Zebrafish and Stem Cell Research

Chapter 38

Zebrafish and Stem Cell Research Emily N. Price* and Leonard I. Zony

Children’s Hospital Boston, MA, USA, yStem Cell Program and Division of Hematology/Oncology, Children’s Hospital and Dana Farber Cancer

*

Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, USA

Chapter Outline Introduction Zebrafish System Morphologic and Embryonic Attributes Blood Formation in the Zebrafish Genetic Screens in Zebrafish Zebrafish Blood Mutants

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INTRODUCTION The utility of the zebrafish system in vertebrate biology has sparked the burgeoning development of a new and noteworthy field. Biologists across the world are taking advantage of Danio rerio for investigations into organogenesis, disease, and development. The small size, ease of care, and rapid generation time are but a few of the aspects making zebrafish a particularly useful model organism. Additionally, the optical transparency of zebrafish embryos is especially suited for investigations into hematopoiesis and stem cell biology (Thisse and Zon, 2002; Bahary and Zon, 1998). Perhaps one of the most significant features of the zebrafish system is the ability to conduct large-scale forward genetic screens in an organism with high homology to the human genome (Driever et al., 1996; Haffter et al., 1996; Barbazuk et al., 2000). Screening imparts the ability to address specific developmental processes without preceding knowledge of what genes may be involved. The Zebrafish Genome project performed by the Wellcome Trust Sanger Institute facilitates the positional cloning of genes uncovered using genetic screens in the zebrafish (http://www.sanger.ac.uk). With regard to stem cells, the zebrafish has largely been used to understand hematopoietic stem cells (HSCs). The success of the system makes it likely that stem cells from other organs will be explored in the zebrafish. This chapter describes the

Chemical Screens in Zebrafish Cell Sorting and Transplantation Disease Models in Zebrafish Future of the System Acknowledgments References

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attributes of zebrafish for investigations into hematopoiesis, strategies for conducting genetic screens in the fish, current research in the field, and finally, the future of the zebrafish system for investigations into stem cell biology.

ZEBRAFISH SYSTEM Morphologic and Embryonic Attributes Zebrafish are relatively small (3e4 cm) and reach sexual maturity between 2 and 3 months. The fish mate yearround, with females mating weekly. Because of the small size of the fish, facilities are able to maintain thousands of individuals in a compact laboratory environment. Few, if any, vertebrate model organisms allow for such population size or ease of care. Furthermore, females lay between 100 and 200 eggs per mating, permitting large-scale genetic screens as well as Mendelian approaches and analysis. Of particular importance to the study of early blood development and HSCs is the unique embryonic morphology of the zebrafish (Bahary and Zon, 1998). Zebrafish embryos develop externally from the one-cell stage and are transparent, permitting embryonic development to be readily viewed under a dissecting microscope. Circulation begins by 24 hours post fertilization (hpf), and the number and morphology of blood cells may be identified under a microscope (Wingert and Zon, 2003).

Handbook of Stem Cells, Two-Volume set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00038-X Copyright Ó 2013 Elsevier Inc. All rights reserved.

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Blood Formation in the Zebrafish The hematopoietic program in vertebrates begins with mesodermal patterning along the dorsaleventral axis (Davidson and Zon, 2000; Mullins et al., 1996). During the process of mesoderm patterning, a subset of cells produces a putative population termed hemangioblasts (Gering et al., 1998). These cells are hypothesized to be a common precursor of blood and vascular lineages because of their close physical association and common gene expression during early development (Gering et al., 1998; Amatruda and Zon, 1999; Liao et al., 1997; Thompson et al., 1998). Unlike many other vertebrates that form primitive HSCs in extraembryonic yolk sac blood islands, the initial site of blood formation in zebrafish is the intermediate cell mass (ICM), an intraembryonic tissue (Thisse and Zon, 2002; Detrich et al., 1995). The zebrafish ICM tissue is analogous to the extraembryonic mammalian blood islands in its role as the primary site of primitive erythropoiesis (Wingert and Zon, 2003; Amatruda and Zon, 1999; Galloway and Zon et al., 2003). As in all vertebrates, a shift in hematopoietic sites occurs in the zebrafish. Cells within the ICM are hypothesized to populate the dorsal mesentery as well as the ventral wall of the dorsal aorta (Gering et al., 1998; Amatruda and Zon, 1999; Liao et al., 1997). This region is thought to be the zebrafish equivalent of the aortaegonademesonephros (AGM) region, an intraembryonic site believed to specify definitive HSCs in vertebrate organisms. These HSCs are then thought to colonize the developing kidney where blood formation continues throughout the juvenile and adult life of the fish (Galloway and Zon et al., 2003). In contrast, the primary sites of definitive hematopoiesis in mammals are the fetal liver and the adult bone marrow. Despite some differences (e.g., fish erythrocytes remain nucleated), zebrafish blood is remarkably similar to mammalian blood with all lineages e erythroid, myeloid, lymphoid, and thrombocytic e represented (Traver et al., 2003a). Thus, the fish is a capable and applicable model organism for the study of hematopoiesis. The identification and isolation of zebrafish homologs of factors identified as critical for normal hematopoiesis and vasculogenesis such as the transcription factors SCL, lmo2, GATA-1, GATA-2, and c-myb has permitted investigations into many aspects of blood and vascular tissue formation (Wingert and Zon, 2003; Gering et al., 1998; Amatruda and Zon, 1999; Liao et al., 1997; Thompson et al., 1998; Traver and Zon, 2002). RNA in situ hybridization studies using whole zebrafish embryos provide gene expression patterns that may be used to investigate the timing and anatomy of hematopoiesis and vasculogenesis (Figure 38.1) (Amatruda and Zon, 1999). The first appearance of vascular and blood markers is seen at the three somite stage (11 hpf) with the expression of

