**4. Commitment to the myeloid lineage**

#### **4.1. Progression of cell development**

From the mouse model we know that the commitment of a pluripotent, self-renewing HSC to a common myeloid progenitor (CMP) is a progression of lineage fate decisions controlled by extracellular cues, such as growth factors, within the hematopoietic niche [46-48], as well as the modulation of intracellular transcription factors [49-52]. The proc‐ ess of committing to a CMP begins with long-term HSCs (LT-HSCs), capable of self-re‐ newal and multi-lineage differentiation. LT-HSCs give rise to short-term HSCs (ST-HSCs) with limited capacity for self-renewal, which then differentiate into multipotent progeni‐ tors (MPPs) with no ability to self-renew, reviewed by [53]. The MPPs can give rise to the CMP or the lymphoid-myeloid primed multipotent progenitors (LMPPs) [54-57]. The CMP can differentiate into megakaryocyte/erythroid progenitor (MEP) or to a granulo‐ cyte/macrophage progenitor (GMP) [58] (Figure 1). The LMPPs can differentiate into a common lymphoid precursor (CLP) that gives rise to T- and B-lymphocytes, or can also give rise to GMPs [54-57, 59, 60], and reviewed in [61].

On the other hand, the "myeloid-based model" of hematopoiesis, in which myeloid poten‐ tial is retained in erythroid, T, and B cell branches even after these lineages have segregated from each other, has been proposed [62]. Notably, there is no CLP in this model [63-65]. Ac‐ cording to this model, hematopoiesis can be understood as follows: specification toward er‐ 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.

**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 colony-stimulating factor receptor.

#### **4.2. Receptors and growth factors**

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

From the mouse model we know that the commitment of a pluripotent, self-renewing HSC to a common myeloid progenitor (CMP) is a progression of lineage fate decisions controlled by extracellular cues, such as growth factors, within the hematopoietic niche [46-48], as well as the modulation of intracellular transcription factors [49-52]. The proc‐ ess of committing to a CMP begins with long-term HSCs (LT-HSCs), capable of self-re‐ newal and multi-lineage differentiation. LT-HSCs give rise to short-term HSCs (ST-HSCs) with limited capacity for self-renewal, which then differentiate into multipotent progeni‐ tors (MPPs) with no ability to self-renew, reviewed by [53]. The MPPs can give rise to the CMP or the lymphoid-myeloid primed multipotent progenitors (LMPPs) [54-57]. The CMP can differentiate into megakaryocyte/erythroid progenitor (MEP) or to a granulo‐ cyte/macrophage progenitor (GMP) [58] (Figure 1). The LMPPs can differentiate into a common lymphoid precursor (CLP) that gives rise to T- and B-lymphocytes, or can also

On the other hand, the "myeloid-based model" of hematopoiesis, in which myeloid poten‐ tial is retained in erythroid, T, and B cell branches even after these lineages have segregated from each other, has been proposed [62]. Notably, there is no CLP in this model [63-65]. Ac‐ cording to this model, hematopoiesis can be understood as follows: specification toward er‐

capable of multi-lineage differentiation, including myelopoiesis [45].

**4. Commitment to the myeloid lineage**

give rise to GMPs [54-57, 59, 60], and reviewed in [61].

**4.1. Progression of cell development**

102 New Advances and Contributions to Fish Biology

#### *4.2.1. Mammalian stem cell factor and Kit receptor*

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 produces two alternatively spliced mRNAs that differ in the presence or absence of exon 6 [71]. Although the two *SCF* splice variants can be expressed in the same tissues, they have tissue specific regulation of expression [71, 76]. Both SCF isoforms are produced as exten‐ sively glycosylated [77, 78] membrane bound forms (mSCF) that can undergo proteolytic cleavage to produce a soluble form of SCF (sSCF) [79, 80]. In human blood, sSCF is at a con‐ centration of 3.0 ± 1.1 ng/mL [77]. Alternatively, mSCF may provide a means for cell-to-cell contact with the stromal cells in the hematopoietic niche [71], and may act to increase the signal strength provided to the HSC/HPCs, reviewed in [81]. Both mSCF and sSCF are capa‐ ble of forming dimers [78, 82] and signal through their receptor, c-KIT.

term HSCs by blocking cell cycling or by inhibiting apoptosis [122, 123]. Furthermore, SCF can synergizing with other growth factors, such as granulocyte-macrophage colony-stimu‐ lating factor (GM-CSF) [124], granulocyte colony-stimulating factor (G-CSF), IL-1, IL-3 [98], IL-6, and IL-7, among others, to promote the proliferation and differentiation of HPCs [125, 126] and reviewed in [101]. Often, the progeny of HPC differentiation depends on the partic‐ ular growth factor and SCF. Lastly, SCF acts as a homing signal to HPCs, such as CFU-GEMM (granulocyte-erythrocyte-macrophage-megakaryocyte), CFU-GM, CFU-Meg

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Whole genome duplication has resulted in two orthologues of *c-KIT* and *SCF* in teleosts. Tel‐ eost orthologues of *c-KIT,* termed kit a (*kita*) and kit b (*kitb*), were first identified in zebrafish and have subsequently been predicted from genomic analysis of *Takifugu rubripes* and *Tet‐ raodon nigroviridis* [128, 129]. The *kita* orthologue has also been identified and characterized in *Carassius auratus* [130]. The two orthologues of mammalian *SCF* are termed kit ligand a (*kitla*) and kit ligand b (*kitlb*) [128, 131]. The *kitla* and *kitlb* have been identified in zebrafish, and predicted in fugu, medaka, and stickleback genomes [131]. The *kitla* orthologue has

Zebrafish *kita*, located on chromosome 20, and *kitb,* located on chromosome 1, are the ortho‐ logues of human and mouse *c-KIT* [128, 129]. Both *kita* and *kitb* genes contain 21 exons, how‐ ever, their respective proteins only retain 55% identity to each other [129]. The partitioning of gene distribution and function was proposed to explain the retention of *kita* and the du‐ plicated gene, *kitb* [128, 129]. From studies on developing zebrafish, *kita* is expressed in hem‐ atopoietic progenitors, melanoblasts and melanocytes derived from the neural crest, along the lateral line, the notochord and pineal gland [128, 129]. The expression of *kitb* occurs by 9 hpf and does not overlap that of *kita*. Instead, *kitb* expression is restricted to the Rohon-Beard neurons, trigeminal ganglia, and otic vesicle [129]. Together, the expression of *kita* and *kitb* approximates that of *c-KIT* in the mouse model system, with the notable exception of *c-*

The *kitla* gene is located on chromosome 25 and the *kitlb* gene is located on chromosome 4 of the zebrafish genome [132]. *Kitla* has 9 exons while *kitlb* has 8 exons [131]. The nine *kitla* exons correspond to the 9 exons of mammalian SCF isoform 1, including exon 6 which al‐ lows for cleavage of membrane bound SCF into a soluble form [131]. However, *kitlb* appears to correspond to SCF isoform 2, in which exon 6 has been spliced out. The expression of *kitla* is first observed at 19 hpf in the zebrafish and is found in the developing tail bud, pineal gland, sensory epithelium of the ear, ventral otic vesicles, and in the somites [131]. Similar to the expression of goldfish *kita*, *kitla* showed constitutive mRNA levels in tissues [130] and this expression pattern was similar to what was observed in adult zebrafish tissues [132]. Goldfish *kitla* showed high levels of mRNA in isolated putative progenitor cells and mono‐ cytes compared to macrophages [130]. Zebrafish *kitlb* mRNA expression was observed in the brain ventricles, ear and cardinal vein plexus and at lower levels in the skin as zebrafish de‐

(megakaryocyte) and burst forming units-erythrocyte (BFU-E) [127]

been identified and characterized in goldfish [130].

