Stem Cells, Vol. 16, No. 2, 89-98,
March 1998
© 1998 AlphaMed Press
Use of the Zebrafish (Danio rerio) to Define Hematopoiesis
Nathan Baharya,b,
Leonard I. Zona
a Children's Hospital, Department of Hematology/Oncology, Howard Hughes Medical Institute, Boston, Massachusetts, USA;
b Dana-Farber Cancer Institute, Department of Medicine, Boston, Massachusetts, USA
Key Words. Zebrafish • Hematopoiesis • Genetic mapping • Genetic screens • Vasculogenesis • Positional cloning
Dr. Leonard I. Zon, Dana-Farber Cancer Institute, Department of Medicine, 44 Binney Street, Boston, MA 02115, USA.
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Abstract
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Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.
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Hematopoiesis
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Mammalian Hematopoiesis
Vertebrate hematopoietic cells are derived from self-renewing multipotential stem cells that are specified early during embryonic development [1]. During early vertebrate development, ventral mesoderm is induced to form tissues of the hematopoietic and vascular systems. In mammals, the primitive or first wave of hematopoiesis originates from cells that have invaginated through the primitive streak during gastrulation and migrated to the yolk sac. These yolk sac blood islands consist of an endodermal layer which supports growth, a central core of primitive erythroid-lineage cells surrounded by vasculogenic cells, and an endothelial layer that surrounds the developing blood island. The embryonic erythroid cells have a distinct morphology and generate only embryonic globins. The finding of blood and vascular cells in the blood islands suggests that a common progenitor, the "hemangioblast," produces both cell types. In addition to ventral (yolk sac) hematopoiesis, most vertebrates have an additional definitive intra-embryonic stem cell population in the dorsal mesentery (AGM—aorta, gonad, mesonephros region) that colonizes the fetal (larval) sites of hematopoiesis. Later, there is a switch to fetal hematopoiesis in the liver, and finally, adult hematopoiesis in the bone marrow.
Factors Involved in Both Primitive and Definitive Mammalian Hematopoiesis
By a variety of experimental methods, several transcription factors, including GATA-1, GATA-2, lmo2, and scl have been implicated in both the primitive and definitive hematopoietic programs. GATA-1 in mammals is expressed primarily in erythrocytes and megakaryocytes and is essential for erythroid development based on mouse knockout experiments [2, 3]. A hematopoietic progenitor defect is seen in mice with a targeted disruption in the GATA-2 gene [4]. Mutant embryos with disruptions of either scl [5] or of the LIM-domain protein lmo2 (previously known as Rbtn2 or ttg-2) [6] which directly interacts with scl [7, 8] do not produce hematopoietic progenitors. Homozygous TGF-ß (Tgfb) [9] demonstrates a semidominant and TGF-ß receptor (Tgfbr2) [10] knockout mice demonstrate a recessive defect in yolk sac hematopoiesis and vasculogenesis, resulting in lethality at about 10.5 days gestation. Embryos homozygous for a knockout of the flk1 gene die in utero between 8.5 and 9.5 days of development as a result of an early defect in the development of hematopoietic and endothelial cells [11].
Factors Involved in Definitive Mammalian Hematopoiesis
Several genes required for definitive hematopoiesis have been described. C-myb is detected in fetal liver, a site of definitive hematopoiesis [12]. In mice homozygous for a c-myb mutation, primitive erythropoiesis is apparently normal, but the generation of all adult blood lineages is defective due to a decrease in the proliferation and number of multipotent progenitor cells [13]. Mutations in the transcription core binding factor cbfa2 (AML1) [14], as well as the cbfb [15], [16], eklf [17, 18], and pu.1 [19] transcription factors, the tyrosine kinase receptor W (c-kit) [20] and the c-kit ligand (Steel) also effect definitive, but not primitive, hematopoiesis.
