Identifications of three novel alleles of Serrate in Drosophila.

The Notch signaling pathway, an evolutionarily highly conserved pathway, participates in various essential physiological processes in organisms. Activation of Notch signaling in the canonical manner requires the combination of ligand and receptor. There are two ligands of Notch in Drosophila: Delta (Dl) and Serrate (Ser). A mutation mf157 is identified for causing nicks of fly wings in genetic analysis from a mutant library (unpublished) that was established previously. Immunofluorescent staining illustrates that mf157 represses the expression of Cut and Wingless (Wg), the targets of Notch signaling. MARCM cloning analysis reveals that mf157 functions at the same level or the upstream of ligands of Notch in signaling sending cells. Sequencing demonstrates that mf157 is a novel allele of the Ser gene. Subsequently, mf553 and mf167 are also identified as new alleles of Ser from our library. Furthermore, the complementary assays and the examination of transcripts confirm the sequencing results. Besides, the repressed phenotypes of Notch signaling were reverted by transposon excision experiments of mf157. In conclusion, we identify three fresh alleles of Ser. Our works supply additional genetic resources for further study of functions of Ser and Notch signaling regulation.

SERRATE: a key factor in coordinated RNA processing in plants.

The SERRATE (SE) protein is involved in the processing of RNA polymerase II (RNAPII) transcripts. It is associated with different complexes engaged in different aspects of plant RNA metabolism, including assemblies involved in transcription, splicing, polyadenylation, miRNA biogenesis, and RNA degradation. SE stability and interactome properties can be influenced by phosphorylation. SE exhibits an intriguing liquid-liquid phase separation property that may be important in the assembly of different RNA-processing bodies. Therefore, we propose that SE seems to participate in the coordination of different RNA-processing steps and can direct the fate of transcripts, targeting them for processing or degradation when they cannot be properly processed or are synthesized in excess.

ASO silencing of a glycosyltransferase, Poglut1 , improves the liver phenotypes in mouse models of Alagille syndrome.

BACKGROUND AND AIMS: Paucity of intrahepatic bile ducts (BDs) is caused by various etiologies and often leads to cholestatic liver disease. For example, in patients with Alagille syndrome (ALGS), which is a genetic disease primarily caused by mutations in jagged 1 ( JAG1) , BD paucity often results in severe cholestasis and liver damage. However, no mechanism-based therapy exists to restore the biliary system in ALGS or other diseases associated with BD paucity. Based on previous genetic observations, we investigated whether postnatal knockdown of the glycosyltransferase gene protein O -glucosyltransferase 1 ( Poglut1) can improve the ALGS liver phenotypes in several mouse models generated by removing one copy of Jag1 in the germline with or without reducing the gene dosage of sex-determining region Y-box 9 in the liver. APPROACH AND RESULTS: Using an ASO established in this study, we show that reducing Poglut1 levels in postnatal livers of ALGS mouse models with moderate to profound biliary abnormalities can significantly improve BD development and biliary tree formation. Importantly, ASO injections prevent liver damage in these models without adverse effects. Furthermore, ASO-mediated Poglut1 knockdown improves biliary tree formation in a different mouse model with no Jag1 mutations. Cell-based signaling assays indicate that reducing POGLUT1 levels or mutating POGLUT1 modification sites on JAG1 increases JAG1 protein level and JAG1-mediated signaling, suggesting a likely mechanism for the observed in vivo rescue. CONCLUSIONS: Our preclinical studies establish ASO-mediated POGLUT1 knockdown as a potential therapeutic strategy for ALGS liver disease and possibly other diseases associated with BD paucity.

Loss of Jagged1 in mature endothelial cells causes vascular dysfunction with alterations in smooth muscle phenotypes.