*KIT* expression in primordial germ cells (PGCs).

velopment progressed [131].

*4.2.3. Teleost Kit and Kit ligand*

The SCF receptor, c-KIT (CD117), was first identified as the cellular oncogene (*c-onc*) equivalent of the viral oncogene (*v-onc*), *v-Kit*, isolated from the Hardy-Zuckerman 4 fe‐ line sarcoma virus [83]. Based on structural analysis, the c-KIT protein was grouped within the Type III tyrosine kinase receptor family that includes colony-stimulating fac‐ tor-1 receptor (CSF-1R), platelet derived growth factor receptor (PDGFR), and FLT3/FLK2 receptor [84-87]. Studies mapped *c-KIT* to the *White* locus (*W*) in the mouse [74, 83], and demonstrated that mice with mutations in the *White* or *Steel* loci exhibit hypopigmenta‐ tion, mast cell deficiency, macrocytic anemia, and sterility, while the complete loss of ei‐ ther of these genes was lethal [74, 88].

The c-KIT protein is primarily found on hematopoietic cells and is a marker of long-term re‐ constituting HSCs in humans [89] and mice [90-92]. c-KIT is expressed on pluripotent and multipotent HSCs and myeloerythroid precursors, but not on differentiating or mature cell types [90-92], with the exception of mast cells [93]. Approximately 2 x 104 c-KIT receptors are found on normal human HPCs [94], and can undergo proteolytic cleavage to release a solu‐ ble form of c-KIT [95-97]. The soluble c-KIT receptor is thought to regulate membrane bound c-KIT activity, *in vivo*, by blocking SCF binding [95, 98].

Binding of homodimeric SCF to c-KIT results in receptor homodimerization, conformational changes in the extracellular and intracellular domains and autophosphorylation of the intra‐ cellular tyrosines (reviewed extensively in [73, 78, 99-105]) leading to a number of downstream signaling pathways that mediate the action of SCF through c-KIT. These signaling pathways include phosphatidylinositol-3-kinase (PI3K), phospholipase Cγ (PLCγ), mem‐ bers of the Janus family of protein tyrosine kinases (JAK) and signal transducers and activa‐ tors of transcription (STATs), Src family members, the Ras/Raf/MAP kinase pathway, and others. The signaling pathway initiated depends on the cell type, and the strength and dura‐ tion of the signal, reviewed in [106-108].

#### *4.2.2. Biological functions of stem cell factor*

SCF and its type III tyrosine kinase receptor c-KIT, are involved in hematopoiesis [81, 107, 108], spermatogenesis [109-111], and development of melanocytes [110, 112-114] and mast cells [93, 96, 115-120]. Within the hematopoietic niche, one role of SCF/c-KIT is to mediate HSC and HPC survival, important for the generation of spleen, interleukin-3 (IL-3), granulo‐ cyte/macrophage, and macrophage colony-forming units (CFU-S, CFU-IL-3, CFU-GM, and CFU-M) [121]. Further studies have confirmed SCF/c-KIT to mediate the survival of longterm HSCs by blocking cell cycling or by inhibiting apoptosis [122, 123]. Furthermore, SCF can synergizing with other growth factors, such as granulocyte-macrophage colony-stimu‐ lating factor (GM-CSF) [124], granulocyte colony-stimulating factor (G-CSF), IL-1, IL-3 [98], IL-6, and IL-7, among others, to promote the proliferation and differentiation of HPCs [125, 126] and reviewed in [101]. Often, the progeny of HPC differentiation depends on the partic‐ ular growth factor and SCF. Lastly, SCF acts as a homing signal to HPCs, such as CFU-GEMM (granulocyte-erythrocyte-macrophage-megakaryocyte), CFU-GM, CFU-Meg (megakaryocyte) and burst forming units-erythrocyte (BFU-E) [127]

#### *4.2.3. Teleost Kit and Kit ligand*

produces two alternatively spliced mRNAs that differ in the presence or absence of exon 6 [71]. Although the two *SCF* splice variants can be expressed in the same tissues, they have tissue specific regulation of expression [71, 76]. Both SCF isoforms are produced as exten‐ sively glycosylated [77, 78] membrane bound forms (mSCF) that can undergo proteolytic cleavage to produce a soluble form of SCF (sSCF) [79, 80]. In human blood, sSCF is at a con‐ centration of 3.0 ± 1.1 ng/mL [77]. Alternatively, mSCF may provide a means for cell-to-cell contact with the stromal cells in the hematopoietic niche [71], and may act to increase the signal strength provided to the HSC/HPCs, reviewed in [81]. Both mSCF and sSCF are capa‐

The SCF receptor, c-KIT (CD117), was first identified as the cellular oncogene (*c-onc*) equivalent of the viral oncogene (*v-onc*), *v-Kit*, isolated from the Hardy-Zuckerman 4 fe‐ line sarcoma virus [83]. Based on structural analysis, the c-KIT protein was grouped within the Type III tyrosine kinase receptor family that includes colony-stimulating fac‐ tor-1 receptor (CSF-1R), platelet derived growth factor receptor (PDGFR), and FLT3/FLK2 receptor [84-87]. Studies mapped *c-KIT* to the *White* locus (*W*) in the mouse [74, 83], and demonstrated that mice with mutations in the *White* or *Steel* loci exhibit hypopigmenta‐ tion, mast cell deficiency, macrocytic anemia, and sterility, while the complete loss of ei‐

The c-KIT protein is primarily found on hematopoietic cells and is a marker of long-term re‐ constituting HSCs in humans [89] and mice [90-92]. c-KIT is expressed on pluripotent and multipotent HSCs and myeloerythroid precursors, but not on differentiating or mature cell

found on normal human HPCs [94], and can undergo proteolytic cleavage to release a solu‐ ble form of c-KIT [95-97]. The soluble c-KIT receptor is thought to regulate membrane bound

Binding of homodimeric SCF to c-KIT results in receptor homodimerization, conformational changes in the extracellular and intracellular domains and autophosphorylation of the intra‐ cellular tyrosines (reviewed extensively in [73, 78, 99-105]) leading to a number of downstream signaling pathways that mediate the action of SCF through c-KIT. These signaling pathways include phosphatidylinositol-3-kinase (PI3K), phospholipase Cγ (PLCγ), mem‐ bers of the Janus family of protein tyrosine kinases (JAK) and signal transducers and activa‐ tors of transcription (STATs), Src family members, the Ras/Raf/MAP kinase pathway, and others. The signaling pathway initiated depends on the cell type, and the strength and dura‐

SCF and its type III tyrosine kinase receptor c-KIT, are involved in hematopoiesis [81, 107, 108], spermatogenesis [109-111], and development of melanocytes [110, 112-114] and mast cells [93, 96, 115-120]. Within the hematopoietic niche, one role of SCF/c-KIT is to mediate HSC and HPC survival, important for the generation of spleen, interleukin-3 (IL-3), granulo‐ cyte/macrophage, and macrophage colony-forming units (CFU-S, CFU-IL-3, CFU-GM, and CFU-M) [121]. Further studies have confirmed SCF/c-KIT to mediate the survival of long-

c-KIT receptors are

ble of forming dimers [78, 82] and signal through their receptor, c-KIT.

types [90-92], with the exception of mast cells [93]. Approximately 2 x 104

c-KIT activity, *in vivo*, by blocking SCF binding [95, 98].

ther of these genes was lethal [74, 88].

104 New Advances and Contributions to Fish Biology

tion of the signal, reviewed in [106-108].

