Davidson, A.J., Dooley, K., Schmidt, B., Paffett-Lugassy, N., White, N., Walderich, B., Odenthal, J., Maischein, H.M., Schulte-Merker, S., Nüsslein-Volhard, C., Thisse, C., Thisse, B., and Zon, L.I. (2001) Functional genomics and mutagenesis screening in the zebrafish identifies genes required for hematopoietic stem cell development. Blood. 98(11):1888.
Hematopoietic stem cells (HSC) arise from ventral mesoderm during embryonic development. The basic helix-loop-helix transcription factor SCL is both necessary and sufficient for inducing HSC fate. To further elucidate the molecular events occurring prior to the onset of scl expression, we performed a high throughput in situ hybridization screen for genes expressed in hematopoietic mesoderm. The expression patterns of over 6000 random embryonic cDNAs were studied and transcripts for 25 genes were detected specifically in developing blood cells. Based on these novel molecular markers we have spatially and temporally defined a new stage in HSC development that occurs during the transition from ventral mesoderm to the induction of scl expression. Of these new markers, the gene draculin (dra) encodes a multi-zinc finger protein and medusa (med) encodes a novel member of the ring finger, B-box, coiled-coiled domain (RBCC) family. Transcripts for dra and med are found throughout ventral mesoderm and then become restricted to a smaller population cells that will ultimately become blood. Expression of scl initiates within these dra+/med+ cells, followed by gata1. A knock-down of dra translation by injection of antisense morpholinos results in an inhibition of blood cell development. Thus, dra+/med+ cells represent a distinct population of "pre-hematopoietic stem" cells. To further genetically dissect this pathway, we undertook a large scale mutagenesis screen for zebrafish mutants that display altered scl expression. A total of 1495 genome equivalents were screened, and 32 potential mutants have been isolated. Of these mutants HS155 displays a complete absence of scl expression and is allelic to the previously identified bloodless mutant cloche. IL239 and HV001 show reduced scl expression and subsequently develop severe anemia. In the IA251 mutant, scl expression is dramatically reduced. Furthermore, the scl+ cells that do develop in the mutant fail to express gata1. These results suggest that the IA251 mutation not only blocks scl induction, but also leads to an arrest in HSC differentiation. In summary, the isolation of new molecular markers has allowed us to visualize a previously uncharacterized stage in the formation of HSCs and the forward genetic screen has identified additional genes required for the genesis of HSCs during embryogenesis.