Barry H. Paw

Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants

The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1−/− mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.

Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia.

Many human anaemias are caused by defects in haemoglobin synthesis. The zebrafish mutant sauternes (sau) has a microcytic, hypochromic anaemia, suggesting that haemoglobin production is perturbed. During embryogenesis, sau mutants have delayed erythroid maturation and abnormal globin gene expression. Using positional cloning techniques, we show that sau encodes the erythroid-specific isoform of δ-aminolevulinate synthase (ALAS2; also known as ALAS-E), the enzyme required for the first step in haem biosynthesis. As mutations in ALAS2 cause congenital sideroblastic anaemia (CSA) in humans, sau represents the first animal model of this disease.

Zebrafish stat3 is expressed in restricted tissues during embryogenesis and stat1 rescues cytokine signaling in a STAT1-deficient human cell line.

Transcription factors of the STAT family are required for cellular responses to multiple signaling molecules. After ligand binding‐induced activation of cognate receptors, STAT proteins are phosphorylated, hetero‐ or homodimerize, and translocate to the nucleus. Subsequent STAT binding to specific DNA elements in the promoters of signal‐responsive genes alters the transcriptional activity of these loci. STAT function has been implicated in the transduction of signals for growth, reproduction, viral defense, and immune regulation. We have isolated and characterized two STAT homologs from the zebrafish Danio rerio. The stat3 gene is expressed in a tissue‐restricted manner during embryogenesis, and larval development with highest levels of transcript are detected in the anterior hypoblast, eyes, cranial sensory ganglia, gut, pharyngeal arches, cranial motor nuclei, and lateral line system. In contrast, the stat1 gene is not expressed during early development. The stat3 gene maps to a chromosomal position syntenic with the mouse and human STAT3 homologs, whereas the stat1 gene does not. Despite a higher rate of evolutionary change in stat1 relative to stat3, the stat1 protein rescues interferon‐signaling functions in a STAT1‐deficient human cell line, indicating that cytokine‐signaling mechanisms are likely to be conserved between fish and tetrapods. Dev Dyn 1999;215:352–370.

Zebrafish kidney stromal cell lines support multilineage hematopoiesis

Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish.

PRIMARY FIBROBLAST CELL CULTURE

Publisher Summary This chapter describes a way to culture stable primary fibroblast cell lines from adult zebrafish caudal fins in a standard tissue culture medium with bovine serum. These primary fibroblasts retain many features of nontransformed cells such as eudiploidy, contact inhibition, and surface adhesion. One of the stable fibroblast lines, AB9, derived from an AB strain, exhibits contact inhibition and surface-growth dependency. A karyotype analysis of these cells showed eudiploid chromosome count (50 chromosomes/diploid cell). The cells have been maintained in continuous culture for over 35 passages without a change in characteristics. A continuous source of stable cultured fish cells offers a variety of applications. In addition, high-quality zebrafish genomic DNA can be easily obtained from these cells. These primary fibroblasts retain the same genetic polymorphism as the original donor fish when a variety of zebrafish genes are analyzed. Therefore, cultured cells derived from mutant zebrafish could prove useful in positional cloning and the identification of mutant genes in much the same way that cultured fibroblasts are in human genetic disease research. In vitro applications of these cultured fibroblasts have been discussed in the chapter.