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GATA-2 and SCL in two stripes of lateral plate mesoderm (Wingert and Zon, 2003; Gering et al., 1998; Amatruda and Zon, 1999). Some cells within these stripes will become blood in the fish. By the 18-somite stage (18 hpf), these expression stripes have fused to form the tubular ICM structure (Gering et al., 1998; Thompson et al., 1998). The ICM is fully formed by 23 hpf just before the commencement of circulation. The presence of c-mybexpressing cells located in the ventral wall of the dorsal aorta at 36 hpf suggests the presence of definitive HSCs in the zebrafish (Thompson et al., 1998). Finally, by 4 days post fertilization (dpf) HSCs have migrated to the developing kidney. Although the discrete steps necessary for the induction of hematopoietic precursors into differentiated blood lineages as well as the program directing the different hematopoietic waves are still unclear, the zebrafish is a powerful model organism with which to begin to unravel the story. It is evident that the process of the differentiation of HSCs into the erythroid, myeloid, and lymphoid lineages is the result of a highly conserved gene program. It is also clear that the zebrafish, like other vertebrates, possesses discrete waves of hematopoietic events characterized by expression of specific and recognized transcription factors. As described later in this chapter, zebrafish have played an important role in uncovering novel genes involved in these programs through large-scale genetic screens.

Genetic Screens in Zebrafish The first large-scale genetic screens in a vertebrate organism were conducted in zebrafish in Boston, MA, and Tu¨bingen, Germany, in the mid-1990s (Driever et al., 1996; Haffter et al., 1996). The ability to conduct screens in a vertebrate organism, in addition to those conducted in nonvertebrates such as Caenorhabditis elegans and Drosophila is clearly an important achievement. Such screens have already yielded scores of mutants with defects in varying stages of blood and vasculature formation, a few of which are described in detail in this chapter. The power of screens in zebrafish is the ability to uncover novel genes involved in specific developmental processes in an unbiased manner. The Boston and Tu¨bingen ventures used F2 screens to uncover developmental mutants (Driever et al., 1996; Haffter et al., 1996; Patton and Zon, 2001). The screens began with the mating of a male fish treated with ethyl nitrosourea (ENU) to a wild-type female (Driever et al., 1996; Haffter et al., 1996). Mutations in the germ line of the ENU-treated male were passed to heterozygous individuals in the F1 generation. The F1 and F2 generations were successively incrossed to obtain an F3 generation. In the presence of a recessive mutation, half of an F2 family should be heterozygous for that mutation. When two F2 fish

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FIGURE 38.1 Expression patterns of specific hematopoietic factors in zebrafish embryos. (A) Whole-embryo in situ hybridization for SCL at 12 hpf shows the stripes of ventral mesoderm and marks putative hemangioblasts. (B) GATA-1 expression in an embryo 24 hpf marks the ICM prior to circulation of erythroblasts. (C) C-myb expression at 36 hpf marks the wall of the dorsal aorta and the zebrafish equivalent of mammalian AGM, shown enlarged in panel D. (Please see CD-ROM for color version of this figure.) (Courtesy of C. Erter.)

are mated and both are heterozygotes for the mutation, their progeny will display 25% wild-type, 50% heterozygous, and 25% homozygous mutant embryos. The Boston and Tu¨bingen screens began with approximately 300 ENU-treated males and uncovered more than 2000 new developmental mutants (Driever et al., 1996; Haffter et al., 1996). An important aspect of the zebrafish is the ease with which mutations may be generated in the fish. The chemical ENU, which causes point mutations throughout much of the genome, is a popular and effective mutagen for zebrafish (Solnica-Krezel et al., 1994; Mullins et al., 1994). However, there are many other chemicals and avenues available to achieve mutations in the fish, all producing slightly different results. Gamma- and X-rays may be used to create large genomic deletions and translocation events as well as point mutations (Walker, 1999). In a newer approach, insertional mutagenesis has been employed in zebrafish (Amsterdam et al., 1999; Golling et al., 2002; Gaiano et al., 1996). Mutations are achieved by injecting retroviruses into the 1000e2000 cell stage embryo. Although the degree of mutations achieved by this approach is significantly lower than by ENU mutagenesis,

