Myelopoiesis during Zebrafish Early Development

Available online at www.sciencedirect.com

Journal of Genetics and Genomics 39 (2012) 435e442

JGG REVIEW

Myelopoiesis during Zebrafish Early Development Jin Xu a, Linsen Du b, Zilong Wen a,* a

State Key Laboratory of Molecular Neuroscience, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China b Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, #12-01, Singapore 117599, Singapore Received 2 May 2012; revised 21 June 2012; accepted 21 June 2012 Available online 4 August 2012

ABSTRACT Myelopoiesis is the process of producing all types of myeloid cells including monocytes/macrophages and granulocytes. Myeloid cells are known to manifest a wide spectrum of activities such as immune surveillance and tissue remodeling. Irregularities in myeloid cell development and their function are known to associate with the onset and the progression of a variety of human disorders such as leukemia. In the past decades, extensive studies have been carried out in various model organisms to elucidate the molecular mechanisms underlying myelopoiesis with the hope that these efforts will yield knowledge translatable into therapies for related diseases. Zebrafish has recently emerged as a prominent animal model for studying myelopoiesis, especially during early embryogenesis, largely owing to its unique properties such as transparent embryonic body and external development. This review introduces the methodologies used in zebrafish research and focuses on the recent research progresses of zebrafish myelopoiesis. KEYWORDS: Zebrafish; Hematopoiesis; Myelopoiesis

1. INTRODUCTION Myeloid cells are basically all non-lymphocyte leukocytes. They are versatile and are involved in development, tissue regeneration, innate and adaptive immune response. Thus, malfunction or dysregulation of myeloid cells could lead to severe human diseases such as leukemia. Due to their critical importance to human health, myeloid cells have been extensively studied in human and mouse. Recently, zebrafish has emerged as a new model animal that complements the study of myeloid cells and provides us new insights of myelopoiesis. Similar to mammals, zebrafish has most of the basic myeloid cell types including monocyte, neutrophil, eosinophil, mast cell and dendritic cell (Herbomel et al., 1999; Willett et al., 1999; Bennett et al., 2001; Lieschke et al., 2001; Dobson et al., 2008; Lugo-Villarino et al., 2010). The conserved myeloid lineage development program between zebrafish and * Corresponding author. Tel: þ852 2358 7294, fax: þ852 2358 1552. E-mail address: [email protected] (Z. Wen).

other vertebrates makes it possible that the discovery in zebrafish could be directly applied to other higher vertebrates. This review will introduce the unique advantages of using zebrafish to study myelopoiesis and the current understanding of myeloid development during zebrafish embryogenesis and highlight some of the recent discoveries. 2. ADVANTAGES AND TECHNIQUES OF ZEBRAFISH TO STUDY MYELOPOIESIS One unique advantage of zebrafish is its transparent body and external development during embryogenesis. Zebrafish embryos could be easily mounted and observed under microscope. Using simple differential interference contrast microscopy (DIC), label free myeloid cells such as macrophages and neutrophils can be directly studied in vivo (Herbomel et al., 1999, 2001; Herbomel and Levraud, 2005; Le Guyader et al., 2008; Colucci-Guyon et al., 2011). In addition, transgenic methods have been well established in zebrafish and many myeloid specific transgenic fish lines have already been

1673-8527/$ - see front matter Copyright Ó 2012, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. http://dx.doi.org/10.1016/j.jgg.2012.06.005