Zebrafish Hematopoiesis
Teleosts such as the zebrafish lack yolk sac blood islands; instead, embryonic hematopoiesis occurs in a relatively dorsal location above the yolk tube. This region is called "the intermediate cell mass" (ICM). The ICM is formed in two paraxial stripes of mesoderm that arise during gastrulation [21-23]. This site is similar to the extraembryonic location of the earliest hematopoietic progenitors on the yolk sac of higher vertebrates. To define the normal sites of hematopoiesis and to examine gene expression within developing blood cells, Thompson and Ransom et al. (submitted), undertook the description of normal embryonic blood development in the ICM using in situ probes for multiple transcription factors and globin. They found lmo2 and GATA-2 in two stripes of presumptive blood progenitors in the ventral mesoderm at the 5-somite stage. Fli1 is expressed in hematopoietic and vascular tissues, and is also expressed early in a similar location. C-myb and GATA-1 are detected after the early markers as the two stripes of progenitors converge to form the ICM. Prior to circulation, ICM cells begin to express large amounts of globin RNA. The early markers lmo2, GATA-2, and fli1 are expressed in the posterior region of the ICM and in several regions of the anterior trunk. In contrast, GATA-1, c-myb, and globin are not detected in these regions. At 48 h of development, c-myb is detected in a line of cells in the ventral wall of the dorsal aorta. These cells are likely to be definitive hematopoietic stem cells in keeping with observations of c-myb expressing hematopoietic cells located in the dorsal aorta in higher vertebrates [12].
Whole-embryo in situ hybridization with scl demonstrates that scl is expressed in developing neural tissues, blood islands, and circulating blood (Liao and Zon, submitted). In the peripheral blood, two distinct waves of scl expression in the ICM are seen. The first occurs before hour 8 and subsides by hour 26. The second wave, which begins at approximately hour 30 and decreases by hour 36, may represent the onset of definitive hematopoiesis. The transcription factors GATA-2, fli1, lmo2, and scl are therefore the earliest defined markers for the vertebrate hematopoietic development program to date.
Modeling Developmental Pathways in Vertebrates
The underlying mechanisms of vertebrate embryology have been difficult to elucidate because of the complexity of genetic studies in these organisms. Although there is a greater than 100-year history in utilizing mice for the analysis of genetic mutations, early developmental pathways such as hematopoiesis have proven inaccessible to mouse genetics because of the intrauterine growth of mouse embryos and their small litter size. What insights have been made utilized the cloning of genes because of their unique temporal-spatial expression or sequence similarity to known regulatory elements, or were based on the translocation of a gene in tumors. Cloning of these translocation breakpoints has led to the discovery of the bcr-abl fusion protein that causes CML [24] and the cloning of scl [7] and lmo2 [25] on the basis of their involvement in T-cell leukemia. The ability to "knock out" a gene in the mouse had also made it possible to study the function of any gene of interest.
One of the most important advancements facilitating these developmental studies was the realization by Nüsslein-Volhard and Wieschaus in the late 1970s that developmentally important genes with broad-ranging vertebrate counterparts could be discovered by saturation mutagenesis in Drosophila melanogaster [26]. Mutagenesis screens do not presuppose an understanding of the temporal-spatial expression, underlying sequence motifs, or functions of these regulatory factors. Cloning of the regulatory elements of any developmental pathway is most efficient by description of their mutant phenotypes during development.
Zebrafish as a Vertebrate Model of Hematopoiesis
Our understanding of hematopoiesis has largely been obtained through the biochemistry and cell biology of hematopoietic cells. The zebrafish, or Danio rerio, is an ideal genetic system for hematopoietic developmental studies. Zebrafish are easy to raise with a short generation time of three months, and the developing embryos are easily studied under a dissecting microscope since they are transparent. Embryos develop rapidly, with a beating heart and visible erythrocytes by 24 h. The females can lay hundreds of eggs at weekly intervals. The organism maintains the diploid state, an important difference from other fish that can be triploid or tetraploid, making genetic analysis difficult.
