**1. Introduction**

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96 New Advances and Contributions to Fish Biology

Macrophages and neutrophils are the sentinel cells of the innate immune response of verte‐ brates, such as bony fish (teleosts). As phagocytic myeloid cells, they are involved in homeo‐ static mechanisms, wound healing, and the detection, elimination and clearance of foreign entities including tumors, virus-infected cells and invading pathogens. Furthermore, macro‐ phages and neutrophils are responsible for producing hundreds of bioactive molecules that are important in pathogen recognition and destruction, cellular communication and activa‐ tion, initiation of an adaptive immune response and later, resolution of an inflammatory re‐ sponse and tissue repair. Neutrophils and macrophages, while essential to survival, have a finite lifespan. Therefore, a manufacturing centre, the hematopoietic niche, is needed for the production of myeloid cells. The hematopoietic niche must maintain basal myeloid cell pro‐ duction levels during homeostasis, yet retain the flexibility to ramp-up cell production in re‐ sponse to physiological demands, such as pathogenic insult. The development of macrophages (monopoiesis) and neutrophils (granulopoiesis) is collectively known as mye‐ lopoiesis, and is regulated by the complex interaction of colony-stimulating factors (CSFs), their receptors, and intracellular transcription factor machinery that control lineage fate de‐ cisions and terminal differentiation events.

Over the past 50 years, research using the mouse model system has culminated in the identi‐ fication of the site(s) of myelopoiesis, the progenitor cell types that give rise to mature mye‐ loid cells, the extracellular and intracellular cues required, and a detailed understanding of the complex intracellular and extracellular milieu of factors that drive this tightly controlled

© 2013 Katzenback et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Katzenback et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

process. From these studies we understand hematopoiesis as an exquisitely fine-tuned, highly regulated, process whereby all blood cells develop from a small number of hemato‐ poietic stem cells (HSCs). HSCs are characterized as long-term repopulating, pluripotent, quiescent cells that undergo symmetrical self-renewal to sustain the population of HSCs within the hematopoietic niche, or asymmetrical division to give rise to hematopoietic pro‐ genitor cells (HPCs) [1]. HPCs can develop along the lymphoid lineage, termed lymphopoi‐ esis, to give rise to B-cells, T-cells, natural killer (NK) cells and dendritic cells (DCs). Alternatively, HPCs can develop along an erythroid lineage, termed erythropoiesis, to give rise to erythrocytes and megakaryocytes, or develop along a myeloid lineage to give rise to granulocytes (neutrophils, basophils, eosinophils, mast cells), mononuclear phagocytes (monocytes and macrophages), and DCs. The lymphoid lineage represents the adaptive arm of the immune response, while the myeloid lineage represents the innate arm of the immune response. Regardless of lineage, the decisions made to commit and develop along a given lineage are controlled by extracellular growth factors and intracellular transcription factors that act in concert to regulate gene and protein expression to achieve the desired outcome.

genetic manipulation. In conjunction, the rapid generation of embryos, embryonic transpar‐ ency, and small embryo size allows for mass screening strategies. The advantages of zebra‐ fish as a model system, and their contributions to hematopoiesis have been extensively reviewed elsewhere [2-5], and thus will not be covered in this review. While the zebrafish is an excellent *in vivo* model, it does not lend itself to *ex-vivo* studies due to the small size of the

Regulation of Teleost Macrophage and Neutrophil Cell Development by Growth Factors and Transcription Factors

http://dx.doi.org/10.5772/53589

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*In vivo* transplantation to test the repopulation activity of donor cells has been the gold standard for the characterization of HSCs and HPCs [6-9]. In cyprinid fish, there is a unique transplantation model system for detecting HSCs and HPCs using clonal ginbuna crucian carp (*Carassius auratus langsdorfii*, S3n strain) and ginbuna-goldfish (*Carassius auratus*) hy‐ brids (S4n strain). Ginbuna crucian carp have advantages for transplantation experiments because they are easily maintained, tolerate handling and are large enough to allow for the collection of sufficient hematopoietic cells. Clonal ginbuna are unisexual triploid fish (all fe‐ male, 3n = 156) that principally reproduce gynogenetically. A unique clone (S3n) can repro‐ duce by not only gynogenesis but also bisexual reproduction. When eggs from the S3n clone are inseminated with UV-irradiated goldfish sperm, triploid clones result. In contrast, when the eggs are inseminated with normal goldfish sperm, tetraploid hybrids (S4n) are obtained [10]. These S4n fish possessed four sets of chromosomes, three from the S3n clone and one from the goldfish. Therefore, when the cells from S3n clones are transferred into S4n recipi‐ ents, transplants are accepted, whereas the reverse transplants are rejected [11, 12]. More‐ over, the donor cells in the recipient tissues are easily distinguished by their difference in

The ginbuna crucian carp model system has been instrumental in serving as a close parallel to the mouse model system in terms of hematopoietic reconstitution experiments to demon‐ strate the existence of HSCs in teleosts. Identification of donor and recipient HSCs/HPCs and characterization of their progeny by ploidy analysis is useful for assessing the multipo‐ tency of different progenitor cell populations. Furthermore, the use of this model system has allowed for the determination of the location of HSCs within the hematopoietic organs of cyprinids. Use of the ginbuan crucian carp system will be particularly important for future work as antibodies are developed against markers on the surface of fish HSCs and HPCs to

The goldfish model system represents a unique opportunity to study myelopoiesis *in vitro*. Firstly, teleost monopoiesis can be examined using the previously developed primary kid‐ ney macrophage (PKM) culture system [14, 15] and has provided information on the growth factors, receptors, and transcription factors involved. Secondly, large numbers of relatively pure neutrophils can be isolated from the goldfish kidney [16] and represents a starting point for studying granulopoiesis in goldfish and will be discussed in the following sections.

fish and the difficulty of isolating sufficient number of cells for *in vitro* studies.

DNA content by flow cytometric analysis (ploidy analysis) [13].

allow for the analysis of the potency of progenitor cell subpopulations.

**2.3. Goldfish model system**

**2.2. Ginbuna crucian carp model system**

When compared to the mechanisms of myelopoiesis in the mouse, studies using lower vertebrates, such as teleosts, have identified both evolutionary conservation as well as di‐ vergence in the mechanisms of myelopoiesis. With over 30,000 identified species, teleosts are the most expansive class of vertebrates, and represent an excellent model system to study the evolution of vertebrate myelopoiesis as they are one of the ancient classes of vertebrates to retain the production of myeloid cells. Within the teleost system, much re‐ search surrounds the characterization of teleost cytokines and receptors involved in in‐ flammation and their cellular targets (primarily macrophages). In comparison, little is known about the mechanisms that govern myeloid cell production. Research on teleost myelopoiesis is hampered by the lack of reagents, the difficulty in isolating appreciable numbers of relatively pure populations of HSCs/HPCs, and in identifying key growth factors important for myeloid cell development due to evolutionary selection pressures. As such, the focus of this review is to provide an overview of the current knowledge of the fish model systems used and the growth factors, receptors and transcription factors involved in teleost myelopoiesis, using information from the mammalian model systems as a scaffold to put the advances into context.
