GDNF signaling through the Ret receptor tyrosine kinase is critical for ureteric bud branching morphogenesis during kidney development, yet few of the downstream genes are currently known. We find that the ETS transcription factors Etv4 and Etv5 are positively regulated by Ret signaling in the ureteric bud tips. Etv4−/−, Etv5+/− mice display either renal agenesis or severe hypodysplasia, while kidney development fails completely in double homozygotes. We identify several genes whose expression in the ureteric bud depends on Etv4 and Etv5, including Cxcr4, Myb, Met, Mmp14. Thus, Etv4 and Etv5 are key components of a gene network downstream of Ret that promotes and controls renal branching morphogenesis.

The trkB family of transmembrane proteins serves as receptors for BDNF and NT-4/5. The family is composed of a tyrosine kinase-containing isoform as well as several alternatively spliced "truncated receptors" with identical extracellular ligandbinding domains but very small intracellular domains. The two best-characterized truncated trkB receptors, designated as trkB.T1 and trkB.T2, contain intracellular domains of only 23 and 21 amino acids, respectively. Although it is known that the tyrosine kinase isoform (trkB.FL) is capable of initiating BDNF and NT-4/5-induced signal transduction, the functional role or roles of the truncated receptors remain enigmatic. At the same time, the potential importance of the truncated receptors in the development, maintenance, and regeneration of the nervous system has been highlighted by recent developmental and injury paradigm investigations. Here we have used trkB cDNA transfected cell lines to demonstrate that both trkB.T1 and trkB.T2 are capable of mediating BDNF-induced signal transduction. More specifically, BDNF activation of either trkB.T1 or trkB.T2 increases the rate of acidic metabolite release from the cell, a common physiological consequence of many signaling pathways. Further, these trkB.T1-and trkB.T2-mediated changes occur with kinetics distinct from changes mediated by trkB.FL, suggesting the participation of at least some unique rate-limiting component or components. Mutational analysis demonstrates that the isoform-specific sequences within the intracellular domains of each receptor are essential for signaling capability. Finally, inhibitor studies suggest that kinases are likely to be involved in the trkB.T1 and trkB.T2 signaling pathways.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

The TF-1 human erythroleukemic cell line exhibits opposing physiological responses toward tumor necrosis factor-␣ (TNF) treatment, dependent upon the mitotic state of the cells. Mitotically active cells in log growth respond to TNF by rapidly undergoing apoptosis whereas TNF exposure stimulates cellular proliferation in mitotically quiescent cells. The concentrationdependent TNF-induced apoptosis was monitored by cellular metabolic activity and confirmed by both DNA epifluorescence and DNA fragmentation. Moreover, these responses could be detected by measuring extracellular acidification activity, enabling rapid prediction (within ϳ 1.5 h of TNF treatment) of the fate of the cell in response to TNF. Growth factor resupplementation of quiescent cells, resulting in reactivation of cell cycling, altered TNF action from a proliferative stimulus to an apoptotic signal. Expression levels of the type II TNF receptor subtype (p75TNFR) were found to correlate with sensitivity to TNF-induced apoptosis. Pretreatment of log growth TF-1 cells with a neutralizing antip75TNFR monoclonal antibody inhibited TNF-induced apoptosis by greater than 80%. Studies utilizing TNF receptor subtype-specific TNF mutants and neutralizing antisera implicated p75TNFR in TNF-dependent apoptotic signaling. These data show a bifunctional physiological role for TNF in TF-1 cells that is dependent on mitotic activity and controlled by the p75TNFR.Cells have the capability of responding to a multitude of signals that it encounters in its extracellular environment. One such signal with widespread pleiotropic actions is the cytokine tumor necrosis factor-␣ (TNF) 1 (1). TNF has been shown to modulate proliferation, differentiation, and apoptotic or necrotic cell death in a number of different cell types (2-4). These disparate responses to TNF are mediated by TNF binding to specific cell surface receptors. Two distinct TNF receptors, type I (p55TNFR) and type II (p75TNFR) (M r 55,000 -60,000 and 70,000 -80,000 in human cells, respectively), have been identified (5, 6), although it remains unclear which of the many responses reported for TNF can be attributed to a specific receptor subtype (4). Moreover, the precise signal transduction pathways for each of these receptor subtypes have yet to be fully delineated. One action of TNF, the induction of apoptosis, is characterized by a discrete set of cellular events regulated by gene expression (7,8). The physiological events accompanying apoptosis include condensation of the chromatin, degradation of DNA through the activation of endogenous nucleases, and dissolution of the cell into small membrane-bound apoptotic vesicles (9, 10). In vivo, these vesicles are phagocytosed by macrophages or other phagocytic cells. Cell death by apoptosis is essential in many physiological processes, including embryonic development of the nervous system (11), oncogenic pathology (12), and clonal selection of hematopoietic cells (13).Conversely, TNF has also been shown to stimulate cellular proliferation in a variety of systems...

