reason why it should not specify them for points outside the plate. If we accept this general specification of co-ordinates by the mesoderm, it becomes considerably easier to understand the peculiar phenomenon of induction by the so-called "complex situation stimuli". Mangold (1931) and Spemann and Schotte (1932) have described experiments in which, if ectoderm of species A is grafted on to species B, this ectoderm is induced to form an organ characteristic of species A and in the position on the host which corresponds to its normal position in species A, although the host species B neither possesses that organ nor any other in that position. The induction here can be more easily considered as the specification of position co-ordinates than as a reaction to a stimulus whose only function is to evocate the organ in question. It seems doubtful, however, if one should conclude from this that "the reaction system can start a definite type of development as a response to an unspecific stimulus" (Rotman, 1935), since the type of development seems to be quite strictly determined by the position co-ordinates, which are themselves definitely specified, that is, are fixed by some agent which acts specifically at each point. This experiment brings up in an acute form the question of what is the theoretical difference between individuation, the specification of a whole system of points making up an I.F., and evocation, the mere calling up of a new type of differentiation as we see it performed by a dead organiser or a solution of a definite substance. It would seem that individuation can hardly be anything other than a co-ordinated system of evocations, in which case we may hope to isolate the various substances concerned in the specification of each point, just as we are now isolating the substance which is common to the specifications of all points within the neural plate. In recent years several cases have been described in which the area of tissue which can perform a given induction is considerably larger than the area which in normal development actually does so. The experiments described in this paper show that the classical induction of the neural plate belongs to the same type. The areas which are capable of inducing have been spoken of {e.g. Holtfreter, 1933 a; Spemann, 1934; Huxley and de Beer, 1934; Rotman, 1935) as "determination fields". The use of the word "field" in this connection is unobjectionable if it denotes some unit-building activity, but it appears sometimes to indicate merely that the boundaries of the area are vague and perhaps that there is some variation 6-2 84 C. H. WADDINGTON in the strength of the inducing stimulus within it. Under abnormal conditions the "field" may induce two or more organs instead of only one, so that it is clear that in this usage no emphasis is being laid on any tendency of the field to form a unit. Similar unanalysed usages of the "field concept" are so common in experimental embryological literature that it would be over-optimistic to expect them to be given up, but it may be suggested that when one speaks of a field one should be able to indicate either what is the end-product of the field activity, i.e. the field should be defined by the equilibrium towards which it tends, as is done for I.F., or one should be able to name the field activity, e.g. one could speak of a diffusion field of an evocator substance. Thus one could define a field by its tendency to produce one complete induction, but hardly by its being an ill-defined area in which an evocator substance is present. Such areas might be referred to by a more neutral term such as "region" or "district". VI. SUMMARY. 1. The just formed side-plate mesoderm from the gastrula in the medium-sized yolk-plug stage can induce the formation of an extra neural plate when implanted into the blastocoel of a young gastrula. 2. The dorsal lip of a young gastrula, substituted for the side-plate mesoderm mentioned in (1), evocates a neural plate. 3. The failure of evocation by the side-plate mesoderm in normal development can only be explained by supposing both that the evocating capacity of the sideplate mesoderm is lower than that of the dorsal lip and that the lateral ectoderm does not react to an evocating stimulus as easily as does the young gastrula ectoderm. 4. The weakness of the evocating capacity of the side-plate mesoderm might be due either to its intrinsic nature or to an active suppression by the rest of the mesoderm. The evidence suggests that both factors may be involved, but it can be shown that the latter factor by itself is not sufficient to explain the facts. 5. Isolated gastrula ectoderm retains its competence to be evocated to neural tissue at least until the age of an open neural plate embryo. Thus the normal loss of competence by the lateral ectoderm is not due to an inherent process. Nor is it caused solely by a continued connection with the neural plate. It cannot be due merely to the presence of side-plate mesoderm; but it may be due to an influence proceeding from side-plate mesoderm which is part of a mesoderm field, or it may be dependent on a preparation of the ectoderm by reason of its connection with the neural plate, or, more probably, by an inherent process, so that by the stage at which the side-plate mesoderm forms, the ectoderm has become such that it reacts with side-plate mesoderm with rapid loss of competence. A Failure of Induction in Normal Development 85 REFERENCES. BAUTZMANN, H. (1926). Arch. EntwMech. Org. 108, 283. (i933)- Arch. EntwMech. Org. 128, 665. HOLTFRETER, J. (1933 a). Arch. EntwMech. Org. 127, 619. (i933 *)• Arch. EntwMech. Org. 129, 669. (1934)- Arch. EntwMech. Org. 132, 307. (1935). S.B. Get. Morph. Phyiiol. MOnchen. HUXLEY, J. and DE BEER, G. R. (1934). Experimental Embryology. Camb. Univ. Press. MACHEMER, H. (1932). Arch. EntwMech. Org. 126, 391. MANGOLD, O. (1931). Naturwissenschaften, 19, 905. MANGOLD, O. and SEIDEL, F. (1927). Arch. EntwMech. Org. I l l , 593. NEEDHAM, J., WADDINGTON, C. H. and NEEDHAM, D. M. (1934). Proc. roy. Soc. B, 114, 393. ROTMAN, E. (1935). Arch. EntwMech. Org. 133, 193. RUUD, G. and SPEMANN, H. (1923). Arch. EntwMech. Org. 62, 95. SPEMANN, H. (1918). Arch. EntwMech. Org. 43, 448. (1924). Arch. EntwMech. Org. 100, 599. (1931)- Arch. EntwMech. Org. 123, 389. (1934). Freiburg tviss. Get. 23. SPEMANN, H. and SCHOTTE, O. (1932). Naturwissenschaften, 20, 463. WADDINGTON, C. H. (1932). PMlos. Trans. B, 221, 179. WADDINGTON, C. H. and SCHMIDT, G. A. (1933). Arch. EntwMech. Org. 128, 522.