**2. Teleost model systems of myelopoiesis**

#### **2.1. Zebrafish model system**

The zebrafish model has been instrumental in advancing our knowledge of the sites of hem‐ atopoiesis/myelopoiesis in teleosts, the development, differentiation and migration of HSCs, and through genetic manipulation, the characterization of the early acting growth factors, receptors and transcription factors involved in hematopoiesis. By far, the major advantage provided by zebrafish is the ease of generating transgenic zebrafish, morphant zebrafish (morpholinos) and knockout zebrafish (zinc finger nucleases), as well as many others, due to 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 fish and the difficulty of isolating sufficient number of cells for *in vitro* studies.

#### **2.2. Ginbuna crucian carp model system**

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.

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

The zebrafish model has been instrumental in advancing our knowledge of the sites of hem‐ atopoiesis/myelopoiesis in teleosts, the development, differentiation and migration of HSCs, and through genetic manipulation, the characterization of the early acting growth factors, receptors and transcription factors involved in hematopoiesis. By far, the major advantage provided by zebrafish is the ease of generating transgenic zebrafish, morphant zebrafish (morpholinos) and knockout zebrafish (zinc finger nucleases), as well as many others, due to

as a scaffold to put the advances into context.

**2.1. Zebrafish model system**

98 New Advances and Contributions to Fish Biology

**2. Teleost model systems of myelopoiesis**

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

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

#### **2.3. Goldfish model system**

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. Together, these two model systems will prove instrumental in understanding the factors that regulate teleost myelopoiesis.

phages *in vitro* in the absence of exogenous growth factors. Thus, the goldfish PKM model system allows for comprehensive analysis of the interactions between developing macro‐

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

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

101

There are two waves of hematopoiesis in vertebrates. The first wave is primitive hematopoi‐ esis and occurs during embryonic development. Definitive hematopoiesis follows primitive hematopoiesis and occurs in the post-natal or adult animal. Primitive and definitive hemato‐ poiesis are different on a temporal scale, a spatial scale, and in the types of cellular progeny generated. With the exception of T-cells, that undergo maturation in the thymus, lympho‐ poiesis and myelopoiesis occur in the major hematopoietic organs. The major hematopoietic

The development of myelopoiesis in fish has primarily been studied using the zebrafish model system. Primitive myelopoiesis is predominated by HPCs with primarily erythroid and myeloid development potential. Initially, primitive hematopoiesis is initiated in the an‐ terior lateral mesoderm (ALM), that gives rise to the rostral blood island (RBI), and in the posterior lateral mesoderm (PLM), that gives rise to the intermediate cell mass (ICM). The RBI is the site of primitive myeloid cell development, generating primarily primitive macro‐ phages that undergo rapid differentiation, lacking or having a very short monocytic stage [23] and a few neutrophils [24], while the ICM is the site of primitive erythroid cell develop‐ ment [25]. This stage of primitive hematopoiesis occurs early during development of zebra‐ fish, approximately 11 hours post fertilization (hpf). Following the onset of circulation, at around 24 hpf, the site of hematopoiesis then switches to the posterior blood island (PBI) [26] and produces multi-lineage progenitor cells capable of producing both primitive eryth‐ roid and myeloid cells [27]. Primitive macrophages act as phagocytes during tissue remodel‐ ing throughout embryonic development and in clearance of bacterial pathogens [23]. While primitive neutrophils also migrate to a site of infection, they were not observed to phagocy‐ tose bacteria [24]. The temporal, spatial and transcriptional control of zebrafish primitive hematopoiesis has been reviewed by [28-30]. Differences in the initial site of hematopoiesis occur between fish species, however, the production of erythrocytes and macrophages dur‐

The onset of definitive myelopoiesis occurs around 36 hpf in the zebrafish. Here, HSCs seed the aorta-gonad-mesonephros (AGM) and the caudal hematopoietic tissue (CHT) [33, 34]. By 48 hpf, the HSCs seed the kidney [33], the final hematopoietic site equivalent to mamma‐

organ of teleosts is the kidney, akin to that of mammalian bone marrow.

phage subpopulations in vitro.

**3. Site of hematopoiesis/myelopoiesis**

**3.1. Two waves of hematopoiesis in vertebrates**

**3.2. Primitive myelopoiesis in teleosts**

ing primitive hematopoiesis is consistent [31, 32].

**3.3. Definitive myelopoiesis in teleosts**

lian bone marrow [35-37].

In the *in vitro* PKM system, small mononuclear cells isolated from the goldfish kidney prolif‐ erated and differentiated over 8-10 days giving rise to three cell sub-populations, R1-, R2 and R3-gated cells [14, 15]. The cytochemical, molecular and functional characterization of these cell sub-populations demonstrated the presence of putative progenitor cells (R1 gate), monocytes (R3 gate), and mature macrophages (R2 gate). These three cell sub-populations in PKM cultures represent distinct junctures of macrophage development simultaneously oc‐ curring *in vitro* [14, 15].

The spontaneous proliferation and differentiation of PKMs suggested the production of endogenous growth factors and prompted the examination of the target cell sub-popula‐ tion(s) upon which they acted and their effects on cell proliferation and differentiation. The putative progenitors (R1 cells) and macrophages (R2 cells), but not monocytes, were determined to be responsible for the production of endogenous growth factors that act in an autocrine and paracrine fashion [15]. Addition of cell-conditioned medium (CCM) to sorted cell populations demonstrated the capacity of putative progenitors and monocytes to proliferate and differentiate in response to endogenous growth factors. However, treat‐ ment of macrophages (R2 cells) with CCM demonstrated their apparent terminal differ‐ entiation, while their capacity to proliferate suggested they were capable of self-renewal [15, 17]. Clearly, different endogenous growth factors present in CCM exert distinct ac‐ tions on macrophage cell sub-populations.

Two pathways of macrophage development were proposed to occur in the PKM cultures. The predominant pathway was classical macrophage development in which progenitor cells differentiated into monocytes and then macrophages [17]. The second was an alternative pathway of macrophage development in which progenitor cells differentiated into macro‐ phages without a prominent monocytic stage [17]. The possible retention of the alternative pathway of macrophage production in addition to the classical pathway may provide a mechanism for rapid generation of macrophages during injury or infection *in vivo*.

The observed kinetics of the PKM cultures suggested three phases of growth. Initially, there is a lag phase (days 1-4) where many cells die, followed by a proliferative phase (days 5-9) where cell numbers rapidly increase [14], and finally, a senescence phase (days 10-14) char‐ acterized by cell clumping and cell apoptosis [17, 18]. Differential cross screening of prolifer‐ ative versus senescence phase PKMs identified a number of differentially expressed genes including those involved in hematopoiesis, signal transduction, transcription, translation and protein processing [19]. The involvement of the identified transcripts in the regulation of cell development [20-22] will be discussed in the following sections.

These seminal observations from PKM cultures established three important ideas regarding goldfish monopoiesis: (1) kidney leukocytes produce their own endogenous growth factors important for driving proliferation and differentiation [14, 15]. (2) Within the population of small leukocyte R1 cells, a population of macrophage progenitor cells must exist. (3) Unlike mammalian systems, the progenitor cell population gives rise to fully differentiated macro‐ phages *in vitro* in the absence of exogenous growth factors. Thus, the goldfish PKM model system allows for comprehensive analysis of the interactions between developing macro‐ phage subpopulations in vitro.