Fertilization elicits a dramatic, transient rise in Ca 2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca 2+ stores have been identified which could, either individually or in combination, initiate Ca 2+ release at fertilization. Inositol 1,4,5-trisphosphate (IP 3 ) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca 2+ transient. Other studies indicate that Ca 2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca 2+ rise and IP 3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP 3 . Translocation of the probe occurred after the Ca 2+ rise was initiated. Earlier, and possibly smaller, IP 3 changes could not be excluded due to limitations in probe sensitivity. High IP 3 levels are maintained during the decline in cytoplasmic Ca 2+ , suggesting that later IP 3 metabolism might not be related to regulation of Ca 2+ , but may function to modulate other PIP 2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.

We used the microphysiometer, a sensitive extracellular pH sensor, to resolve luminal (or apical) H+ secretion and basolateral release of OH- as well as liberation of acidic metabolites in rabbit gastric glands. Stimulation of glands via the adenosine 3',5'-cyclic monophosphate pathway produced a biphasic change in the extracellular acidification rate (EAR): after an initial transient decrease below the unstimulated baseline (-40.9 +/- 3.4%), the EAR increased to a steady-state maximal plateau (+98.1 +/- 5.3%) within 30 min (n = 37). We interpret the biphasic EAR profile as an initial excess of basolaterally released OH- followed by delayed luminal efflux of simultaneously produced H+. The elevated EAR at steady state reflected liberation of metabolic acid attributed to H(+)-K(+)-ATPase enzymatic activity. The presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid prevented OH- release and reduced steady-state EAR. Basolateral OH- release and steady-state EAR were also inhibited by the H(+)-K(+)-ATPase inactivators omeprazole and SCH-28080. Inhibition of Na+/H+ exchange did not reduce steady-state EAR and did not affect apical H+ production, as judged by the accumulation of the weak base aminopyrine. Sodium thiocyanate (1 mM), which short circuits intraluminal H+ accumulation, blocked OH- release, demonstrating its dependence on H(+)-OH- separation at the apical membrane. A computerized model was developed to illustrate how the observed biphasic EAR profile would result from a delayed luminal efflux of H+ due to transitory intraluminal compartmentalization.

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The GAPF technique identifies palindromes associated with gene amplicons in cancers; however, the initial publication over stimated the frequency of palindrome formation and the frequency of palindromes occurring in similar locations among different cancers. The performance of the modified technique is discussed in the accompanying Correspondence 1 . In the version of this article initially published, the sentence under Table 1 on p. 1296 should stop after the words "and 430A arrays," and the words "or >3 if represented on only one array" should be removed. In addition, on p. 1298, the second parenthesis after "Tg(Hoxb7-myrVenus)" is missing. These errors have been corrected in the HTML and PDF versions of the article. c o r r i g e n d a a n d e r r ata nature genetics | volume 42 | number 4 | april 2010 361