BACKGROUND: Notch signaling is an evolutionarily conserved pathway that functions via direct cell-cell contact. The Notch ligand Jagged1 (Jag1) has been extensively studied in vascular development, particularly for its role in smooth muscle cell maturation. Endothelial cell-expressed Jag1 is essential for blood vessel formation by signaling to nascent vascular smooth muscle cells and promoting their differentiation. Given the established importance of Jag1 in endothelial cell/smooth muscle crosstalk during development, we sought to determine the extent of this communication in the adult vasculature for blood vessel function and homeostasis. METHODS: We conditionally deleted Jag1 in endothelial cells of adult mice and examined the phenotypic consequences on smooth muscle cells of the vasculature. RESULTS: Our results show that genetic loss of Jag1 in endothelial cells has a significant impact on Notch signaling and vascular smooth muscle function in mature blood vessels. Endothelial cell-specific deletion of Jag1 causes a concomitant loss of JAG1 and NOTCH3 expression in vascular smooth muscle cells, resulting in a transition to a less differentiated state. Aortic vascular smooth muscle cells isolated from the endothelial cell-specific Jag1 deficient mice retain an altered phenotype in culture with fixed changes in gene expression and reduced Notch signaling. Utilizing comparative RNA-sequence analysis, we found that Jag1 deficiency preferentially affects extracellular matrix and adhesion protein gene expression. Vasoreactivity studies revealed a reduced contractile response and impaired agonist-induced relaxation in endothelial cell Jag1-deficient aortas compared to controls. CONCLUSIONS: These data are the first to demonstrate that Jag1 in adult endothelial cells is required for the regulation and homeostasis of smooth muscle cell function in arterial vessels partially through the autoregulation of Notch signaling and cell matrix/adhesion components in smooth muscle cells.

Notch signaling patterns head horn shape in the bull-headed dung beetle Onthophagus taurus.

Size and shape constitute fundamental aspects in the description of morphology. Yet while the developmental-genetic underpinnings of trait size, in particular with regard to scaling relationships, are increasingly well understood, those of shape remain largely elusive. Here we investigate the potential function of the Notch signaling pathway in instructing the shape of beetle horns, a highly diversified and evolutionarily novel morphological structure. We focused on the bull-headed dung beetle Onthophagus taurus due to the wide range of horn sizes and shapes present among males in this species, in order to assess the potential function of Notch signaling in the specification of horn shape alongside the regulation of shape changes with allometry. Using RNA interference-mediated transcript depletion of Notch and its ligands, we document a highly conserved role of Notch signaling in general appendage formation. By integrating our functional genetic approach with a geometric morphometric analysis, we find that Notch signaling moderately but consistently affects horn shape, and does so differently for the horns of minor, intermediate-sized, and major males. Our results suggest that the function of Notch signaling during head horn formation may vary in a complex manner across male morphs, and highlights the power of integrating functional genetic and geometric morphometric approaches in analyzing subtle but nevertheless biologically important phenotypes in the face of significant allometric variation.

Alternative isoforms of BmYki have different transcriptional co-activator activity in the silkworm, Bombyx mori.

Yorki (Yki), a transcriptional co-activator that is a key component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. Bombyx mori Yki3 (BmYki3), with 445 amino acid residues, facilitates cell migration and cell division, and enlarges cultured cell and wing disc size. In this study, cellular localization, transcriptional co-activator activity, cell migration, cell cycle, and cell size were characterized in alternative isoforms of BmYki. BmYki1 and BmYki3 are mainly located in the cytoplasm and nucleus, respectively, while, BmYki2 is located in both the cytoplasm and nucleus. The mutation BmYki1S97A (S97mutated to A) was transported from the cytoplasm to nucleus. Cell migration, cell cycle, and cell size could be enhanced by BmYki, however, the effect of BmYki1 and BmYki2 on cell proliferation was less compared to BmYki3. Moreover, wing discs could be enlarged by overexpressing BmYki1 or BmYki2 isoforms. Dual-luciferase reporter assay showed that BmYki3 had the highest activity to B. mori ovarian tumor gene. In BmN cells overexpressing one of the BmYki isoforms, expression levels of kibra ortholog (kibra), inhibitor of apoptosis protein (iap), four-jointed (fj), expanded (ex), crumbs (crb) and BMP and activin membrane-bound inhibitor homolog (Bmpr) genes were upregulated, while those of α-catenin (α-cat), decapentaplegic (dpp), serrate (serr) and signal transducer and activator of transcription (stat) genes were down-regulated. There was some difference in the regulation of gene expression between different isoforms. These results suggested that the activity of BmYki isoforms was different in the silkworm.