the ease of cloning the mutated gene makes it an exciting resource for the field (Golling et al., 2002). In addition to the F2 diploid screens conducted in Tu¨bingen and Boston, haploid and gynogenetic diploid screens have been designed and carried out in zebrafish (Walker, 1999; Streisinger et al., 1981). Zebrafish embryos are able to survive 3 dpf as haploid organisms (Walker, 1999). This allows a shortcut to uncovering mutations, since recessive alleles in F1 fish may be uncovered in a single generation as opposed to the two generations needed in diploid screens. To create viable haploid embryos, a female fish is gently squeezed to release her eggs. Sperm from male fish is harvested from testes and UV-inactivated before adding to the unfertilized eggs. Because the UV-irradiated sperm is unable to contribute any genetic material to the eggs, the embryos develop with only the genetic material of the mother. Haploid embryos have the same body plan as diploids; however, they are shorter in length and possess some developmental problems as well as a lifespan of only 3e5 days. Nonetheless, haploid screens have successfully uncovered many types of developmental mutations and have proved an extremely significant resource (Walker, 1999; Streisinger et al., 1981).

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In addition to haploid embryos, gynogenetic embryos may be used in zebrafish screens. By applying early hydrostatic pressure or heat shock, unfertilized eggs from female fish can be forced to become diploid, comprised solely of the mother’s genetic material (Traver and Zon, 2002; Nasevicius and Ekker, 2000). Like haploid embryos, gynogenetic diploid embryos represent the genome of one female fish, and the large size of the female’s clutch allows the implementation of Mendelian genetics. Gynogenetic diploid embryos will survive to adulthood, unlike haploid embryos, and allow screening for later developmental abnormalities in the fish. This method bypasses the lethal haploinsufficiency of haploid embryos but maintains identical genomic information. With the use of haploid and gynogenetic embryos, the genome of a female fish may be screened for recessive mutations displaying a phenotype in her progeny. These approaches provide a less burdensome method for uncovering mutations involved in specific developmental pathways. New techniques have been pioneered for reverse genetics in zebrafish. The zebrafish field has greatly benefited from the use of morpholinos, which are antisense oligonucleotides that are injected into the one cell embryo (Nasevicius and Ekker, 2000). Morpholinos have been generated to hundreds of zebrafish genes, and the knockdown approach recapitulates the mutant phenotype. For instance, a knockdown of GATA1 leads to a bloodless embryo (Paik and Zon, 2010). Morpholinos are generated with two methods. It is possible to target the 50 -region of the RNA near the ATG, and this will prevent translation, or the splicing junctions can be targeted and splicing is then aberrant. Many investigators in the field evaluate gene function with these approaches. Recently, it has been possible to create knockout zebrafish with two methods. TILLING (Target Induced Local Lesions in Genomes) relies on sequencing exons of a candidate gene from a large number of ENU-derived male fish DNA samples (Mouillesseaux and Chen, 2011). This brute force approach has led to a number of mutant zebrafish. A p53 mutant was derived by this technique (Winkler et al., 2011). A newer technique is the zinc-finger approach. Targeted zinc fingers make use of the FOK1 enzyme, and this cleaves DNA at the sites specific for the attached zinc fingers (Zhu et al., 2011). The flk1 gene was targeted in this approach, producing mutant fish with angiogenesis defects.

Zebrafish Blood Mutants Many blood mutants have been uncovered in large-scale screens, and several others have occurred spontaneously or have been discovered while screening for mutations in other developmental areas (Driever et al., 1996; Haffter et al., 1996; Donovan et al., 2000; Dooley and Zon, 2000). Characterization and attempts to clone these genes has