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generated (Table 1) (Ward et al., 2003; Hsu et al., 2004; Mathias et al., 2006; Renshaw et al., 2006; Hall et al., 2007; Peri and Nusslein-Volhard, 2008; Ellett et al., 2011; Wittamer et al., 2011). For example, the behavior of macrophage and neutrophil under stimulation or steady state has been monitored in vivo under fluorescent microscope using such transgenic approaches (Mathias et al., 2006; Renshaw et al., 2006; Hall et al., 2007; Zhang et al., 2008; Ellett et al., 2011). Besides, fluorescent dextran has been used to label specific cells and trace their fate, which improve our understanding of the lineage relationship between different cell types (Serluca and Fishman, 1999, 2001; Keegan et al., 2004; Murayama et al., 2006; Vogeli et al., 2006; Bertrand et al., 2007; Jin et al., 2007, 2009; Schoenebeck et al., 2007; Le Guyader et al., 2008; Warga et al., 2009). Another unique advantage of zebrafish is its large progeny number. Each pair of adult zebrafish could produce hundreds of progenies per week, which makes zebrafish an ideal model for genetic and chemical screening. ENU-induced mutagenesis and forward genetic screen generated many mutants affecting various developmental processes including myelopoiesis (Driever et al., 1996; Haffter et al., 1996; Dai et al., 2010). In particular, such unbiased approaches provide opportunities to identify novel factors involved in myelopoiesis. High throughput chemical screening using zebrafish is also valuable to discover new drugs (Peterson et al., 2000, 2004). Zebrafish makes it possible to conduct large scale in vivo test of chemical function at low cost and provide potential novel treatment to human diseases like leukemia. Reverse genetic methods have also been established after decades of development. The most traditional reverse genetic method is morpholino knockdown. Morpholinos are modified anti-sense oligomers that can resist nuclease digestion (Nasevicius and Ekker, 2000). Morpholinos could attenuate normal gene function either by blocking protein translation or by interfering RNA splicing (Nasevicius and Ekker, 2000; Draper et al., 2001). The effect of morpholinos could last for a few days, which offer a suitable time window to study early developmental processes such as myelopoiesis. To permanently abrogate the function of one particular gene, the targeting induced local lesions in genomes (TILLING) method is developed in zebrafish (Wienholds et al., 2002, 2003). The TILLING method couples ENU-induced mutagenesis and DNA sequencing to identify mutants with mutations in the

target gene. However, the chance to recover the targeted zebrafish mutant largely depends on the efficiency of mutagenesis and the number of F1 progenies sequenced. Recently, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) methods were reported as a more robust method to produce mutants of interest (Doyon et al., 2008; Huang et al., 2011; Sander et al., 2011). The catalytic domain of Fok I endonuclease is fused with zinc finger (ZF) DNA recognition domain or transcription activator-like effectors (TALEs), and mRNAs of such fusion proteins once injected into zebrafish embryos can create target specific mutations in both somatic and germ cells. Heterozygous mutants could then be identified in the next generation. Taken together, these genetic approaches also make zebrafish a powerful vertebrate model to investigate gene functions.

3. MYELOID LINEAGE DEVELOPMENT DURING ZEBRAFISH EMBRYOGENESIS Similar to mammals, zebrafish hematopoiesis consists of successive waves which originate from anatomically different locations, giving rise to different myeloid lineages that are controlled by different genetic programs (Fig. 1) (Ellett and Lieschke, 2010; Jing and Zon, 2011). The earliest wave, namely primitive hematopoiesis, occurs in two locations: the anterior lateral mesoderm (ALM) and posterior lateral mesoderm (PLM). The ALM and PLM later form rostral blood island (RBI) and intermediate cell mass (ICM), respectively. This primitive hematopoiesis mainly produces myeloid cells and erythrocytes. The second wave of hematopoiesis, or the intermediate hematopoiesis occurring in the posterior blood island (PBI), produces committed erythromyeloid progenitors (EMPs) capable of giving rise to erythroid and myeloid lineage cells (Bertrand et al., 2007). Both the primitive and intermediate hematopoiesis cannot sustain for long time. Only the last wave, namely definitive hematopoiesis, is able to produce hematopoietic stem cells (HSCs) that can generate all types of hematopoietic cells for the whole life span. Zebrafish HSCs emerge in the ventral wall of dorsal aorta (VDA), and later migrate to the caudal hematopoietic tissue (CHT) and finally colonized the kidney. Interestingly, myeloid cells of definitive wave are differentiated from both VDA and CHT (Jin et al., 2009).

Table 1 Transgenic lines used to study zebrafish myelopoiesis Transgenic line

Labeled cell type

Reference

Tg(mpeg1:EGFP), Tg(mpeg1:mCherry)

Macrophage

Ellett et al., 2011

Tg(lyz:EGFP), Tg(lyz:DsRED2)

Neutrophil

Hall et al., 2007

Tg(-5.3spi1:EGFP)gl21, Tg(-9.0spi1:EGFP)zdf11

Myeloid progenitor

Ward et al., 2003; Hsu et al., 2004

Tg(mpx:GFP)

Neutrophil

Mathias et al., 2006; Renshaw et al., 2006

Tg(apoeb:lynEGFP)

Microglia

Peri and Nusslein-Volhard, 2008

Tg(mhc2dab:EGFP), Tg(mhc2dab:mCherry)

Antigen-presenting cell, macrophage, dendritic cell and B cell

Wittamer et al., 2011

J. Xu et al. / Journal of Genetics and Genomics 39 (2012) 435e442

437

A ICM VDA

Kidney

PBI RBI

CHT 22 hpf

32 hpf

4 dpf

B RBI

ICM

PBI

Hemangioblast/hematopoietic precursor EMP

Hemangioblast/hematopoietic precursor cloche

gata1

gata1

etsrp tif1

scl alk8

bmp

Myeloid precursor

tif1

pu.1

Erythrocyte

Sumoylated Cebpα Neutrophil

pu.1

Erythrocyte

Myeloid cell

notch1b

gata2

Mast cell

gfi1.1 pu.1

gcsf

gata1

VDA

CHT

HSC

HSC

spi-1l High level

Low level pu.1

gata1

runx1

tif1

pu.1

irf8 Neutrophil

Macrophage

Myeloid cell

Erythrocyte

Myeloid cell

Lymphocyte

Fig. 1. Schematic diagram of myelopoiesis during early zebrafish development. A: different origins of myelopoiesis. B: lineage differentiation and gene regulation of myelopoiesis. Blue arrows indicate lineage differentiation and black arrows depict gene regulation of myelopoiesis. BRI, rostral blood island; ICM, intermediate cell mass; PBI, posterior blood island; VDA, ventral wall of dorsal aorta; CHT, caudal hematopoietic tissue.

3.1. Hemangioblast Hemangioblast is defined as the bipotential precursor that can differentiate into both hematopoietic and endothelial cells. Vogeli et al. (2006) performed lineage tracing experiments in zebrafish and thus provided the in vivo evidence for the existence of hemangioblast in vertebrates. They injected 2,3-dimethyl 2,3dinitrobutane (DMNB)-caged fluorescein (FITC) dextran into zebrafish embryos at one cell stage and used ultraviolet laser to uncage the DMNB caged FITC dextran at single cell level when the embryos were at gastrulation stage. The descendants of the uncaged cells were traced at 30 hours post-fertilization (hpf). The result showed that a portion of uncaged single cells gave rise to only erythrocytes and endothelial cells but not to other cell types (Vogeli et al., 2006). Another similar lineage tracing experiment was performed later by Warga et al. (2009), except that they directly injected lineage tracer dye into single cells at mid-blastula stage. They found that not only erythrocytes but also some macrophages shared common precursors with

endothelial cells, suggesting that at least some macrophages were derived from hemangioblast like precursors. 3.2. Myelopoiesis in primitive hematopoiesis In primitive hematopoiesis, hematopoietic precursor markers such as gata2, lmo2 and scl can be detected in the ALM at early somite stages (Liao et al., 1998; Thompson et al., 1998). The expression of pu.1, the master regulator of myeloid development, can be detected in the ALM later at 6-somite stage (Lieschke et al., 2002). As fish grow up, the ALM evolves into the RBI by 16 hpf (Ellett and Lieschke, 2010). Meanwhile, the pu.1 positive myeloid progenitors migrate rostrally and then spread on the yolk sac (Bennett et al., 2001; Lieschke et al., 2002). This process requires granulocyte colony-stimulating factor receptor (GCSFR) signaling (Liongue et al., 2009). During migration, these myeloid progenitors start to turn on pan-leukocyte marker lcp1 and other markers for monocyte and neutrophil lineages such as csf1ra, mfap4, mpeg1, ptpn6, cxcr3.2, lyc and mpo

