Macrophages and neutrophils are the sentinel cells of the innate immune response of vertebrates, such as bony fish (teleosts). As phagocytic myeloid cells, they are involved in homeostatic mechanisms, wound healing, and the detection, elimination and clearance of foreign entities including tumors, virus-infected cells and invading pathogens. Furthermore, macrophages and neutrophils are responsible for producing hundreds of bioactive molecules that are important in pathogen recognition and destruction, cellular communication and activation, initiation of an adaptive immune response and later, resolution of an inflammatory response and tissue repair. Neutrophils and macrophages, while essential to survival, have a finite lifespan. Therefore, a manufacturing centre, the hematopoietic niche, is needed for the production of myeloid cells. The hematopoietic niche must maintain basal myeloid cell production levels during homeostasis, yet retain the flexibility to ramp-up cell production in response to physiological demands, such as pathogenic insult. The development of macrophages (monopoiesis) and neutrophils (granulopoiesis) is collectively known as myelopoiesis, and is regulated by the complex interaction of colony-stimulating factors (CSFs), their receptors, and intracellular transcription factor machinery that control lineage fate decisions and terminal differentiation events.
Over the past 50 years, research using the mouse model system has culminated in the identification of the site(s) of myelopoiesis, the progenitor cell types that give rise to mature myeloid cells, the extracellular and intracellular cues required, and a detailed understanding of the complex intracellular and extracellular milieu of factors that drive this tightly controlled process. From these studies we understand hematopoiesis as an exquisitely fine-tuned, highly regulated, process whereby all blood cells develop from a small number of hematopoietic stem cells (HSCs). HSCs are characterized as long-term repopulating, pluripotent, quiescent cells that undergo symmetrical self-renewal to sustain the population of HSCs within the hematopoietic niche, or asymmetrical division to give rise to hematopoietic progenitor cells (HPCs) . HPCs can develop along the lymphoid lineage, termed lymphopoiesis, to give rise to B-cells, T-cells, natural killer (NK) cells and dendritic cells (DCs). Alternatively, HPCs can develop along an erythroid lineage, termed erythropoiesis, to give rise to erythrocytes and megakaryocytes, or develop along a myeloid lineage to give rise to granulocytes (neutrophils, basophils, eosinophils, mast cells), mononuclear phagocytes (monocytes and macrophages), and DCs. The lymphoid lineage represents the adaptive arm of the immune response, while the myeloid lineage represents the innate arm of the immune response. Regardless of lineage, the decisions made to commit and develop along a given lineage are controlled by extracellular growth factors and intracellular transcription factors that act in concert to regulate gene and protein expression to achieve the desired outcome.
When compared to the mechanisms of myelopoiesis in the mouse, studies using lower vertebrates, such as teleosts, have identified both evolutionary conservation as well as divergence in the mechanisms of myelopoiesis. With over 30,000 identified species, teleosts are the most expansive class of vertebrates, and represent an excellent model system to study the evolution of vertebrate myelopoiesis as they are one of the ancient classes of vertebrates to retain the production of myeloid cells. Within the teleost system, much research surrounds the characterization of teleost cytokines and receptors involved in inflammation and their cellular targets (primarily macrophages). In comparison, little is known about the mechanisms that govern myeloid cell production. Research on teleost myelopoiesis is hampered by the lack of reagents, the difficulty in isolating appreciable numbers of relatively pure populations of HSCs/HPCs, and in identifying key growth factors important for myeloid cell development due to evolutionary selection pressures. As such, the focus of this review is to provide an overview of the current knowledge of the fish model systems used and the growth factors, receptors and transcription factors involved in teleost myelopoiesis, using information from the mammalian model systems as a scaffold to put the advances into context.