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Mutagenesis
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Induction of Mutations in the Zebrafish
There are four commonly employed methods for introducing mutations in zebrafish sperm which are then used to fertilize eggs in vitro. N-ethyl-N-nitrosourea (ENU) induces point mutations, gamma irradiation (which in turn induces deletions and/or gene rearrangements), insertional mutagenesis, and UV irradiation.
ENU Mutagenesis
Mutagenesis of male zebrafish with ENU consistently induces point mutations. Mutations in premeiotic stem cells are not transmitted by mature sperm until at least two weeks after treatment, allowing the selective mutation of either pre- or postmeiotic gametes. Prespermatogonial (premeiotic) mutagenesis with ENU has been investigated extensively and yields a mutagenesis rate of ~0.1-0.2% [27-28], although the rates of mutagenesis vary many fold at individual loci. Much higher rates approaching 2% are seen in postmeiotic mutagenesis [29], that is, outcrossing of the mutagenized males within three weeks of ENU treatment.
Gamma Mutagenesis
Gamma irradiation typically induces mutation rates approaching 1%. The types of mutations that can be seen range from point mutations to large deletions, inversions, and translocations. A number of studies have analyzed radiation-induced mutations in the hprt locus and have found that >50% are large deletions. This can confound the analysis of a mutation because more than one gene may contribute to the observed phenotype. Deletion of essential genes linked to the mutated gene of interest can also make recovery and propagation of a gamma-induced mutation difficult. Several studies have documented potentially interesting zebrafish developmental mutants induced by irradiation [30, 31] but no systematic gamma irradiation screen to identify hematopoietic mutants has been carried out to date.
Insertional Mutagenesis
P-element insertional mutagenesis in Drosophila melanogaster has greatly facilitated the cloning of genes disrupted by the insertional tag. Insertional mutagenesis utilizing proviral vectors is a promising new technique for generating developmental mutations in the zebrafish [32, 33]. Approximately 1 in 70 proviral insertions creates a developmental mutant that can be rapidly cloned using the proviral insertion as a genetic marker. Allende et al. [34] utilized this technique to clone three mutants with a variety of developmental abnormalities. The overall rate of mutagenesis is, however, approximately 10-fold lower than with ENU, although higher efficiency vectors hold out the promise of being able to carry out a saturation insertional screen in the zebrafish. The recent isolation of a transposable element related to a SINE element in the zebrafish holds the promise of a mutagenic approach analogous to P-elements in Drosophila if further characterization of the element proves it feasible [35].
These varied and complementary approaches to mutagenesis in the zebrafish demonstrate its utility in searching for mutations to uncover the steps of a complex mechanism such as hematopoiesis. It is also possible to generate mutations in a specific gene by gamma mutagenesis ( Fig. 1) [29]. Gamma-mutagenized sperm is used to fertilize wild-type eggs. Female offspring are anesthetized and their eggs squeezed and fertilized with UV-irradiated sperm. The sperm does not make a genetic contribution because of inactivation of the DNA by UV light to the now haploid offspring. These haploid offspring, which can live up to six days, are then rapidly screened by polymerase chain reaction (PCR) for deletions in the gene of interest. Although only a small fraction of the recovered mutations may be small enough to disrupt a single gene, larger deletions represent true null phenotypes. Point mutants of the gene of interest can then be recovered by a subsequent noncomplementation assay with ENU-treated fish.
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Figure 1. Generation of zebrafish harboring a specific mutation by PCR analysis of gamma-generated deletions in haploids. Female zebrafish are anesthetized and their eggs squeezed. Sperm which have been gamma-irradiated are then used to fertilize the eggs in vitro. F1 females are then squeezed for eggs which are fertilized in vitro with UV-inactivated sperm, blocking any DNA contribution by the sperm. The fertilized eggs undergo meiosis II like normally fertilized eggs, but because of the lack of paternal DNA contribution, a haploid offspring results. The resultant haploids can live up to six days. Deletions in a particular gene are then found by PCR analysis using gene-specific primers. If a clutch of embryos is found to contain fish harboring a deletion of the gene of interest, the F1 female is outcrossed to recover it. Point mutants of the gene of interest can then be recovered by a subsequent noncomplementation assay with ENU-treated fish.