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(Herbomel et al., 1999, 2001; Bennett et al., 2001; Liu and Wen, 2002; Zakrzewska et al., 2010). Morphologically distinguishable macrophages are observed as early as 22 hpf on the yolk sac. They are found to remove apoptotic red blood cells as early as 26 hpf and by 30 hpf they are capable of phagocytosing invaded bacteria (Herbomel et al., 1999). Some macrophages migrate into the cephalic mesenchyme from 22 hpf onwards via a csf1ra dependent manner. These macrophages can further emigrate to epithelial tissues and may eventually become various resident macrophages such as microglia (Herbomel et al., 2001; Peri and Nusslein-Volhard, 2008). Morphologically distinguishable neutrophils appear later than macrophages. Electron microscopy (EM) has shown that maturing granulocytes can be detected at 34 hpf (Willett et al., 1999). In consistent with the EM result, the sudan black B (SB), a lipid marker for granules in granulocytes, can be detected at about 33e35 hpf and the first granules are observed under video-enhanced differential interference contrast (VE-DIC) microscopy at 35 hpf. By 48 hpf, neutrophils can be reliably detected by all three methods (Lieschke et al., 2001; Le Guyader et al., 2008). Zebrafish neutrophils possess limited ability to phagocytose fluid-borne bacteria, but they can quickly migrate to wounded or infected tissues and efficiently remove surface-associated bacteria (Lieschke et al., 2001; Le Guyader et al., 2008; Colucci-Guyon et al., 2011). It is generally believed that both macrophage and neutrophil are generated from the RBI. However, Warga et al. (2009) proposed that neutrophil was generated from the ICM region but not the RBI region based on their lineage tracing experiment. So far, lineage tracing experiments performed by us and other groups have clearly shown that the RBI does generate neutrophils (Le Guyader et al., 2008; Jin et al., 2012). Thus, this discrepancy is probably due to the fact that Warga et al. (2009) considered lcp1 as a macrophage specific marker that actually also marks neutrophils. In addition to neutrophil and monocyte lineages, the RBI may also generate mast cells. cpa5, a marker for mast cells, is first detected at 24 hpf. Major cpa5 positive cell population appears on the yolk sac whereas minor population presents in the ICM (Dobson et al., 2008). Because blood circulation starts at around 24 hpf, those cpa5 positive cells in the ICM may be brought from yolk sac by circulation. Alternatively, they could be generated in the ICM in situ. Recently, cpa5 positive cells have been found to express lmo2 at both 28 hpf and 48 hpf, raising the possibility that early mast cells may arise from EMPs (Daas et al., 2012). Myelopoiesis in primitive hematopoiesis is also proposed to occur in the posterior part of ICM, where neutrophils are produced (Warga et al., 2009). Interestingly, the posterior ICM is anatomically close to the PBI, a site anatomically for intermediate hematopoiesis. Whether myeloid cells generated in the ICM belong to primitive hematopoiesis or intermediate hematopoiesis remain to be clarified.

hematopoietic cells in the ICM (Thompson et al., 1998). Traver’s group carried out extensive studies of hematopoiesis in the PBI. By a combination of gene expression pattern, in vitro culture, transplantation and lineage tracing experiment, they concluded that PBI generates EMPs which are independent of HSCs. EMPs exist between 24 and 48 hpf and can generate myeloid cells in the PBI (Bertrand et al., 2007).