2. Teleost model systems of myelopoiesis
2.1. Zebrafish model system
The zebrafish model has been instrumental in advancing our knowledge of the sites of hematopoiesis/myelopoiesis in teleosts, the development, differentiation and migration of HSCs, and through genetic manipulation, the characterization of the early acting growth factors, receptors and transcription factors involved in hematopoiesis. By far, the major advantage provided by zebrafish is the ease of generating transgenic zebrafish, morphant zebrafish (morpholinos) and knockout zebrafish (zinc finger nucleases), as well as many others, due to genetic manipulation. In conjunction, the rapid generation of embryos, embryonic transparency, and small embryo size allows for mass screening strategies. The advantages of zebrafish as a model system, and their contributions to hematopoiesis have been extensively reviewed elsewhere [2-5], and thus will not be covered in this review. While the zebrafish is an excellent
2.2. Ginbuna crucian carp model system
The ginbuna crucian carp model system has been instrumental in serving as a close parallel to the mouse model system in terms of hematopoietic reconstitution experiments to demonstrate the existence of HSCs in teleosts. Identification of donor and recipient HSCs/HPCs and characterization of their progeny by ploidy analysis is useful for assessing the multipotency of different progenitor cell populations. Furthermore, the use of this model system has allowed for the determination of the location of HSCs within the hematopoietic organs of cyprinids. Use of the ginbuan crucian carp system will be particularly important for future work as antibodies are developed against markers on the surface of fish HSCs and HPCs to allow for the analysis of the potency of progenitor cell subpopulations.
2.3. Goldfish model system
The goldfish model system represents a unique opportunity to study myelopoiesis
The spontaneous proliferation and differentiation of PKMs suggested the production of endogenous growth factors and prompted the examination of the target cell sub-population(s) upon which they acted and their effects on cell proliferation and differentiation. The putative progenitors (R1 cells) and macrophages (R2 cells), but not monocytes, were determined to be responsible for the production of endogenous growth factors that act in an autocrine and paracrine fashion . Addition of cell-conditioned medium (CCM) to sorted cell populations demonstrated the capacity of putative progenitors and monocytes to proliferate and differentiate in response to endogenous growth factors. However, treatment of macrophages (R2 cells) with CCM demonstrated their apparent terminal differentiation, while their capacity to proliferate suggested they were capable of self-renewal [15, 17]. Clearly, different endogenous growth factors present in CCM exert distinct actions on macrophage cell sub-populations.
Two pathways of macrophage development were proposed to occur in the PKM cultures. The predominant pathway was classical macrophage development in which progenitor cells differentiated into monocytes and then macrophages . The second was an alternative pathway of macrophage development in which progenitor cells differentiated into macrophages without a prominent monocytic stage . The possible retention of the alternative pathway of macrophage production in addition to the classical pathway may provide a mechanism for rapid generation of macrophages during injury or infection
The observed kinetics of the PKM cultures suggested three phases of growth. Initially, there is a lag phase (days 1-4) where many cells die, followed by a proliferative phase (days 5-9) where cell numbers rapidly increase , and finally, a senescence phase (days 10-14) characterized by cell clumping and cell apoptosis [17, 18]. Differential cross screening of proliferative versus senescence phase PKMs identified a number of differentially expressed genes including those involved in hematopoiesis, signal transduction, transcription, translation and protein processing . The involvement of the identified transcripts in the regulation of cell development [20-22] will be discussed in the following sections.
These seminal observations from PKM cultures established three important ideas regarding goldfish monopoiesis: (1) kidney leukocytes produce their own endogenous growth factors important for driving proliferation and differentiation [14, 15]. (2) Within the population of small leukocyte R1 cells, a population of macrophage progenitor cells must exist. (3) Unlike mammalian systems, the progenitor cell population gives rise to fully differentiated macrophages
3. Site of hematopoiesis/myelopoiesis
3.1. Two waves of hematopoiesis in vertebrates
There are two waves of hematopoiesis in vertebrates. The first wave is primitive hematopoiesis and occurs during embryonic development. Definitive hematopoiesis follows primitive hematopoiesis and occurs in the post-natal or adult animal. Primitive and definitive hematopoiesis are different on a temporal scale, a spatial scale, and in the types of cellular progeny generated. With the exception of T-cells, that undergo maturation in the thymus, lymphopoiesis and myelopoiesis occur in the major hematopoietic organs. The major hematopoietic organ of teleosts is the kidney, akin to that of mammalian bone marrow.
