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Examines the mutation in zebrafish gata1 causing bloodless phenotype in vlad tepes. Isolation of mutants through chemical mutagenesis screening; Expression on hematopoietic stem cell markers; Importance of transcription factors in mammalian hematopoietic development.

Summary: Background: This study aimed to further investigate the role of circulating TGF-β1 during radiation therapy (RT) in predicting radiation-induced lung toxicity (RILT). Methods and materials: Patients with stages I–III non-small cell lung cancer treated with RT based therapy were included in this study. Platelet poor plasma was obtained pre-RT, at 2 and 4 weeks during-RT, and at the end of RT. TGF-β1 was measured using an enzyme-linked immunosorbent assay. The primary endpoint for RILT was ≥grade 2 radiation pneumonitis or fibrosis. Results: Twenty-six patients with a minimum follow-up of 12 months were included. Six patients (23.1%) experienced ≥grade 2 RILT. There was no significant difference in absolute TGF-β1 levels pre-RT, at 2 and 4 weeks during-RT, or at the end of RT between patients with and without RILT. The TGF-β1 ratios (over the pre-RT levels) for patients with and without RILT at 2, 4 weeks during-, and the end of RT were 2.8±2.2 and 1.0±0.6 (P =0.123), 2.3±1.3 and 0.8±0.5 (P =0.001), 1.5±0.9 and 0.8±0.5 (P =0.098), respectively. Using 2.0 as a cut-off, the TGF-β1 ratio at 4 weeks during-RT predicted RILT with a sensitivity and specificity of 66.7% and 95.0%, respectively. Conclusion: Elevation of plasma TGF-β1 level 4 weeks during-RT is significantly predictive of RILT. The role of plasma TGF-β1 in predicting RILT deserves further study. [Copyright &y& Elsevier]

The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhdmi149), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function. [ABSTRACT FROM AUTHOR]

The stem cell leukemia (SCL) transcription factor is essential for vertebrate hematopoiesis. Using the powerful zebrafish model for embryonic analysis, we compared the effects of either reducing or ablating Scl using morpholino-modified antisense RNAs. Ablation of Scl resulted in the loss of primitive and definitive hematopoiesis, consistent with its essential role in these processes. Interestingly, in embryos with severely reduced Scl levels, erythroid progenitors expressing gata1 and embryonic globin developed. Erythroid maturation was deficient in these Scl hypomorphs, supporting that Scl was required both for the erythroid specification and for the maturation steps, with maturation requiring higher Scl levels than specification. Although all hematopoietic functions were rescued by wild-type Scl mRNA, an Scl DNA binding mutant rescued primitive and definitive hematopoiesis but did not rescue primitive erythroid maturation. Together, we showed that there is a distinct Scl hypomorphic phenotype and demonstrated that distinct functions are required for the roles of Scl in the specification and differentiation of primitive and definitive hematopoietic lineages. Our results revealed that Scl participates in multiple processes requiring different levels and functions. Further, we identified an Scl hypomorphic phenotype distinct from the null state. [ABSTRACT FROM AUTHOR]

Gata1 is a prototype transcription factor that regulates hematopolesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3'-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene. [ABSTRACT FROM AUTHOR]