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provided valuable information into hematopoiesis and its genetic program. Some mutants have also proved useful as new models for human blood diseases (Kimmel et al., 1989; Griffin et al., 1998). The blood mutants have been divided into discrete categories based on their stage of disruption in the hematopoietic design. Categories of blood mutants include mesoderm patterning mutants, hematopoietic stem cell mutants, committed progenitor mutants, proliferation and/or maintenance mutants, hypochromic mutants, and photosensitive mutants (Wingert and Zon, 2003). Mutants involved in the first three categories are described in this section. In the category of mesoderm patterning is the zebrafish mutant spadetail (spt), which is a homolog of tbx16, a T-box transcription factor (Davidson and Zon, 2000; Davidson et al., 2003; Stainier et al., 1995). Tbx16 is necessary for correct release from mesoderm-inducing signals as well as proper formation of endoderm (Davidson and Zon, 2000). The phenotype of spt is one of incorrect trunk formation, poorly formed dorsal somites, undifferentiated blood, disorganized dorsal vessel formation, and other characteristics indicating severe mesoderm and endoderm tissue deficiencies (Wingert and Zon, 2003; Thompson et al., 1998; Stainier et al., 1995). Through studying the interaction of paraxial mesoderm (PM), trunk intermediate mesoderm (IM), and labeled cell transplantation it was found that spt/ tbx16 is required cell autonomously within RBCs. Spt/tbx16 affects RBC development by directing the positioning of PM in the IM. When transplanted into a wild-type fish, both wild-type and spt cells were able to form mesoderm and endothelial but only wild-type labeled RBCs were observed. This indicates that the disruption in spt is specific to hematopoietic trunk intermediate mesoderm (IM) (Rohde et al., 2004). It was also shown that spt/tbx16 is required in the paraxial mesoderm to help position it next to the IM, creating the environment for RBC formation. Wild-type IM cells that were transplanted into spt trunks did not express GATA-1 unless they were in proximity to wild-type trunk PM (Rohde et al., 2004). This indicates that the interaction of PM and IM is a critical step to form embryonic RBCs in zebrafish. Other mesoderm-patterning mutants have been identified in zebrafish, including swirl (swr) and snailhouse (snh), which have increased dorsal tissue leading to scarcity of ventral blood (Wingert and Zon, 2003; Mullins et al., 1996). Both swr and snh phenotypes are caused by mutations in members of the bone morphogenetic protein (BMP) subgroup belonging to the TGF superfamily of growth factors (Davidson and Zon, 2000). The dorsalized phenotype of swr is caused by a mutation in bmp2b, and the snh mutation is in bmp7. BMP signaling is critical for correct mesodermal patterning along the ventral axis, plays a key role in the antagonistic relationship between dorsalizing and ventralizing signals during mesodermal patterning, and

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leads to the correct formation of blood. Whereas these mutants are not blood mutants directly, spt, swr, and snh are prime examples of the importance of correct mesoderm patterning for both proper blood and vessel formation. The kugelig (kgg) mutant exhibits an embryonic lethal phenotype because of a mutation in the cdx4 gene belonging to the caudal family of homeobox genes implicated in anteroposterior (AP) patterning (Liao et al., 1998). Homozygous mutant kgg embryos develop abnormal AP patterning resulting in severe tail defects as well as acute anemia within 1 dpf. Blood cell numbers recover by 5 dpf in the mutants; however, all embryos die by 10 dpf. The large family of hox genes is known to play a pivotal role in AP patterning; however, only overexpression of certain hox genes (hoxb7a and hoxa9a but not hoxb8a) can rescue the mutant phenotype. These results imply that specific hox genes play unique roles in hematopoietic cell fate specification. The additional failure of SCL overexpression to rescue the mutant phenotype implies that cdx4 as well as downstream hox genes are required for the posterior mesoderm to become “competent” for blood formation. A compensation interaction between cdx1a, another member of the zebrafish cdx family, and cdx4 has been shown through morpholino knockdown in the kgg mutants. Cdx1 and cdx4 act together to form embryonic erythroid cells, definitive HSCs, and regulate the expression of hox genes, supporting the importance of the cdxehox pathway in directing blood cell fate (Davidson and Zon, 2006). While cdx4/ alone resulted in the GATA-1 levels only reaching 40e50% of wild-type levels by the 18 somite stage, in embryos deficient for cdx1 and cdx4 there was a complete absence of GATA-1 expression or earlier markers such as draculin. The injected morphant embryos had a shortened hoxa9a expression domain and a small population of SCLþ ICM precursors that are most likely angioblasts since they do not express draculin (Davidson and Zon, 200). There is a partial rescue of the GATA-1 expression through injection of hoxa9a mRNA but the embryos retain their truncated phenotype indicating the blood cell formation and posterior truncation pathways can be separated. In the injected morphants there was an absence of runx1a expression in the artery indicating that the formation of HSCs is also affected by the knockdown of both cdx1a and cdx4 (Davidson and Zon, 200). A chemical screen was performed investigating chemicals that increase GATA-1 expression in kgg. Two chemicals, both members of the psoralen family, bergapten and 8-methoxypsoralen, were shown to rescue GATA-1 in kgg. The treatment with bergapten also rescues pu.1 expression. To test if bergapten was acting through the hox gene pathway, levels of RNA expression in the control and bergapten-treated embyros were measured and there was no change between them. Bergapten is acting on a mechanism that is independent of the hox genes to rescue the GATA-1 expression in kgg (Paik et al., 2010).