3.2. Primitive myelopoiesis in teleosts
The development of myelopoiesis in fish has primarily been studied using the zebrafish model system. Primitive myelopoiesis is predominated by HPCs with primarily erythroid and myeloid development potential. Initially, primitive hematopoiesis is initiated in the anterior lateral mesoderm (ALM), that gives rise to the rostral blood island (RBI), and in the posterior lateral mesoderm (PLM), that gives rise to the intermediate cell mass (ICM). The RBI is the site of primitive myeloid cell development, generating primarily primitive macrophages that undergo rapid differentiation, lacking or having a very short monocytic stage  and a few neutrophils , while the ICM is the site of primitive erythroid cell development . This stage of primitive hematopoiesis occurs early during development of zebrafish, approximately 11 hours post fertilization (hpf). Following the onset of circulation, at around 24 hpf, the site of hematopoiesis then switches to the posterior blood island (PBI)  and produces multi-lineage progenitor cells capable of producing both primitive erythroid and myeloid cells . Primitive macrophages act as phagocytes during tissue remodeling throughout embryonic development and in clearance of bacterial pathogens . While primitive neutrophils also migrate to a site of infection, they were not observed to phagocytose bacteria . The temporal, spatial and transcriptional control of zebrafish primitive hematopoiesis has been reviewed by [28-30]. Differences in the initial site of hematopoiesis occur between fish species, however, the production of erythrocytes and macrophages during primitive hematopoiesis is consistent [31, 32].
3.3. Definitive myelopoiesis in teleosts
The onset of definitive myelopoiesis occurs around 36 hpf in the zebrafish. Here, HSCs seed the aorta-gonad-mesonephros (AGM) and the caudal hematopoietic tissue (CHT) [33, 34]. By 48 hpf, the HSCs seed the kidney , the final hematopoietic site equivalent to mammalian bone marrow [35-37].
The existence of teleost kidney HSCs and HPCs capable of generating all hematopoietic lineages was demonstrated using transplantation studies in zebrafish and ginbuna crucian carp. Transplantation of whole kidney marrow from
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 process of committing to a CMP begins with long-term HSCs (LT-HSCs), capable of self-renewal 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 progenitors (MPPs) with no ability to self-renew, reviewed by . 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 granulocyte/macrophage progenitor (GMP)  (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 .
On the other hand, the “myeloid-based model” of hematopoiesis, in which myeloid potential is retained in erythroid, T, and B cell branches even after these lineages have segregated from each other, has been proposed . Notably, there is no CLP in this model [63-65]. According to this model, hematopoiesis can be understood as follows: specification toward erythroid, 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 paradigm 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.