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Introduction of transgenes into zebrafish is also possible with relatively high efficiency; approximately 5% of embryos injected at the one-cell stage will carry transgenes in their germlines. Lin et al. have created a stable line of transgenic fish with fluorescent blood by the introduction of the green fluorescent protein gene under the control of the GATA-1 promotor [36].
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Zebrafish Genetics
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Genetic Screens for Zebrafish Hematopoietic Mutants
Two spontaneous zebrafish mutants, cloche (clom39) and spadetail, have recently been investigated because of their defects in hematopoietic development. Homozygous clo embryos lack endocardial cells, head and trunk endothelial cells, and blood, but retain some tail endothelial cells [37, 38] ( Fig. 2). GATA-1 and GATA-2 are not expressed in the ICM of clo mutant embryos [37]. In cloche homozygotes, expression of flk1 is delayed compared to wild-type embryos and is expressed only in the lower trunk and tail. Furthermore, these flk1-positive cells do not express another endothelial marker, tie-1, suggesting an early block in endothelial differentiation in clo mutants [39].
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Figure 2. Photomicrograph of sibling 72-h wild-type (top) and cloche (clo) (bottom) embryos. In the top panel, the arrow points to the wild-type embryo's cardiac atrium, which is filled with red blood cells. In contrast, the cloche embryo demonstrates an enlarged atrium that lacks a normal cardiac endothelium and no visible red blood cells. The slightly smaller eyes and head of the 72-h-old cloche mutant compared to its wild-type sibling is indicative of a mild developmental delay that affects the cloche mutants.
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In order to better define the block in hematopoietic development in cloche (clom39) and spadetail embryos, Thompson and Ransom et al. (submitted) studied the expression of a number of markers of hematopoiesis and vascular development in cloche and spadetail. In clo, they found that the expression of ttg2, fli1, c-myb, flk1, flt4, GATA-1, and GATA-2 was almost entirely eliminated in the ICM region, indicating that the clo mutation disrupts both primitive and definitive hematopoiesis. The lack of expression of the vascular markers fli1, flk1, and flt4, defects in cardiac and vascular endothelial cells, and defective hematopoiesis support the idea that the defect in cloche may lie in a hemangioblast, a hypothesized common vascular and hematopoietic precursor. Recent studies (Trede et al., personal communication) have demonstrated a lack of expression of the lymphoid specific markers rag-1 and ikaros in homozygous cloche embryos as well. Because of the combination of erythroid, lymphoid, and vascular arrest seen in cloche, it appears that cloche represents the earliest mutation characterized to date in the hematopoietic program. Further studies in cloche will certainly provide further insights into early hematopoiesis and vasculogenesis.
The expression patterns in spt embryos is different from those of the clo embryos. Few or no GATA-1 or c-myb-positive blood cells are seen in spt embryos. In contrast to clo embryos, spt embryos do show extensive but disorganized ttg2, fli1, flk1, and flt4 staining throughout their bodies. Because fli1, flk1, and flt4 expression in spt embryos is seen, the early phases of vasculogenesis likely occur in spt embryos. As ttg2 is expressed in spt embryos, ttg2 may have a role in the genesis of vasculogenic cells. Thus, the spt mutation appears to affect the development of both primitive and definitive hematopoietic cells, while it does not affect the expression of vascular genes. The cells affected by spt may represent a subset of cells affected by the clo mutation.
Although both cloche and spadetail underlie important early stem cell defects, further mutations are needed in order to better understand the many steps involved in hematopoietic stem cell differentiation.
