Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34+ cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

The transcription factors Scl and Lmo2 are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl ؊/؊ embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of hemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important hematopoietic regulators is lost, reduced, or delayed, panendothelial gene expression is down-regulated, and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in hemangioblast development within a multiprotein complex, as shown for erythropoiesis. Consistent with this, we find that scl morphants cannot be rescued by a non-Lmo2-binding form of Scl but can be rescued by non-DNAbinding forms, suggesting tethering to target genes through DNA-binding part- IntroductionLmo2 was first discovered through its homology to the T-cell oncogene lmo1 and has been found to be activated by chromosomal translocations in several T-cell leukemias. 1 Like its partner in erythroid cells, scl, during normal development lmo2 is specifically expressed in hematopoietic progenitors, erythroid cells, and megakaryocytes, and in endothelial cells. 2,3 Targeted gene ablation in mice revealed an essential role for Lmo2 in primitive hematopoiesis, with lmo2 Ϫ/Ϫ embryos dying at embryonic day (E) 9 to 10 due to severe anemia. 2 Lmo2 Ϫ/Ϫ embryonic stem (ES) cells were unable to contribute to any hematopoietic lineage in chimeras, indicating a critical requirement for Lmo2 in definitive hematopoiesis. 4 In addition, although lmo2 Ϫ/Ϫ ES cells were initially able to contribute to normal primary capillary plexus formation, they were unable to contribute to the formation of larger vessels, and chimeras with a large contribution of lmo2 Ϫ/Ϫ ES cells exhibited disrupted vascular organization from E9 onwards, indicating a role for Lmo2 in angiogenesis. 3 The Lmo2 protein comprises 2 LIM zinc finger-like protein interaction domains and has been found to act as a bridging molecule between the Scl/E47 heterodimer and Gata1/2 in the formation of DNA-binding complexes. 5-10 A specific phenylalanine residue in helix 2 of the Scl basic helix-loop-helix (bHLH) domain has been shown to be essential in vitro for hematopoietic activity of Scl. However, rather than being important for heterodimerization with E47, this residue promotes protein interactions with Lmo2. 11 Remarkably, introduction of a phenylalanine residue at the corresponding position in the myogenic bHLH, MyoD, was sufficient to confer hematopoietic activity, rescuing hematopoietic development in scl Ϫ/Ϫ ES cells. The interaction between Scl and Lmo2 is therefore essential for initiation of hematopoiesis in ES cells. However, the in vivo requirement for this interact...

Stem cells share the defining characteristics of self-renewal, which maintains or expands the stem-cell pool, and multi-lineage differentiation, which generates and regenerates tissues. Stem-cell self-renewal and differentiation are influenced by the convergence of intrinsic cellular signals and extrinsic microenvironmental cues from the surrounding stem-cell niche, but the specific signals involved are poorly understood. Recently, several studies have sought to identify the genetic mechanisms that underlie the stem-cell phenotype. Such a molecular road map of stem-cell function should lead to an understanding of the true potential of stem cells.

Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow CD34+CD33−CD38−Rholoc-kit+ cells, enriched for hematopoietic stem/progenitor cells with CD34+CD33−CD38−Rhohi cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.

BackgroundUnderstanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins – representing the vertebrate secretome – are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown.ResultsWe identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%–3% estimate from random gene mutagenesis studies.Conclusion(s)This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.