*4.2.2. Biological functions of stem cell factor*

Whole genome duplication has resulted in two orthologues of *c-KIT* and *SCF* in teleosts. Tel‐ eost orthologues of *c-KIT,* termed kit a (*kita*) and kit b (*kitb*), were first identified in zebrafish and have subsequently been predicted from genomic analysis of *Takifugu rubripes* and *Tet‐ raodon nigroviridis* [128, 129]. The *kita* orthologue has also been identified and characterized in *Carassius auratus* [130]. The two orthologues of mammalian *SCF* are termed kit ligand a (*kitla*) and kit ligand b (*kitlb*) [128, 131]. The *kitla* and *kitlb* have been identified in zebrafish, and predicted in fugu, medaka, and stickleback genomes [131]. The *kitla* orthologue has been identified and characterized in goldfish [130].

Zebrafish *kita*, located on chromosome 20, and *kitb,* located on chromosome 1, are the ortho‐ logues of human and mouse *c-KIT* [128, 129]. Both *kita* and *kitb* genes contain 21 exons, how‐ ever, their respective proteins only retain 55% identity to each other [129]. The partitioning of gene distribution and function was proposed to explain the retention of *kita* and the du‐ plicated gene, *kitb* [128, 129]. From studies on developing zebrafish, *kita* is expressed in hem‐ atopoietic progenitors, melanoblasts and melanocytes derived from the neural crest, along the lateral line, the notochord and pineal gland [128, 129]. The expression of *kitb* occurs by 9 hpf and does not overlap that of *kita*. Instead, *kitb* expression is restricted to the Rohon-Beard neurons, trigeminal ganglia, and otic vesicle [129]. Together, the expression of *kita* and *kitb* approximates that of *c-KIT* in the mouse model system, with the notable exception of *c-KIT* expression in primordial germ cells (PGCs).

The *kitla* gene is located on chromosome 25 and the *kitlb* gene is located on chromosome 4 of the zebrafish genome [132]. *Kitla* has 9 exons while *kitlb* has 8 exons [131]. The nine *kitla* exons correspond to the 9 exons of mammalian SCF isoform 1, including exon 6 which al‐ lows for cleavage of membrane bound SCF into a soluble form [131]. However, *kitlb* appears to correspond to SCF isoform 2, in which exon 6 has been spliced out. The expression of *kitla* is first observed at 19 hpf in the zebrafish and is found in the developing tail bud, pineal gland, sensory epithelium of the ear, ventral otic vesicles, and in the somites [131]. Similar to the expression of goldfish *kita*, *kitla* showed constitutive mRNA levels in tissues [130] and this expression pattern was similar to what was observed in adult zebrafish tissues [132]. Goldfish *kitla* showed high levels of mRNA in isolated putative progenitor cells and mono‐ cytes compared to macrophages [130]. Zebrafish *kitlb* mRNA expression was observed in the brain ventricles, ear and cardinal vein plexus and at lower levels in the skin as zebrafish de‐ velopment progressed [131].

### *4.2.4. Biological functions of teleost kit ligands and receptors*

Based on the non-overlapping expression of *kita* and *kitb*, the functional roles of c-KIT in mammals may be partitioned between teleost KITA and KITB. The zebrafish mutant *sparse,* shown to map to *kita* [128], or *kit*w34 mutants [133] show defects in their pigmentation pat‐ tern. Zebrafish KITA was shown to be involved in the dispersion and maintenance of mela‐ nocytes [128], and may play a transient role in melanocyte differentiation when melanoblast development is perturbed [134]. Furthermore, knock-down of zebrafish *kitla* or *kitlb* using morpholinos supported the involvement of KITLA in the migration and survival of melano‐ cytes [131]. Teleost *kit* expression in melanocytes has been implicated in the pigment pattern formation in a number of fish species [128, 135-137] and suggests that the functions in mye‐ locyte development have been partitioned to the *kita* orthologue.

culties in identifying the IL-3 orthologue in teleosts due to the low sequence conservation of IL-3 observed between mammals, or may represent the evolutionary loss of IL-3 in teleosts. As IL-3 and IL-3R have not been identified in teleosts, IL-3 and IL-3R will not be discussed here. The structure, function and regulation of mammalian IL-3 and its receptor have been

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GM-CSF shares redundancy with IL-3 in terms of its function. However, GM-CSF acts on a more mature population of HPCs and has been associated with the formation of both granu‐ locyte and macrophage colonies from CFU-GM [147, 148]. GM-CSF is produced by activated T-lymphocytes [147, 149], endothelial cells [150], and lung fibroblasts [151] and suggests the importance of GM-CSF during emergency hematopoiesis. GM-CSF promotes the survival, proliferation and differentiation of GMPs [147, 148, 152]. Furthermore, GM-CSF is chemoat‐ tractive to immature and mature neutrophils *in vitro* and *in vivo* [153, 154] and enhances neutrophil anti-microbial functions and neutrophil survival [155]. GM-CSF can also promote monocytes to differentiate into inflammatory dendritic cells [156, 157]. The GM-CSF recep‐ tor (GM-CSFR) is composed of heterodimeric alpha and beta chains as described for IL-3. Since the βc chain is common to IL-3, IL-5 and GM-CSF, the βc chain signals through JAK/

Similar to that of *IL-3*, *GM-CSF* has not been identified in teleosts (Figure 1). The close prox‐ imity of *IL-3* and *GM-CSF* on the same chromosome may suggest that a genomic deletion occurred on this chromosome, subsequent to the divergence of fish and mammals. The hem‐ atopoietic CSFs that compensate for the loss of IL-3 and GM-CSF in teleosts are not known.

Commitment of LT-HSCs to the myeloid lineage is an intricate regulation of the transcrip‐ tion factors expressed, their relative levels to one another, and their expression on a tempo‐ ral scale. Transcription factors (TFs) can act antagonistically or co-operatively. Thus, the presence or absence of a TF partner, or the relative levels of a TF to its antagonistic counter‐ part, determine lineage fate decisions. Furthermore, the expression of a transcription factor in an HSC does not exert the same effect as when it is expressed in a committed progenitor cell. The transcriptional regulation of mammalian hematopoiesis/myelopoiesis has been ex‐ tensively reviewed elsewhere [159-162], and will only be briefly described here for the pur‐ pose of putting advances in the teleost model systems into context. A visual representation

*MAFB*, a bZIP transcription factor family member, is highly expressed in LT-HSCs, but not in MPPs, CMPs, or GMPs and was recently found to be involved in restricting proliferation and myeloid lineage differentiation of LT-HSCs [163]. MAFB-/- LT-HSCs showed increased

of which stages these transcription factors are important is shown in Figure 2.

*4.2.6. Granulocyte-macrophage colony-stimulating factor/Granulocyte-macrophage colony-*

extensively reviewed elsewhere by [144-146].

STAT, MAPK, and PI3K pathways [145, 158].

*stimulating factor receptor*

**4.3. Transcription factors**

*4.3.1. MafB*

The role of teleost *kita/kitla and kitb/kitlb* during hematopoiesis is not clear. Examination of hematopoiesis in zebrafish *sparse* mutants revealed no obvious defects in hematopoiesis dur‐ ing development. Although, slight decreases in promyelocyte and neutrophil cell numbers, and slight increases in band cells and monocytes were observed in the kidney [128]. In addi‐ tion, zebrafish injected with *kitla* morpholinos or *kitlb* morpholinos also did not show defects in hematopoiesis. However, studies in the goldfish model system demonstrated the expres‐ sion of *kita* mRNA in isolated kidney progenitor cells, and the functional role of goldfish KI‐ TLA in progenitor cell chemotaxis, proliferation, and maintenance [130]. Taken together, these data suggest that KITA and KITLA proteins play a central role in myelopoiesis (Figure 1). However, redundancy between the two ligands and receptors may account for the ab‐ sence of hematopoietic defects in the zebrafish system, or there may be redundancy with an‐ other tyrosine kinase receptor. Additionally, the absence of hematopoietic defects in the zebrafish may represent KIT-independent and KIT-dependent stages of hematopoiesis. The function of KITLB and KITB during hematopoiesis in teleosts remains to be determined.