As part of a large scale chemical mutagenesis screen of the zebrafish genome achieved in the laboratory of Dr. Nüsslein-Volhard, 39 mutants with defects in hematopoiesis were found, comprising a total of 26 complementation groups (17 groups are described in [40], and our lab has subsequently found another nine). Investigations of these mutations in our laboratory have demonstrated that they represent a variety of defects that affect the cascade of steps for hematopoiesis. Further chemical and radiation mutational screens are under way in many labs, and additional hematopoietic developmental mutants are continually being found.
Since developing blood cells have a standard size, morphology, rate of development, and hemoglobinization, these factors were used in classifying the mutants isolated from the Nüsslein-Volhard ENU screen. Four broad classifications of mutants were found: bloodless mutants (a category which includes the cloche and spadetail mutants), mutants with decreasing blood counts, hypochromic mutants, and photosensitive mutants.
One new bloodless mutant, moonshine (mon), has been described from this screen. There are eight recessive mon alleles, seven of which are embryonic lethal. At the start of circulation, there are several dozen blood cells, but never more than approximately 100 cells at four days of age. Additional phenotypic characteristics include jagged fin edges and increased iridophores. The relative strength of the different mon alleles correlates with the amount of GATA-1 expression as detected by whole-mount in situ hybridization. Unlike cloche and spadetail homozygotes, moonshine homozygotes demonstrate no detectable difference in GATA-2 expression. These data suggest that the moonshine gene product facilitates development of GATA-1-expressing hematopoietic precursors.
Nine distinct complementation groups, chablis (cha, 2 alleles), frascati (frs), retsina (ret, 2 alleles), thunderbird (tbr, 1 allele), merlot (mot, 2 alleles), riesling (ris, 1 allele), grenache (gre, 1 allele), pinotage (pnt, 1 allele), and cabernet (cab, 1 allele) were described in which mutants initially have normal numbers of erythroid cells but then have a rapid deterioration in number beginning on days 2-4. These mutations share normal GATA-1 expression at 24 h. Blood collected from these mutants reveals cells blocked at various stages of early erythroid differentiation. It appears based on the decreasing number of cells and block in erythroid maturation demonstrated by these mutants that these mutations may affect erythroid cell proliferation and differentiation.
Initially, the embryos of five mutants, zinfandel (zin, 1 allele), chardonnay (cdy, 1 allele), weibherbst (weh, 2 alleles), sauternes (sau, 2 alleles), and chianti (cia, 1 allele) have normal numbers of erythroid cells, but at about two to three days of development show a decreased number of erythroid cells and decreased hemoglobin expression (hypochromic). One of these mutations, zinfandel, is dominant, while the rest are recessive. Studies on peripheral blood reveal cells blocked at the proerythroblast stage of differentiation and may underlie genes required for hematopoietic differentiation.
Mapping of Hematopoietic Zebrafish Mutants
Once a zebrafish with a hematopoietic defect is obtained, mapping of the underlying genetic defect and positional strategies can be employed to isolate the gene ( Fig. 3). Because of homology between the zebrafish and human genomes, initial mapping studies may yield a cloned human candidate for the defect of interest.
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Figure 3. Positional cloning strategies in the zebrafish. Females harboring the mutation of interest (usually on an AB background) are mated to wild-type males. Heterozygous F1 female carriers are then anesthetized, their eggs squeezed, and fertilized in vitro with UV-inactivated sperm. The resultant haploids are then scored for the mutant phenotyped and initially mapped to the genome using any one of a variety of methods. Once the genomic position is known, candidate genes can be ruled out on the basis of map location. Close flanking markers are utilized in conjunction with YAC/PAC/BAC libraries as the starting point for a chromosomal walk and cloning of the mutant gene.
