RNA helicase Belle/DDX3 regulates transgene expression in Drosophila

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dcr-1 also rescued the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we showed that genetic inhibition of Bel function led to the de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited. Our findings have demonstrated a novel involvement of Bel in regulating transgene expression and its loss triggers a transgene silencing mechanism mediated by protein regulators implicated in RNA processing, piRNA biogenesis, chromatin remodeling and the microRNA pathway

Uif, a Large Transmembrane Protein with EGF-Like Repeats, Can Antagonize Notch Signaling in Drosophila

<div><h3>Background</h3><p>Notch signaling is a highly conserved pathway in multi-cellular organisms ranging from flies to humans. It controls a variety of developmental processes by stimulating the expression of its target genes in a highly specific manner both spatially and temporally. The diversity, specificity and sensitivity of the Notch signaling output are regulated at distinct levels, particularly at the level of ligand-receptor interactions.</p> <h3>Methodology/Principal Findings</h3><p>Here, we report that the <em>Drosophila</em> gene <em>uninflatable</em> (<em>uif</em>), which encodes a large transmembrane protein with eighteen EGF-like repeats in its extracellular domain, can antagonize the canonical Notch signaling pathway. Overexpression of Uif or ectopic expression of a neomorphic form of Uif, Uif*, causes Notch signaling defects in both the wing and the sensory organ precursors. Further experiments suggest that ectopic expression of Uif* inhibits Notch signaling <em>in cis</em> and acts at a step that is dependent on the extracellular domain of Notch. Our results suggest that Uif can alter the accessibility of the Notch extracellular domain to its ligands during Notch activation.</p> <h3>Conclusions/Significance</h3><p>Our study shows that Uif can modulate Notch activity, illustrating the importance of a delicate regulation of this signaling pathway for normal patterning.</p> </div

New insights into the ambivalent role of YAP/TAZ in human cancers

Abstract Hippo signaling was first identified in Drosophila as a key controller of organ size by regulating cell proliferation and anti-apoptosis. Subsequent studies have shown that this pathway is highly conserved in mammals, and its dysregulation is implicated in multiple events of cancer development and progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) (hereafter YAP/TAZ) are the downstream effectors of the Hippo pathway. YAP/TAZ overexpression or activation is sufficient to induce tumor initiation and progression, as well as recurrence and therapeutic resistance. However, there is growing evidence that YAP/TAZ also exert a tumor-suppressive function in a context-dependent manner. Therefore, caution should be taken when targeting Hippo signaling in clinical trials in the future. In this review article, we will first give an overview of YAP/TAZ and their oncogenic roles in various cancers and then systematically summarize the tumor-suppressive functions of YAP/TAZ in different contexts. Based on these findings, we will further discuss the clinical implications of YAP/TAZ-based tumor targeted therapy and potential future directions. Graphical Abstrac

A single-cell atlas of adult Drosophila ovary identifies transcriptional programs and somatic cell lineage regulating oogenesis.

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, we identified the transcriptional trajectory of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. We further identify expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to our identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types

Uif* enhances <i>cis</i> inhibition of Notch signaling by its ligands.

<p>(A and A′) <i>dpp-Gal4>UAS-Ser</i> leads to both <i>cis</i> inhibition (in the inner region of the <i>dpp-Gal4</i> expressing, GFP⁺ domain in the ventral part of the disc; marked by the asterisk) and <i>trans</i> activation of Wg (in cells neighboring to the <i>dpp-Gal4</i> expressing domain in the ventral compartment of wing disc; marked by arrowheads). The <i>cis</i> inhibition is incomplete and, thus, Wg expression (arrows) is detected in the outer region of the <i>dpp-Gal4</i> expressing domain. Coexpession of Uif* enhances <i>cis</i> inhibition, leading to Wg reduction inside the <i>dpp-Gal4</i> expressing domain, without affecting <i>trans</i> activation (arrowheads in B and B′). Expression of Dl by <i>dpp-Gal4</i> causes Wg expression mainly in the dorsal compartment both inside and outside of the <i>dpp-Gal4</i> regions (C). Wg protein level inside of the <i>dpp-Gal4</i> expressing domain is lower, reflective of <i>cis</i> inhibition (arrow in C). When Uif* is coexpressed, this <i>cis</i> inhibition is enhanced, leading to a nearly complete loss of Wg expression inside of the <i>dpp-Gal4</i> expression domains (asterisks in D and D′), without affecting <i>trans</i> activation (outside of <i>dpp-Gal4</i> expression domain; arrowheads in D and D′). GFP (green) marks the domain where <i>dpp-Gal4</i> is expressed (A′, B′, C′ and D′). See the main text for further details.</p

Full-length wt Uif antagonizes Notch signaling.

