**5. Commitment of bi-potent myeloid progenitors to the macrophage or neutrophil lineage**

#### **5.1. Macrophage development**

#### *5.1.1. Progression of cell development*

In mammalian systems, the progression of macrophage development proceeds from a committed macrophage progenitor, monoblast, promonocyte, monocyte and then to a mature tissue macrophage, reviewed by [205-207] (Figure 1). While the presence of a uni‐ potent committed macrophage progenitor has yet to be unequivocally demonstrated in the teleost systems, progenitor/precursor cells that give rise to monocytes and macro‐ phages have been demonstrated. *In vitro*, a spontaneous proliferating trout RTS-11 cell line has two predominant cell types; a round non-adherent cell type that appears to be a pre-monocyte or myeloid precursor and an adherent macrophage-like cell, arising from the non-adherent cell type [208]. The cultivation of trout kidney progenitor-like cells de‐ veloped a trout primary kidney monocyte culture that contained progenitor cells, prom‐ onocyte-like cells, and monocytes [209]. Furthermore, the generation of goldfish primary kidney macrophage cultures demonstrated that small mononuclear cells became mono‐ cytes and mature macrophages, *in vitro*. In the zebrafish model system, whole kidney marrow was added to a kidney fibroblast layer and was shown to maintain HPCs and precursor cells that then differentiated into myeloid and lymphoid cells [210]. Recently, the development of a zebrafish methylcellulose colony forming unit assay suggested the presence of a common erythro-myeloid HPC [211]. *In vivo* studies, primarily in the zebra‐ fish, have demonstrated that monocytes/ macrophages arise from the hematopoietic or‐ gan [45, 212-215], migrate to various tissues [216], and both primitive and definitive macrophages are motile, migrate to the site of insult, and readily phagocytose particles or pathogens [23, 45, 217-219]. The identification of progenitor cells that are capable of differentiating into monocytes and macrophages suggests a conserved macrophage differ‐ entiation pathway in vertebrates.

#### *5.1.2. Receptors and growth factors*

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

**5. Commitment of bi-potent myeloid progenitors to the macrophage or**

In mammalian systems, the progression of macrophage development proceeds from a committed macrophage progenitor, monoblast, promonocyte, monocyte and then to a mature tissue macrophage, reviewed by [205-207] (Figure 1). While the presence of a uni‐ potent committed macrophage progenitor has yet to be unequivocally demonstrated in the teleost systems, progenitor/precursor cells that give rise to monocytes and macro‐ phages have been demonstrated. *In vitro*, a spontaneous proliferating trout RTS-11 cell line has two predominant cell types; a round non-adherent cell type that appears to be a pre-monocyte or myeloid precursor and an adherent macrophage-like cell, arising from the non-adherent cell type [208]. The cultivation of trout kidney progenitor-like cells de‐ veloped a trout primary kidney monocyte culture that contained progenitor cells, prom‐ onocyte-like cells, and monocytes [209]. Furthermore, the generation of goldfish primary kidney macrophage cultures demonstrated that small mononuclear cells became mono‐ cytes and mature macrophages, *in vitro*. In the zebrafish model system, whole kidney marrow was added to a kidney fibroblast layer and was shown to maintain HPCs and precursor cells that then differentiated into myeloid and lymphoid cells [210]. Recently, the development of a zebrafish methylcellulose colony forming unit assay suggested the presence of a common erythro-myeloid HPC [211]. *In vivo* studies, primarily in the zebra‐ fish, have demonstrated that monocytes/ macrophages arise from the hematopoietic or‐ gan [45, 212-215], migrate to various tissues [216], and both primitive and definitive macrophages are motile, migrate to the site of insult, and readily phagocytose particles or pathogens [23, 45, 217-219]. The identification of progenitor cells that are capable of differentiating into monocytes and macrophages suggests a conserved macrophage differ‐

cell differentiation [204].

112 New Advances and Contributions to Fish Biology

**neutrophil lineage**

**5.1. Macrophage development**

*5.1.1. Progression of cell development*

entiation pathway in vertebrates.

#### *5.1.2.1. Colony-stimulating factor-1*

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‐ ment, differentiation and activation of macrophages and their precursors.

#### *5.1.2.2. Interleukin-34*

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

#### *5.1.2.3. Colony-stimulating factor-1 receptor*

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 progressively increased with macrophage differentiation [144].

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

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‐ taxis, phagocytosis of pathogens, and the production of antimicrobial agents, reviewed by [162, 238]. CSF-1 is a pleiotropic cytokine and functions in a number of other biological sys‐ tems such as regulation of macrophage and osteoclast numbers, bone remodeling, tooth pro‐ duction and fertility and breast development [241-245]

the MCSFs [249]. Whether MCSF-1 and MCSF-2 are functionally redundant or functionally

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115

The *csf-1r* sequences have been identified in a number of teleost species including puffer fish [250, 251], zebrafish [252], rainbow trout [253], gilthead seabream [254] and goldfish [20]. CSF-1R protein appears to be a marker of monocytes and macrophages in teleosts [20, 254, 255] (Figure 1). Analysis of the puffer fish *csf-1r* gene shows a 21 exon gene structure in fish, same as in mammals. However, the puffer fish *csf-1r* gene only spans 10.5 kbp versus the mammalian 55 kbp, due to the decrease in the size of the intronic sequences [250]. The *csf-1r* mRNA open reading frame encodes for a 975 aa protein, with a signal peptide, an extracel‐ lular domain with 10 conserved cysteine residues characteristic of immunoglobulin do‐ mains, transmembrane domain, and an intracellular kinase domain with an interruption of 70 bp [250]. While CSF-1R of puffer fish is only 39% similar to human CSF-1R, the kinase domain is considerably more conserved, particularly in the motifs associated with signaling.

A second *csf-1r* gene (*csf-1r-2*) was also identified in puffer fish, and linked with a second *pdgfrb* (*pdgfrb-2*). The *csf-1r-2* gene was comprised of 22 exons and had a different intronexon organization than *csf-1r-1* [251]. Despite the similar protein structure of the two CSF-1Rs, the amino acid sequences were only 39% identical. The *csf-1r* mRNAs were differ‐ entially expressed in tissues. The *csf-1r-1* was expressed in blood, brain, eye, gill, heart, kid‐ ney, ovary, skin, and spleen, while *csf-1r-2* was expressed in the blood, brain, eye, gill, heart,

The duplication of *csf-1r* genes was also observed in cichlids, the green-spotted pufferfish, medaka, and *Tetraodon* (found on chromosomes 1 and 7), with the *csf-1r-2* duplicated genes appearing to have undergone evolutionary selection or diversification while the *csf-1r-1* gene appeared to resemble that of the ancestral gene [256]. It was proposed that the fish spe‐ cific whole genome duplication generated the two paralogues of *csf-1r* in fish, as well as two *pdgfrb* and *kit* genes, and that *kit* and *csf-1r-2* may have been retained to play a role in the survival, migration and differentiation of melanocytes and xanthophores, important pig‐

The *panther* (*fms*) mutant zebrafish have a defect in the *csf-1r* gene, and mutant fish fail to develop their characteristic pigment pattern of black and yellow stripes. The CSF-1R was found to be important in the survival, migration and differentiation of precursors to yellow xanthophores in zebrafish [257, 258]. However, unlike that of the CSF-1R-/- mice, there were no reports of hematopoietic defects in *panther* zebrafish. The lack of hematopoietic defects may be due to the presence of another *csf-1r* gene, a low level of *csf-1r* expression, or a differ‐ ential requirement for CSF-1R during embryonic macrophage development versus adult macrophage development in teleosts. However, CSF-1R was shown to be important in the migration of primitive macrophages to tissues, such as the brain, retina and epidermis upon comparing primitive macrophage distribution and migration in wild-type and *panther* ze‐ brafish [252]. Furthermore, *csf-1r* mRNA was detected in inflammatory macrophages from 3

partitioned (sub-functionalization), remains to be determined.