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Another interesting blood mutant is cloche (clo), which exhibits severe depletion of blood and vasculature as well as reduced expression of transcription factors necessary for both blood and vessel formation (Liao et al., 1997, 2002). The abnormally low expression of SCL, lmo2, GATA-2, and GATA-1 suggests that clo has a loss of blood progenitors (Liao et al., 1997). clo embryos have severely reduced expression of flk1 or tie1, both shown to be expressed by endothelial progenitors. The almost complete lack of blood and vasculature in clo suggests that the clo gene product may be responsible for the maintenance or differentiation of the putative hemangioblast (Thompson et al., 1998; Liao et al., 2002). Interestingly, overexpression of SCL has been shown to partially rescue GATA-1, flk1, and tie1 expression in clo but SCL has been ruled out as a candidate based on linkage studies (Long et al., 1997). Additionally, flk1 and hhex have been shown to rescue expression of some important transcription factors but have also been shown to map to different linkage groups from the clo gene. The clo gene has been shown to act upstream of flk1 in the program of endothelial cell dedifferentiation (Liao et al., 1997). In addition to the study of rescuing erythroid and vascular cells, the rescue of myeloid cells has also been investigated. When clo mutants were injected with SCL mRNA, l-plastin, pu.1, and mpo expression was observed. To investigate if pu.1 alone was sufficient to rescue the myleoid lineage in cloche, pu.1 mRNA was injected into mutants. pu.1 MRNA injection led to expression of pu.1, l-plastin, and mpo staining but not of GATA-1 or alpha globin (Rhodes et al., 2005). This indicates that pu.1 expression is sufficient to drive myeloid expression of a progenitor cell but does not rescue erythropoiesis. The bloodless (bls) mutant, like clo, may be placed in the category of HSC mutants. Embryos carrying the bls mutation possess almost no erythroid cells at the onset of circulation but contain normal macrophages and carry out normal, though delayed, lymphopoiesis (Wingert and Zon, 2003; Traver et al., 2003a). Although dominant, the bls mutant is not penetrant in its display of an almost complete lack of primitive erythrocytes. Corresponding with the lack of embryonic erythroid cells are decreased levels of SCL and GATA-l expression in bls. Levels of GATA-2 expression are also reduced but present in bls mutants, likely because of the presence of GATA-2-expressing endothelial precursors in the ICM. The bls mutant does possess normal vessel formation and has normal flk1 expression during embryogenesis. Interestingly, bls embryos produce normal red blood cells at approximately 5 dpf after the initiation of the definitive wave of hematopoiesis. These results suggest that the bls gene product is responsible for the maintenance or production of hematopoietic precursors during primitive hematopoiesis but does not affect the production of HSCs. A partial rescue of GATA-1 positive cells can be obtained through overexpression of SCL in bls mutants. However,

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these GATA-1 positive cells never differentiated to mature red cells. Overexpression of GATA-1 or bmp4 failed to rescue GATA-1 expression in bls mutants. These results and the fact that bmp4 does not induce SCL expression indicate that bls acts downstream of bmp4 in maintaining the expression of SCL (Liao et al., 2002). Through reciprocal cell transplantation bloodless was shown to act in a non-cell autonomous manner. Neither bls nor transplanted wild-type cells were able to differentiate when placed in bls hosts. However, when bls donor cells were transplanted into a wild-type host, GATA-1 expressing hematopoietic progenitors derived from the mutant bls cells were observed (Liao et al., 2002). This mutant should provide much insight into the method of differentiation of stem cells from precursors and for the connection between primitive and definitive waves of embryonic hematopoiesis. In the moonshine (mon) mutant, the primitive erythroid lineage is specified normally but during development a severe anemia occurs. This results in embryonic lethality by 7 dpf. The mon gene encodes for TIFIg (transcriptional intermediary factor 1 gamma), which is ubiquitously expressed in the embryo, yet strongly expressed in blood tissues. Around 1/500 mon mutants will survive to adult but have severe anemia indicating that TIFIg is also critical for definitive erythropoiesis. To elucidate the function of TIFIg during erythropoiesis, the first genetic modifier screen in zebrafish haploids was conducted to find a suppressor mutant for mon. A mutant called sunshine (sun) was discovered which restores erythroid lineages in moonshine. Sun has a mutation in cdc73 gene that encodes for a subunit within the PAF complex, which is associated with pol II during elongation. Pausing and stalling of RNA polymerase II (pol2) has been identified in genome-wide studies and is thought to be used by the cell to temporarily stall transcription while maintaining its identity and viability (Bai et al., 2010). Pol II elongation is inhibited by PAF and DSIF. In wild-type blood cells TIFIg recruits FACT and p-TEFb to the SCL complex that is bound to TIFIg. p-TEFb, a positive elongation factor, phosphorylates DSIF and allows pol2 to proceed with elongation. With the loss of TIFIg, p-TEFb and FACT are not recruited, phosphorylation does not occur, and elongation is paused. In a double mutant for both TIFIg and DSIF/PAF there is no pausing of pol2 and elongation proceeds leading to rescued erythroid development. Suppressor screens on mutants such as mon, which define critical steps in both hematopoiesis and organogenesis, are very important. Through these screens, interacting pathways may be identified and may have clinical implications (Bai et al., 2010).