Lastly, c-KIT plays a role in the development of primordial germ cells (PGCs) in mice. Examination of primordial germ cell development in fish revealed that *kita* and *kitb* ex‐ pression was not detected in PGCs, and suggests teleost KITs do not play a role in the development of PGCs [128, 129]. However, it appears that *kita*, *kitb*, *kitla* and *kitlb* play a role in ovarian folliculogenesis in zebrafish and provides evidence of neofunctionalization of these genes [132].

#### *4.2.5. Interleukin-3 and Interleukin-3 receptor*

Interleukin-3 (IL-3) is a multi-lineage colony-stimulating factor (multi-CSF) that acts through the IL-3 receptor alpha and common beta chain on multipotent erythro/myeloid HPCs to promote their self renewal, proliferation and differentiation [138-140]. IL-3 can also act on committed myeloid progenitors to promote their proliferation and differentiation [138-142]. Interestingly, *IL-3*, *IL-4*, *IL-5* and *GM-CSF* are all found on chromosome 5q in humans. The close proximity of the CSFs on the chromosome, along with their similar structure and func‐ tion may suggest they arose from a common ancestral gene [143]. However, genes encoding IL-3 and the specific IL-3 receptor alpha (IL-3Rα) have not been identified in any teleosts to date, despite genome sequencing (Figure 1). The lack of IL-3 in teleosts may be due to diffi‐ culties in identifying the IL-3 orthologue in teleosts due to the low sequence conservation of IL-3 observed between mammals, or may represent the evolutionary loss of IL-3 in teleosts. As IL-3 and IL-3R have not been identified in teleosts, IL-3 and IL-3R will not be discussed here. The structure, function and regulation of mammalian IL-3 and its receptor have been extensively reviewed elsewhere by [144-146].

#### *4.2.6. Granulocyte-macrophage colony-stimulating factor/Granulocyte-macrophage colonystimulating factor receptor*

GM-CSF shares redundancy with IL-3 in terms of its function. However, GM-CSF acts on a more mature population of HPCs and has been associated with the formation of both granu‐ locyte and macrophage colonies from CFU-GM [147, 148]. GM-CSF is produced by activated T-lymphocytes [147, 149], endothelial cells [150], and lung fibroblasts [151] and suggests the importance of GM-CSF during emergency hematopoiesis. GM-CSF promotes the survival, proliferation and differentiation of GMPs [147, 148, 152]. Furthermore, GM-CSF is chemoat‐ tractive to immature and mature neutrophils *in vitro* and *in vivo* [153, 154] and enhances neutrophil anti-microbial functions and neutrophil survival [155]. GM-CSF can also promote monocytes to differentiate into inflammatory dendritic cells [156, 157]. The GM-CSF recep‐ tor (GM-CSFR) is composed of heterodimeric alpha and beta chains as described for IL-3. Since the βc chain is common to IL-3, IL-5 and GM-CSF, the βc chain signals through JAK/ STAT, MAPK, and PI3K pathways [145, 158].

Similar to that of *IL-3*, *GM-CSF* has not been identified in teleosts (Figure 1). The close prox‐ imity of *IL-3* and *GM-CSF* on the same chromosome may suggest that a genomic deletion occurred on this chromosome, subsequent to the divergence of fish and mammals. The hem‐ atopoietic CSFs that compensate for the loss of IL-3 and GM-CSF in teleosts are not known.

#### **4.3. Transcription factors**

*4.2.4. Biological functions of teleost kit ligands and receptors*

106 New Advances and Contributions to Fish Biology

locyte development have been partitioned to the *kita* orthologue.

of these genes [132].

*4.2.5. Interleukin-3 and Interleukin-3 receptor*

Based on the non-overlapping expression of *kita* and *kitb*, the functional roles of c-KIT in mammals may be partitioned between teleost KITA and KITB. The zebrafish mutant *sparse,* shown to map to *kita* [128], or *kit*w34 mutants [133] show defects in their pigmentation pat‐ tern. Zebrafish KITA was shown to be involved in the dispersion and maintenance of mela‐ nocytes [128], and may play a transient role in melanocyte differentiation when melanoblast development is perturbed [134]. Furthermore, knock-down of zebrafish *kitla* or *kitlb* using morpholinos supported the involvement of KITLA in the migration and survival of melano‐ cytes [131]. Teleost *kit* expression in melanocytes has been implicated in the pigment pattern formation in a number of fish species [128, 135-137] and suggests that the functions in mye‐

The role of teleost *kita/kitla and kitb/kitlb* during hematopoiesis is not clear. Examination of hematopoiesis in zebrafish *sparse* mutants revealed no obvious defects in hematopoiesis dur‐ ing development. Although, slight decreases in promyelocyte and neutrophil cell numbers, and slight increases in band cells and monocytes were observed in the kidney [128]. In addi‐ tion, zebrafish injected with *kitla* morpholinos or *kitlb* morpholinos also did not show defects in hematopoiesis. However, studies in the goldfish model system demonstrated the expres‐ sion of *kita* mRNA in isolated kidney progenitor cells, and the functional role of goldfish KI‐ TLA in progenitor cell chemotaxis, proliferation, and maintenance [130]. Taken together, these data suggest that KITA and KITLA proteins play a central role in myelopoiesis (Figure 1). However, redundancy between the two ligands and receptors may account for the ab‐ sence of hematopoietic defects in the zebrafish system, or there may be redundancy with an‐ other tyrosine kinase receptor. Additionally, the absence of hematopoietic defects in the zebrafish may represent KIT-independent and KIT-dependent stages of hematopoiesis. The function of KITLB and KITB during hematopoiesis in teleosts remains to be determined.

Lastly, c-KIT plays a role in the development of primordial germ cells (PGCs) in mice. Examination of primordial germ cell development in fish revealed that *kita* and *kitb* ex‐ pression was not detected in PGCs, and suggests teleost KITs do not play a role in the development of PGCs [128, 129]. However, it appears that *kita*, *kitb*, *kitla* and *kitlb* play a role in ovarian folliculogenesis in zebrafish and provides evidence of neofunctionalization

Interleukin-3 (IL-3) is a multi-lineage colony-stimulating factor (multi-CSF) that acts through the IL-3 receptor alpha and common beta chain on multipotent erythro/myeloid HPCs to promote their self renewal, proliferation and differentiation [138-140]. IL-3 can also act on committed myeloid progenitors to promote their proliferation and differentiation [138-142]. Interestingly, *IL-3*, *IL-4*, *IL-5* and *GM-CSF* are all found on chromosome 5q in humans. The close proximity of the CSFs on the chromosome, along with their similar structure and func‐ tion may suggest they arose from a common ancestral gene [143]. However, genes encoding IL-3 and the specific IL-3 receptor alpha (IL-3Rα) have not been identified in any teleosts to date, despite genome sequencing (Figure 1). The lack of IL-3 in teleosts may be due to diffi‐ Commitment of LT-HSCs to the myeloid lineage is an intricate regulation of the transcrip‐ tion factors expressed, their relative levels to one another, and their expression on a tempo‐ ral scale. Transcription factors (TFs) can act antagonistically or co-operatively. Thus, the presence or absence of a TF partner, or the relative levels of a TF to its antagonistic counter‐ part, determine lineage fate decisions. Furthermore, the expression of a transcription factor in an HSC does not exert the same effect as when it is expressed in a committed progenitor cell. The transcriptional regulation of mammalian hematopoiesis/myelopoiesis has been ex‐ tensively reviewed elsewhere [159-162], and will only be briefly described here for the pur‐ pose of putting advances in the teleost model systems into context. A visual representation of which stages these transcription factors are important is shown in Figure 2.