*5.1.2.6. Teleost colony-stimulating factor-1 receptor*

The fish *csf-1r* gene was linked with *pdgfrb-1* [250].

kidney, liver, muscle, skin, spleen and testis. [251].

ment cells involved in fish coloration patterns [256].

#### *5.1.2.5. Teleost colony stimulating factor-1*

Teleost *csf-1* (*mcsf*) was first identified in the goldfish as a 600 bp mRNA transcript that was present at high levels in spleen tissue, monocytes, and phorbol ester-activated mono‐ cytes [246]. The *csf-1* transcript encoded for a 199 aa precursor protein, with the mature CSF-1 protein predicted to have a molecular weight of 22 kDa. The goldfish CSF-1 has 27% aa identity to human CSF-1 [246]. Alignment of goldfish CSF-1 with mammalian CSF-1s showed conservation of four cysteine residues required for protein folding, simi‐ lar to that of mammalian CSF-1 [246]. Ligand-receptor binding studies demonstrated that homodimeric CSF-1 could bind to soluble CSF-1R (see teleost CSF-1R section below, Fig‐ ure 1). Functional characterization of a recombinant goldfish CSF-1 was shown to induce monocyte proliferation and differentiation (Figure 1), which was abrogated in the pres‐ ence of sCSF-1R or in monocytes transfected with *csf-1r* RNAi oligos [246, 247]. Recombi‐ nant goldfish CSF-1 also aided in the long-term survival of mature macrophages *in vitro* [247]. The recombinant CSF-1 protein was chemoattractive to PKMs, and promoted their ability to perform phagocytosis and produce antimicrobial compounds [248], suggesting a pro-inflammatory role for CSF-1 in goldfish.

Two *csf-1* genes were later identified in trout and zebrafish, termed *mcsf-1* and *mcsf-2*, and a second goldfish *mcsf* transcript was identified [249]. The trout and zebrafish *mcsf-1* genes en‐ coded for proteins of 593 and 526 aa, the trout and zebrafish *mcsf-2* genes encoded for pro‐ teins of 276 and 284 aa, respectively, while the goldfish *mcsf* gene encoded for a 544 aa protein [249]. All of the identified transcripts possessed a signal peptide, a CSF-1 domain, a transmembrane domain, and a short cytoplasmic domain [249]. However, the N-terminal re‐ gion of all teleost CSF-1 proteins showed high homology (46-88%), consistent with the im‐ portant role of the CSF-1 N-terminal portion for biological function.

The genomic structure of the identified *mcsf*s also differed. The zebrafish *mcsf-1*, found on chromosome 11, possessed seven exons and *mcsf-2*, found on chromosome 8, pos‐ sessed nine exons. Based on syntenic analysis, the two *mcsf* genes appeared to have arose through a chromosomal or genome duplication [249]. Examination of the intron-exon structure of trout *mcsf*s showed *mcsf-1* to possess 10 exons and 9 introns, and *mcsf-2* to have 9 exons and 8 introns [249].

Along with differing genomic organizations, trout *mcsf-1/-2* are differentially expressed in tissues. The *mcsf-1* transcript was predominantly expressed in the spleen, intestine and brain, while *mcsf-2* was predominantly expressed in the head kidney, gills, muscle and liver [249]. While a recombinant trout MCSF-1 protein was produced and demonstrated to induce the proliferation of head kidney macrophages, a recombinant trout MCSF-2 protein was not produced to examine whether there was differential regulation of macrophage function by the MCSFs [249]. Whether MCSF-1 and MCSF-2 are functionally redundant or functionally partitioned (sub-functionalization), remains to be determined.

#### *5.1.2.6. Teleost colony-stimulating factor-1 receptor*

taxis, phagocytosis of pathogens, and the production of antimicrobial agents, reviewed by [162, 238]. CSF-1 is a pleiotropic cytokine and functions in a number of other biological sys‐ tems such as regulation of macrophage and osteoclast numbers, bone remodeling, tooth pro‐

Teleost *csf-1* (*mcsf*) was first identified in the goldfish as a 600 bp mRNA transcript that was present at high levels in spleen tissue, monocytes, and phorbol ester-activated mono‐ cytes [246]. The *csf-1* transcript encoded for a 199 aa precursor protein, with the mature CSF-1 protein predicted to have a molecular weight of 22 kDa. The goldfish CSF-1 has 27% aa identity to human CSF-1 [246]. Alignment of goldfish CSF-1 with mammalian CSF-1s showed conservation of four cysteine residues required for protein folding, simi‐ lar to that of mammalian CSF-1 [246]. Ligand-receptor binding studies demonstrated that homodimeric CSF-1 could bind to soluble CSF-1R (see teleost CSF-1R section below, Fig‐ ure 1). Functional characterization of a recombinant goldfish CSF-1 was shown to induce monocyte proliferation and differentiation (Figure 1), which was abrogated in the pres‐ ence of sCSF-1R or in monocytes transfected with *csf-1r* RNAi oligos [246, 247]. Recombi‐ nant goldfish CSF-1 also aided in the long-term survival of mature macrophages *in vitro* [247]. The recombinant CSF-1 protein was chemoattractive to PKMs, and promoted their ability to perform phagocytosis and produce antimicrobial compounds [248], suggesting

Two *csf-1* genes were later identified in trout and zebrafish, termed *mcsf-1* and *mcsf-2*, and a second goldfish *mcsf* transcript was identified [249]. The trout and zebrafish *mcsf-1* genes en‐ coded for proteins of 593 and 526 aa, the trout and zebrafish *mcsf-2* genes encoded for pro‐ teins of 276 and 284 aa, respectively, while the goldfish *mcsf* gene encoded for a 544 aa protein [249]. All of the identified transcripts possessed a signal peptide, a CSF-1 domain, a transmembrane domain, and a short cytoplasmic domain [249]. However, the N-terminal re‐ gion of all teleost CSF-1 proteins showed high homology (46-88%), consistent with the im‐

The genomic structure of the identified *mcsf*s also differed. The zebrafish *mcsf-1*, found on chromosome 11, possessed seven exons and *mcsf-2*, found on chromosome 8, pos‐ sessed nine exons. Based on syntenic analysis, the two *mcsf* genes appeared to have arose through a chromosomal or genome duplication [249]. Examination of the intron-exon structure of trout *mcsf*s showed *mcsf-1* to possess 10 exons and 9 introns, and *mcsf-2* to

Along with differing genomic organizations, trout *mcsf-1/-2* are differentially expressed in tissues. The *mcsf-1* transcript was predominantly expressed in the spleen, intestine and brain, while *mcsf-2* was predominantly expressed in the head kidney, gills, muscle and liver [249]. While a recombinant trout MCSF-1 protein was produced and demonstrated to induce the proliferation of head kidney macrophages, a recombinant trout MCSF-2 protein was not produced to examine whether there was differential regulation of macrophage function by

duction and fertility and breast development [241-245]

*5.1.2.5. Teleost colony stimulating factor-1*

114 New Advances and Contributions to Fish Biology

a pro-inflammatory role for CSF-1 in goldfish.

have 9 exons and 8 introns [249].

portant role of the CSF-1 N-terminal portion for biological function.

The *csf-1r* sequences have been identified in a number of teleost species including puffer fish [250, 251], zebrafish [252], rainbow trout [253], gilthead seabream [254] and goldfish [20]. CSF-1R protein appears to be a marker of monocytes and macrophages in teleosts [20, 254, 255] (Figure 1). Analysis of the puffer fish *csf-1r* gene shows a 21 exon gene structure in fish, same as in mammals. However, the puffer fish *csf-1r* gene only spans 10.5 kbp versus the mammalian 55 kbp, due to the decrease in the size of the intronic sequences [250]. The *csf-1r* mRNA open reading frame encodes for a 975 aa protein, with a signal peptide, an extracel‐ lular domain with 10 conserved cysteine residues characteristic of immunoglobulin do‐ mains, transmembrane domain, and an intracellular kinase domain with an interruption of 70 bp [250]. While CSF-1R of puffer fish is only 39% similar to human CSF-1R, the kinase domain is considerably more conserved, particularly in the motifs associated with signaling. The fish *csf-1r* gene was linked with *pdgfrb-1* [250].