4.2. Receptors and growth factors
4.2.1. Mammalian stem cell factor and Kit receptor
Stem cell factor (SCF) was identified [70-72] as short-chain four-helix bundle  encoded by the
The SCF receptor, c-KIT (CD117), was first identified as the cellular oncogene (
The c-KIT protein is primarily found on hematopoietic cells and is a marker of long-term reconstituting HSCs in humans  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 . Approximately 2 x 104 c-KIT receptors are found on normal human HPCs , and can undergo proteolytic cleavage to release a soluble form of c-KIT [95-97]. The soluble c-KIT receptor is thought to regulate membrane bound c-KIT activity,
Binding of homodimeric SCF to c-KIT results in receptor homodimerization, conformational changes in the extracellular and intracellular domains and autophosphorylation of the intracellular tyrosines (reviewed extensively in [73, 78, 99-105]) leading to a number of down-stream signaling pathways that mediate the action of SCF through c-KIT. These signaling pathways include phosphatidylinositol-3-kinase (PI3K), phospholipase Cγ (PLCγ), members of the Janus family of protein tyrosine kinases (JAK) and signal transducers and activators 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 duration 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), granulocyte/macrophage, and macrophage colony-forming units (CFU-S, CFU-IL-3, CFU-GM, and CFU-M) . Further studies have confirmed SCF/c-KIT to mediate the survival of long-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-stimulating factor (GM-CSF) , granulocyte colony-stimulating factor (G-CSF), IL-1, IL-3 , IL-6, and IL-7, among others, to promote the proliferation and differentiation of HPCs [125, 126] and reviewed in . Often, the progeny of HPC differentiation depends on the particular 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) 
4.2.3. Teleost Kit and Kit ligand
Whole genome duplication has resulted in two orthologues of
4.2.4. Biological functions of teleost kit ligands and receptors
Based on the non-overlapping expression of
The role of teleost
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
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,
4.2.6. Granulocyte-macrophage colony-stimulating factor/Granulocyte-macrophage colony-stimulating 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 granulocyte and macrophage colonies from CFU-GM [147, 148]. GM-CSF is produced by activated T-lymphocytes [147, 149], endothelial cells , and lung fibroblasts  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 chemoattractive to immature and mature neutrophils
Similar to that of
4.3. Transcription factors
Commitment of LT-HSCs to the myeloid lineage is an intricate regulation of the transcription factors expressed, their relative levels to one another, and their expression on a temporal 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 counterpart, 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 extensively reviewed elsewhere [159-162], and will only be briefly described here for the purpose 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.
In zebrafish, the
CCAAT/enhancer binding proteins (C/EBPs) are members of the family of transcription factors that contain a C-terminal basic leucine zipper domain (bZIP) comprised of a basic region involved in DNA binding and a leucine zipper domain involved in protein interactions . Six members of the C/EBP family have been identified in mammals: alpha, beta, gamma, delta, epsilon and zeta . 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 . C/EBPα is capable of binding to the
The zebrafish CEBPα orthologue showed 66% amino acid identity to human C/EBPα, while the bZIP domains showed 99% amino acid identity . In zebrafish,
Two studies have examined the function of CEBPα in zebrafish primitive myelopoiesis. The injection of a deletion mutant of
The orthologues of C/EBPδ, C/EBPγ and C/EBPε exist in teleosts. The
The Ets transcription family member PU.1 is well known as the master transcriptional regulator of mammalian myelopoiesis through an antagonistic relationship with GATA1, recently reviewed by . 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 . At the C-terminus, PU.1 has an Ets domain important for binding the DNA consensus sequence AAAG(A/C/G)GGAAG . Mice deficient in PU.1 (
PU.1 also plays a role at the GMP stage to regulate commitment to a granulocyte or macrophage 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
An orthologue of PU.1 has been identified in teleosts. In the Japanese flounder,
A pu.1-like gene (spi-1 like,
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 unipotent committed macrophage progenitor has yet to be unequivocally demonstrated in the teleost systems, progenitor/precursor cells that give rise to monocytes and macrophages have been demonstrated.
5.1.2. Receptors and growth factors
18.104.22.168. 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]. Alternative splicing of
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 motifs . 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] . 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.
22.214.171.124. Colony-stimulating factor-1 receptor
126.96.36.199. 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 chemotaxis, 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 systems such as regulation of macrophage and osteoclast numbers, bone remodeling, tooth production and fertility and breast development [241-245]
188.8.131.52. Teleost colony stimulating factor-1
The genomic structure of the identified
Along with differing genomic organizations, trout
184.108.40.206. Teleost colony-stimulating factor-1 receptor
The duplication of
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 glycosylation sites, a transmembrane domain, and an intracellular tyrosine kinase domain . The mRNA of mCSF-1R could be detected in progenitor, monocyte and macrophage subpopulations, and an antibody produced against the first two Ig domains of CSF-1R was able to recognize monocytes and macrophages . However, unlike mammalian neutrophils, zebrafish and goldfish neutrophils do not appear to express mRNA for
5.2. Neutrophil development
5.2.1. Progression of cell development
Following the commitment of the CFU-GM to a committed granulocyte progenitor cell, terminal differentiation through a promyelocyte, myelocyte, and metamyelocyte stages occur to give rise to a mature neutrophil, and are regulated through growth factor and transcription factor signaling, reviewed by  (Figure 1). Similar to that of mammals, the differentiation 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 hydrogen peroxide attractant produced by cells at the site of damage . However, the responding neutrophils had low phagocytic activity , or engulfed small fragments of the pathogen .
