Mouse embryos lacking the receptor tyrosine kinase, Flk1, die without mature endothelial and hematopoietic cells. To investigate the role of Flk1 during vasculogenesis and hematopoiesis, we examined the developmental potential of Flk1-/- embryonic stem cells in chimeras. We show that Flk1 is required cell autonomously for endothelial development. Furthermore, Flk1-/- cells do not contribute to primitive hematopoiesis in chimeric yolk sacs or definitive hematopoiesis in adult chimeras and chimeric fetal livers. We also demonstrate that cells lacking Flk1 are unable to reach the correct location to form blood islands, suggesting that Flk1 is involved in the movement of cells from the posterior primitive streak to the yolk sac and, possibly, to the intraembryonic sites of early hematopoiesis.

No abstract

Background: Functional relationships between the microRNA and cellular hypoxia response pathways are unknown. Results: Dicer is down-regulated in chronic hypoxia; this mechanism maintains the induction of hypoxia-inducible factor-␣ subunits and hypoxia-responsive genes. Conclusion: Loss of Dicer-dependent microRNA regulation is important for maintaining the concerted cellular response to hypoxia. Significance: Altogether, we provide a newer perspective into the post-transcriptional pathways that regulate the cellular hypoxic response.

SUMMARYThe Wilms' tumor suppressor 1 (WT1) gene encodes a DNA-and RNA-binding protein that plays an essential role in nephron progenitor differentiation during renal development. To identify WT1 target genes that might regulate nephron progenitor differentiation in vivo, we performed chromatin immunoprecipitation (ChIP) coupled to mouse promoter microarray (ChIP-chip) using chromatin prepared from embryonic mouse kidney tissue. We identified 1663 genes bound by WT1, 86% of which contain a previously identified, conserved, high-affinity WT1 binding site. To investigate functional interactions between WT1 and candidate target genes in nephron progenitors, we used a novel, modified WT1 morpholino loss-of-function model in embryonic mouse kidney explants to knock down WT1 expression in nephron progenitors ex vivo. Low doses of WT1 morpholino resulted in reduced WT1 target gene expression specifically in nephron progenitors, whereas high doses of WT1 morpholino arrested kidney explant development and were associated with increased nephron progenitor cell apoptosis, reminiscent of the phenotype observed in Wt1 -/-embryos. Collectively, our results provide a comprehensive description of endogenous WT1 target genes in nephron progenitor cells in vivo, as well as insights into the transcriptional signaling networks controlled by WT1 that might direct nephron progenitor fate during renal development.

Nuclear hormone receptors and homeodomain proteins are two classes of transcription factor that regulate major developmental processes. Both depend on interactions with other proteins for specificity and activity. The Drosophila gene fushi tarazu (ftz), which encodes a homeodomain protein (Ftz), is required zygotically for the formation of alternate segments in the developing embryo. Here we show that the orphan nuclear receptor alphaFtz-F1 (ref. 3), which is deposited in the egg during oogenesis, is an obligatory cofactor for Ftz. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. This interaction suggests that other nuclear receptor/homeodomain protein interactions maybe important and common in developing organisms.

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS) −/− mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS −/− mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS −/− mice, despite comparable reductions in tissue PO 2 . Paradoxically, nNOS −/− mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S -nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S -nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Understanding the mechanisms that regulate nephron progenitors during kidney development should aid development of therapies for renal failure. MicroRNAs, which modulate gene expression through post-transcriptional repression of specific target mRNAs, contribute to the differentiation of stem cells, but their role in nephrogenesis is incompletely understood. Here, we found that the loss of miRNAs in nephron progenitors results in a premature depletion of this population during kidney development. Increased apoptosis and expression of the pro-apoptotic protein Bim accompanied this depletion. Profiling of miRNA expression during nephrogenesis identified several highly expressed miRNAs (miR10a, miR-106b, miR-17-5p) in nephron progenitors that are either known or predicted to target Bim. We propose that modulation of apoptosis by miRNAs may determine congenital nephron endowment. Furthermore, our data implicate the pro-apoptotic protein Bim as a miRNA target in nephron progenitors. Kidney development begins with the outgrowth of the ureteric bud from the Wolffian duct into the metanephric mesenchyme. 1,2 The metanephric mesenchyme condenses as a tight "cap" of nephron progenitors around the tip of the ureteric bud and the ureteric bud branches to form the collecting system. Nephron progenitors have the capacity to selfrenew to generate the full complement of nephrons and to differentiate into the multiple cell types required to form the nephron. This process continues in an iterative fashion during nephrogenesis such that the most immature cells are present in the subcapsular cortex of the developing kidney, termed the nephrogenic zone.MicroRNAs (miRNAs) are a group of endogenous, small noncoding RNAs that function by causing the post-transcriptional repression of their respective target mRNAs. The first suggestion that miRNAs are critical in stem cell populations came from the observation that embryos that are null for Dicer, an enzyme required for the production of

Endothelial-derived nitric oxide (NO) is classically viewed as a regulator of vasomotor tone. NO plays an important role in regulating O(2) delivery through paracrine control of vasomotor tone locally and cardiovascular and respiratory responses centrally. Very soon after the cloning and functional characterization of the endothelial nitric oxide synthase (eNOS), studies on the interaction between O(2) and NO made the paradoxical finding that hypoxia led to decreases in eNOS expression and function. Why would decreases in O(2) content in tissues elicit a loss of a potent endothelial-derived vasodilator? We now know that restricting our view of NO as a regulator of vasomotor tone or blood pressure limited deeper levels of mechanistic insight. Exciting new studies indicate that functional interactions between NO and O(2) exhibit profound complexity and are relevant to diseases states, especially those associated with hypoxia in tissues. NOS isoforms catalytically require O(2). Hypoxia regulates steady-state expression of the mRNA and protein abundance of the NOS enzymes. Animals genetically deficient in NOS isoforms have perturbations in their ability to adapt to changes in O(2) supply or demand. Most interestingly, the intracellular pathways for O(2) sensing that evolved to ensure an appropriate balance of O(2) delivery and utilization intersect with NO signaling networks. Recent studies demonstrate that hypoxia-inducible factor (HIF) stabilization and transcriptional activity is achieved through two parallel pathways: (1) a decrease in O(2)-dependent prolyl hydroxylation of HIF and (2) S-nitrosylation of HIF pathway components. Recent findings support a role for S-nitrosothiols as hypoxia-mimetics in certain biological and/or disease settings, such as living at high altitude, exposure to small molecules that can bind NO, or anemia.