A second *csf-1r* gene (*csf-1r-2*) was also identified in puffer fish, and linked with a second *pdgfrb* (*pdgfrb-2*). The *csf-1r-2* gene was comprised of 22 exons and had a different intronexon organization than *csf-1r-1* [251]. Despite the similar protein structure of the two CSF-1Rs, the amino acid sequences were only 39% identical. The *csf-1r* mRNAs were differ‐ entially expressed in tissues. The *csf-1r-1* was expressed in blood, brain, eye, gill, heart, kid‐ ney, ovary, skin, and spleen, while *csf-1r-2* was expressed in the blood, brain, eye, gill, heart, kidney, liver, muscle, skin, spleen and testis. [251].

The duplication of *csf-1r* genes was also observed in cichlids, the green-spotted pufferfish, medaka, and *Tetraodon* (found on chromosomes 1 and 7), with the *csf-1r-2* duplicated genes appearing to have undergone evolutionary selection or diversification while the *csf-1r-1* gene appeared to resemble that of the ancestral gene [256]. It was proposed that the fish spe‐ cific whole genome duplication generated the two paralogues of *csf-1r* in fish, as well as two *pdgfrb* and *kit* genes, and that *kit* and *csf-1r-2* may have been retained to play a role in the survival, migration and differentiation of melanocytes and xanthophores, important pig‐ ment cells involved in fish coloration patterns [256].

The *panther* (*fms*) mutant zebrafish have a defect in the *csf-1r* gene, and mutant fish fail to develop their characteristic pigment pattern of black and yellow stripes. The CSF-1R was found to be important in the survival, migration and differentiation of precursors to yellow xanthophores in zebrafish [257, 258]. However, unlike that of the CSF-1R-/- mice, there were no reports of hematopoietic defects in *panther* zebrafish. The lack of hematopoietic defects may be due to the presence of another *csf-1r* gene, a low level of *csf-1r* expression, or a differ‐ ential requirement for CSF-1R during embryonic macrophage development versus adult macrophage development in teleosts. However, CSF-1R was shown to be important in the migration of primitive macrophages to tissues, such as the brain, retina and epidermis upon comparing primitive macrophage distribution and migration in wild-type and *panther* ze‐ brafish [252]. Furthermore, *csf-1r* mRNA was detected in inflammatory macrophages from 3 dpf zebrafish embryos [219]. Taken together, these results support a role for CSF-1R in tele‐ ost macrophage biology.

tems for studying progenitor cell to neutrophil differentiation. The identification of func‐ tional neutrophils and their precursors suggests the presence of a committed granulocyte

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

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117

Neutrophils contribute to both innate and adaptive immune responses. They are capable of chemotaxis, phagocytosis, antimicrobial molecule production, and formation of extracellular traps [262-267]. Upon activation, neutrophils produce a number of chemokines, pro-inflam‐ matory and anti-inflammatory cytokines, as well as the colony-stimulating factors G-CSF, CSF-1, GM-CSF, IL3 and SCF, reviewed by [268, 269]. However, neutrophils are short lived,

GCSF, a member of the class I cytokine family, is the primary CSF that mediates the prolifer‐ ation, differentiation, survival and activation of neutrophils and their progenitors, and has been reviewed extensively by [144, 270]. The transcription of *GCSF* is controlled by an up‐ stream promoter region with a tumor necrosis factor alpha response region that is bound by NF-kB p65 and NF-IL6, reviewed elsewhere by [144, 271]. As such, GCSF can be produced by activated monocytes/macrophages, neutrophils, fibroblasts and endothelial cells in re‐ sponse to a number of pro-inflammatory stimuli, reviewed elsewhere by [144, 270, 271]. In humans, the normal GCSF concentration in blood ranges from 30-162 pg/mL, and can be

The protein structure of GCSFR is comprised of a signal peptide, an immunoglobulin-like domain, a cytokine receptor homology (CRH) domain containing the class I cytokine recep‐ tor superfamily motif W-S-X-W-S, three fibronectin domains, a transmembrane domain, and an intracellular cytoplasmic signaling domain containing three motifs termed Box 1, Box 2, and Box 3, important for signal transduction [270, 275]. Based on their protein structure and conserved motifs, the human and mouse integral membrane GCSFR proteins were placed in

While there are reports of GCSFR on other hematopoietic cells such as monocytes [276] and lymphocytes, as well as some non-hematopoietic cells, GCSFR is primarily found on neutro‐ phils and their precursors [270, 277]. Neutrophils up-regulate their levels of GCSFR as they differentiate from progenitor cell to mature neutrophil, with 50-500 GCSF receptors per cell [278]. Structural analysis showed GCSF forms a homodimer, binds two GCSFRs, and leads to receptor homodimerization in a 2:2 complex [279-281]. Binding of a homodimeric GCSF to two GCSF receptors triggers intracellular signaling through the JAK/STAT, Ras/Raf/Erk, or PI3K pathways [275, 277, 282]. These signaling pathways ultimately lead to the migration, survival, proliferation, and differentiation of neutrophils. Control of GCSFR signaling in neutrophils is modulated through (1) transcriptional activation of the *GCSFR* by AP-1, AP-2,

progenitor cell in teleosts.

*5.2.2. Receptors and growth factors*

*5.2.2.1. Granulocyte colony-stimulating factor*

6-90 hrs, and need to be continuously replaced.

*5.2.2.2. Granulocyte colony-stimulating factor receptor*

the type I cytokine receptor family.

massively up-regulated during infection up to 3200 pg/mL [272-274].

A full-length *csf-1r* cDNA sequence was identified in trout, with an open reading frame of 2904 bp encoding for a 967 aa protein, predicted to be ~109 kDa. Trout CSF-1R had 40% aa identity to that of human and mouse, and 54% and 52% identity to that of puffer fish and zebrafish CSF-1R [253]. The trout *csf-1r* gene was similar to that of the ancestral gene, and mRNA was found in the head-kidney, spleen, blood, ovary, and showed lower mRNA lev‐ els in the liver, brain, heart, muscle, gill, and skin [253]. Southern blotting revealed two bands in each lane, suggestive of a second *csf-1r* gene in trout. However, a second *csf-1r* gene was never identified.

CSF-1R was also identified in goldfish as a 975 aa integral membrane bound protein (mCSF-1R) that possessed the five Ig extracellular domains with multiple N-linked glycosy‐ lation sites, a transmembrane domain, and an intracellular tyrosine kinase domain [20]. The mRNA of mCSF-1R could be detected in progenitor, monocyte and macrophage subpopula‐ tions, and an antibody produced against the first two Ig domains of CSF-1R was able to rec‐ ognize monocytes and macrophages [20]. However, unlike mammalian neutrophils, zebrafish and goldfish neutrophils do not appear to express mRNA for *csf-1r* [16, 219]. Addi‐ tionally, alternative splicing of the *csf-1r* transcript encoded for a soluble form of the CSF-1R (sCSF-1R), possessing only the D1 and D2 Ig domains, important for binding of CSF-1. The s*csf-1r* mRNA was expressed by leukocytes within the progenitor and macrophage popula‐ tions, but not in the monocyte subpopulation [20]. Furthermore, addition of a recombinant purified sCSF-1R dampened the proliferation of spontaneously growing and differentiating PKMs [20]. The increased production of the sCSF-1R by PKMs during senescence phase sug‐ gested that sCSF-1R was involved in the negative regulation of CSF-1 signaling through mCSF-1R [20, 246] (Figure 1).