Chemical Screens in Zebrafish Hematopoietic stem cells are controlled and regulated through a series of growth factors, signaling molecules, and

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transcription factors. Transplantation of hematopoietic cells including HSCs is a therapy for patients with leukemia or lymphoma. It is critical that the HSCs migrate to niches for successful engraftment and repopulation (Durand and Zon, 2010). Through a chemical screen of biologically active compounds on zebrafish embryos it was found that chemicals that increase prostaglandin (PG) E2 synthesis increased HSCs. A derivative of PGE2, 16,16-dimethylPGE2 (dm PGE2), shows an increase in runx1þ/cymbþ AGM HSCs in zebrafish (North et al., 2007). PGE2 in zebrafish is regulated by Cox1 and Cox2. Morpholino knockdown of Cox1 and 2 decreases the levels of prostaglandins leading to a decrease in AGM HSCs. Co-injection of the morpholino and dmPGE2 lead to a rescue of HSCs. These results confirm that PGE2 signaling regulates HSCs in the AGM. A kidney marrow irradiation recovery assay was performed in fish and showed that marrow repopulation was enhanced after exposure to dmPGE2. Upregulation of other stem, progenitor, and endothelial markers occurred after this treatment and a decrease of kidney marrow recovery was shown in the presence of a cox inhibitor. PGE2 also is important with controlling kidney marrow homeostasis. A limiting dilution competitive repopulation assay was performed to see the effects of dmPGE2 on HSC reconstitution. Whole bone marrow was treated with dmPGE2 and transplanted into mice. The increase in short-term repopulating HSCs was fourfold higher at 12 weeks post-transplantation and long-term repopulating cells were 2.3-fold enhanced at 24 weeks post-transplantation. The addition of dmPGE2 to the WBM leads to a significant increase in engraftment and repopulation (North et al., 2007). Clinical trials are under way to test PGE2 as a therapy to enhance the engraftment and success of core blood transplantations. Patients receive two cord blood transfusions, one is treated with dmPGE2 and the other serves as a control. The relative blood cell engraftment overtime is monitored to see if dmPGE2 has a positive effect on the transplant (North et al., 2007). This story is an excellent example of how ideas can be developed through the fish system, fine-tuned in the mouse and then emerge as potential therapeutic uses in human patients.

Cell Sorting and Transplantation New techniques have been carried out in zebrafish allowing differentiated blood cell populations to be sorted using flow cytometry (Thisse et al., 2003). Samples have been collected from the kidney, spleen, and blood and analyzed by their light scatter characteristics. Importantly, kidney tissue in the zebrafish, the site of adult definitive hematopoiesis, reveals distinct scatter populations when examined using this method.

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Scatter populations of kidney tissue correspond to the major blood lineages and can be used to isolate pure populations of mature erythroid cells, myelomonocytic cells, lymphoid cells, and immature precursors (Traver and Zon, 2002). The percentage of cells in each category obtained by morphologic cell counts in normal adults corresponds to the percentage of each cell type found using flow cytometry. Thus, the use of scatter profiles to evaluate and quantitate discrete populations of differentiated cells is an accurate and important tool in both adult wild-type and mutant zebrafish. Cell sorting may be used to characterize the blood mutants obtained through genetic screens in the fish (Traver and Zon, 2002). The light scatter populations of mutant fish may be compared to those of wild-type fish, enabling the quantification of population deficits or increases in specific blood lineages in the mutant fish. Many primitive blood mutants exhibit a homozygous lethal phenotype, making the analysis of adult homozygous mutants impossible. The analysis of blood lineages of adult heterozygous fish carrying only one copy of the mutant allele, however, can be revealed using this technique. This technique has exposed several cases of haploinsufficiency, suggesting that many genes required for embryonic and primitive hematopoiesis are additionally important in the adult fish. The technique of flow cytometry also enables the collection and purification of green fluorescent protein (GFP)-tagged cells in zebrafish transgenic lines (Traver and Zon, 2002). The creation of transgenic lines has become common in zebrafish studies as it allows visualization of specific tissues in the transparent embryo (Figure 38.2) (Traver et al., 2003b). GFP lines have been created with the GATA-1 promoter to produce fluorescent erythroid cells and the lmo2 promoter to create zebrafish with fluorescent green blood and vasculature (Brownlie et al., 1998). GFPtagged cells in these transgenic fish can be isolated from the rest of the population of cells in the fish. Once isolated, purified blood lineages may be collected and transplanted into recipient fish (Bai et al., 2010). Transplantation assays may be used to measure many aspects of hematopoiesis in mutant and non-mutant fish alike. In one assay, HSC activity in mutants is characterized by injecting kidney cells harvested from lines of fish in which the GATA-1 promoter drives GFP. The donor transgenic HSCs will produce GFPþ erythrocytes, which, because of their short half-lives, are continuously made and replenished from upstream stem and progenitor cells. Many transplant recipients possess GFPþ blood cells up to 6 months after transplantation, indicating the existence of a long-term repopulating stem cell in the zebrafish kidney. A major advancement in zebrafish transplantation and the study of HSCs is the generation of histocompatible