#### *4.3.1. MafB*

*MAFB*, a bZIP transcription factor family member, is highly expressed in LT-HSCs, but not in MPPs, CMPs, or GMPs and was recently found to be involved in restricting proliferation and myeloid lineage differentiation of LT-HSCs [163]. MAFB-/- LT-HSCs showed increased proliferative activity and gave rise to large numbers of primarily myeloid progeny in a mouse repopulation assay [163]. The MAFB-/- HSCs had higher proliferative ability and gives rise to greater numbers of myeloid progeny in response to CSF-1 compared to wild type HSCs, *in vitro*. Furthermore, *in vitro* studies demonstrated that treatment of MAFB-/- HSCs with CSF-1 led to the rapid activation of *PU.1* transcription that suggested MAFB must be down-regulated to allow expression of *PU.1* in MPPs [163]. It appears that MAFB plays an important role in antagonizing the expression of PU.1 and the commitment of MPPs to CMPs. Furthermore, MAFB has been shown to bind ETS-1 though its zipper-bind‐ ing domain and can act to repress erythroid lineage commitment in CMPs [164].

*4.3.2. C/EBPs*

EBPε, and C/EBPδ.

hpf, the majority of the *cebpa*<sup>+</sup>

granulocytes, but *cebpa*<sup>+</sup>

an increase in *gata1*<sup>+</sup>

CCAAT/enhancer binding proteins (C/EBPs) are members of the family of transcription fac‐ tors that contain a C-terminal basic leucine zipper domain (bZIP) comprised of a basic re‐ gion involved in DNA binding and a leucine zipper domain involved in protein interactions [166]. Six members of the C/EBP family have been identified in mammals: alpha, beta, gam‐ ma, delta, epsilon and zeta [167]. Orthologues of the C/EBP family of transcription factors have been identified in teleosts [168-171], corresponding to C/EBPα, C/EBPβ, C/EBPγ, C/

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

Expressed in HSCs, CMPs and GMPs [172, 173], C/EBPα has been shown to be involved in directing granulocyte cell fate and terminal differentiation of neutrophils, along with C/ EBPε. Mice deficient in C/EBPα show diminished numbers of CFU-GM, CFU-M, CFU-G, macrophages and neutrophils [174, 175]. The loss of myeloid cells in C/EBPα deficient mice is reflective of the role that CEBPα plays in determining the fate of a CMP to a GMP lineage versus an MEP lineage [176]. C/EBPα is capable of binding to the *PU.1* promoter [175] and up-regulating *PU.1* expression, to dictate a GMP cell fate [175, 177] (see discussion on PU.1 below). The increase in C/EBPα in GMPs has been shown to inhibit monocyte/macrophage differentiation [178] and initiate differentiation along the granulocyte lineage by regulating

The zebrafish CEBPα orthologue showed 66% amino acid identity to human C/EBPα, while the bZIP domains showed 99% amino acid identity [168]. In zebrafish, *cebpa* was expressed in myeloid cells on the surface of the yolk sac during embryogenesis [168]. At 16 hpf, a pop‐ ulation of blood cells co-expressed the transcription factors *gata1*, *pu.1* and *cebpa*, and by 22

*cebpa* [182]. Furthermore, *cebpa* was co-expressed with myeloperoxidase (*mpo*), a marker for

tions likely represent distinct junctures in myeloid cell development: erythromyeloid cells, GMPs and committed neutrophils and their precursors, respectively. The expression of *cebpa* with these additional markers mirrors the importance of C/EBPα in the mammalian system in which C/EBPα is important for committment to a myeloid lineage versus an erythroid lin‐ eage, to a granulocyte lineage over a macrophage lineage, and in terminal differentiation of neutrophils. An orthologue of *cebpa* was also identified in Japanese flounder and mRNA was observed in the head and posterior kidney, spleen, liver, gill, heart, brain, skin, intraper‐ itoneal cells, and weakly in the intestine, muscle and PBLs [171]. However, expression of

Two studies have examined the function of CEBPα in zebrafish primitive myelopoiesis. The injection of a deletion mutant of *cebpa* into zebrafish embryos functioned as a dominant-neg‐ ative mutation and blocked the production of full-length CEBPα. These embryos exhibited

intermediate cell mass at 26 hpf, reflective of an erythroid progenitor cell expansion. This expression corresponded to a subsequent increase in circulating erythrocytes based on the increase in *α-hemoglobin* expression, indicative of erythrocytes [182]. However, the expres‐ sions of the myeloid specific genes, *mpo* and *l-plastin*, were normal [182]. Based on the pat‐

cells co-expressed *pu.1*, however, not all *pu.1+*

cells did not always express *mpo* [182]. These three cell sub-popula‐

expression in the posterior lateral plate mesoderm at 22 hpf and in the

cells expressed

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

109

*GCSFR*, elastase and myeloperoxidase gene expression [179-181].

*cebpa* in isolated cells populations was not performed.

**Figure 2.** Transcription factors involved in goldfish myelopoiesis. Goldfish transcription factors shown in lower case lettering are up-regulated, goldfish transcription factors shown in bold are down-regulated, transcription factors that are important in cellular differentiation in mammalian systmes but have yet to be studied in the teleost system are shown in italics. The dashed arrow denotes the alternative pathway of macrophage development in teleosts. Question marks denote unknown transcription factors involved in the alternative pathway of macrophage development. Aster‐ isks mark differences between teleosts and mammals. Abbreviations used: (1) Cellular stages: HSC, hematopoietic stem cell; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; M, monocytic precursor; G, granulocytic precursor. (2) Transcription factors: c-MYB, cellular myelobastosis oncogene; EGR-1, early growth re‐ sponse-1; MAFB, musculoaponeurotic fibrosarcoma oncogene homologue B; GATA2, GATA binding protein 2; IRF8, interferon regulatory factor 8; CEBPα, CCAAT/enhancer-binding protein alpha; GFI1, growth factor independent 1; RUNX1, runt-related transcription factor 1.

In zebrafish, the *mafb* orthologue has been identified and mRNA was found expressed in the blood forming regions of the developing embryo [165]. However, the role of MAFB in zebra‐ fish HSCs has not yet been assessed.

### *4.3.2. C/EBPs*

proliferative activity and gave rise to large numbers of primarily myeloid progeny in a mouse repopulation assay [163]. The MAFB-/- HSCs had higher proliferative ability and gives rise to greater numbers of myeloid progeny in response to CSF-1 compared to wild type HSCs, *in vitro*. Furthermore, *in vitro* studies demonstrated that treatment of MAFB-/- HSCs with CSF-1 led to the rapid activation of *PU.1* transcription that suggested MAFB must be down-regulated to allow expression of *PU.1* in MPPs [163]. It appears that MAFB plays an important role in antagonizing the expression of PU.1 and the commitment of MPPs to CMPs. Furthermore, MAFB has been shown to bind ETS-1 though its zipper-bind‐

**Figure 2.** Transcription factors involved in goldfish myelopoiesis. Goldfish transcription factors shown in lower case lettering are up-regulated, goldfish transcription factors shown in bold are down-regulated, transcription factors that are important in cellular differentiation in mammalian systmes but have yet to be studied in the teleost system are shown in italics. The dashed arrow denotes the alternative pathway of macrophage development in teleosts. Question marks denote unknown transcription factors involved in the alternative pathway of macrophage development. Aster‐ isks mark differences between teleosts and mammals. Abbreviations used: (1) Cellular stages: HSC, hematopoietic stem cell; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; M, monocytic precursor; G, granulocytic precursor. (2) Transcription factors: c-MYB, cellular myelobastosis oncogene; EGR-1, early growth re‐ sponse-1; MAFB, musculoaponeurotic fibrosarcoma oncogene homologue B; GATA2, GATA binding protein 2; IRF8, interferon regulatory factor 8; CEBPα, CCAAT/enhancer-binding protein alpha; GFI1, growth factor independent 1;

In zebrafish, the *mafb* orthologue has been identified and mRNA was found expressed in the blood forming regions of the developing embryo [165]. However, the role of MAFB in zebra‐

RUNX1, runt-related transcription factor 1.

108 New Advances and Contributions to Fish Biology

fish HSCs has not yet been assessed.

ing domain and can act to repress erythroid lineage commitment in CMPs [164].

CCAAT/enhancer binding proteins (C/EBPs) are members of the family of transcription fac‐ tors that contain a C-terminal basic leucine zipper domain (bZIP) comprised of a basic re‐ gion involved in DNA binding and a leucine zipper domain involved in protein interactions [166]. Six members of the C/EBP family have been identified in mammals: alpha, beta, gam‐ ma, delta, epsilon and zeta [167]. Orthologues of the C/EBP family of transcription factors have been identified in teleosts [168-171], corresponding to C/EBPα, C/EBPβ, C/EBPγ, C/ EBPε, and C/EBPδ.

Expressed in HSCs, CMPs and GMPs [172, 173], C/EBPα has been shown to be involved in directing granulocyte cell fate and terminal differentiation of neutrophils, along with C/ EBPε. Mice deficient in C/EBPα show diminished numbers of CFU-GM, CFU-M, CFU-G, macrophages and neutrophils [174, 175]. The loss of myeloid cells in C/EBPα deficient mice is reflective of the role that CEBPα plays in determining the fate of a CMP to a GMP lineage versus an MEP lineage [176]. C/EBPα is capable of binding to the *PU.1* promoter [175] and up-regulating *PU.1* expression, to dictate a GMP cell fate [175, 177] (see discussion on PU.1 below). The increase in C/EBPα in GMPs has been shown to inhibit monocyte/macrophage differentiation [178] and initiate differentiation along the granulocyte lineage by regulating *GCSFR*, elastase and myeloperoxidase gene expression [179-181].

The zebrafish CEBPα orthologue showed 66% amino acid identity to human C/EBPα, while the bZIP domains showed 99% amino acid identity [168]. In zebrafish, *cebpa* was expressed in myeloid cells on the surface of the yolk sac during embryogenesis [168]. At 16 hpf, a pop‐ ulation of blood cells co-expressed the transcription factors *gata1*, *pu.1* and *cebpa*, and by 22 hpf, the majority of the *cebpa*<sup>+</sup> cells co-expressed *pu.1*, however, not all *pu.1+* cells expressed *cebpa* [182]. Furthermore, *cebpa* was co-expressed with myeloperoxidase (*mpo*), a marker for granulocytes, but *cebpa*<sup>+</sup> cells did not always express *mpo* [182]. These three cell sub-popula‐ tions likely represent distinct junctures in myeloid cell development: erythromyeloid cells, GMPs and committed neutrophils and their precursors, respectively. The expression of *cebpa* with these additional markers mirrors the importance of C/EBPα in the mammalian system in which C/EBPα is important for committment to a myeloid lineage versus an erythroid lin‐ eage, to a granulocyte lineage over a macrophage lineage, and in terminal differentiation of neutrophils. An orthologue of *cebpa* was also identified in Japanese flounder and mRNA was observed in the head and posterior kidney, spleen, liver, gill, heart, brain, skin, intraper‐ itoneal cells, and weakly in the intestine, muscle and PBLs [171]. However, expression of *cebpa* in isolated cells populations was not performed.

Two studies have examined the function of CEBPα in zebrafish primitive myelopoiesis. The injection of a deletion mutant of *cebpa* into zebrafish embryos functioned as a dominant-neg‐ ative mutation and blocked the production of full-length CEBPα. These embryos exhibited an increase in *gata1*<sup>+</sup> expression in the posterior lateral plate mesoderm at 22 hpf and in the intermediate cell mass at 26 hpf, reflective of an erythroid progenitor cell expansion. This expression corresponded to a subsequent increase in circulating erythrocytes based on the increase in *α-hemoglobin* expression, indicative of erythrocytes [182]. However, the expres‐ sions of the myeloid specific genes, *mpo* and *l-plastin*, were normal [182]. Based on the pat‐ tern of expression, it was suggested that PU.1 acts upstream or in parallel with C/EBPα during zebrafish primitive myelopoiesis [182]. Recently, it has been shown that the sumoy‐ lation (a post-translational protein modification) of zebrafish CEBPα inhibited CEBPα tran‐ scriptional activity and its ability to interact with and repress GATA1, thus driving lineage commitment of a myelo-erythroid progenitor to that of the erythroid lineage [183]. Taken to‐ gether, these studies demonstrate the conserved role of CEBPα in the commitment of a CMP to a GMP. However, due to the toxicity of *cebpa* morpholinos to zebrafish embryos, knock‐ down experiments could not be performed.

cells, T-cells, monocytes/macrophages as well as severely reduced numbers of granulocytes [186-190]. *PU.1* is expressed in HSCs, CLPs and at varying levels in CMPs, increasing as these progenitors are induced to differentiate into monocytes/macrophages and neutrophils [191]. At the CMP stage, PU.1 antagonizes with GATA1 to determine whether the CMP commits to a GMP or a MEP. PU.1 binds to GATA1 and inhibits GATA1 from binding to and initiating transcriptional activation of a number of erythroid genes that are important for commitment to an erythroid lineage [184, 192, 193]. The reverse is also true; GATA1 can bind to PU.1 and inhibit the binding of PU.1 and transcriptional activation of a number of myeloid genes [184, 192, 193], including to the promoters of *CSF-1R* [194-196] and *GCSFR* genes [181, 196, 197]. Therefore, the lineage fate decision along a GMP or a MEP fate is a

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

PU.1 also plays a role at the GMP stage to regulate commitment to a granulocyte or mac‐ rophage lineage. Increased levels of PU.1 at the GMP stage, along with AP-1 association, drives a monocyte cell fate, while lower levels of PU.1 drives granulocyte cell fate [175, 177]. Furthermore, PU.1 induces *EGR-2* and *NAB-2* expression [177]. The EGR-2/NAB-2 transcription factors function to repress neutrophil genes by antagonizing GFI1, an im‐ portant transcription factor in the initiation of neutrophil differentiation [177], discussed

An orthologue of PU.1 has been identified in teleosts. In the Japanese flounder, *pu.1* mRNA was detected in the head and posterior kidney, spleen, heart, PBLs, intraperitoneal cells, and weakly in the intestine and gill, but was absent from the liver, skin, muscle and brain [171]. In zebrafish, *pu.1* was identified as a single gene copy and analysis of the predicted protein sequence showed the conserved transactivation, PEST, and DNA-binding domains. Al‐ though the overall amino acid identity to other PU.1 proteins was 48-53%, the DNA-binding domain of zebrafish PU.1 showed 83% amino acid identity to mammalian PU.1 [198]. Ex‐ amination of the zebrafish *pu.1* promoter region predicted potential binding sites for PU.1 and CEBPα [199]. The expression of *pu.1* is first detected at 12 hpf in blood cells from the PLM, later in the ICM, and finally in the kidney, and these *pu.1+* blood cells give rise to mye‐

sors, monocytes/macrophages and neutrophils during both primitive and definitive

Knockdown of *pu.1* in zebrafish using morpholinos showed a large reduction in the number of cells positive for *mpo* and *l-plastin* mRNA, markers of granulocytes and monocytes/macro‐ phages [201, 202]. In addition to the loss of myeloid cells, an increase in *gata1* expression was

morphants failed to develop mature erythrocytes and showed an increase in the number of

