Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance.

BackgroundEarly events in vertebrate liver development have been the major focus in previous studies, however, late events of liver organogenesis remain poorly understood. Liver vasculogenesis in vertebrates occurs through the interaction of endoderm-derived liver epithelium and mesoderm-derived endothelial cells (ECs). In zebrafish, although it has been found that ECs are not required for liver budding, how and when the spatio-temporal pattern of liver growth is coordinated with ECs remains to be elucidated.ResultsTo study the process of liver development and vasculogenesis in vivo, a two-color transgenic zebrafish line Tg(lfabf:dsRed; elaA:EGFP) was generated and named LiPan for liver-specific expression of DsRed RFP and exocrine pancreas-specific expression of GFP. Using the LiPan line, we first followed the dynamic development of liver from live embryos to adult and showed the formation of three distinct yet connected liver lobes during development. The LiPan line was then crossed with Tg(fli1:EGFP)y1 and vascular development in the liver was traced in vivo. Liver vasculogenesis started at 55–58 hpf when ECs first surrounded hepatocytes from the liver bud surface and then invaded the liver to form sinusoids and later the vascular network. Using a novel non-invasive and label-free fluorescence correction spectroscopy, we detected blood circulation in the liver starting at ~72 hpf. To analyze the roles of ECs and blood circulation in liver development, both cloche mutants (lacking ECs) and Tnnt2 morphants (no blood circulation) were employed. We found that until 70 hpf liver growth and morphogenesis depended on ECs and nascent sinusoids. After 72 hpf, a functional sinusoidal network was essential for continued liver growth. An absence of blood circulation in Tnnt2 morphants caused defects in liver vasculature and small liver.ConclusionThere are two phases of liver development in zebrafish, budding and growth. In the growth phase, there are three distinct stages: avascular growth between 50–55 hpf, where ECs are not required; endothelium-dependent growth, where ECs or sinusoids are required for liver growth between 55–72 hpf before blood circulation in liver sinusoids; and circulation-dependent growth, where the circulation is essential to maintain vascular network and to support continued liver growth after 72 hpf.

BackgroundThe choroid plexus (ChP), a component of the blood-brain barrier (BBB), produces the cerebrospinal fluid (CSF) and as a result plays a role in (i) protecting and nurturing the brain as well as (ii) in coordinating neuronal migration during neurodevelopment. Until now ChP development was not analyzed in living vertebrates due to technical problems.Methodology/Principal FindingsWe have analyzed the formation of the fourth ventricle ChP of zebrafish in the GFP-tagged enhancer trap transgenic line SqET33-E20 (Gateways) by a combination of in vivo imaging, histology and mutant analysis. This process includes the formation of the tela choroidea (TC), the recruitment of cells from rhombic lips and, finally, the coalescence of TC resulting in formation of ChP. In Notch-deficient mib mutants the first phase of this process is affected with premature GFP expression, deficient cell recruitment into TC and abnormal patterning of ChP. In Hedgehog-deficient smu mutants the second phase of the ChP morphogenesis lacks cell recruitment and TC cells undergo apoptosis.Conclusions/SignificanceThis study is the first to demonstrate the formation of ChP in vivo revealing a role of Notch and Hedgehog signalling pathways during different developmental phases of this process.

Background: Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome.