UsingTriturus pyrrhogaster embryos, the effects of uninduced cells on the differentiation of induced cells were investigated. The inducing stimulus was given to the presumptive ectoderm of early gastrulae by treatment with protein sooution from guinea pig bone-marrow. Mesodermal induction was evoked in the ectodermal explants. After the treatment, some of the ectodermal explants were cut into pieces 1/8 of their original size and combined with untreated presumptive ectoderm. Mesodermal tissues were differentiated in the combined explants too, but the mesodermal tissues evoked in these combined ectodermal explants were different in their regional characters from these in uncombined explants; dorsal structures, such as notochrod and muscle, were observed predominatly in the latter, whereas the dominant structures observed in the former were ventral ones, such as mesothelium and mesenchyme. The shifting of the regional characters in the combined explants was regarded as the result of an unknown effect from the uninduced cells.
J A P A N Using newt, Cyiiops pyrrliogaster., embryos, the production and/or transmission of a homoiogcnetic effect of the inddction within a presumptive ectoderm was investigated. The primary inductor was the swimbladder of a crucian carp, Caracius auratus. In the first experiment, a piece of presumptive ectoderm was isolated from an early gastrula and one-third of its inner surface was placed in contact with the swimbladder for 30 min. After removal of the inductor, the ectodermal piece was allowed to stand by itself for several hours (pre-cultivation), and then it was divided into three parts of equal size, i.e. the end that had been placed in contact with the swimbladder (PI), the next middle (P2) and the end part (P3).The tissues produced in each part were examined after 10 days' cultivation in Holtfreter's solution. The induction was evoked not only in PI, but also in P2 and P3 which had been free from the inductor. The incidences of tissue differentiation in P2 and P3 increased with the lapse of the pre-cultivation time, and the rise in P3 came after P2. These results suggested that the mesodermal tissues in the parts not placed in contact with the swimbladder (P2 and P3) were evoked by the homoiogenetic stimulus which came from PI and P2, respectively.In the next experiment, P2 of the ectodermal piece was substituted by an aged ectoderm which had lost its primary competence. In this system, the mesodermal induction was not evoked in P3. This suggested that the production of the homoiogenetic activity of the ectoderm might be associated with its cornpetenece.In the analysis of the primary induction system, the main line of research has been concerned with heterogenetic induction, in which the inductor (the archenteron roof) and the induced tissue (the neural plate) belong to different germ layers. However, besides the heterogenetic nature of primary induction, its homoiogenetic or assimilatory aspect can be seen in the pioneering work on primary induction by SPEMANN and MANGOLD (I). The secondary primodium consisted partly of the light tissue of the organizer (Triton cristatus) and partly of the pigmented materials of the host (T. tcreniatus). The homoiogenetic nature of this induction was clearly established by the discovery of neural induction by the neural tissue (2). Recently, an analytical approach to homoiogenetic induction was made by DEUCHAR (3). She made a combination experiment on a small number of induced cells (labelled with 3H-TdR) with a much larger number of uninduced cells (unlabelled) of Xenopus embryo, and she discovered that the participation of the uninduced cells in the formation of neural tissues in the induced cells had taken place in her system. The same was demonstrated by RASILO and LEIKOLA (4) with a quailchick system.
