A recent study in zebrafish presents a potentially important new system for studying the process of stem cell biology and hematopoiesis, the process by which blood is produced.

The zebrafish has proved to be a valuable model for studying hematopoiesis. This species is ideal for large-scale mutagenesis studies, because considerable numbers of zebrafish can be raised and maintained simultaneously. Furthermore, the mature blood cells produced by the adult zebrafish closely resemble their mammalian counterparts, and arise from a process very similar to the hematopoietic pathway seen in mammals. Finally, zebrafish embryos are transparent, allowing for easy observation of blood formation.

Researchers have previously identified dozens of hematopoietic mutants in the zebrafish, although many of these mutations produce similar phenotypes or result in embryonic lethality for homozygotes, and thus remain poorly characterized. In an effort to establish better methods for studying such zebrafish mutants, a group of researchers led by Leonard Zon from the Children's Hospital Boston (Boston, MA) developed a technique for transplantation of hematopoietic stem cells derived from whole-kidney marrow (WKM), which is the zebrafish equivalent of mammalian bone marrow and the source of all blood cell production.

Zon's group began by developing a system for characterizing the relative proportions of different cell types in wild-type WKM, using flow cytometry to sort blood cells from a variety of fish lines expressing green fluorescent protein (GFP) from different blood cell-specific promoters; they then compared these findings to WKM derived from different anemic mutant fish lines (Nat. Immunol., December 2003). Their findings indicated that several of these mutations were lethal to embryos as a result of defects that blocked the maturation of precursor cells for certain blood cell lineages, a phenotype that was mildly apparent even in seemingly normal heterozygotes.

Zon and his colleagues developed a transgenic line expressing GFP from an erythroid-specific promoter, and transplanted WKM thus labeled into moonshine (mon/mon) and vlad tepes (gata1−/−) mutants, lines that normally die by 14 days after fertilization. In both lines, there was rescue of normal blood production, and the transparency of the embryos allowed the researchers to visualize easily the migration and growth of the labeled cells.

The researchers also generated double-transgenic zebrafish, which produced GFP-tagged leukocytes and erythroid cells labeled with dsRED; after transplantation of WKM from this transgenic line, the researchers could easily track the migration of the two cell types. They found that the dsRED+ cells remained largely in circulation, while the GFP+ cells achieved two different fates, forming both lymphocytes and myelomonocytic cells, both of which could be easily identified by their visual characteristics.

According to Zon, this work marks two important achievements: the ability to sort blood cell types easily by flow cytometry, and the technique for marrow transplantation. He tells Lab Animal, “This work establishes the basis for a genetic approach for studying marrow homing, engraftment, proliferation, and differentiation.” Given the advantages of zebrafish embryos as a mutant model, “it is [now] possible to study individual genes and their role in short-term and long-term reconstitution.” He indicates that also on the horizon are additional transplantation and gene expression profiling experiments, which they anticipate will yield yet more insights into the genetics of stem cell biology and hematopoiesis.