3.3. Myelopoiesis in intermediate hematopoiesis

4.2. Specification of myeloid cell lineages

The most posterior part of the ICM, namely the PBI, generates hematopoietic cells that are different from primitive

Specification of primitive hematopoietic progenitors into either myeloid or erythroid lineage is controlled by

3.4. Myelopoiesis in definitive hematopoiesis The appearance of HSCs marks the initiation of definitive hematopoiesis. In zebrafish, HSCs are generated in the VDA from about 1 day post fertilization (dpf) to 2.5 dpf. By powerful live imaging technique, HSCs have been shown to arise from hemogenic endothelial cells via a process termed endothelial hematopoietic transition (EHT) (Bertrand et al., 2010; Kissa and Herbomel, 2010). These HSCs further migrate to CHT and eventually reach the kidney, the organ maintaining hematopoiesis throughout the life span of zebrafish, at about 4e5 dpf (Willett et al., 1999; Murayama et al., 2006; Jin et al., 2007; Kissa et al., 2008). Different from definitive erythrocytes, which predominantly arise in the CHT, definitive myeloid cells can be generated from both the VDA and the CHT. lyc positive myeloid cells produced by definitive hematopoiesis were detected in the VDA as early as 2 dpf (Jin et al., 2009). Eventually definitive myeloid cells also appear in the kidney (Willett et al., 1999). 4. GENETIC CONTROL OF MYELOPOIESIS DURING ZEBRAFISH EMBRYOGENESIS 4.1. From hemangioblast to hematopoietic cells The genetic regulation of myeloid development has been studied in zebrafish embryos. Two parallel pathways have been proposed to be involved in the induction of primitive myeloid lineages in zebrafish. One pathway is the clocheeetsrpescl pathway. The cloche mutant shows defects in both hematopoietic and endothelial lineages. Therefore, the cloche gene may be required as early as hemangioblast (Stainier et al., 1995). etsrp and scl have been shown to act downstream of cloche to regulate hematopoietic and endothelial development (Liao et al., 1998; Sumanas and Lin, 2006; Liu and Patient, 2008). In addition, scl has been shown to function downstream of etsrp to regulate primitive myelopoiesis. Overexpression of scl can rescue myelopoiesis in the cloche mutant and etsrp morphants (Rhodes et al., 2005; Sumanas et al., 2008). The BmpeAlk8 is another pathway involved in myelopoiesis. The Bmp receptor Alk8 has been shown to specifically regulate myelopoiesis in the RBI but not erythropoiesis in the ICM. Absence of alk8 leads to loss of pu.1 expression, whereas constitutively activated alk8 can increase pu.1 expression, suggesting an instructive role of the BmpeAlk8 pathway in myelopoiesis (Hogan et al., 2006).

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orchestrating the expression of a series of key transcription factors. pu.1, the key master regulator of myelopoiesis, is essential for establishment of myeloid lineage. It is well known that pu.1 and gata1 negatively regulate each other and the interplay of these two factors determines myeloid versus erythroid cell fate (Galloway et al., 2005; Rhodes et al., 2005). In addition, tif1g has been shown to affect erythroid versus myeloid cell fate by controlling the expression of pu.1 and gata1 (Monteiro et al., 2011). Interestingly, tif1g can either suppress or promote pu.1 expression under different hematopoietic cellular contexts. For example, tif1g suppresses pu.1 expression in CHT, but promote its expression in the RBI, ICM and EMP (Monteiro et al., 2011). bik1f, a factor required for erythropoiesis, has been shown to have synergistic effect with gata1 to suppress the expression of myeloid genes in ICM (Kitaguchi et al., 2009). gfi1.1 is another factor that affects the erythroid versus myeloid lineage choice. Blocking gfi1.1 function by morpholino knockdown leads to reduced expression of gata1 and increased expression of pu.1, whereas overexpression of gfi1.1 results in enhanced expression of gata1 and inhibition of pu.1 expression (Wei et al., 2008). In addition to the transcriptional control, post-translational modification is also important for myeloideerythroid lineage choice. Recently, it has been reported that hyposumoylation of C/ebpa leads to expansion of myeloid lineage at the expense of erythroid lineage, suggesting that sumoylation of C/ebpa is important for proper differentiation of erythroidemyeloid progenitors (Yuan et al., 2011). 4.3. From myeloid progenitor to differentiated myeloid cells Once specified as myeloid-restricted cells, these myeloid progenitors require additional factors to promote their maturation and differentiation. Some factors are required for panmyeloid development, whereas others are only involved in specific lineage choice or maturation of certain myeloid subtypes. For example, spi-1l is an ETS factor just recently identified. It functions as an intrinsic factor downstream of pu.1 to promote myeloid development (Bukrinsky et al., 2009). Extrinsic factors also play important roles for myelopoiesis in zebrafish. Granulocyte colony-stimulating factor (GCSF) is shown to be critical for the development and migration of myeloid cells in the RBI (Liongue et al., 2009). Myeloid progenitors are capable of differentiating into macrophages, neutrophils, and mast cells during embryonic development of zebrafish. The cell fate choice between neutrophils and macrophages has been extensively studied in zebrafish. It has been shown that high level of Pu.1 favors macrophage fate, whereas low level of Pu.1 prefers neutrophil fate during primitive hematopoiesis (Su et al., 2007; Jin et al., 2012). In addition, Runx1 promotes neutrophil but suppresses macrophage fate by regulating pu.1 expression in a negative feedback loop. Absence of Runx1 leads to increased macrophage numbers at the expense of neutrophils, whereas increasing Runx1 expands neutrophil population at the expense of macrophages (Jin et al., 2012). In contrast to runx1,