*gata1* or *pu.1* morphants, respectively, suggesting the conversion of progenitors to an alter‐ nate lineage [201, 202]. Microarray analysis of genes regulated by PU.1 revealed the regula‐ tion of ~250 genes, including *cebpa*, *csf-1r* and myeloid-specific peroxidase *(mpx)*, among others [203]. Taken together, PU.1 has a conserved role in dictating a myeloid lineage, op‐

posing GATA1 and the transcriptional activation of erythroid genes.

cells represents myeloid HPCs, myeloid precur‐

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

111

cells gave rise to mature erythrocytes [201]. Conversely, *gata1*

cells [201, 202]. Ectopic expression of *pu.1* or *gata1* was observed in

balancing act in timing and relative protein levels of PU.1 and GATA1.

in section 5.3.2.

loid cells [198-200]. The population of *pu.1+*

myelopoiesis in the zebrafish [24, 200].

observed, and these *gata1*<sup>+</sup>

and *l-plastin+*

*pu.1+*

*, mpo+*

*Cebpb* was identified in rainbow trout as a single intron-less gene and the predicted CEBPβ protein showed 30-34% amino acid identity to mammalian C/EBPβ [169]. The *cebpb* mRNA was detected in the head and posterior kidney, spleen, liver, gill, intestine, muscle and pe‐ ripheral blood leukocytes (PBLs) [169]. Japanese flounder CEBPβ also showed a low (33-38%) amino acid identity to other vertebrate sequences, but retained 95% amino acid identity in the bZIP domain. The *cebpb* mRNA was expressed in the head and posterior kid‐ ney, liver, gill, brain, peritoneal cavity fluid and PBLs, with low mRNA levels in the heart, intestine, mucus, eye and spleen [170]. In zebrafish, CEBPβ showed 49% amino acid identity to human C/EBPβ and *cebpb mRNA* was detected in cells on the surface of the yolk sac, cor‐ responding to the myeloid cells that normally spread over the yolk sac early in embryogene‐ sis [168]. A *cebpb* transcript was also identified in a differential cross-screen of goldfish proliferative phase and senescence phase PKMs, and was up-regulated in goldfish mono‐ cytes, and expressed in low levels in progenitors and macrophages [19]. However, the func‐ tional role of CEBPβ has not been examined in teleost myelopoiesis.

The orthologues of C/EBPδ, C/EBPγ and C/EBPε exist in teleosts. The *cebpd* and *cebpg* tran‐ scripts were identified in zebrafish and show a ubiquitous expression pattern in embryos [168]. CEBPδ and CEBPγ showed 57 and 50% identity to their human counterparts on the amino acid level. However, their bZIP domains showed higher conservation to their human counterparts, with 86% and 76% amino acid identity, respectively [168]. The *cebpe* ortho‐ logue was identified in Japanese flounder and its corresponding predicted protein had a 27% overall amino acid identity and a 90% amino acid identity in the bZIP domain com‐ pared to the mammalian counterparts, but failed to cluster with other *cebpe* sequences in phylogenetic analysis [170]. The *cebpe* mRNA was detected in the head and posterior kidney, spleen, brain, peritoneal cavity fluid and at low levels in the PBLs. However, the functional role of these C/EBPs in teleost myelopoiesis is unknown.

#### *4.3.3. PU.1*

The Ets transcription family member PU.1 is well known as the master transcriptional regu‐ lator of mammalian myelopoiesis through an antagonistic relationship with GATA1, recent‐ ly reviewed by [184]. At the N-terminus, PU.1 comprises of an acidic domain and a glutamine rich domain that are involved in activation of transcription, and a PEST domain important for protein interactions [184]. At the C-terminus, PU.1 has an Ets domain impor‐ tant for binding the DNA consensus sequence AAAG(A/C/G)GGAAG [185]. Mice deficient in PU.1 (*PU.1*-/-) have reduced CLPs, and GMPs, increased numbers of MEPs, and lack B- cells, T-cells, monocytes/macrophages as well as severely reduced numbers of granulocytes [186-190]. *PU.1* is expressed in HSCs, CLPs and at varying levels in CMPs, increasing as these progenitors are induced to differentiate into monocytes/macrophages and neutrophils [191]. At the CMP stage, PU.1 antagonizes with GATA1 to determine whether the CMP commits to a GMP or a MEP. PU.1 binds to GATA1 and inhibits GATA1 from binding to and initiating transcriptional activation of a number of erythroid genes that are important for commitment to an erythroid lineage [184, 192, 193]. The reverse is also true; GATA1 can bind to PU.1 and inhibit the binding of PU.1 and transcriptional activation of a number of myeloid genes [184, 192, 193], including to the promoters of *CSF-1R* [194-196] and *GCSFR* genes [181, 196, 197]. Therefore, the lineage fate decision along a GMP or a MEP fate is a balancing act in timing and relative protein levels of PU.1 and GATA1.

tern of expression, it was suggested that PU.1 acts upstream or in parallel with C/EBPα during zebrafish primitive myelopoiesis [182]. Recently, it has been shown that the sumoy‐ lation (a post-translational protein modification) of zebrafish CEBPα inhibited CEBPα tran‐ scriptional activity and its ability to interact with and repress GATA1, thus driving lineage commitment of a myelo-erythroid progenitor to that of the erythroid lineage [183]. Taken to‐ gether, these studies demonstrate the conserved role of CEBPα in the commitment of a CMP to a GMP. However, due to the toxicity of *cebpa* morpholinos to zebrafish embryos, knock‐

*Cebpb* was identified in rainbow trout as a single intron-less gene and the predicted CEBPβ protein showed 30-34% amino acid identity to mammalian C/EBPβ [169]. The *cebpb* mRNA was detected in the head and posterior kidney, spleen, liver, gill, intestine, muscle and pe‐ ripheral blood leukocytes (PBLs) [169]. Japanese flounder CEBPβ also showed a low (33-38%) amino acid identity to other vertebrate sequences, but retained 95% amino acid identity in the bZIP domain. The *cebpb* mRNA was expressed in the head and posterior kid‐ ney, liver, gill, brain, peritoneal cavity fluid and PBLs, with low mRNA levels in the heart, intestine, mucus, eye and spleen [170]. In zebrafish, CEBPβ showed 49% amino acid identity to human C/EBPβ and *cebpb mRNA* was detected in cells on the surface of the yolk sac, cor‐ responding to the myeloid cells that normally spread over the yolk sac early in embryogene‐ sis [168]. A *cebpb* transcript was also identified in a differential cross-screen of goldfish proliferative phase and senescence phase PKMs, and was up-regulated in goldfish mono‐ cytes, and expressed in low levels in progenitors and macrophages [19]. However, the func‐

The orthologues of C/EBPδ, C/EBPγ and C/EBPε exist in teleosts. The *cebpd* and *cebpg* tran‐ scripts were identified in zebrafish and show a ubiquitous expression pattern in embryos [168]. CEBPδ and CEBPγ showed 57 and 50% identity to their human counterparts on the amino acid level. However, their bZIP domains showed higher conservation to their human counterparts, with 86% and 76% amino acid identity, respectively [168]. The *cebpe* ortho‐ logue was identified in Japanese flounder and its corresponding predicted protein had a 27% overall amino acid identity and a 90% amino acid identity in the bZIP domain com‐ pared to the mammalian counterparts, but failed to cluster with other *cebpe* sequences in phylogenetic analysis [170]. The *cebpe* mRNA was detected in the head and posterior kidney, spleen, brain, peritoneal cavity fluid and at low levels in the PBLs. However, the functional

The Ets transcription family member PU.1 is well known as the master transcriptional regu‐ lator of mammalian myelopoiesis through an antagonistic relationship with GATA1, recent‐ ly reviewed by [184]. At the N-terminus, PU.1 comprises of an acidic domain and a glutamine rich domain that are involved in activation of transcription, and a PEST domain important for protein interactions [184]. At the C-terminus, PU.1 has an Ets domain impor‐ tant for binding the DNA consensus sequence AAAG(A/C/G)GGAAG [185]. Mice deficient in PU.1 (*PU.1*-/-) have reduced CLPs, and GMPs, increased numbers of MEPs, and lack B-

tional role of CEBPβ has not been examined in teleost myelopoiesis.

role of these C/EBPs in teleost myelopoiesis is unknown.