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Several techniques are available for mapping and positional cloning in the zebrafish. The zebrafish map consists of three overlapping systems of markers: anchoring genes, sequence-specific length polymorphisms (SSLP, CA, or tandem repeat markers) and RAPDs (random amplified polymorphic DNA). SSLPs are arrays of tandem repeats (for instance, dinucleotide CA repeats) which vary in length between animal strains. SSLPs are amplified by the PCR using unique flanking oligos followed by either agarose or denaturing acrylamide electrophoresis to detect the strain-specific allele size differences [41, 42]. RAPDs are very short (usually 10-mers) oligos that can simultaneously amplify by PCR, multiple (4-12) loci [43]. Detection of the variably amplified products is accomplished by agarose electrophoresis. A map of the zebrafish genome consisting of 1,000 CA repeats has been created and has a projected saturation of 6,000 markers (<1 cM resolution) within the next several years. The current RAPD map consists of ~700 RAPD loci derived from 200 primers. These two maps are quickly being integrated into each other, and anchoring gene loci are being placed on the combined map. In addition, over 80 genes known to be important in mammalian hematopoiesis, some of which might be candidates based on the characterization of an hematopoietic mutant, have been cloned in zebrafish by our laboratory. These reagents make the zebrafish an extremely robust genetic system for the characterization and cloning of mutant phenotypes.
Generation of Haploid and Gynogenetic Diploid Zebrafish
Although the zebrafish is a diploid organism, haploids can live several days, and maternally homozygous diploid fish can be produced by applying early pressure (EP) to inhibit the second meiotic division after fertilization with UV-inactivated sperm ( Fig. 4). Analogous to the creation of the maternal diploids, diploid androgenotes can be obtained by UV inactivation of the egg, subsequent fertilization by normal sperm, and application of EP to inhibit the second meiotic division [44].
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Figure 4. Schema for generation of haploids or gynogenetic diploids in the zebrafish. For either embryo, a female, usually on the AB strain, carrying the mutation of interest, is mated to a wild-type male. Heterozygous F1 offspring females are then anesthetized and their eggs squeezed. By fertilization with UV inactivated sperm, no paternal genetic contribution is made. If the resultant embryos are not subjected to further treatment, haploids result as meiosis II results in a reduction of the DNA content from 2n to n. If the offspring are subjected to high pressures shortly after fertilization, meiosis II is inhibited and the result is a diploid offspring whose genetic contribution is solely from the female (gynogenetic diploid). In an analogous fashion, fertilization of UV inactivated eggs by unaltered sperm can result in the production of diploid androgenotes.
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The ability to create gynogenetic diploids allows the rapid assignment of a gene to a particular chromosome while obtaining information about its distance from the centromere ( Fig. 5). In order to genetically localize a mutation, female offspring of a mutant x AB (wild-type) outcross are squeezed and fertilized with sperm from a genetically unrelated (wild-type) male. F1 heterozygous mut/+ females are identified by random matings between the mut.AB F1 offspring. Obligate carriers should be detected at approximately a 25% rate. F1 mut/+ females are subsequently squeezed, and gynogenetically diploid embryos derived from them. Based on the equation [distance in cM = 50 (1 - (2 x mutant number/total number of embryos))], the mut-centromere distance is approximately calculated. Because the second meiotic division has been inhibited in creating gynogenetic diploids, the region of the chromosome between the centromere and the mutation cannot have recombined. Markers proximal to the mutant (so-called centromeric markers) when polymorphic between the background and wild-type strain will be necessarily homozygous background strain in mutants and wild-type in unaffected embryos. Centromeric markers have been defined for all 20 zebrafish chromosomes, and thus, chromosomal localization and distance from the centromere can be rapidly assigned.
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Figure 5. Generation of gynogenetic diploids by application of EP to developing embryos. Eggs from a female heterozygous for the desired mutation undergo meiosis I inside the adult fish. During meiosis I, (a) the chromosomal material is duplicated and (b) recombination occurs. The homologous chromosomal partners normally remain attached until after fertilization when they would separate during the reductional division of meiosis II.