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zebrafish lines. In human bone marrow transplantation higher rates of donor cell engraftment and recipient survival are seen with matching donor and recipient’s major histocompatibility complex (MHC). Utilizing previously defined haplotypes, the sequences from six MHC class I U genes and five additional U genes were sequenced. Primers were developed from these sequences to categorize siblings from a cross of Tg(b-actin) and AB. Based on these genotyping results, whole kidney marrow (WKM) was taken from GFPþ donors and transplanted into irradiated GFP recipients who were either matched or unmatched to the donors. The levels of donor chimerism and engraftment were measured 14e16 weeks later, 87% of matched recipients showed in engraftment and there was a significantly higher rate of donor chimerism in the matched recipients (de Jong et al., 2011). The success of immune-matched transplantation will expand the use of the zebrafish in the study of diseases. While the optical clarity of the zebrafish embryos is useful for imaging and analysis, this advantage is lost as the animal matures and pigmented melanocytes and reflective iridophores develop. A line of zebrafish called Casper has been developed, which remains transparent throughout adulthood. Organs such as the heart, liver, gallbladder, and individual eggs in the female can be observed using a stereomicroscope. This model can be utilized to track GFP-labeled transplanted HSCs.

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understanding the behavior of HSCs. Tracing in vivo can be done using caged rhodamine-dextran, which can be activated on a single cell level and then their migration can be traced. Using fluorescently labeled cells from whole kidney marrow, adults and embryos can be transplanted to see effects of signaling pathways or genes on the honing and migration of stem cells (Li and Zon, 2011). In addition to these assays developed with the use of flow cytometry, purified blood lineages are being used to create novel reagents for zebrafish studies (Traver and Zon, 2002; Traver et al., 2003a). cDNA libraries have been generated from purified myeloid, lymphoid, and precursor cell populations. Thousands of clones from these libraries are being sequenced to identify zebrafish orthologs of mammalian genes as well as novel genes. The use of transgenic animals also allows the generation of cDNA libraries from specific lineages as described previously. From these libraries, in situ probes can be formed to screen for expression patterns and locations (Galloway and Zon, 2003; Thisse et al., 2003).

Disease Models in Zebrafish FIGURE 38.3 GATA-1 promoter driving GFP in transgenic zebrafish embryo at 24 hpf. (A) The transgenic embryo as seen under a dissecting scope. (B) Under fluorescence, the GATA-1 driving GFP expression localizes to the ICM. (Please see CD-ROM for color version of this figure.) (GATA-1 GFP line courtesy of S. Lin; photo courtesy of R. Wingert.)

Distribution and engraftment in the kidney tissues can be observed without sacrificing the animal by using fluorescent microscopy. Through forward and side scatter and measurements of the GFP of kidney marrow in both wildtype and Casper recipients reveals similar patterns of reconstitution and florescence. When Caspers are injected with tumor cells the metastatic progression and engraftment can be monitored. Tracking of movement and development of early tumors would be impossible to image in the adult fish, but can be done with the Casper. The appearance of dissemination from the tumor at the injection site can be seen as early as 5e10 days in Casper whereas metastasis in wild-type fish is not detectable. The Casper fish is an innovation in zebrafish transplantation which will provide advances to those studying the engraftment and honing of stem cells, metastasis, and analysis of tumor growth which can now be conducted in vivo without sacrificing the animal (White et al., 2009). Through utilizing the Casper and fluorescent lines of transgenic fish recent protocols have been developed for in vivo imaging of HSC transplantation, and stem cell fate tracing and migration (Li and Zon, 2011). Through well-defined markers and a similarity to mammalian hematopoiesis zebrafish hematopoiesis is a crucial tool in

