The appearance and extension of neural differentiation tendencies in the neurectoderm of the early chick embryo.

1. Recently the direct analysis of neural induction in the early chick embryo has become possible by the perfection of a technique for mechanical germ layer separation (HARA, 1961). This technique made it possible to interrupt the inductive process at various stages of development by separating the ectoderm from the inducing mesoderm anterior toHENSEN'S node. By subsequently culturing pieces of the isolated ectodermin vivo a study was made of the temporal sequence of appearance and the spatial extension of neural differentiation tendencies in the prospective neural ectoderm. 2. An area of the blastoderm anterior to the node was carefully freed of endo- and mesoderm with tungsten needles, and the remaining ectoderm was then cut into pieces of definite sizes with a glass needle. These pieces were culturedin vivo by means of transplantation into the coelome of 2 1/2 days old host embryos in order to assess their self-differentiation. 3. Donor blastoderms of four carefully defined stages (I-IV) were used: the nearly definitive and definitive streak stages, and the early and medium headprocess stages. The ectodermal area concerned was divided into a median zone and two lateral zones, and each of these zones was subdivided into two, three, or four anteroposterior areas, according to the stage used. The areas were designated with the letters 'A'-'D' for the median ones, and 'LA'-'LD' for the lateral ones. The 'A', 'LA', 'B', and 'LB' areas together always included the prospective prosencephalic region of the neurectoderm, whereas the remaining areas included the prospective mesencephalic region and part of the prospective rhombencephalic region (cf. Figs. 14, 16, 18, 20). 4. A total of 903 grafts prepared from 114 donor blastoderms were transplanted intracoelomically; 202 grafts were lost due to the death of the host embryos; out of the remaining 701 grafts 304 were recovered and studied histologically after 12 days of culturing. 5. There was a marked difference in the rates of recovery between grafts from the lateral and anteriormost median areas containing peripheral parts of the prospective neural anlage, and grafts from the more posterior median areas. The possible reasons for this difference were discussed. 6. The regional neural structures differentiating in the grafts were identified with the help of criteria established byHARA (1961) and extended in the course of the present study. 7. The results may be summarized as follows (cf. Figs. 14, 16, 18, 20): a. In all stages the relative numbers of grafts forming neural structures were lower in the lateral and anteriormost median graft areas than in the more posterior median areas. They generally increased from stage to stage in all graft areas, ranging from 0% in the anterolateral areas of stage I and II, to 100% in the posteromedian areas from stage III onwards. Among the lateral areas in any one stage it was always the area located at the level of the prechordal mesoderm (the 'LB' area) which showed the highest relative number of grafts forming neural structures. b. The median 'A' and 'B' grafts, representing a part of the prospective prosencephalic region, essentially showed prosencephalic differentiation only. Prosencephalic differentiation also occurred in the more posterior 'C' and 'D' grafts, representing a part of the prospective mesencephalon and rhombencephalon. In addition, more posterior neural structures appeared in these grafts, and this became more pronounced with each successive stage. c. All the lateral grafts from all stages showed prosencephalic differentiation only, except for the 'LC' grafts in stage IV, in which mesencephalic differentiation was encountered as well. 8. The results were interpreted in terms of the "activation-transformation" hypothesis of neural induction put forward byNIEUWKOOP (1952) on the basis of experiments carried out with amphibian embryos. The results complement those ofHARA (1961), leading to the following dynamic picture of the origin of the pattern of neural organization in the chick embryo. As a result of the first contact between the neurectoderm and the most anterior (prechordal) mesoderm, which exerts an almost exclusively "activating" action, a wave of activation spreads centrifugally through the ectoderm from the area of contact. As a consequence a neural field (activation field) is set up in the neurectoderm. Activation leads to the appearance of prosencephalic differentiation tendencies in the ectoderm. During the formation of the head-process the more posterior axial mesoderm (prospective notochordal material) is laid down craniocaudally in front of the node. At the same time the corresponding parts of the future neural plate shift from the left and right towards the midline, where they come under the inductive influence of the recently formed notochordal mesoderm. This mesoderm also possesses activating capacities, and consequently the activation field in the ectoderm gradually extends caudally. In the more posterior regions the activation field initially extends less far laterally than in the anterior regions, possibly because the activating action of the notochordal mesoderm is weaker than that of the prechordal mesoderm, but also because the notochordal mesoderm is laid down later, so that its activating action starts later. The activation field thus initially remains widest at the level of the prechordal mesoderm. Besides activating capacities, the notochordal mesoderm also possesses "transforming" capacities. Consequently, in the ectoderm overlying the notochordal mesoderm the wave of activation is succeeded by a wave of transformation, which likewise extends caudally and laterally, and which gradually converts the prosencephalic differentiation tendencies of the ectoderm into differentiation tendencies for more caudal parts of the central nervous system. The transforming influence of the notochordal mesoderm increases in caudal direction. However, since the more caudal parts of this mesoderm are laid down later than the more cranial parts, in the more posterior regions of the neurectoderm the transforming action initially spreads less far laterally than in the more anterior regions. 9. This interpretation leads to the conclusion that striking similarities exist between the neural induction processes in avian and amphibian embryos, notwithstanding the pronounced differences in their mechanisms of germ layer formation.