*4.3.3. PU.1*

down experiments could not be performed.

110 New Advances and Contributions to Fish Biology

PU.1 also plays a role at the GMP stage to regulate commitment to a granulocyte or mac‐ rophage lineage. Increased levels of PU.1 at the GMP stage, along with AP-1 association, drives a monocyte cell fate, while lower levels of PU.1 drives granulocyte cell fate [175, 177]. Furthermore, PU.1 induces *EGR-2* and *NAB-2* expression [177]. The EGR-2/NAB-2 transcription factors function to repress neutrophil genes by antagonizing GFI1, an im‐ portant transcription factor in the initiation of neutrophil differentiation [177], discussed in section 5.3.2.

An orthologue of PU.1 has been identified in teleosts. In the Japanese flounder, *pu.1* mRNA was detected in the head and posterior kidney, spleen, heart, PBLs, intraperitoneal cells, and weakly in the intestine and gill, but was absent from the liver, skin, muscle and brain [171]. In zebrafish, *pu.1* was identified as a single gene copy and analysis of the predicted protein sequence showed the conserved transactivation, PEST, and DNA-binding domains. Al‐ though the overall amino acid identity to other PU.1 proteins was 48-53%, the DNA-binding domain of zebrafish PU.1 showed 83% amino acid identity to mammalian PU.1 [198]. Ex‐ amination of the zebrafish *pu.1* promoter region predicted potential binding sites for PU.1 and CEBPα [199]. The expression of *pu.1* is first detected at 12 hpf in blood cells from the PLM, later in the ICM, and finally in the kidney, and these *pu.1+* blood cells give rise to mye‐ loid cells [198-200]. The population of *pu.1+* cells represents myeloid HPCs, myeloid precur‐ sors, monocytes/macrophages and neutrophils during both primitive and definitive myelopoiesis in the zebrafish [24, 200].

Knockdown of *pu.1* in zebrafish using morpholinos showed a large reduction in the number of cells positive for *mpo* and *l-plastin* mRNA, markers of granulocytes and monocytes/macro‐ phages [201, 202]. In addition to the loss of myeloid cells, an increase in *gata1* expression was observed, and these *gata1*<sup>+</sup> cells gave rise to mature erythrocytes [201]. Conversely, *gata1* morphants failed to develop mature erythrocytes and showed an increase in the number of *pu.1+ , mpo+* and *l-plastin+* cells [201, 202]. Ectopic expression of *pu.1* or *gata1* was observed in *gata1* or *pu.1* morphants, respectively, suggesting the conversion of progenitors to an alter‐ nate lineage [201, 202]. Microarray analysis of genes regulated by PU.1 revealed the regula‐ tion of ~250 genes, including *cebpa*, *csf-1r* and myeloid-specific peroxidase *(mpx)*, among others [203]. Taken together, PU.1 has a conserved role in dictating a myeloid lineage, op‐ posing GATA1 and the transcriptional activation of erythroid genes.

A pu.1-like gene (spi-1 like, *spi-1l*) was also identified in zebrafish. The predicted amino acid sequence of SPI-1l showed 45% amino acid identity to zebrafish PU.1, and retained all three domains [204]. *In situ* hybridization revealed a population of blood cells positive for *pu.1* and *spi-1l*, in addition to a population of single positive *pu.1* cells [204]. Howev‐ er, only a few single-positive *spi-1l* cells were observed. *Spi-1l* morphants showed a loss of *mpx* and *l-plastin* positive cells, indicative of a loss in granulocytes and monocytes/ macrophages [204]. Unlike *pu.1* morphants, no change in *gata1* expression was observed, suggesting that SPI-1l acts downstream of PU.1, and plays an important role in myeloid cell differentiation [204].

*5.1.2. Receptors and growth factors*

*5.1.2.1. Colony-stimulating factor-1*

*5.1.2.2. Interleukin-34*

The central growth factor that regulates the survival, proliferation, and differentiation of macrophages and their precursors is colony-stimulating factor-1 (CSF-1) [220-223]. Alterna‐ tive splicing of *CSF-1* transcripts leads to production of a secreted glycoprotein, a secreted proteoglycan, or a membrane-bound glycoprotein that can be proteolytically cleaved from the surface, reviewed by [144, 221]. However, only the first 149-150 aa of the N-terminal por‐ tion of the CSF-1 core protein has shown to be important for biological function [224, 225]. CSF-1 homodimers, are covalently linked by an interchain disulphide bond to form a dimer [226] that then binds the CSF-1 receptor. CSF-1 is produced by an array of cell types includ‐ ing fibroblasts, endothelial cells, and bone marrow stromal cells, reviewed by [144]. In addi‐ tion, activated T-cells [227-229], monocytes, macrophages [230, 231], fibroblasts, and endothelial cells [144] can produce CSF-1. CSF-1 production by activated cell types suggests a role for CSF-1 at the site of inflammation, which may be necessary for the rapid recruit‐

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Recently, IL-34 was identified as another growth factor involved in mediating macrophage development in mammals, in addition to CSF-1 [232-234]. The IL-34 protein does not show homology to any other human protein and or contain any known conserved structural mo‐ tifs [232]. Homodimeric IL-34 binds to CSF-1R, although with a different affinity than that of CSF-1, and to different sites on the receptor [232, 233] [235]. The hierarchy in binding of the CSF-1R ligands may provide a mechanism for differential signaling depending on the

The *CSF-1R* gene, shown to map to the proto-oncogene *c-fms*, is a member of the type III ty‐ rosine kinase family of receptors [236, 237], reviewed by [238]. The binding of homodimeric CSF-1 to CSF-1R, triggers receptor homodimerization and activation [239]. Receptor activa‐ tion triggers autophosphorylation of the intracellular tyrosine residues and activation of JAK/STAT, PI3K/Akt, and MAPK pathways, as well as pathways for receptor-mediated in‐ ternalization and destruction, reviewed by [162, 238, 240]. Within the hematopoietic system, CSF-1R protein is primarily found on macrophages and their precursors and has been used as a marker of cells along the macrophage lineage in mammalian systems [222, 237]. CSF-1R

In addition to the regulation of survival, proliferation, and differentiation of macrophages and their precursors [220-223], CSF-1 has been shown to exert pro-inflammatory effects on monocytes and macrophages. These effects include the enhancement of macrophage chemo‐

ment, differentiation and activation of macrophages and their precursors.

bound ligand. To date, IL-34 has not been identified in teleosts.

progressively increased with macrophage differentiation [144].

*5.1.2.4. Biological functions of colony stimulating factor-1*

*5.1.2.3. Colony-stimulating factor-1 receptor*
