Yuko Wakamatsu

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.

Developmental Genetics of Medaka

Small laboratory fish such as medaka and zebrafish have been used for studies of development and genetics in vertebrates for many years. Their advantages for use in these studies are as follows. First, their body systems are the simplest among vertebrates; second, the fact that they reproduce through external fertilization and the transparency of their eggs facilitates observation and manipulation of embryos; third, their small size, 2 to 3 cm in body length, and short generation time, 2 to 3 months, are convenient for genetic studies. These advantages of laboratory fish, however, have not been fully utilized until comparatively recently. In the last few years, the potential of laboratory fish as models for developmental genetic studies has been reassessed, based on the vast success of studies using Drosophila, C. elegans, and mice in this field. The most powerful tool in the success of Drosophila and C. elegans in developmental genetic studies has been large-scale mutagenesis and identification of mutant genes based on detailed genetic maps. For researchers achieving success in these invertebrates, laboratory fish have become strikingly attractive as vertebrate models for developmental genetic studies. Large-scale mutagenesis has been realized in zebrafish (24, 33), and efforts to construct genetic maps have begaun using DNA markers in zebrafish (15, 28) and medaka (20, 32). On the other hand, mice have been used with great success in vertebrate developmental genetic studies in the last decade, due to the discovery of gene transfer techniques, transgenesis (9) and gene targeting (4). These techniques have become powerful tools for elucidating genetic mechanisms of development through alteration of genomes by transfer of cloned and well-characterized genes. However, difficulties in direct observation of embryos due to the fact that mice

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.

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.

TWO TYPES OF MELANOMAS IN A NEW EXPERIMENTAL SYSTEM OF PLATYFISHSWORDTAIL HYBRIDS

A new experimental system for investigating platyfish‐swordtail hybrid melanomas was developed using ornamental platyfish (Xiphophorus maculatus) and swordtails (Xiphophorus helleri). The incidence of melanomas in the hybrids was studied in relation to individual age factors. Based on the age of the onset of the melanomas, two types of the melanomas were discerned and were designated as the fry and the adult melanomas, respectively. The fry melanomas were characterized by early onset, wide‐ranged lesions, an invasive nature owing to densely pigmented cells, and a tendency toward regression. The adult melanomas were characterized by occurrence in the later stage of life, a small number of primary sites, and rapid, expanding, and invasive growth due to sparsely pigmented cells.

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.