#### **5.2. Neutrophil development**

#### *5.2.1. Progression of cell development*

Following the commitment of the CFU-GM to a committed granulocyte progenitor cell, ter‐ minal differentiation through a promyelocyte, myelocyte, and metamyelocyte stages occur to give rise to a mature neutrophil, and are regulated through growth factor and transcrip‐ tion factor signaling, reviewed by [259] (Figure 1). Similar to that of mammals, the differen‐ tiation of fish neutrophils appears to occur through various stages, based on morphological and cytochemical characteristics, and include the promyelocyte, myelocyte, metamyelocyte and the mature neutrophil, which sometimes had a segmented nucleus [45, 212, 213, 215, 260]. These neutrophils were shown to migrate from the hematopoietic organ to the site of wounding, pathogen injection, or transformed cell injection [24, 45, 261], in response to a hy‐ drogen peroxide attractant produced by cells at the site of damage [217]. However, the re‐ sponding neutrophils had low phagocytic activity [24], or engulfed small fragments of the pathogen [217]. *In vitro*, treatment of zebrafish kidney marrow cells with G-CSF gave rise to CFU-GM in a methylcellulose assay [211]. However, there is a lack of *in vitro* culture sys‐ tems for studying progenitor cell to neutrophil differentiation. The identification of func‐ tional neutrophils and their precursors suggests the presence of a committed granulocyte progenitor cell in teleosts.

#### *5.2.2. Receptors and growth factors*

dpf zebrafish embryos [219]. Taken together, these results support a role for CSF-1R in tele‐

A full-length *csf-1r* cDNA sequence was identified in trout, with an open reading frame of 2904 bp encoding for a 967 aa protein, predicted to be ~109 kDa. Trout CSF-1R had 40% aa identity to that of human and mouse, and 54% and 52% identity to that of puffer fish and zebrafish CSF-1R [253]. The trout *csf-1r* gene was similar to that of the ancestral gene, and mRNA was found in the head-kidney, spleen, blood, ovary, and showed lower mRNA lev‐ els in the liver, brain, heart, muscle, gill, and skin [253]. Southern blotting revealed two bands in each lane, suggestive of a second *csf-1r* gene in trout. However, a second *csf-1r* gene

CSF-1R was also identified in goldfish as a 975 aa integral membrane bound protein (mCSF-1R) that possessed the five Ig extracellular domains with multiple N-linked glycosy‐ lation sites, a transmembrane domain, and an intracellular tyrosine kinase domain [20]. The mRNA of mCSF-1R could be detected in progenitor, monocyte and macrophage subpopula‐ tions, and an antibody produced against the first two Ig domains of CSF-1R was able to rec‐ ognize monocytes and macrophages [20]. However, unlike mammalian neutrophils, zebrafish and goldfish neutrophils do not appear to express mRNA for *csf-1r* [16, 219]. Addi‐ tionally, alternative splicing of the *csf-1r* transcript encoded for a soluble form of the CSF-1R (sCSF-1R), possessing only the D1 and D2 Ig domains, important for binding of CSF-1. The s*csf-1r* mRNA was expressed by leukocytes within the progenitor and macrophage popula‐ tions, but not in the monocyte subpopulation [20]. Furthermore, addition of a recombinant purified sCSF-1R dampened the proliferation of spontaneously growing and differentiating PKMs [20]. The increased production of the sCSF-1R by PKMs during senescence phase sug‐ gested that sCSF-1R was involved in the negative regulation of CSF-1 signaling through

Following the commitment of the CFU-GM to a committed granulocyte progenitor cell, ter‐ minal differentiation through a promyelocyte, myelocyte, and metamyelocyte stages occur to give rise to a mature neutrophil, and are regulated through growth factor and transcrip‐ tion factor signaling, reviewed by [259] (Figure 1). Similar to that of mammals, the differen‐ tiation of fish neutrophils appears to occur through various stages, based on morphological and cytochemical characteristics, and include the promyelocyte, myelocyte, metamyelocyte and the mature neutrophil, which sometimes had a segmented nucleus [45, 212, 213, 215, 260]. These neutrophils were shown to migrate from the hematopoietic organ to the site of wounding, pathogen injection, or transformed cell injection [24, 45, 261], in response to a hy‐ drogen peroxide attractant produced by cells at the site of damage [217]. However, the re‐ sponding neutrophils had low phagocytic activity [24], or engulfed small fragments of the pathogen [217]. *In vitro*, treatment of zebrafish kidney marrow cells with G-CSF gave rise to CFU-GM in a methylcellulose assay [211]. However, there is a lack of *in vitro* culture sys‐

ost macrophage biology.

116 New Advances and Contributions to Fish Biology

was never identified.

mCSF-1R [20, 246] (Figure 1).

**5.2. Neutrophil development**

*5.2.1. Progression of cell development*

### *5.2.2.1. Granulocyte colony-stimulating factor*

Neutrophils contribute to both innate and adaptive immune responses. They are capable of chemotaxis, phagocytosis, antimicrobial molecule production, and formation of extracellular traps [262-267]. Upon activation, neutrophils produce a number of chemokines, pro-inflam‐ matory and anti-inflammatory cytokines, as well as the colony-stimulating factors G-CSF, CSF-1, GM-CSF, IL3 and SCF, reviewed by [268, 269]. However, neutrophils are short lived, 6-90 hrs, and need to be continuously replaced.

GCSF, a member of the class I cytokine family, is the primary CSF that mediates the prolifer‐ ation, differentiation, survival and activation of neutrophils and their progenitors, and has been reviewed extensively by [144, 270]. The transcription of *GCSF* is controlled by an up‐ stream promoter region with a tumor necrosis factor alpha response region that is bound by NF-kB p65 and NF-IL6, reviewed elsewhere by [144, 271]. As such, GCSF can be produced by activated monocytes/macrophages, neutrophils, fibroblasts and endothelial cells in re‐ sponse to a number of pro-inflammatory stimuli, reviewed elsewhere by [144, 270, 271]. In humans, the normal GCSF concentration in blood ranges from 30-162 pg/mL, and can be massively up-regulated during infection up to 3200 pg/mL [272-274].

#### *5.2.2.2. Granulocyte colony-stimulating factor receptor*

The protein structure of GCSFR is comprised of a signal peptide, an immunoglobulin-like domain, a cytokine receptor homology (CRH) domain containing the class I cytokine recep‐ tor superfamily motif W-S-X-W-S, three fibronectin domains, a transmembrane domain, and an intracellular cytoplasmic signaling domain containing three motifs termed Box 1, Box 2, and Box 3, important for signal transduction [270, 275]. Based on their protein structure and conserved motifs, the human and mouse integral membrane GCSFR proteins were placed in the type I cytokine receptor family.

While there are reports of GCSFR on other hematopoietic cells such as monocytes [276] and lymphocytes, as well as some non-hematopoietic cells, GCSFR is primarily found on neutro‐ phils and their precursors [270, 277]. Neutrophils up-regulate their levels of GCSFR as they differentiate from progenitor cell to mature neutrophil, with 50-500 GCSF receptors per cell [278]. Structural analysis showed GCSF forms a homodimer, binds two GCSFRs, and leads to receptor homodimerization in a 2:2 complex [279-281]. Binding of a homodimeric GCSF to two GCSF receptors triggers intracellular signaling through the JAK/STAT, Ras/Raf/Erk, or PI3K pathways [275, 277, 282]. These signaling pathways ultimately lead to the migration, survival, proliferation, and differentiation of neutrophils. Control of GCSFR signaling in neutrophils is modulated through (1) transcriptional activation of the *GCSFR* by AP-1, AP-2,

C/EBPα, NF-IL6, GATA-1, and PU.1/SPI1 transcription factors [181, 197], (2) the production of a soluble receptor through alternative splicing [275], and (3) cleavage of surface GCSFR by elastase [283].