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irf8 is necessary for macrophage fate determination. Suppression of Irf8 function causes loss of macrophages and increases the number of neutrophils, whereas overexpression of Irf8 increases macrophage cell number at the expense of neutrophil development (Li et al., 2011). It is not clear yet how the mast cell fate is established in zebrafish. Recently, it has been shown that gata2 functions downstream of Notch signaling to regulate mast cell development. In addition, pu.1 also plays important roles in mast cell development, but it is independent of the Notchegata2 signaling pathway (Daas et al., 2012). 5. CONCLUSION A well-regulated and orchestrated formation process of myeloid cells is critical for human health, and thus myelopoiesis has been of intensive research interest among clinicians and biologists. The unique advantages of zebrafish and recent technical advance have made zebrafish an ideal animal model system to study myelopoiesis, and indeed a lot of recent progress in this field improves our understanding about this basic process. As a newly developed model animal, the potential of zebrafish system is only partially explored. With the powerful live imaging technique and gene manipulation methods, it is possible to unravel novel mechanisms controlling this process. Besides being an excellent model for developmental hematopoiesis, zebrafish also demonstrates the power to understand human myeloid disorders and develop new therapies. For example, using forward genetic approach to screen zebrafish mutants with defects in myelopoiesis offers the opportunity to identify novel leukemia genes, given that most leukemia genes have important roles in normal hematopoiesis (Craven et al., 2005; Payne et al., 2011). Reverse genetic method in zebrafish is also invaluable for investigating selected genes that are known to be involved in leukemia in mammals (Dayyani et al., 2008; Bolli et al., 2010). Indeed, there is an exponential growth of the mutation list from leukemia and other myeloid disorder patients due to the availability of high throughput sequencing technology in recent years, yet it is much more challenge to analyze biological significance of such large number of mutations in vivo. Zebrafish provides a suitable platform to quickly assess the function of the leukemia associated mutations in the vertebrate context, and help to discriminate the passenger and driver mutations. Besides, many leukemia models using transgenic zebrafish expressing mammalian oncogenes or fusion genes have been established. Although many of those are modeling lymphoblastic leukemia (ALL), several were shown to be good models for myeloid leukemia and myeloproliferative disorder (AML/MDS) (Le et al., 2007; Yeh et al., 2008; Zhuravleva et al., 2008; Forrester et al., 2011). Moreover, such fish model provides a unique system to conduct drug screening for targeted therapies (Yeh et al., 2009). For example, one recent study showed that a novel anti-leukemia compound with little toxicity identified in zebrafish screening had potential to be a new highly targeted therapy for humans e even those resistant to conventional therapies (Ridges et al.,

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