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  • Developmental Engineering of Fishes

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  • Intro

    •  Various breeds of plants have been produced and used in agriculture. Methods of breeding technology include gene modification (gene transfer and genome editing), chromosomal set manipulation, utilization of nuclear/cytoplasmic hybrids, and the induction of chimeric individuals, which are collectively called “developmental engineering.” Plant cells improved by developmental engineering techniques can be cultured to transform them into individuals and then be used as agricultural varieties, because of their “pluripotency” of single plant cells that allows them to differentiate into various cell types.. What about animals? At present, a single animal cell cannot be cultured to produce an individual, and gametes (eggs/sperm) must be generated and undergo the process of fertilization. Therefore, even if an individual possesses a useful gene, it cannot be utilized without going through the gametes. Individuals, of course, can be generated by nuclear transplantation, but the success rate is very low. However, it has been shown that if a “germ-line chimera” is generated by transplanting germ-line cells (donors), which differentiate into eggs and sperms, into another individual (host), then the gamete of the donor can be generated. This method, called “germ-line chimera,” may lead to the improvement of fish breeds, and the Nanae Freshwater Station of Hokkaido University is researching developmental engineering using various fishes as materials.

  • Mechanism and utilization of various cells produced from a fertilized egg, which is a single cell

    • A fertilized egg is a single cell. As it progresses in cell division, not only does the number of cells increase but various cells with different properties are created. What mechanisms produce such different properties of the cells? To conduct research on developmental engineering, we must understand the process by which the body is created from an actual fertilized egg. The elucidation of such mechanisms may allow us to produce individuals from isolated cells. The actual body has “somatic line cells” that make up the body and “germ-line cells” that produce gametes for the next generation.

  • Differentiation of the somatic line

    • In the fertilized egg of a fish, the number of cells increases exponentially, leading to the formation of a blastocyst with approximately 1,000 cells. At this stage, the fate of the cells is not determined. Therefore, the development proceeds normally even if the cells of the embryo are removed or cells are transplanted from another embryo (Figure 1. Embryos obtained by cutting and replacing the blastocyst of a goldfish. Figure 2. Embryos in which the blastodisc cells are added or removed.）


    • Figure 1．Blastodisc transplantation at the blastocyst stage. A) Red-stained and unstained transparent embryos. B) Cutting the upper side of stained and unstained blastocysts. C) Immediately after cutting. D) Immediately after replacing the cut part and transplanting it. E) Restored blastodisc.


    • Figure 2．Embryos where the entire blastodisc is transplanted to the animal pole side of another embryo using transplantation similar to Figure 1. Bottom (from left): transplanted embryo, embryo with the upper part of blastodisc removed, and untreated embryo. All develop normally.

    • The direction of the differentiation of blastodisc cells in the blastocyst stage is determined by the induction signal from the yolk side. The yolk is also called the yolk cell because the nucleus also enters the yolk side during the division process of the fertilized egg. When the blastodisc of the anaphase blastocyst stage is separated from the yolk cell, rotated 180° horizontally and reattached to the yolk cell, many individuals with two bodies are born from this embryo. This is because the body is made up of two groups of cells that have started building the body before or after being separated from the yolk cell (Figure 3．Individual with two axes generated from an embryo in which the blastodisc of the anaphase blastocyst of a goldfish is separated, rotated 180°, and reattached.)


    • Figure 3．A–E) Embryos in which the blastodisc in the blastocyst stage was separated from the yolk cell and rotated 180° horizontally and then reattached. A–D were operated on during the metaphase blastocyst stage, while B and E were operated on in the anaphase blastocyst stage. F is an embryo that was rotated 360° and reattached, and G is an untreated embryo.

    • The body-inducing power of these yolk cells originates in the embryo immediately after fertilization. Immediately after fertilization, if the embryo is cut in half between the animal pole side with the nucleus and cytoplasm and the plant pole side with a lot of yolk, embryonic formation no longer occurs, although the cells increase and grow longer, accompanied by the production of some cells. Furthermore, if it is cut closer to the animal pole side, it only becomes a cell mass. This indicates that the factors that shape future individuals are localized in the isolated plant pole side of the egg immediately after fertilization.


    • Figure 4. When a 2-cell stage embryo is pushed through with a silkworm gut, it is divided into the animal pole hemisphere with the cytoplasm and the plant pole hemisphere with a lot of yolk.


    • Figure 5．When the animal pole hemisphere in the early cleavage stage is isolated and cultured, the embryo does not form a normal shape and it becomes an embryo with rotational symmetry.


    • Figure 6．Removal of the cytoplasm at the 1-cell stage leads to the formation of an embryo with only a cell mass (K and L).

  • Differentiation of germ-line cells

    • Germ-line cells of fishes arise from cells that have taken up the special cytoplasm called “germ cytoplasm” stored in the egg cytoplasm. The germ cytoplasm gathers on both sides of the early cleavage furrow during cleavage. As cell division progresses, only the cells that have taken up this cytoplasm differentiate into germ cells. Early germ cells are called primordial germ cells (PGCs). The germ cytoplasm contains proteins and mRNA. The physical removal of this germ cytoplasm or inhibition of mRNA translation into protein leads to the loss of germ cells, resulting in infertile individuals. Moreover, with the microinjection of an artificial mRNA that imitates germ cytoplasm into a fertilized egg, it is possible to confer fluorescence on the germ cytoplasm (Figure 8) or make the germ cells glow (Figure 9).