kocytes, spleen, gill, intestine and muscle [291]. However, black rockfish *gcsf-2* was ubiqui‐ tously expressed in the peripheral blood leukocytes, head and trunk kidney, spleen, gill, intestine, muscle, liver and brain [291]. Although both *gcsf-1* and *gcsf-2* black rockfish mRNA levels were upregulated in PBLs treated with LPS or ConA/PMA, differential kinet‐ ics and levels of expression were observed between the two *gcsfs* [291]. It appears that *gcsf-1* may be rapidly induced with sustained levels following stimulation, whereas *gcsf-2* is only slightly upregulated and showed a drastic increase in mRNA levels after ConA/PMA treat‐ ment for 24 hrs [291]. Taken together, these data suggest that GCSF-1 may play an important role during inflammation, although functional studies are required to determine the roles of

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119

Functional studies on fish GCSF-1 are limited. Only two manuscripts report on the func‐ tion of GCSF-1 and both utilize the zebrafish model system. *In vitro*, precursor cells from whole kidney marrow were sorted, plated in a methylcellulose colony forming unit assay and treated with either GCSF or a combination of GCSF and erythropoietin (EPO). While both treatments led to CFUs containing granulocytes and macrophages, the combination of GCSF and EPO also supported the formation of erythroid CFUs [211]. *In vivo*, mor‐ pholino mediated knockdown of *gcsfr* in zebrafish showed a decrease in numbers and migration of cells expressing both neutrophil and macrophage specific transcripts, during both primitive and definitive hematopoiesis in the zebrafish embryo. However, a popula‐ tion of myeloid cells remained, despite morpholino mediated knockdown of *gcsfr*, sug‐ gesting the presence of a GCSFR-independent pathway of myeloid cell development and migration [292]. Injection of wild-type zebrafish with *gcsf* mRNA increased the number of myeloid and *gcsfr*+ cells, while injection of *gcsf* mRNA into *gcsfr* morpholino zebrafish did not result in an increase in myeloid cell numbers [292]. These studies suggested GCSF-1 participates in myeloid cell development, similar to that observed in mammalian systems (Figure 1). No functional studies have been performed using GCSF-2, and the

The *gcsfr* has been identified in zebrafish [292], goldfish [293], and trout (AJ616901). Only one gene copy has been identified, although Southern blotting for goldfish *gcsfr* suggested the presence of more than one gene [293]. Analysis of the upstream promoter region of the 16 exon zebrafish *gcsfr* gene showed conserved putative sites for binding of the transcription factors HOXA5, PU.1 and CEBP family members [292], similar to the human *gcsfr* promoter region. These data suggest the conserved regulation of *gcsfr* gene expression in teleosts.

The predicted protein structure of zebrafish and goldfish GCSFRs is conserved across vertebrates. The teleost GCSFR extracellular domain is comprised of a signal peptide, an Ig-like domain, a cytokine homology domain containing the WSXWS motif and four cys‐ teine residues, and three fibronectin domains. Following the transmembrane region, the intracellular region contains predicted Box1, Box2, and Box 3 signaling motifs and 6 tyro‐ sine residues [292, 293], shown to be involved in receptor activation and internalization

GCSF-1 and GCSF-2 in teleost granulopoiesis and inflammation.

role(s) of GCSF-2 in myelopoiesis remain to be elucidated.

*5.2.2.5. Teleost granulocyte colony-stimulating factor receptor*

in higher vertebrates.

#### *5.2.2.3. Biological activity of granulocyte colony stimulating factor*

The targeted gene disruption of *GCSF* and *GCSFR* has demonstrated the important func‐ tional roles of GCSF *in vivo*. GCSF and GCSFR deficient mice display severe neutropenia (70%-88% reduction in circulating neutrophils), reduction in monocyte and macrophage numbers, and ~50% reduction in the numbers of neutrophil precursors present in the bone marrow [284, 285] [282, 286] and are unable to control *Listeria monocytogenes* infec‐ tions [284, 285]. GCSF treatment of bone marrow cells, *in vitro*, induced CFU activity to produce mainly neutrophil colonies [287] and promoted the proliferation of neutrophil precursors [270]. The release of mature neutrophils, their terminal differentiation, surviv‐ al, and activation, is also mediated by GCSF *in vitro* and *in vivo*, reviewed by [270]. Last‐ ly, GCSF has been used in the clinical setting to increase peripheral blood neutrophil numbers for treatment of disease and for stem cell mobilization from the bone marrow into the peripheral blood, reviewed by [288, 289].

#### *5.2.2.4. Teleost granulocyte colony-stimulating factor*

The teleost *gcsf* gene was first identified in Japanese flounder, fugu, and the green-spotted pufferfish [290]. Both the fugu and green-spotted pufferfish have two *gcsf* genes, termed *gcsf-1* and *gcsf-2*, while only an orthologue of *gcsf-2* was identified in flounder [290]. Phylo‐ genetic analysis of vertebrate *gcsf*s predicted fish *gcsf-1* to be the ancestral gene, while *gcsf-2* was predicted to be the duplicated gene. Alignment of the fish GCSFs with human and mouse GCSF showed low identity, ranging from no significant identity to 34% amino acid identity [290]. Despite the low amino acid identity of fish to mammalian GCSF, all fish *gcsf* genes retained a 5 exon/ 4 intron structure with a conserved tumor necrosis factor alpha re‐ sponse element in the promoter region. Furthermore, the predicted transcripts have an open reading frame of 561-636 bp, corresponding to a predicted protein of 20-23 kDa, and 4-5 AU rich sequences in their 3' UTRs shown to be involved in mRNA instability and degradation [290]. Determination of the ratio of synonymous to asynonymous nucleotide substitutions (Ks/Ka) in fish *gcsf* genes ranged from 0.467 to 0.961 with an average of 0.793, demonstrating that positive selection was occurring in GCSFs of fish (and chicken) [290]. Two *gcsf* genes were also identified in the black rockfish (*Sebastes schlegelii*) [291] and in zebrafish [292] (O. Svoboda and P. Bartunek, personal communication), while only one *gcsf* gene has been iden‐ tified in trout (NM\_001195184).

Flounder *gcsf-2* mRNA levels were highest in the spleen, kidney, and gill. However, *gcsf-2* mRNA was still detected in the brain, eyes, heart, peripheral blood leukocytes, ovary, skin, and stomach, but was not detected in intestine, liver, or muscle tissue [290]. As expected, *gcsf-2* mRNA levels were up-regulated in kidney and peripheral blood leukocytes following treatment with lipopolysaccharide (LPS) or a mixture of concanavalin A and phorbol esters (ConA/PMA) [290]. The black rockfish *gcsf-1* showed expression in the peripheral blood leu‐ kocytes, spleen, gill, intestine and muscle [291]. However, black rockfish *gcsf-2* was ubiqui‐ tously expressed in the peripheral blood leukocytes, head and trunk kidney, spleen, gill, intestine, muscle, liver and brain [291]. Although both *gcsf-1* and *gcsf-2* black rockfish mRNA levels were upregulated in PBLs treated with LPS or ConA/PMA, differential kinet‐ ics and levels of expression were observed between the two *gcsfs* [291]. It appears that *gcsf-1* may be rapidly induced with sustained levels following stimulation, whereas *gcsf-2* is only slightly upregulated and showed a drastic increase in mRNA levels after ConA/PMA treat‐ ment for 24 hrs [291]. Taken together, these data suggest that GCSF-1 may play an important role during inflammation, although functional studies are required to determine the roles of GCSF-1 and GCSF-2 in teleost granulopoiesis and inflammation.

