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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.
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
The change in the capacity to form neural structures was quantitatively analyzed in both intact and isolated ectoderms of Cynops pyrrhogasrer gastrula. The frequency of explants with induced neural structures abruptly decreases between stage 12c and stage 13b in intact ectoderm, and between 12 hr and 18 hr preculture in isolated ectoderm. The quantitative analysis also made clear that the size of the cell population of induced neural structures was gradually reduced with the aging of the ectoderm. The authors simultaneously examined the cell proliferation of early gastrula ectoderm and confirmed that all ectodermal cells divided at least once within 18 hr at 2 3 T , after which the neural competence of the ectoderm completely disappeared.The relationships between neural competence and cell lineage (cell generation) of the ectoderm are discussed in the light of these findings.It has been generally accepted that the young gastyla ectoderm of an amphibian embryo is pluripotent and there is no difference in the reactivity between the various parts of the ectoderm. The pluripotent reactivity gradually decreases with each corresponding stage of the developmental process, and then the reactivity of each ectodermal part becomes restricted to certain areas which correspond to their future places in the embryo.It is also a well-known fact that the capacity of the ectoderm to form neural structures progressively decreases during the gastrulation process and the neural competence is finally lost at the end of gastrulation (1, 21, 22,25). Furthermore, many investigations (6, 11, 19,26,33) have shown that the neural competence bf the isolated ectoderm gradually disappeared with the aging of the ectoderm in a way similar to that of, in vivo. Some of these studies (1 1, 22) demonstrated that the size of the induced neural structures was reduced with the disappearance of the neural competence of the ectoderm. As for the mechanism for the loss of the competence of the ectoderm, NIEUWKOOP (22) suggested the relationships between the metabolic activity and the loss of the neural competence. KAWAKAMI et al. (15), who tried to analyze primary induction at the cellular level by electrophoretically separating the ectodermal cells, imphasized the possibility that the changes in the competence of presumptive ectoderm must be due to a shift of cell susceptibility to the killing capacity of inducing agents. More recently, GRUNZ (8) reported that the rate of reduction of neural competence was delayed by cycloheximid, a specific inhibitor of protein synthesis. These facts suggest that the loss of neural competence might be a reflection of the metabolic changes of the ectodermal cells themselves.It seems, therefore, most important to analyze neural competence in relation to the cytological and physiological natures of the ectodermal cells. Unfortunately, there have been few studies which have examined the exact cytological and physiological changes in the ectodermal cells during gastrulation. Recently, cytological and physiological changes hav...
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