<p>Overexpression of wt Uif by <i>dpp-Gal4</i>><i>GS11655</i> leads to a significant reduction in the levels of both Cut and, to a lesser degree, Wg. (A) and (C) show Cut and Wg expression patterns in wing discs from the <i>dpp-Gal4/+</i> control flies, respectively. (B) and (D) show the Cut and Wg levels in wing discs from <i>dpp-Gal4</i>><i>GS11655</i> flies, respectively (see regions pointed by arrows). GFP in (A′–D′) shows the expression pattern of <i>dpp-Gal4</i>. A notched wing detected in a <i>dpp-Gal4</i>><i>GS11655</i> adult fly (F), compared with a <i>dpp-Gal4/+</i> control wing (E). Flies were reared at 29°C.</p

Uif* reduces the expression of Notch target genes.

<p>Expression of Notch target genes, Cut (A and B), Wg (C and D), vg^BE-lacZ (E and F) and E(spl)mβ–lacZ (G and H), in the third instar wing discs of wild type larvae, with (B, D, F and H) or without (A, C, E and G) Uif* overexpression. Genotypes are: (A and C) <i>dpp-Gal4 UAS-GFP/+</i>; (B and D) <i>dpp-Gal4 UAS-GFP/UAS-Uif</i>*; (E) <i>dpp-Gal4 UAS-GFP/vg^BE-lacZ</i>; (F) <i>dpp-Gal4 UAS-GFP/vg^BE-lacZ UAS-Uif</i>*; (G) <i>E(spl)mβ–lacZ/+; dpp-Gal4 UAS-GFP/+</i> and (H) <i>E(spl)mβ–lacZ/+; dpp-Gal4 UAS-GFP/UAS-Uif*</i>. Arrows indicate a loss or a decreased expression of the Notch target genes at the AP boundary of the wing discs where Uif* was expressed under the control of <i>dpp-Gal4</i> (B, D, F and H).</p

The inhibitory effect of Uif* is dependent on the extracellular domain of Notch.

<p>Ectopic expression of the full-length Notch (N^FL) under the control of <i>dpp-Gal4</i> induces aberrant Wg (red) expression at the AP boundary where it intersects with the DV boundary (white arrow in A) (A and A′). GFP (green) marks <i>dpp-Gal4</i> positive cells (A′, B′, C′ and D′). Coexpression of Uif* with N^FL reduces the ectopic induction of Notch signaling mediated by N^FL at the intersection between AP and DV boundaries (white arrow in B) (B and B′). Ectopic expression of the membrane tethered active version of Notch (N^ECN) induces Wg (red) expression in the <i>dpp-Gal4</i> region that is marked by GFP (green) (C and C′). Coexpression of Uif* does not alter the Wg expression that is induced by N^ECN (D and D′).</p

Ectopic expression of Uif* causes phenotypes that are characteristic of Notch signaling defects.

<p>(A) A wt adult wing. (B and C) Targeted Uif* expression under the control of <i>en-Gal4</i> causes loss of wing margin structures in the posterior wing compartment. These two panels show different expressivity, ranging from a partial loss of wing margin (arrow in B) to an almost complete loss of the posterior wing margin (black line in C). (D) A <i>dpp-Gal4>Uif*</i> adult wing shows wing margin loss (arrow) at the most distal tip area of the wing and an occasional loss of the anterior cross vein (arrowhead). (E and F) Thickened veins, which resemble an aspect of the Notch loss of function phenotypes (particularly veins III and V, arrows), observed in adult wings of <i>A9-Gal4</i>><i>Uif*</i> (E) and <i>MS1096-Gal4>Uif*</i> (F) flies. (G–I) Expression of Uif* in the notal region causes losses of sensory bristles. (G) A wt adult notum with a regular pattern of sensory bristles. (H) The notum of <i>Eq-Gal4>Uif*</i> flies shows random losses of microchaeta (asterisks). (I) Expression of Uif* in the notum controlled by <i>pnr-Gal4</i> leads to a great loss of sensory bristles (rectangle).</p

Expression of Notch target genes rescues Uif*-induced defects.

<p>(A) A wt wing. Expression of Uif* under the control of <i>A9-Gal4</i> causes thickened vein phenotype with broadened veins III and V (arrows in B). This defect can be significantly alleviated by coexpression of a Notch downstream component, E(spl)mβ (arrows in D). (C) shows control wing of <i>A9-Gal4</i>>E(spl)mβ flies. A small and rough eye phenotype (F) in <i>GMR-Gal4</i>><i>Uif*</i> flies is significantly rescued by coexpression of E(spl)m7 (H). (E) and (G) show control eyes of <i>GMR-Gal4/+</i> and <i>GMR-Gal4>E(spl)m7</i> flies, respectively.</p