The more distal a mutation lies on a chromosome (c), the more likely it becomes that a recombination event can take place between it and its centromere. (d) An anesthetized heterozygous female's eggs are squeezed, (e) fertilized by sperm whose DNA is rendered incapable of providing genetic material by UV irradiation. Although its DNA cannot contribute genetic material, sperm prepared in such a manner still permits the egg to undergo the fertilization process.
If nothing further were done to the embryos, then haploid embryos would result since meiosis II would reduce the 2n DNA content to n. With application of very high pressures (f) within 2 min 15 sec after fertilization, meiosis II fails to occur as the mitotic spindle complex is disassociated at these high pressures. The resultant viable embryos therefore inherit a normal 2n complement of DNA entirely from their mothers (gynogenetic diploids). The further distal a mutation is located, the more likely a recombination event will occur in the mutation—centromere interval and the fewer mutant embryos will result. The more proximal a mutation lies, the less likely it is to recombine with respect to its centromere, and the percentage of mutants will rise towards the theoretical 50% maximum. The centromeric-mutant distance is given by the equation [distance in cM = 50 (1 - (2 x mutant number/total number of embryos))]. Assignment to a particular chromosome can be rapidly accomplished by analyzing pools of such embryos' linkage to centromeric markers. Mutant embryos would be expected to demonstrate only the mutant allele centromeric markers, while wild-type embryos could demonstrate either mostly wild-type alleles (when the mutation is close to the centromere) or, if located more distally, a mixture of wild-type and mutant alleles.
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Positional Cloning in the Zebrafish
Once assigned to a particular chromosome, candidates that are on that chromosome can be considered and others excluded. Most mutations will require the generation of closely linked markers that can be used as a prelude to "walking" and the eventual cloning of the gene underlying a specific mutant. Both RAPDs and tandem repeat markers have been successfully utilized as the starting point for the physical mapping and walking stages in positional cloning strategies ( Fig. 3). In our laboratory, AFLP (amplified fragment length polymorphism) has been successfully used to quickly obtain close (<0.25 cM) flanking markers as the next step in a positional cloning approach to these mutants ( Fig. 6) [45, 46]. In AFLP, high-quality embryo DNA is digested with MseI and EcoRI and linkers ligated to the DNA. A preamplification step using the EcoRI and MseI linkers plus a single nucleotide is performed to reduce the complexity of the DNA fragments. Subsequently, a specific amplification is carried out using the same preamplification primers, one of which is radiolabeled, this time containing a two-nucleotide overhang. Separation of the fragments on a standard denaturing acrylamide gel demonstrates about 125 bands per primer pair, of which 25-30 segregate. By initial screening of pools of mutant and wild-type embryos and then further evaluation of individual members of those pools, closely linked markers can be isolated. Because of the density of segregating markers in AFLP, it is feasible to search >25,000 loci (<0.25 cM resolution) in a matter of weeks. Physical mapping and cloning of a mutant gene can then be accomplished in a relatively rapid fashion as high-quality PAC, BAC, and YAC libraries already exist.
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Figure 6. Generating tightly linked markers by AFLP. High-quality genomic DNA is digested with EcoRI and MseI. EcoRI and MseI adapters are then ligated to the digested DNA. To reduce the complexity of the digested DNA, the resultant mixture is amplified by the PCR, using the linkers containing a 1bp extension (preamplification). These products are then themselves amplified using the preamplification oligos, one of which is radiolabeled for ease of detection, containing a 2 bp extension. These reactions result in a mixture of approximately 125 bands, of which 25-30 segregate in a particular cross, that are easily separable on standard acrylamide gels. By employing two preamplification EcoRI and MseI primers, and 16 each specific EcoRI and MseI primers, a total of ~25 loci/pair specific primers X 256 (162) specific primers x 4 (22) preamplification primers or ~25,000 loci can be tested for linkage in pools of mutant and wild-type animals. Bands found in either mutant only or wild-type embryo pools can then be tested on individual embryos to prove linkage. Given the approximate 3,000 cM size of the zebrafish genome, loci within 0.25cM can thus be quickly isolated. The tightly linked loci are then excised from the acrylamide gel, subcloned into an appropriate vector, and sequenced to make marker-specific primers/probes.