Functional studies on fish GCSF-1 are limited. Only two manuscripts report on the func‐ tion of GCSF-1 and both utilize the zebrafish model system. *In vitro*, precursor cells from whole kidney marrow were sorted, plated in a methylcellulose colony forming unit assay and treated with either GCSF or a combination of GCSF and erythropoietin (EPO). While both treatments led to CFUs containing granulocytes and macrophages, the combination of GCSF and EPO also supported the formation of erythroid CFUs [211]. *In vivo*, mor‐ pholino mediated knockdown of *gcsfr* in zebrafish showed a decrease in numbers and migration of cells expressing both neutrophil and macrophage specific transcripts, during both primitive and definitive hematopoiesis in the zebrafish embryo. However, a popula‐ tion of myeloid cells remained, despite morpholino mediated knockdown of *gcsfr*, sug‐ gesting the presence of a GCSFR-independent pathway of myeloid cell development and migration [292]. Injection of wild-type zebrafish with *gcsf* mRNA increased the number of myeloid and *gcsfr*+ cells, while injection of *gcsf* mRNA into *gcsfr* morpholino zebrafish did not result in an increase in myeloid cell numbers [292]. These studies suggested GCSF-1 participates in myeloid cell development, similar to that observed in mammalian systems (Figure 1). No functional studies have been performed using GCSF-2, and the role(s) of GCSF-2 in myelopoiesis remain to be elucidated.

#### *5.2.2.5. Teleost granulocyte colony-stimulating factor receptor*

C/EBPα, NF-IL6, GATA-1, and PU.1/SPI1 transcription factors [181, 197], (2) the production of a soluble receptor through alternative splicing [275], and (3) cleavage of surface GCSFR

The targeted gene disruption of *GCSF* and *GCSFR* has demonstrated the important func‐ tional roles of GCSF *in vivo*. GCSF and GCSFR deficient mice display severe neutropenia (70%-88% reduction in circulating neutrophils), reduction in monocyte and macrophage numbers, and ~50% reduction in the numbers of neutrophil precursors present in the bone marrow [284, 285] [282, 286] and are unable to control *Listeria monocytogenes* infec‐ tions [284, 285]. GCSF treatment of bone marrow cells, *in vitro*, induced CFU activity to produce mainly neutrophil colonies [287] and promoted the proliferation of neutrophil precursors [270]. The release of mature neutrophils, their terminal differentiation, surviv‐ al, and activation, is also mediated by GCSF *in vitro* and *in vivo*, reviewed by [270]. Last‐ ly, GCSF has been used in the clinical setting to increase peripheral blood neutrophil numbers for treatment of disease and for stem cell mobilization from the bone marrow

The teleost *gcsf* gene was first identified in Japanese flounder, fugu, and the green-spotted pufferfish [290]. Both the fugu and green-spotted pufferfish have two *gcsf* genes, termed *gcsf-1* and *gcsf-2*, while only an orthologue of *gcsf-2* was identified in flounder [290]. Phylo‐ genetic analysis of vertebrate *gcsf*s predicted fish *gcsf-1* to be the ancestral gene, while *gcsf-2* was predicted to be the duplicated gene. Alignment of the fish GCSFs with human and mouse GCSF showed low identity, ranging from no significant identity to 34% amino acid identity [290]. Despite the low amino acid identity of fish to mammalian GCSF, all fish *gcsf* genes retained a 5 exon/ 4 intron structure with a conserved tumor necrosis factor alpha re‐ sponse element in the promoter region. Furthermore, the predicted transcripts have an open reading frame of 561-636 bp, corresponding to a predicted protein of 20-23 kDa, and 4-5 AU rich sequences in their 3' UTRs shown to be involved in mRNA instability and degradation [290]. Determination of the ratio of synonymous to asynonymous nucleotide substitutions (Ks/Ka) in fish *gcsf* genes ranged from 0.467 to 0.961 with an average of 0.793, demonstrating that positive selection was occurring in GCSFs of fish (and chicken) [290]. Two *gcsf* genes were also identified in the black rockfish (*Sebastes schlegelii*) [291] and in zebrafish [292] (O. Svoboda and P. Bartunek, personal communication), while only one *gcsf* gene has been iden‐

Flounder *gcsf-2* mRNA levels were highest in the spleen, kidney, and gill. However, *gcsf-2* mRNA was still detected in the brain, eyes, heart, peripheral blood leukocytes, ovary, skin, and stomach, but was not detected in intestine, liver, or muscle tissue [290]. As expected, *gcsf-2* mRNA levels were up-regulated in kidney and peripheral blood leukocytes following treatment with lipopolysaccharide (LPS) or a mixture of concanavalin A and phorbol esters (ConA/PMA) [290]. The black rockfish *gcsf-1* showed expression in the peripheral blood leu‐

*5.2.2.3. Biological activity of granulocyte colony stimulating factor*

into the peripheral blood, reviewed by [288, 289].

*5.2.2.4. Teleost granulocyte colony-stimulating factor*

tified in trout (NM\_001195184).

by elastase [283].

118 New Advances and Contributions to Fish Biology

The *gcsfr* has been identified in zebrafish [292], goldfish [293], and trout (AJ616901). Only one gene copy has been identified, although Southern blotting for goldfish *gcsfr* suggested the presence of more than one gene [293]. Analysis of the upstream promoter region of the 16 exon zebrafish *gcsfr* gene showed conserved putative sites for binding of the transcription factors HOXA5, PU.1 and CEBP family members [292], similar to the human *gcsfr* promoter region. These data suggest the conserved regulation of *gcsfr* gene expression in teleosts.

The predicted protein structure of zebrafish and goldfish GCSFRs is conserved across vertebrates. The teleost GCSFR extracellular domain is comprised of a signal peptide, an Ig-like domain, a cytokine homology domain containing the WSXWS motif and four cys‐ teine residues, and three fibronectin domains. Following the transmembrane region, the intracellular region contains predicted Box1, Box2, and Box 3 signaling motifs and 6 tyro‐ sine residues [292, 293], shown to be involved in receptor activation and internalization in higher vertebrates.

In zebrafish, *gcsfr* mRNA is expressed by 14 hpf in the RBI, followed by the yolk sac, the ICM, and finally in the kidney by 96 hpf, consistent with the production of neutrophils dur‐ ing primitive and definitive hematopoiesis. In adult goldfish, *gcsfr* mRNA levels were high‐ est in kidney and spleen, followed by the gill, intestine, heart, brain and blood [293]. The *gcsfr* mRNA was highly expressed in goldfish neutrophils and was up-regulated in response to mitogens or pathogens [293] (Figure 1).

310, 312] and acts by activating Ras guanine nucleotide releasing protein 1 (RasGRP1) which is necessary for activating Ras in the Ras/MEK/Erk pathway that is initiated during GCSF signaling [313]. The expression of GFI1 is sustained during differentiation and the transcrip‐ tion factor functions by blocking the expression of *EGR-2/NAB-2*, effectively antagonizing the EGR1/2 transcription factor and preventing initiation of a monocytic differentiation pathway, thereby promoting neutrophil differentiation [177, 312]. Like that of PU.1 and GA‐ TA1, GFI1 and EGR-1/EGR-2 act as an antagonistic pair to regulate neutrophil versus macro‐

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

In zebrafish, two *gfi1* genes have been identified, termed *gfi1* and *gfi1.1*. *gfi1* is primarily ex‐ pressed in neural tissues, and not in the hematopoietic system [314], suggesting that this is not the functional orthologue of mammalian *GFI1*. However, *gfi1.1* was expressed in the dif‐ ferent hematopoietic organs of the developing zebrafish embryo, suggesting that *gfi1.1* is ex‐ pressed in hematopoietic cells [315]. Zebrafish *gfi1.1* morphants displayed a three-fold

crease in *mpo* expression [315]. These data are consistent with the known functional role of mammalian GFI1, suggesting that zebrafish GFI1.1, and not zebrafish GFI1, is the functional

Interferon response factor-8 (IRF-8, also known as ICSBP) is one out of nine members of the IRF transcription factor family and is characterized by an N-terminal DNA binding domain and a C-terminus IRF association domain that can associate with other IRF or Ets transcrip‐ tion family members [316, 317]. IRF8-/- mice and BXH-2 mice with a mutation in their IRF association domain show a drastic expansion of granulocytes at the expense of macrophages [318, 319]. Enforced expression of *IRF8* in myeloid progenitor cells *in vitro* led to the induced expression of a number of macrophage lineage differentiation transcripts including *CSF-1R* and *EGR1*. Additionally, enforced expression of *IRF8* in myeloid cell lines prevented their differentiation into granulocytes when treated with GCSF [320]. It is clear from the *in vivo* and *in vitro* studies that IRF8 promotes the commitment of myeloid progenitors along the