Zebrafish mutants have been shown to resemble human diseases including hematopoietic, cardiovascular, and kidney disorders. Although other organisms such as Caenorhabditis elegans and Drosophila have been extensively studied with respect to developmental processes and may be used in screens, they do not address many of the morphologic aspects unique to vertebrates. The zebrafish, however, is a vertebrate system and thus may be used to address developmental processes in the kidney, in multilineage hematopoiesis, in notochord, as well as in neural crest cells, among others (Griffin et al., 1998). Zebrafish mutants have played an important role not only in clarifying vertebrate-specific developmental processes but also as models for specific human disorders. The hematopoietic mutant known as sauternes (sau) exhibits delayed maturation of erythroid cells and abnormal globin expression (Brownlie et al., 1998). The sau mutant fish develops a microcytic hypochromic anemia. Cloning of the sau gene revealed the gene product to be aminolevulinate synthase (ALAS2), an enzyme required for the first step of heme biosynthesis. Mirroring the fish genetic disease, humans with a mutation in ALAS2 gene have been found to have congenital sideroblastic anemias (Edgar and Wickramasinghe, 1998). Sau is the first animal model of this disease. Another hematopoietic mutant acting as a model of a human disease is yquem (yqe) (Dooley and Zon, 2000; Wang et al., 1998). The mutant yqetp61 is homozygous for a mutation in the gene encoding uroporphyrinogen decarboxylase (UROD), an enzyme involved in the heme biosynthetic pathway (Wang et al., 1998). Humans with

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homozygous deficiencies of UROD develop hepatoerythropoietic porphyria. The yqetp61 zebrafish develop a photosensitive porphyria syndrome. The zebrafish mutant known as retsina (ret) has been shown to exhibit an erythroid-specific mitotic defect with dyserythropoiesis comparable to human congenital dyserythropoietic anemia (Paw et al., 2003). This work identifies a gene (slc4a1) encoding an erythroid-specific cytoskeletal protein necessary for correct mitotic divisions of erythroid cells. In addition to modeling a human disease, this study demonstrates the notion of cell-specific mitotic adaptation. Positional cloning of the zebrafish mutant wiessherbst identified the gene ferroportin1, a transmembrane protein responsible for the transport of iron from maternal yolk stores into circulation, solving the puzzle of the elusive iron exporter (Kimmel et al., 1989). This gene was first identified in zebrafish. Later research determined that humans with mutations in ferroportin1 develop the human disease type IV hemochromatosis (Njajou et al., 2001). Further studies are being conducted in zebrafish to tie mutant phenotypes to known as well as undocumented human disorders. The use of the zebrafish system to gain understanding of human disease states has been proved and evolves as technologies advance. Screens continue to uncover mutant phenotypes with specific developmental abnormalities, many of which will play roles in deciphering human disease states.

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transgenic lines expressing GFP have been used as a visual tool and stand to aid the purification of specific cell populations involved in regeneration events (Poss et al., 2003). In addition to the fin regeneration studies, heart regeneration has been examined in zebrafish (Poss et al., 2002). The processes of dedifferentiation, patterning, and proliferation, as well as the existence of pluripotent cells in adult tissues, are being examined in zebrafish and remain extremely compelling for the field of stem cell biology. Although at a very early stage, regeneration studies may also address human tissue repair, stem cell transplantation, and even tissue engineering (Slack, 2003). The applications for the utilization of the zebrafish system are rapidly expanding with new technology and innovation. Through chemical screens, the specific effects of new drugs are being discovered. Chromatin factors have also been studied for their effect on hematopoiesis. The use of Chip-seq has helped us to explore the interactions of proteins to DNA and leads to the discovery of similarities across genomes. New technology in transgenic lines, imaging, and bioinformatics has been used in investigations of hematopoiesis and stem cell biology. Further dissection of developmental processes and their connections to human disease will surely benefit from the utility of this model organism.

ACKNOWLEDGMENTS Future of the System In one study, transgenic zebrafish lines were created that resulted in acute T-cell leukemia in the fish (Langenau et al., 2003). In this investigation, transgenic fish were created with the lymphoid-specific RAG2 promoter driving mouse c-myc, a gene known to function in the pathogenesis of leukemias and lymphomas. Additionally, a chimeric transgene consisting of myc fused to GFP allowed the visualization of the spread of tumors from the thymus of affected fish. Tumors in these transgenic fish spread from origins in the thymus to skeletal muscle and abdominal organs. The leukemic transgenic fish from this study may be used for suppressoreenhancer genetic screens to identify components either lessening or worsening the leukemic progress. Another body of work focused on zebrafish regeneration (Poss et al., 2003). The zebrafish is able to regenerate fins, a unique characteristic of some lower vertebrates. Little is known about the molecular and cellular processes involved in regeneration, and genetic screens have recently been employed to investigate the molecular mechanism. Because many of the genes involved in regeneration are necessary for embryonic development, temperature-sensitive mutants have been screened for and successfully identified (Johnson and Weston, 1995). In addition,

We thank Caroline Burns for providing Figure 38.1 and Rebecca Wingert and Shou Lin for providing Figure 38.2. We thank Xiaoying Bai, Michelle Lin, and Elizabeth Paik for critical review of this manuscript and beneficial discussions. (E.N. Price is supported by grants from the National Institutes of Health. L.I. Zon is an Investigator of the Howard Hughes Medical Institute.)

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