    • Figure 7．Staining of vasa mRNA in the cytoplasm during the early cleavage stage revealed its accumulation on both sides of the cleavage furrow (a–e).


    • Figure 8． The distribution of the germ cytoplasm can be examined by conferring fluorescence on Bucky ball protein.


    • Figure 9． Embryos of pond smelt with GFP-fluorescent primordial germ cells.

  • Chimeric organisms

    •  A chimera is defined as an organism in which the cells generated from multiple fertilized eggs form an individual. In other words, they are individuals in which the cells of one fertilized egg are transplanted into another fertilized egg, including the one in which the blastodisc is transplanted into another blastocyst as shown above. Among the chimeras, an individual in which the somatic line cells are composed of cells derived from multiple fertilized eggs is called a “somatic cell chimera,” while an individual in which the germ-line cells are composed of cells derived from multiple fertilized eggs is called “germ-line chimera.”

    • Germ-line chimera

      Because the blastocyst cells of fishes are pluripotent, the transplantation of cells from other embryos can induce chimeras. In such a case, the transplantation of germ-line cells produces not only a germ-line chimera but also a somatic cell chimera. Thus, as shown in Figure 10, the transplantation of the blastodisc of the crucian carp into the blastodisc of the goldfish produces a chimera with both goldfish and crucian carp cells, which lays both goldfish and crucian carp eggs. Therefore, it produces an individual fish that lays two types of eggs.

      In addition, as shown in the differentiation of germ-line cells, fluorescent PGCs can be produced by microinjecting artificial mRNA into a fertilized egg. The transplantation of these germ cells into other embryos can induce germ-line chimera as shown in Figure 11.

      Furthermore, a gonad can be formed even from a single PGC when one PGC is transplanted from a donor after blocking the production of PGCs in the host (Figure 12）.


    • Figure 10. Transplantation of a crucian carp embryo into a goldfish embryo produces a “chimeric individual” with red and black pigments. This individual lays both goldfish and crucian carp eggs. Therefore, it is not only a germ-line chimera but also a somatic cell chimera. PGC: primordial germ cell.


    • Figure 11. A germ-line chimeric individual in which only fluorescent germ cells were transplanted. Fluorescent germ cells autonomously migrate to the gonads in the transplanted individual.


    • Figure 12．From left: a gonad of an untreated control individual (Wild), a gonad of acontrol individual whose primordial germ cell (PGC) production was blocked (MO.cont), and a gonad of the individual in which a PGC was transplanted to another individual whose PGC production was blocked (SPT-chimera). A pair of developed gonads were found in the Wild individual, but not in MO.cont. Furthermore, only one gonad was developed in the SPT-chimera.

  • Gamete production by surrogate

    • As mentioned above, it has been found that donor gametes are formed in germ-line chimera in which a PGC was transplanted to another embryo whose PGC production was blocked. The production of culture seedlings would be dramatically improved if the gametes of various fishes could be produced in fish that are easily maintained, such as goldfish (Figure 13). However, this is not as easy as it sounds. Despite the successful production of gametes of closely related species, the gametes of genetically distant fish have not been produced. Although there are many possible reasons for this, fish such as eels may become cheaper to eat if such a barrier in gamete production could be removed.


    • Figure 13．Schematic diagram of seedling production by a surrogate. The primordial germ cell is removed from the fertilized egg of the donor (a flatfish in this diagram) and transplanted into the embryo of the host (a goldfish in this diagram) to produce gametes of the flatfish in the goldfish.

  • Chromosome manipulation


    • Figure 14．Technology to suppress cell division is essential for increasing the number of chromosomes. The color photo on the left shows the first cleavage of Oncorhynchus masou.

  • Nuclear transplantation

    • In cows and sheep, it has become possible to transplant somatic cell nuclei into egg cells to produce individuals, and such a technique is called nuclear transplantation. It is difficult to obtain a genetically homogeneous population in mammals due to the small number of their offspring. Therefore, to maintain the traits of certain individuals that are useful for humans, such as delicious meat and better milk production, we are making cloned individuals that are transplanted with the nucleus of the individuals with the traits. In fish, however, clonal populations can be induced by performing the chromosome manipulation mentioned above, and nuclear transplantation has not been highly regarded.

      However, after the death of a large number of fish due to disease, surviving individuals may have genes for disease resistance. Regenerating such individuals and establishing their strains are important tasks in fish breeding. However, this requires the generation of a gamete from the surviving individuals, and I would have to say it is difficult to obtain a gamete from an individual who survived the disease. Thus, can we produce the first stage of a germ-line, which is the PGC, from the somatic cells of such individuals?

      In fish, the microinjection of many sperms into one egg induces embryonic development, although at a low rate. Then, can we isolate a PGC from this embryo? Figure 13 shows the external morphology of an embryo that emerged from an egg in which a large amount of sperm had been microinjected into an unfertilized egg. It has become clear that this embryo also has blastomeres with germ cytoplasm. Therefore, we are working on extracting such blastomeres and inducing germ-line chimera in our research.


    • Figure15. 

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