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Summary and Future Directions
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Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the final peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the generation of hematopoietic mutants. In this regard, the zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing such mutational events. A large number of hematopoietic mutants in zebrafish have been generated and characterized to date. The strength of the zebrafish system is illustrated by the fact that these hematopoietic mutants represent many of the proposed steps of both the primitive and definitive hematopoietic programs. Early stem cell defects are seen in mutants such as cloche, spadetail, and moonshine, while other mutants appear to have defects in differentiation and proliferation of erythroid cells. The ENU screen accomplished by Dr. Nusslein and colleagues was not designed to specifically find early hematopoietic mutants. However, more refined in situ screens using early stem cell markers are under way, and preliminary data (Trede et al., personal communication) suggest that many new stem cell, lymphoid/myeloid/erythroid-specific mutants are being isolated. These new mutants can be quickly characterized and their underlying genetic defect described by positional techniques.
The zebrafish is also particularly suited for studying early hematopoiesis because of the wide variety of manipulations that can be accomplished with it. In situ studies can be utilized to help order and delineate the sequence of events that mark stem cell differentiation. Cell autonomy of a mutation can be tested. Our lab has demonstrated that the layering of collagenase-treated donor kidney cells onto the yolk sac of two-day-old zebrafish embryos can reconstitute defective hematopoiesis, a process analogous to bone marrow transplantation in humans(Guo and Zon, personal communication). Transplantation between mutant and wt embryos can be carried out. If wild type kidney cannot contribute to embryonic blood in a mutant host, then the mutation causes a non-cell autonomous defect that acts on the blood cells. The ability to generate null mutants of a desired gene allows the rapid assessment of a particular gene's contribution to hematopoietic stem cell development.
Injection of cDNAs and RNA into mutant vertebrate embryos has been used to rescue a variety of genetic mutations. Recently, injection of a zebrafish lambda phage clone containing a wild-type floating head gene was used to partially correct the development of the floating head mutant (William Talbot, personal communication). In this manner, injection of DNA into zebrafish embryos can be used to rescue a mutant, supporting the argument that the injected fragment contains the wild-type allele of the mutant gene. In other animal systems such as Drosophila and C. Elegans, such rescue assays are often employed to prove that a cloned gene underlies the mutation of interest. In the zebrafish, a wide variety of hematopoietic mutants now exists, and new ones are rapidly being described. These mutants encompass early hematopoietic development as well as later differentiation and proliferation of the stem cell compartment. Cloned genes already known to effect hematopoiesis can be injected into these mutant or wild-type embryos, and their effect on specific components of hematopoietic stem cell development explored.
Our ability to manipulate and/or target the stem cell compartment for therapeutic benefit will hinge upon our deeper understanding of the processes which regulate stem cell development. Since hematopoiesis is based upon the differentiation and expansion of stem cells, a better understanding of early hematopoiesis will provide crucial insights into issues of stem cell development needed to translate our knowledge into effective novel therapies.
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Acknowledgments
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We would like to thank Dorothy Giarla for her help in the preparation of this manuscript. This work was supported by a National Institutes of Health Centers in Excellence Grant P50 DK49216-03 and Dana-Farber Cancer Institute Institutional National Research Service Award 5T32CA09172-22 (NB). L. I. Zon is an Associate Investigator in the Howard Hughes Medical Institute. N. Bahary is a recipient of a Howard Hughes Medical Institute Physician Postdoctoral Fellowship.
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References
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accepted for publication December 12, 1997.
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Abstract
Hematopoiesis