The homologue of *irf-8* was identified in rainbow trout [321] and zebrafish [322] with 53-55% amino acid identity to human IRF8 [321, 322]. In trout tissues, *irf8* mRNA was detected in the spleen, head kidney, gill, brain, intestine, skin, muscle, and liver [321] and mRNA levels could be up-regulated in splenocytes upon treatment with Poly I:C, PMA, PHA and recombinant IL-15. However, the role of IRF8 in GMP fate decisions or during macrophage development was not assessed. In zebrafish developing embryos, *irf8* mRNA was first detected in the rostral blood island, the site of primitive myelopoiesis, and was co-expressed with *csf-1r* mRNA, but not in cells positive for *mpx*, suggesting that *irf8* is expressed in cells committed to the macrophage lineage [322]. In zebrafish *irf8* morphants, *csf-1r+* cells were absent, while *mpx*<sup>+</sup> cells and mature neutrophils were in‐ creased by approximately three-fold, suggesting IRF8 is required for macrophage devel‐ opment. This phenotype could be rescued by injecting embryos with *irf8* mRNA.

cells, along with an increase in *l-plastin* expression and a de‐

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121

phage lineage fate.

increase in the number of *pu.1+*

orthologue of mammalian GFI.

*5.3.3. Interferon response factor-8 (IRF-8)*

macrophage lineage at the expense of the granulocyte lineage.

#### **5.3. Transcription factors**

In addition to the transcription factors described in section 4.3, there are a number of transcription factors downstream that participate in determining GMP fate decisions and that play a role in macrophage and neutrophil cell development, reviewed by [51, 294]. A visual representation of the stage(s) in which these transcription factors are important are shown in Figure 2.

#### *5.3.1. Early growth response (Egr)*

The four Egr proteins, EGR1 [295, 296], EGR2 [297], EGR3 [298] and EGR4 [299], are members of the zinc finger transcription factor family and have an N-terminus activation domain, a repressor domain capable of binding to NAB1/2, and a DNA binding domain comprised of three zinc fingers that bind to the GC rich sequence, 5'-GCGGGGGC'3' [300]. EGR1 promotes commitment to the macrophage lineage at the expense of granulo‐ cytic lineage [301, 302] and has been shown to be essential for myeloblast differentiation into monocytes/macrophages [303, 304]. Treatment of mouse bone marrow cells with CSF-1 has been shown to induce *EGR1* mRNA levels by 6-7 fold three hours post treat‐ ment, as well as *EGR2* and *EGR3* mRNA levels by 2-4 fold [305]. Although *EGR*-/- mice display normal macrophage development [306], it is thought that there is redundancy amongst the Egr transcription factors. Consistent with this idea, EGR2 is also abundant in monoblasts and monocytes [307], and may be involved in monocyte differentiation. Although a zebrafish orthologue of *EGR1* has been identified [308], the role of *egr1* in tel‐ eost macrophage development has not been examined.

#### *5.3.2. Growth factor independence 1 (Gfi1)*

Growth factor independence 1 (GFI1) is a zinc finger transcription factor comprised of an Nterminal Snail/Gfi1 (SNAG) domain that is involved in recruiting proteins to modify histo‐ nes, and a C-terminal domain containing six zinc fingers involved in DNA recognition [309]. *GFI1* is expressed in T-cells, B-cells, mature granulocytes and activated macrophages [310, 311]. GFI1-/- mice showed slight defects in lymphocyte development, increased monocyte and monocyte precursor numbers, an absence of granulocytes and were highly susceptible to infections [310, 311]. Furthermore, myeloid progenitors from GFI1-/- mice did not differen‐ tiate into mature granulocytes in the presence of GCSF *in vitro* [310] or *in vivo* [311]. C/EBPα can up-regulate *GFI1* expression, promoting a neutrophil cell fate, and GFI1 also acts as a negative regulator on *PU.1* to decrease its expression [177, 180]. This lower level of *PU.1* drives granulocyte cell fate [175, 177]. GFI1 is important for neutrophil differentiation [177, 310, 312] and acts by activating Ras guanine nucleotide releasing protein 1 (RasGRP1) which is necessary for activating Ras in the Ras/MEK/Erk pathway that is initiated during GCSF signaling [313]. The expression of GFI1 is sustained during differentiation and the transcrip‐ tion factor functions by blocking the expression of *EGR-2/NAB-2*, effectively antagonizing the EGR1/2 transcription factor and preventing initiation of a monocytic differentiation pathway, thereby promoting neutrophil differentiation [177, 312]. Like that of PU.1 and GA‐ TA1, GFI1 and EGR-1/EGR-2 act as an antagonistic pair to regulate neutrophil versus macro‐ phage lineage fate.

In zebrafish, two *gfi1* genes have been identified, termed *gfi1* and *gfi1.1*. *gfi1* is primarily ex‐ pressed in neural tissues, and not in the hematopoietic system [314], suggesting that this is not the functional orthologue of mammalian *GFI1*. However, *gfi1.1* was expressed in the dif‐ ferent hematopoietic organs of the developing zebrafish embryo, suggesting that *gfi1.1* is ex‐ pressed in hematopoietic cells [315]. Zebrafish *gfi1.1* morphants displayed a three-fold increase in the number of *pu.1+* cells, along with an increase in *l-plastin* expression and a de‐ crease in *mpo* expression [315]. These data are consistent with the known functional role of mammalian GFI1, suggesting that zebrafish GFI1.1, and not zebrafish GFI1, is the functional orthologue of mammalian GFI.

#### *5.3.3. Interferon response factor-8 (IRF-8)*

In zebrafish, *gcsfr* mRNA is expressed by 14 hpf in the RBI, followed by the yolk sac, the ICM, and finally in the kidney by 96 hpf, consistent with the production of neutrophils dur‐ ing primitive and definitive hematopoiesis. In adult goldfish, *gcsfr* mRNA levels were high‐ est in kidney and spleen, followed by the gill, intestine, heart, brain and blood [293]. The *gcsfr* mRNA was highly expressed in goldfish neutrophils and was up-regulated in response

In addition to the transcription factors described in section 4.3, there are a number of transcription factors downstream that participate in determining GMP fate decisions and that play a role in macrophage and neutrophil cell development, reviewed by [51, 294]. A visual representation of the stage(s) in which these transcription factors are important

The four Egr proteins, EGR1 [295, 296], EGR2 [297], EGR3 [298] and EGR4 [299], are members of the zinc finger transcription factor family and have an N-terminus activation domain, a repressor domain capable of binding to NAB1/2, and a DNA binding domain comprised of three zinc fingers that bind to the GC rich sequence, 5'-GCGGGGGC'3' [300]. EGR1 promotes commitment to the macrophage lineage at the expense of granulo‐ cytic lineage [301, 302] and has been shown to be essential for myeloblast differentiation into monocytes/macrophages [303, 304]. Treatment of mouse bone marrow cells with CSF-1 has been shown to induce *EGR1* mRNA levels by 6-7 fold three hours post treat‐ ment, as well as *EGR2* and *EGR3* mRNA levels by 2-4 fold [305]. Although *EGR*-/- mice display normal macrophage development [306], it is thought that there is redundancy amongst the Egr transcription factors. Consistent with this idea, EGR2 is also abundant in monoblasts and monocytes [307], and may be involved in monocyte differentiation. Although a zebrafish orthologue of *EGR1* has been identified [308], the role of *egr1* in tel‐

Growth factor independence 1 (GFI1) is a zinc finger transcription factor comprised of an Nterminal Snail/Gfi1 (SNAG) domain that is involved in recruiting proteins to modify histo‐ nes, and a C-terminal domain containing six zinc fingers involved in DNA recognition [309]. *GFI1* is expressed in T-cells, B-cells, mature granulocytes and activated macrophages [310, 311]. GFI1-/- mice showed slight defects in lymphocyte development, increased monocyte and monocyte precursor numbers, an absence of granulocytes and were highly susceptible to infections [310, 311]. Furthermore, myeloid progenitors from GFI1-/- mice did not differen‐ tiate into mature granulocytes in the presence of GCSF *in vitro* [310] or *in vivo* [311]. C/EBPα can up-regulate *GFI1* expression, promoting a neutrophil cell fate, and GFI1 also acts as a negative regulator on *PU.1* to decrease its expression [177, 180]. This lower level of *PU.1* drives granulocyte cell fate [175, 177]. GFI1 is important for neutrophil differentiation [177,

to mitogens or pathogens [293] (Figure 1).

