Kenjiro Ozato

Production of transgenic fish: introduction and expression of chicken δ-crystallin gene in medaka embryos

To produce a model of transgenic fish, recombinant plasmids containing chicken delta-crystallin gene were microinjected into the oocyte nucleus of a small teleost, medaka (Oryzias latipes). About 50% of the microinjected oocytes developed to 7-day-old embryos. By Southern blotting delta-crystallin gene was detected in 4 of 8 embryos, and, by Western blotting, delta-crystallin polypeptides in 5 of 16. In 1 of 6 examined histologically, delta-crystallin DNA was detected in all the tissues, and delta-crystallin polypeptides, in many of the tissues including the lens. Thus, the exogenous gene and/or its products were detected in 10 of 30 embryos examined. This is the first report of successful production of transgenic fish.

Fish Hereditary Melanoma Cell Lines of Different Degrees of Cell Differentiation

Four cell lines including two sublines were established from hereditary melanomas in interspecific hybrids between platyfish (Xiphophorus maculatus) carrying the Sp gene and swordtails (X. helleri) and maintained in vitro for more than 34 months. Cells in each cell line grew randomly across each other with an apparent lack of contact inhibition of growth and at a population doubling time of 50 to 72 hr. They retained the characteristics of young pigment cells in regard to ultrastructure, tyrosinase activity, the DOPA and combined DOPA‐premelanin reactions. In the degree of differentiation, the cells of the three cell lines seemed comparable to early melanocytes close to melanoblasts, and those of the remaining one cell line seemed comparable to young melanocytes but were in a more differentiated state than the early melanocytes. Colony forming ability on plastic plates was at a level of 10% in the three cell lines but only 1% in the one cell line. All four cell lines failed to form colonies in soft agar. Chromosome analysis revealed that these four cell lines were heteroploid with many abnormal figures of chromosomes and double minute chromosomes. None of the cell lines showed transplantability to fish.

Effects of ACTH, adenyl compounds, and methylxanthines on goldfish erythrophores in culture

Abstract Effects of ACTH, nucleotides, nucleosides, bases, and methylxanthines on pigment dispersion were studied in goldfish erythrophores cultured in vitro . ACTH induced pigment dispersion. The action of ACTH was mimicked by cAMP and dbcAMP at concentrations of 10 −4 and 10 −3 M , respectively. Theophylline and caffeine, inhibitors of phosphodiesterase, were effective at 5 × 10 −3 M . On the other hand, adenosine and adenine nucleotides including 2′, 3′-cAMP were also effective at a concentration as low as 10 −5 M .

Stage-dependent expression of the chicken δ-crystallin gene in transgenic fish embryos

To study the regulation of gene expression of vertebrate crystallin genes, the chicken delta-crystallin gene was introduced into a small freshwater fish, medaka (Oryzias latipes), which lacks this gene, and its expression was examined immunohistologically at several developmental stages before hatching. The gene expression was detected in the central fiber cells of the lens at an early stage, showing a stage-dependent expression. In non-lens tissues, the expression was barely detectable before tissue differentiation. It first became substantial mainly in mesodermal tissues and then later in a greater variety of tissues, including ectodermal and endodermal ones. Thus, the non-lens expression of delta-crystallin was also stage-dependent, with the stage being dependent on the tissue type. These results from lens and non-lens tissues are discussed in relation to tissue differentiation and two categories of delta-crystallin expression.

Cell Cycle in the Primitive Streak and the Notochord of Early Chick Embryos

In respect to cell proliferation in early chick embryos, several investigations have been done by means of counting the number of mitotic figures, and they revealed the different mitotic activity in the different regions of the embryo (DERRICK, 1937; BELLAIRS, 1955 and 1957; EMANUELSSON, 1962, 1965). The present study was performed to investigate the cycle of the cells in the process of morphogenetic movement in early chick embryos, especially of cells immigrating through the primitive streak. For this purpose, besides counting the number of mitotic figures, autoradiographic observations were carried out on the embryos explanted on a nutrient medium containing -thymidine.

Induction of xanthophores from non-pigmented dermal cells of xanthic goldfish in vitro.

To identify precursor cells of xanthophores (xanthoblasts), non-pigmented cells without any phenotypic traits as pigment cells were isolated from the dermal tissue of xanthic goldfish with an adult color pattern and cultured in a medium containing 1 mM db-cAMP or 0.25 U/ml ACTH and 10% carp serum. These non-pigmented cells differentiated into xanthophores which showed a dendritic morphology and contained a large quantity of fluorescent pteridines and numerous vesicular inclusions. Sepiapterin was the major component, and the vesicles contained fuzzy material in addition to small membranous elements. The fluorescent pattern and the morphological characteristics indicated that the differentiated pigment cells were xanthophores of larval type.

Cellular Heterogeneity in the Late‐onset Form of Hereditary Melanomas in the Xiphophorus Fish Hybrids

The late‐onset form of melanomas occurring in the Xiphophorus, fish hybrids carrying a macro‐melanophore gene Sp was investigated for its cellular heterogeneity. The melanoma tissues were dissociated enzymatically and cultured for a short term. The cultured melanoma cells were characterized according to cell size, cell shape, pigmentation, and response to epinephrine. The melanoma cells were considerably heterogeneous in these phenotypic traits. Various combinations of these heterogeneous cells gave a great heterogeneity to individual melanomas. The stability of the phenotypic traits was followed during the course of tumor growth. Cell size and cell shape were stable, but pigmentation and response to epinephrine varied. The results are discussed in relation to cell differentiation and tumor progression.

The Degree of Differentiation in Early‐ and Late‐onset Forms of Platyfish‐swordtail Hybrid Melanomas: A Morphological and Physiological Study in Primary Culture

The early‐ and late‐onset forms of platyfish‐swordtail hybrid melanomas (fry and adult melanomas) and the macromelanophores of platyfish and melanotic hybrids were cultured and characterized according to their cellular morphology and physiology. The fry melanomas contained many large and broad cells. The pigmentation of these cells was somewhat less than that of the macromelanophores of platyfish. Most of the fry melanoma cells responded rapidly to 10−6M epinephrine, exhibiting reversible melanosome aggregation. The adult melanomas consisted of small, dendritic, and sparsely pigmented cells. The physiological response of these adult melanoma cells varied widely from tumor to tumor. These findings are discussed in relation to the differentiation of fish melanophores.

CELL CYCLE IN THE PRIMARY EMBRYONIC INDUCTION OF CHICK EMBRYOS

It was shown in the previous paper (OZATO, 1969) that labelling indices (LI) indicated characteristic distribution pattern along the notochord a t different stages of chick embryos labelled with 3Hthymidine and observed by autoradiography. LI was lowest in the prenotochordal cells immediately after invagination and was increased gradually after migration anteriorwards. Since an important role of the notochord in the primary embryonic induction in chick embryos has been well established (review : WADDINGTON, 19521, it is an interesting subject to examine a correlation in cell cycle between the reacting neural tissue and the inductive notochord. In the present work, distribution pattern of LI along the ventral portion of the neural tissue was compared with that along the underlying notochord.