The Hippo Signaling Pathway Components Lats and Yap Pattern Tead4 Activity to Distinguish Mouse Trophectoderm from Inner Cell Mass

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation

The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties

Extensive post-transcriptional regulation of microRNAs and its implications for cancer

MicroRNAs (miRNAs) are short, noncoding RNAs that post-transcriptionally regulate gene expression. While hundreds of mammalian miRNA genes have been identified, little is known about the pathways that regulate the production of active miRNA species. Here we show that a large fraction of miRNA genes are regulated post-transcriptionally. During early mouse development, many miRNA primary transcripts, including the Let-7 family, are present at high levels but are not processed by the enzyme Drosha. An analysis of gene expression in primary tumors indicates that the widespread down-regulation of miRNAs observed in cancer is due to a failure at the Drosha processing step. These data uncover a novel regulatory step in miRNA function and provide a mechanism for miRNA down-regulation in cancer

H1 Linker Histones Are Essential for Mouse Development and Affect Nucleosome Spacing In Vivo

Most eukaryotic cells contain nearly equimolar amounts of nucleosomes and H1 linker histones. Despite their abundance and the potential functional specialization of H1 subtypes in multicellular organisms, gene inactivation studies have failed to reveal essential functions for linker histones in vivo. Moreover, in vitro studies suggest that H1 subtypes may not be absolutely required for assembly of chromosomes or nuclei. By sequentially inactivating the genes for three mouse H1 subtypes (H1c, H1d, and H1e), we showed that linker histones are essential for mammalian development. Embryos lacking the three H1 subtypes die by mid-gestation with a broad range of defects. Triple-H1-null embryos have about 50% of the normal ratio of H1 to nucleosomes. Mice null for five of these six H1 alleles are viable but are underrepresented in litters and are much smaller than their littermates. Marked reductions in H1 content were found in certain tissues of these mice and in another compound H1 mutant. These results demonstrate that the total amount of H1 is crucial for proper embryonic development. Extensive reduction of H1 in certain tissues did not lead to changes in nuclear size, but it did result in global shortening of the spacing between nucleosomes

Defects in Yolk Sac Vasculogenesis, Chorioallantoic Fusion, and Embryonic Axis Elongation in Mice with Targeted Disruption of Yap65

YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yap(tm1Smil) allele (Yap(−/−)) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostaining, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in Yap(−/−) embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper. Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap(−/−) embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithelium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation

Amino acid metabolites that regulate G protein signaling during osmotic stress

<div><p>All cells respond to osmotic stress by implementing molecular signaling events to protect the organism. Failure to properly adapt can lead to pathologies such as hypertension and ischemia-reperfusion injury. Mitogen-activated protein kinases (MAPKs) are activated in response to osmotic stress, as well as by signals acting through G protein-coupled receptors (GPCRs). For proper adaptation, the action of these kinases must be coordinated. To identify second messengers of stress adaptation, we conducted a mass spectrometry-based global metabolomics profiling analysis, quantifying nearly 300 metabolites in the yeast <i>S</i>. <i>cerevisiae</i>. We show that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the MAPK Hog1. Ectopic addition of these BCAA derivatives promotes phosphorylation of the G protein α subunit and dampens G protein-dependent transcription, similar to that seen in response to osmotic stress. Conversely, genetic ablation of Hog1 activity or the BCAA-regulatory enzymes leads to diminished phosphorylation of Gα and increased transcription. Taken together, our results define a new class of candidate second messengers that mediate cross talk between osmotic stress and GPCR signaling pathways.</p></div

Proteolysis of ribulose-1,5-bisphosphate carboxylase oxygenase in citrus leaf extracts

Peer reviewe

BCAA derivatives diminish MAPK-dependent gene transcription.

<p>(A) Addition of BCAA derivatives or KCl dampens α-factor pheromone-induced gene transcription (P_FUS1-GFP). The dampening capacity of each BCAA derivative is correlated with Gpa1 phosphorylation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006829#pgen.1006829.g004" target="_blank">Fig 4</a>). (B) Genetic ablation of the AMPK kinases increases basal gene transcription, consistent with reduced Gpa1 phosphorylation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006829#pgen.1006829.ref021" target="_blank">21</a>]. Correspondingly, the kinase mutants abrogate any ability of the metabolites to suppress basal signaling and limit their ability to suppress pheromone signaling (41% reduction in wildtype vs. 26% reduction in the kinase mutant strain). Data are presented as mean ± standard deviation, N = 4.</p

Phosphorylation of Gpa1 in response to osmotic stress occurs in a Hog1-dependent, pH-independent manner.

<p>Western blot analysis reveals that Gpa1 and Snf1 are phosphorylated (p-Gpa1 and p-Snf1) in response to (A) glucose (Glc) limitation (“High” = 2% glucose, “Low” = 0.05% glucose), (B) heat shock (42°C), or (C) osmotic stress (0.5 M KCl). Note that Hog1 is phosphorylated (p-Hog1) in response to heat shock or osmotic stress, but not glucose limitation. Intracellular pH (insets) decreases in response to glucose limitation or heat shock (shaded area), but not osmotic stress. Hog1 catalytic activity (<i>hog1</i>^<i>K52R</i>) is required for phosphorylation of Gpa1 but not Snf1. Diploid, control cells lacking Gpa1. <i>reg1</i>Δ, control cells lacking Gpa1 phosphatase. Hog1, Snf1, and Load correspond to gels probed with Hog1, poly-His, and G6PDH antibodies, respectively. Data were quantified based on band intensity, and are presented as mean ± standard deviation, N = 3.</p

BCAA derivatives promote Gpa1 phosphorylation.

<p>(A) Ectopic addition of 2-hydroxyisocaproate (HIC) promotes phosphorylation of Gpa1 but not Hog1 or Snf1. (B) Ectopic addition of the BCAA derivatives promotes Gpa1 phosphorylation while intracellular pH is unaffected (inset). (C, D) Ectopic addition of HIC promotes Gpa1 phosphorylation in wild-type and Hog1-deficient cells. Osmotic stress promotes Gpa1 phosphorylation only in wild-type cells. Data presented as mean ± standard deviation, N = 3.</p