Akio S. Suzuki

Studies on the formation and state of determination of the trunk organizer in the newt,Cynops pyrrhogaster

SummaryThe exact localization of the presumptive trunk organizer was determined by means of vital staining at the initiation of gastrulation (0 h embryo) and subsequently in 6, 9, 12 and 24 h embryos.The progressive changes in the self-differentiation and inductive capacity of the trunk organizer were studied in isolation cultures (sitting drop) and in sandwich cultures with competent gastrula ectoderm. In the 0 and 6 h embryo cultures the excised trunk organizer predominantly formed atypical ectoderm. A dramatic change in differentiation and inductive capacity occurred in the 9 h embryo. The positive cases — 83% of the isolation and 50% of the sandwich cultures — mainly formed notochord and somites, accompanied by spinal cord and hindbrain in the sandwich cultures. Although no further change in self-differentiation occurred from that time onwards, a gradual increase in inductive capacity was recognized.

Germ Layer Interactions in Pattern Formation of Amphibian Mesoderm during Primary Embryonic Induction

Mesodermal differentiation of dorsal marginal zone (DMZ) before and after invagination was analyzed by a series of combination experiments with different kinds of ectoderm.

TEMPORAL RELATIONS BETWEEN EXTENSION OF ARCHENTERON ROOF AND REALIZATION OF NEURAL INDUCTION DURING GASTRULATION OF NEWT EMBRYO

This investigation was performed in order to analyze the basic relationships between the archenteron roof and the overlying ectoderm in primary induction in the Cynopus (Triturus) pyrrhogaster embryo.

ANALYSIS OF CELL PROLIFERATION DURING EARLY EMBRYOGENESIS

Cell proliferation was examined during early embryogenesis of the newt (Triturus pyrrhogaster) by various methods. After the two‐cell stage, at 23°C, the blastomere (cell) number per whole embryo increased logarithmically until the mid‐blastula stage (for about 19 hr) and the rate of increase slowed down in and after the late blastula stage. On the other hand, the synchronous cleavage of the blastomeres at the animal pole continued for 18 hr until the twelfth cleavage (mid‐blastula) and the transition from synchronous to asynchronous division occurred abruptly at and after the thirteenth cell division (late blastula). The study also showed that the presumptive neuro‐ectoderm consisted mainly of cells of the fifteenth generation (G‐15) at the onset of gastrulation (pigment stage).

Studies on the formation and state of determination of the trunk organizer in the newt,Cynops pyrrhogaster: IV. The association of the neural-inducing activity with the mesodermization of the trunk organizer

SummaryThe stepwise process of the formation and determination of mesoderm inCynops pyrrhogaster was analyzed. The presumptive ectoderm (PE) of the early gastrula was transformed into mesoderm within 12 h when transplanted into the upper half of the dorsal marginal zone of the same stage. The self-differentiation capacity and the neural-inducing activity of this newly mesodermized PE (MPE) were examined by both isolation and sandwich cultures.The MPE showed self-differentiation for notochord and muscle in the isolation culture. In the sandwich culture, the MPE made contact with the PE of the successive gastrula stages. The MPE was capable of inducing neural tissues even in the PE of the mid-gastrula, which has high neural competence but loses it within a short period of 6 h.These results show that firstly the mesodermization of the PE is completed within 12 h and secondly both the self-differentiation capacity and the neural-inducing activity are established immediately after the mesodermization of the PE.

NEURAL COMPETENCE AND CELL LINEAGE OF GASTRULA ECTODERM OF NEWT EMBRYO

The change in the capacity to form neural structures was quantitatively analyzed in both intact and isolated ectoderms of Cynops pyrrhogaster 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 23°C, after which the neural competence of the ectoderm completely disappeared.

Two essential processes in the formation of a dorsal axis during gastrulation ofCynops embryo

The isolated upper marginal zone from the initial stage ofCynops gastrulation is not yet determined to form the dorsal axis mesoderm: notochord and muscle. In this experiment, we will indicate where the dorsal mesoderm-inducing activity is localized in the very early gastrula, and what is an important event for specification of the dorsal axis mesoderm during gastrulation. Recombination experiments showed that dorsal mesoderm-inducing activity was localized definitively in the endodermal epithelium (EE) of the lower marginal zone, with a dorso-ventral gradient; and the EE itself differentiated into endodermal tissues, mainly pharyngeal endoderm. Nevertheless, when dorsal EE alone was transplanted into the ventral region, a secondary axis with dorsal mesoderm was barely formed. However, when dorsal EE was transplanted with the bottle cells which by themselves were incapable of mesoderm induction, a second axis with well-developed dorsal mesoderm was observed. When the animal half with the lower marginal zone was rotated 180° and recombined with the vegetal half, most of the rotated embryos formed only one dorsal axis at the primary blastopore side. The present results suggest that there are at least two essential processes in dorsal axis formation: mesoderm induction of the upper marginal zone by endodermal epithelium of the lower marginal zone, and dorsalization of the upper dorsal marginal zone evoked during involution.

MITOTIC ACTIVITY AND CELL PROLIFERATION IN PRIMARY INDUCTION OF NEWT EMBRYO

Mitotic activity and cell proliferation of newt (Triturus pyrrhogaster) embryo were examined with special reference to primary induction.

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

SummaryFor analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 103 and 30 × 103 Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level.

Direct evidence of an essential role for extended involution in the specification of a dorsal marginal mesoderm during Cynops gastrulation

It has been indicated that specification of the dorsal marginal mesoderm of the Cynops gastrula is established by vertical interactions with other layers, which occur during its extended involution. In the present study, when the prospective notochordal area of the early gastrula was almost completely removed together with the dorsal mesoderm‐inducing endoderm and most of the bottle cells, the D‐less gastrulas still formed the dorsal axis with a well‐differentiated notochord; in half of them, where the involution occurred bi‐laterally, twin axes were observed. On the other hand, when the wound of a D‐less gastrula was repaired by transplanting the ventral marginal zone and ectoderm, the formation of the dorsal axis was inhibited if the involution of the lateral marginal zone was prevented by the transplanted piece. The present study suggests that: (i) cells having dorsal mesoderm‐forming potency distribute farther laterally than the fate map; and (ii) the extended involution plays an essential role in the specification of the dorsal marginal mesoderm, especially in notochordal differentiation in normal Cynops embryogenesis.