5.2.2. Receptors and growth factors
220.127.116.11. 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-inflammatory 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 proliferation, differentiation, survival and activation of neutrophils and their progenitors, and has been reviewed extensively by [144, 270]. The transcription of
18.104.22.168. 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 receptor 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  and lymphocytes, as well as some non-hematopoietic cells, GCSFR is primarily found on neutrophils 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 . 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
22.214.171.124. Biological activity of granulocyte colony stimulating factor
The targeted gene disruption of
126.96.36.199. Teleost granulocyte colony-stimulating factor
Functional studies on fish GCSF-1 are limited. Only two manuscripts report on the function of GCSF-1 and both utilize the zebrafish model system.
188.8.131.52. Teleost granulocyte colony-stimulating factor receptor
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 cysteine residues, and three fibronectin domains. Following the transmembrane region, the intracellular region contains predicted Box1, Box2, and Box 3 signaling motifs and 6 tyrosine residues [292, 293], shown to be involved in receptor activation and internalization in higher vertebrates.
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 , EGR3  and EGR4 , 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’ . EGR1 promotes commitment to the macrophage lineage at the expense of granulocytic 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
5.3.2. Growth factor independence 1 (Gfi1)
Growth factor independence 1 (GFI1) is a zinc finger transcription factor comprised of an N-terminal Snail/Gfi1 (SNAG) domain that is involved in recruiting proteins to modify histones, and a C-terminal domain containing six zinc fingers involved in DNA recognition .
In zebrafish, two
5.3.3. Interferon response factor-8 (IRF-8)
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 transcription 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
The homologue of
In addition to the previously described role of MAFB in HSCs and CMPs (see section 4.3.1),
Studies examining the role of MAFB in teleost myelopoiesis are limited. In the goldfish PKM system, a
Myelopoiesis is an orchestration of a multitude of growth factors and transcription factors that control cell fate decisions and differentiation along a chosen cell lineage. It is evident that there exists some functional redundancy in the action of myelopoietic growth factors, most likely put in place to ensure the production of these critical innate immune cells. Studies have focused on examining the regulation of myelopoiesis in the mouse model system, and have only just begun in the teleost model system.
The divergence of teleosts and mammals occurred approximately 400-450 Mya, thus teleosts represent one of the most basal groups of vertebrates . Comparison of soluble factors and their receptors in teleosts and mammals show retention of many of important hematopoietic growth factors and receptors, including PDGFR [250, 251, 327], c-KIT [128-130], FLT3 (accession number DQ317446), CSF-1R [20, 250-254], GCSFR [292, 293], and their ligands PDGF , KIT ligand [128, 130, 131], CSF-1 [246, 249], and GCSF [290-292], although FLK2, the ligand to FLT3, has not yet been reported. However, it appears that teleosts do not possess the key myeloid growth factors IL-3 and GM-CSF, and their cognate receptors. In addition, teleosts possess all of the TF families required for hematopoiesis in higher vertebrates, reviewed in [28, 30]. Based on studies performed to date, the regulation of hematopoiesis is largely similar between mammals and teleosts. However, teleosts often possess a 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 sub-functionalization or neo-functionalization making the identification of teleost orthologues difficult. By developing an understanding of the soluble mediators, receptors, and the intracellular machinery that govern teleost myelopoiesis, we may be better equipped to develop strategies to promote host defense against pathogens, particularly in aquaculture in which fish are predisposed to infection.
This work was supported by a grant from Natural Sciences and Engineering Research Council 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 Science (JSPS) research fellowship.