IN VITRO DIFFERENTIATION OF CHICK EMBRYONIC LENS EPITHELIAL CELLS INTO FIBER CELLS.

The processes of fiber-cell formation in the lens epithelium of 9-day-old chick embryo in vitro were studied. Mitotic activity was enhanced during the first 12 hr, but with a drop at the 4th hour of cultivation. After the 24th hour, when the cells began to elongate, almost no mitotic figures or incorporation of 3 H-thymidine into the nuclei were observed. α- and δ-crystallin were contained in and synthesized by the newly isolated lens epithelium. The content and syntheses had diminished by the 12th hour. In the earlier phase of cultivation, both fiber cell formation and crystallin synthesis were suppressed by treatment with Actinomycin D, but after the 12th hour they were resistant to the antibiotic. The correlation between cell division and fiber-cell differentiation in the lens epithelium in vitro is discussed and compared with that reported in Wolffian lens regeneration and in developing bovine lens.

DIFFERENCE IN INDUCTIVE EFFECT OF LIVER TISSUES WITH AND WITHOUT PERISINUSOIDAL BASEMENT MEMBRANE.

Differential inductive capacities among liver tissues of several animals were examined by anticipating the correlation between the capacity and the completness of perisinusoidal basement membrane. The reacting tissue was competent ectoderm of gastrula of Triturus pyrrhogaster, and the inductive effects of livers on the ectoderm were tested by explantation method. The inductive effect of livers being devoid of the membrane (chick and guinea pig) was neural and the tissues having the dense well-developed membrane (reptiles) produced an assembly of neural and meso-dermal tissues, such as notochord and somite or muscle. The livers with the membrane being of intermediate grade of development (calf, Triturus and mouse) induce mesodermal tissues, but not frequently, together with neural tissue or alone. The liver tissue was more active in mesodermal induction in proportion to the completeness of the perisinusoidal basement membrane. On the basis of these data the difference in inductive capacity among liver tissues from different kinds of animals were discussed.

STAGING OF NEWT BLASTULA EMBRYOS AND FIRST APPEARANCE OF PRIMARY MESODERMAL COMPETENCE.

Using the swimbladder of the crusian carp (Carrasius auratus) as an inductor, the first appearance of mesodermal competence in the presumptive ectoderm of the newt (Triturus pyrrhogaster) blastula was investigated. The time course of embryonic development before the gastrula stage was determined by counting the number of surface cells on a 0.25 mm line at the animal pole. Pregastrula embryos with 2-3, 4-5, 6-7 and 7-8 cells roughly correspond to those at 14, 14-12, 8-6 and 4-0 hr before the beginning of gastrulation. Using presumptive ectoderm of the early gastrula stage, 15 min was found to be the minimum time of contact necessary for the realization of induction. The reactivity of the presumptive ectoderm from pregastrula embryos was tested by 30 min contact. Presumptive ectoderm up to the 4-5 cell stage did not react to the inductor. It may become competent within the next 4-8 hr, since the ectoderm from embryos in the 6-7 cell stage was reactive.

INDUCTIVE CAPACITIES OF GLOMERULAR BASEMENT MEMBRANES AND DENTINE MATRIX.

The inductive capacities of the basement membranes of calf kidney glomeruli and the dentine matrix of the incisors of 23-day rabbit fetuses were examined on the presumptive ectoderm of Triturus gastrulae. The basement membranes caused almost entirely neural induction and the dentine matrix caused mesodermal induction. These findings suggest that intercellular substances play an important role in the inductive effects of heterologous tissues.

VEGETALIZATION OF PRESUMPTIVE ECTODERM OF NEWT GASTRULAE IN A TRANSFILTER EXPERIMENT WITH FISH SWIMBLADDER AS THE INDUCTOR.

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 µm thick, with nominal pore sizes of 0.05, 0.1, 0.6, 0.8, 3.0 and 8.0 µm 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 µm 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.

Vegetalising factor extracted from the fish swimbladder and tested on presumptive ectoderm ofTriturus embryos.

Vegetalising factor was isolated from swimbladder of crusian carp (Carassius auratus) by solubilishing with 8 M urea the precipitate obtained after digesting the swimbladder with collagenase. The urea-soluble fraction vegetalised isolated presumptive ectoderm ofTriturus gastrula and produced both undifferentiated mesodermal and endodermal cells. Brief heating of the fraction changed its capacity to produce organised mesodermal tissues, such as notochord and somite, and the frequency of induction of undifferentiated cells was reduced. By inserting the urea-soluble fraction into the blastocoel of an early gastrula, embryos without epidermis were obtained. Some of the embryos consisted of undifferentiated mesodermal and endodermal cells, but in the remaining ones small fragments of notochord, small numbers of somites and pronephros developed, enclosed by endodermal cells.