120 New Advances and Contributions to Fish Biology

**5.3. Transcription factors**

are shown in Figure 2.

*5.3.1. Early growth response (Egr)*

eost macrophage development has not been examined.

*5.3.2. Growth factor independence 1 (Gfi1)*

Interferon response factor-8 (IRF-8, also known as ICSBP) is one out of nine members of the IRF transcription factor family and is characterized by an N-terminal DNA binding domain and a C-terminus IRF association domain that can associate with other IRF or Ets transcrip‐ tion family members [316, 317]. IRF8-/- mice and BXH-2 mice with a mutation in their IRF association domain show a drastic expansion of granulocytes at the expense of macrophages [318, 319]. Enforced expression of *IRF8* in myeloid progenitor cells *in vitro* led to the induced expression of a number of macrophage lineage differentiation transcripts including *CSF-1R* and *EGR1*. Additionally, enforced expression of *IRF8* in myeloid cell lines prevented their differentiation into granulocytes when treated with GCSF [320]. It is clear from the *in vivo* and *in vitro* studies that IRF8 promotes the commitment of myeloid progenitors along the macrophage lineage at the expense of the granulocyte lineage.

The homologue of *irf-8* was identified in rainbow trout [321] and zebrafish [322] with 53-55% amino acid identity to human IRF8 [321, 322]. In trout tissues, *irf8* mRNA was detected in the spleen, head kidney, gill, brain, intestine, skin, muscle, and liver [321] and mRNA levels could be up-regulated in splenocytes upon treatment with Poly I:C, PMA, PHA and recombinant IL-15. However, the role of IRF8 in GMP fate decisions or during macrophage development was not assessed. In zebrafish developing embryos, *irf8* mRNA was first detected in the rostral blood island, the site of primitive myelopoiesis, and was co-expressed with *csf-1r* mRNA, but not in cells positive for *mpx*, suggesting that *irf8* is expressed in cells committed to the macrophage lineage [322]. In zebrafish *irf8* morphants, *csf-1r+* cells were absent, while *mpx*<sup>+</sup> cells and mature neutrophils were in‐ creased by approximately three-fold, suggesting IRF8 is required for macrophage devel‐ opment. This phenotype could be rescued by injecting embryos with *irf8* mRNA. Conversely, the over-expression of *irf8* mRNA in zebrafish resulted in an increase in macrophages by approximately 50% and a decrease in neutrophil numbers by about 40% [322]. These data are similar to those of the mammalian system and suggest a conserved role for IRF8 in determining macrophage over neutrophil cell lineage during primitive myelopoiesis. However, whether IRF8 plays the same role during definitive myelopoiesis in teleosts remains to be determined.

number of gene duplications for many of the soluble factors, receptors, and to some extent, transcription factors as a result of a teleost-specific whole genome duplication predicted to have occurred approximately 350 Mya, and is believed to be responsible for the radiation of teleosts [329, 330]. Many of these teleost genes are rapidly evolving, often undergoing subfunctionalization or neo-functionalization making the identification of teleost orthologues difficult. By developing an understanding of the soluble mediators, receptors, and the intra‐ cellular machinery that govern teleost myelopoiesis, we may be better equipped to develop strategies to promote host defense against pathogens, particularly in aquaculture in which

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This work was supported by a grant from Natural Sciences and Engineering Research Coun‐ cil of Canada (NSERC) to MB. BAK was supported by NSERC and Alberta Ingenuity Fund (AIF) doctoral scholarships, FK was supported by a Japan Society for the Promotion of Sci‐

**ABC** ATP-binding cassette; **AGM** aorta-gonad-mesonephros; **ALM** anterior lateral meso‐ derm; **ATP** adenosine triphosphate; **BFU-E** burst-forming unit-erythrocyte; **bZIP** basic leu‐ cine zipper domain; **CCM** cell-conditioned medium; **C/EBPs** CCAAT/enhancer binding proteins; **CFU** colony forming unit; **CFU-G** colony-forming unit-granulocyte; **CFU-GEMM** colony-forming unit granulocyte/erythrocyte/macrophage/megakaryocyte; **CFU-GM** colo‐ ny-forming unit-granulocyte/macrophage; **CFU-M** colony-forming unit-macrophage; **CFU-Meg** colony-forming unit-megakaryocyte; **CFU-S** colony-forming unit-spleen; **CHT** caudal hematopoietic tissue; **CLP** common lymphoid progenitor; **CMP** common myeloid progeni‐ tor; **ConA** concanavalin A; **CRH** cytokine receptor homology; **CSFs** colony-stimulating fac‐ tors; **CSF-1** colony-stimulating factor 1; **CSF-1R** colony-stimulating factor-1 receptor; **DCs** dendritic cells; **EGR/egr** early growth response; **EPO** erythropoietin; **GCSF** granulocyte col‐ ony-stimulating factor; **GFI1** growth factor independence 1; **GM-CSF** granulocyte/macro‐ phage colony-stimulating factor; **GM-CSFR** granulocyte/macrophage colony-stimulating factor receptor; **GM-CSFRα** granulocyte/macrophage colony-stimulating factor receptor al‐ pha chain; **GMP** granulocyte/macrophage progenitor; **HPCs** hematopoietic progenitor cells; **hpf** hours post fertilization; **HSCs** hematopoietic stem cells; **IL** interleukin; **IL-3Rα** interleu‐ kin-3 receptor alpha; **IRF** interferon response factor; **JAK** Janus family of protein tyrosine kinases; **Ks/Ka** ratio of synonymous to asynonymous nucleotide substitutions; **LMPPs** lym‐ phoid-myeloid primed multipotent progenitors; **LPS** lipopolysaccharide; **LT-HSCs** longterm hematopoietic stem cells; **MCSF** macrophage colony-stimulating factor; **mCSF-1R** membrane-bound colony-stimulating factor-1 receptor; **MEP** megakaryocyte/erythroid pro‐ genitor; **MMP** matrix metalloprotease; **mpo** myeloperoxidase; **MPPs** multipotent progeni‐

fish are predisposed to infection.

ence (JSPS) research fellowship.

**List of abbreviations:**

**Acknowledgements**

#### *5.3.4. MafB*

In addition to the previously described role of MAFB in HSCs and CMPs (see section 4.3.1), *MAFB* is highly expressed in monocytes and macrophages [164, 323] and has been shown to induce differentiation of myeloblasts into monocytes and macrophages [163, 307, 324, 325]. Furthermore, MAFB and c-MAF double knockout mice displayed differen‐ tiated macrophages that were capable of proliferating in response to CSF-1 in semi-solid and liquid culture [325]. Therefore, it appears that *MAFB* expression is sustained in mon‐ ocytes and macrophages in order to prevent proliferation in these terminally differentiat‐ ed cell populations.

Studies examining the role of MAFB in teleost myelopoiesis are limited. In the goldfish PKM system, a *mafb* transcript was identified and showed increasing mRNA levels with macro‐ phage development [19]. The increasing mRNA levels of *mafb* during macrophage differen‐ tiation are similar to what has been observed in mammalian systems and suggest that MAFB may play a role in teleost macrophage differentiation.
