**3. Site of hematopoiesis/myelopoiesis**

Together, these two model systems will prove instrumental in understanding the factors

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‐

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‐

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

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

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‐

mechanism for rapid generation of macrophages during injury or infection *in vivo*.

of cell development [20-22] will be discussed in the following sections.

that regulate teleost myelopoiesis.

100 New Advances and Contributions to Fish Biology

tions on macrophage cell sub-populations.

curring *in vitro* [14, 15].

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

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

#### **3.2. Primitive myelopoiesis in teleosts**

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‐ ing primitive hematopoiesis is consistent [31, 32].

#### **3.3. Definitive myelopoiesis in teleosts**

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‐ lian bone marrow [35-37].

The existence of teleost kidney HSCs and HPCs capable of generating all hematopoietic line‐ ages was demonstrated using transplantation studies in zebrafish and ginbuna crucian carp. Transplantation of whole kidney marrow from *gata1*eGFP zebrafish into pre-thymic *vlad tepes* (*gata1*-/-) zebrafish [37] or whole kidney marrow from *β-actineGFP* zebrafish into lethally irradi‐ ated zebrafish [38], resulted in rescue of the phenotype and produced lymphoid and mye‐ loid cell types suggestive of the presence of HSCs capable of long-term reconstitution. However, these studies were complicated by the use of whole kidney marrow during trans‐ plantation. Using ginbuna crucian carp, HSCs, found to be associated with the trunk kidney renal tubules, were identifiable by their ability to efflux Hoechst 33342 using the ATP-bind‐ ing cassette (ABC) transporter, ABCG2a, and HPCs were identified by their ability to efflux rhodamine 123 by another ABC transporter, P-glycoprotein [39-42]. HSCs, consisting of 0.33% ± 0.15 of the total body kidney cells, were capable of engraftment and long-term pro‐ duction (>9 months) of all hemopoietic progeny, including erythrocytes, granulocytes, mon‐ ocytes, thrombocytes and lymphocytes [40, 41, 43]. HPCs, while they could also give rise to all hemopoietic progeny, were only capable of short-term reconstitution [42]. However, en‐ graftment of donor HSCs and HPCs only occurred in anemia-induced or gamma irradiated recipients [40, 43, 44] suggesting that space within the hematopoietic niche is required for successful engraftment of HSCs to occur [40, 43]. Experiments using zebrafish and ginbuna crucian carp provide strong evidence that the teleost trunk kidney contains HSCs and HPCs capable of multi-lineage differentiation, including myelopoiesis [45].

ythroid, T, and B cell lineages proceeds on a basis of a prototypical developmental program to construct myeloid cells [66, 67]. Indeed, several findings in teleosts are supportive of the myeloid-based model [68, 69]. In the future, the myeloid-based model may bring a para‐ digm shift in the concept of blood cell lineage development. In the following sections the key growth factors and transcription factors studied in the teleost system will be discussed.

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

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

103

**Figure 1.** Growth factors and their receptors involved in goldfish myelopoiesis. Goldfish growth factors are shown in uppercase lettering, goldfish growth factor receptors/surface receptors are shown in uppercase italics lettering, and growth factors and their receptors important in mammalian myelopoiesis, but have yet to be identified in teleosts are shown in uppercase **italics**. The dashed arrow denotes the alternative pathway of macrophage development in gold‐ fish, the solid curved arrows denote negative regulation of macrophage development by sCSF-1R. Question marks de‐ note the hypothesized role of growth factors or receptors and further studies are required to test the hypothesis. Asterisks mark differences between teleosts and mammals. Abbreviations used: (1) **Cellular stages**: HSC, hemato‐ poietic stem cell; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; M, monocytic precur‐ sor; G, granulocytic precursor. (2) **Growth factors**: KITLA, kit ligand a; IL-3, interleukin 3; GM-CSF, granulocytemacrophage colony-stimulating factor; CSF-1, colony-stimulating factor 1 (macrophage colony-stimulating factor); GCSF, granulocyte colony-stimulating factor; GF, growth factor. **Receptors**: IL-3R, interleukin 3 receptor; GM-CSFR, granulocyte-macrophage colony-stimulating factor receptor; CSF-1R, colony-stimulating factor-1 receptor (macro‐ phage colony-stimulating factor receptor); sCSF-1R, soluble colony-stimulating factor-1 receptor; GCSFR, granulocyte

Stem cell factor (SCF) was identified [70-72] as short-chain four-helix bundle [73] encoded by the *Steel* locus in the mouse [74]. Mutations in the *Steel* locus were associated with defects in stromal cells, and resulted in reduced numbers of HSCs and HPCs [75]. The *SCF* gene

colony-stimulating factor receptor.

**4.2. Receptors and growth factors**

*4.2.1. Mammalian stem cell factor and Kit receptor*