Using embryos of the Japanese newt, Cynops pyrrhogaster, homoiogenetic and heterogenetic induction were investigated in the partially mesodermalized presumptive ectoderm. Half of the isolated presumptive ectoderm was placed in contact with the swimbladder of the crucian carp, Carasius auratus: for 15 or 60 min, while the other half was stained with Nile blue sulfate at the same time. The distribution of the stained cells in the tissues evoked in the explants was examined after cultivation for 10 days.This indicates homoiogenetic induction by the primarily induced part of the ectoderm on the other half. The neural and epidermal tissues in the explants were composed of stained cells only, except in one case. We conclude that the neural tissues.are derived from cells not placed in contact with the swimbladder and that they are induced by the primarily induced part of the ectoderm.Some mesodermal tissues were composed of both stained and unstained cells.The idea of "homoiogenetic induction" was originally proposed by MANGOLD and SPEMANN (8) with regard to neural induction, and many papers on the homoiogenetic nature of neural induction have been published (1, 5, 6, 7, 10,19,22). As for mesodermal induction, the homoiogenetic nature was demonstrated by implantation and explantation experiments (3, 1 1, 13, 16, 17). Since homoiogenetic or assimilatory induction may be caused by cell to cell transmission of an inducing stimulus (15), the analysis of this transmission as well as that of layer to layer (14, 20) may offer an useful key for understanding primary induction.Recently, KAWAKAMI (2) demonstrated the strong mesodermal inducing activity of the swimbladder of the crucian carp. Since the area of contact between the isolated presumptive ectoderm and the swimbladder can be manually controlled due to their sheet-like form, the swimbladder is a useful tool for the analysis of propagation of the inducing stimulus within a reactor.In the present study, half of the isolated presumptive ectoderm of newt gastrula was placed in contact with the swimbladder and at the same time, the other half was stained with Nile blue sulfate to distinguish both parts in one explant. MATERIALS AND METHODSEmbryos of the Japanese newt, Cynops pyrrhogaster, were used for all experiments. The presumptive ectoderm of an early gastrula (stage 12a (9)) was isolated manually ( 1 . 0~ 1.5 mm in size) and used as the reactor.
The diffusibility of the vegetalizing factor was examined by a transfilter culture using an ethanol-fixed swimbladder of the crucian carp (Carassius auratus) as the inductor and presumptive ectoderm from gastrulae of Cynops pyrrhogaster as the responding tissue. Nucleopore filters, about 12-14 pm thick, with nominal pore sizes of 0.05, 0.1, 0.6, 0.8, 3.0 and 8.0 pm were interposed between the interacting tissues. The responding pieces of ectoderm were removed from the assemblies after contact for 0.5, 1, 3, or 24 hr and cultured in Holtfreter's solution for 10 days at 20°C.The inductions observed were almost entirely mesodermal, although masses of endoderm-like yolky cells were seen in explants and neural tissues in a few cases. Filter membranes with pores of 0.05 to 8.0 pm did not interfere with the vegetalizing effect.Under an electron microscope, small cytoplasmic cones of the responding cells of the presumptive ectoderm were observed in the pores of the interposed filter after 3 hr's contact. The cones grew longer as the cultivation time increased, but even after 24 hr there was no contact between the interacting tissues. Since 3 hr's contact between the interacting tissues was sufficient to cause full vegetalization on the transfilter culture with the swimbladder, the formation of the cytoplasmic outgrowths had no significance in the induction.The transfilter method, which was first developed by GROBSTEIN (5, 6 ) in pioneer work on the mechanisms of development of the salivary gland, has been employed in experiments on various organs (1, 4, 19, 24, 25). The method was devised to test whether cell-to-cell contact between interacting tissues in induction systems is necessary for induction. Experiments have shown that direct contact between interacting tissues is not usually essential, although induction of the kidney tubule by the spinal cord may require close cellular interaction (22).Just before development of the neural plate in amphibian embryos, the notochordal anlage and the presumptive neuro-ectoderm come into contact, suggesting that cell contact may be significant in primary induction (23). This possibility has been tested using several methods which prevented direct contact between competent ectoderm and inductive materials (2, 3, 7, 9). NIU and TWITTY (13) also examined the effects of treating small pieces of presumptive ectoderm with a medium conditioned by previous cultivation of axial mesoderm. In some of these earlier experiments, no inductions occurred (3, 7) and in others, the results were either questionable or only neural cells without organized tissue were induced (2, 13). However, applying a modification of GROBSTEIN'S technique in studies on primary induction in amphibian embryos (14, 17, 20), induction of the brain was observed using a dorsal blastopore lip as the inductor. TOIVONEN et al. (20) employed a new type of filter membrane, the Nucleopore
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