Detection of MicroRNA Processing Intermediates Through RNA Ligation Approaches.

MicroRNAs (miRNA) are small RNAs of 20-22 nt that regulate diverse biological pathways through the modulation of gene expression. miRNAs recognize target RNAs by base complementarity and guide them to degradation or translational arrest. They are transcribed as longer precursors with extensive secondary structures. In plants, these precursors are processed by a complex harboring DICER-LIKE1 (DCL1), which cuts on the precursor stem region to release the mature miRNA together with the miRNA*. In both plants and animals, the miRNA precursors contain spatial clues that determine the position of the miRNA along their sequences. DCL1 is assisted by several proteins, such as the double-stranded RNA binding protein, HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). The precise biogenesis of miRNAs is of utter importance since it determines the exact nucleotide sequence of the mature small RNAs and therefore the identity of the target genes. miRNA processing itself can be regulated and therefore can determine the final small RNA levels and activity. Here, we describe methods to analyze miRNA processing intermediates in plants. These approaches can be used in wild-type or mutant plants, as well as in plants grown under different conditions, allowing a molecular characterization of the miRNA biogenesis from the RNA precursor perspective.

SERRATE, a miRNA biogenesis factor, affects viroid infection in Nicotiana benthamiana and Nicotiana tabacum.

Viroids are plant infecting, non - coding RNA molecules of economic importance. Potato spindle tuber viroid (PSTVd), the type species of Pospiviroidae family, has been shown to be affected by specific RNA silencing pathways. Dicer like 1 (DCL1), a key player in micro RNA (miRNA) pathway has been previously linked with PSTVd infectivity. In this report we aim to further dissect the interaction between the miRNA pathway and Pospiviroid virulence. We mainly focused on the Zinc-finger protein SERRATE (SE) a co-factor of DCL1 and core component of miRNA pathway. We generated Nicotiana tabacum and Nicotiana benthamiana SE knock-down plants exhibiting considerable miRNA reduction and strong phenotypic abnormalities. PSTVd infection of SE suppressed plants resulted in a significant viroid reduction, especially at the initial infection stages. This positive correlation between SE levels and viroid infectivity underlines its role in PSTVd life cycle and reveals the importance of the miRNA pathway upon viroid infection.

Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila.

The Drosophila glucoside xylosyltransferase Shams xylosylates Notch and inhibits Notch signaling in specific contexts including wing vein development. However, the molecular mechanisms underlying context-specificity of the shams phenotype is not known. Considering the role of Delta-Notch signaling in wing vein formation, we hypothesized that Shams might affect Delta-mediated Notch signaling in Drosophila. Using genetic interaction studies, we find that altering the gene dosage of Delta affects the wing vein and head bristle phenotypes caused by loss of Shams or by mutations in the Notch xylosylation sites. Clonal analysis suggests that loss of shams promotes Delta-mediated Notch activation. Further, Notch trans-activation by ectopically overexpressed Delta shows a dramatic increase upon loss of shams. In agreement with the above in vivo observations, cell aggregation and ligand-receptor binding assays show that shams knock-down in Notch-expressing cells enhances the binding between Notch and trans-Delta without affecting the binding between Notch and trans-Serrate and cell surface levels of Notch. Loss of Shams does not impair the cis-inhibition of Notch by ectopic overexpression of ligands in vivo or the interaction of Notch and cis-ligands in S2 cells. Nevertheless, removing one copy of endogenous ligands mimics the effects of loss shams on Notch trans-activation by ectopic Delta. This favors the notion that trans-activation of Notch by Delta overcomes the cis-inhibition of Notch by endogenous ligands upon loss of shams. Taken together, our data suggest that xylosylation selectively impedes the binding of Notch with trans-Delta without affecting its binding with cis-ligands and thereby assists in determining the balance of Notch receptor's response to cis-ligands vs. trans-Delta during Drosophila development.