Mapping of complex regulatory elements by pufferfish/zebrafish transgenesis.

V hematopoiesis is a richly complex developmental system in which many transcription factors have essential and nonredundant roles (1). Some factors act as prominent controllers of differentiation in specific cell lineages, others are needed for stem cell generation, and others are needed for both. In this system, precise levels of one transcription factor relative to another in the same cell can control the direction of cell lineage choices, proliferation vs. apoptosis, lineage-specific malignant transformation, and the timing and sites of stem cell generation (2–9). Much has been learned in the past decade about the way the prominent transcription factors associated with particular hematopoietic cell types act on their target genes to execute lineage-specific differentiation programs. But lineage choice itself, the way progeny of the same pluripotent precursor adopt diverse fates, is less well understood. Its mechanism ultimately depends on the regulation of the key transcription factor genes themselves. There is very little i n f o r m a t i o n about the cisand trans-acting elements that control expression of most of these genes. Furthermore, the genes encoding the relevant transcription factors are often large, and the sequences needed for correct expression in transgenic mice can be dispersed over several hundred kilobases (10), making the full regulatory system hard to define. It would be extremely valuable to devise a shortcut to help map the regulatory regions for such genes. In a paper in this issue of PNAS (11), the gene encoding an essential hematopoietic transcription factor, SCL, is used to illustrate a strategy that may provide such a shortcut. To map regulatory elements, two things are needed: first, the assurance that the essential regulatory sequences are actually present in the DNA to be tested, and second, an assay system that allows their functional activity to be read out. Even the first of these conditions is hard to satisfy for large genes with complex patterns of expression, because enhancer modules (12) can be dispersed among introns and sometimes distant flanking regions. The second condition can also be difficult to meet for regulatory elements of mammalian genes that act in multiple embryonic and adult developmental contexts, because cell lines cannot duplicate the developmental shifts in expression, and mammalian transgenesis is costly and slow. Barton et al. (11) suggest that, by focusing on teleost fish as sources of both genes and assay systems, both sets of conditions can be met. The first element of this strategy is to clone the gene of interest from the pufferfish (Fugu rubripes), which has a genome about eight times smaller than that of a mammal. Nevertheless, coding sequences and intronyexon structures appear to be conserved: thus, essential regulatory elements may also be squeezed much closer to the genes they regulate than in mammalian genomes (13– 17). At least some of the Fugu regulatory sequences remain similar enough to those in mammals to be recognizable by sequence andyor by function in transgenic rats or mice (16, 18). Of course, only regulatory elements that serve similar functions in fish and mammals are likely to be conserved, but studies of blood development in frogs and fish generally suggest that key hematopoietic transcription factor genes are used in encouragingly similar ways (19–22). Recent studies in cartilaginous fish and lampreys further suggest that some detailed aspects of transcription factor use in blood development may be shared among all jawed vertebrates (23, 24). Whereas transgenic rodents can often read out the regulatory information in pufferfish DNA, they are too expensive, variable, and slow to map the borders of such elements purely on the basis of function. Pufferfish themselves are not yet adapted to gene transfer, or even to experimental embryology. Therefore, Barton et al. have taken advantage of the strong experimental embryology of another teleost, the zebrafish (Danio rerio). For example, the zebrafish GATA-1 59f lanking region is able to drive apparently correct expression of a green fluorescent protein (GFP) transgene in transgenic zebrafish erythrocytes (25). By injecting pufferfish genomic cosmids into zebrafish zygotes and testing their expression in the resulting embryos by in situ hybridization, a rapid determination can be made of the location of sequences controlling expression in each of several embryonic domains at once (11). Thus, noncoding DNA regions can be scanned relatively easily for the sequences that are necessary or sufficient to drive a wide range of tissuespecific expression patterns. At a minimum, this approach should define most cis-regulatory elements of these pufferfish genes that are mutually compatible in the two kinds of teleost fish. The gene used to illustrate this strategy is the SCL (Tal1) gene, which encodes a hematopoietic transcription factor with diverse developmental roles and complex regulation. SCL controls generation of hematopoietic stem cells of adult and fetal types, and both primitive hemangioblast and endothelial cell differentiation (26). SCL-deficient mice have defects in endothelial morphogenesis as well as a complete block in both primitive and definitive hematopoiesis. In later stages of mammalian hematopoietic differentiation, SCL overexpression drives erythromyeloid precursors toward erythroid and megakaryocytic fates at the expense of myeloid fates. In addition, SCL has several major expression sites in the central nervous system where its roles are less known.