9 research outputs found
Additional file 1: Figure S1. of Improved transcription and translation with L-leucine stimulation of mTORC1 in Roberts syndrome
Quantitation of Western blotting in Fig. 1a and b. Figure S2. Gene transcription and translation pattern of WT cells and ESCO2-Corrected cells. Figure S3. The boxplots display the show expression of mRNAs with 5’TOP sequences (a), PRTE sequences (b), and Babel genes (c). Figure S4. Motif and GO terms associated with genes with increased translational efficiency upon L-leucine treatment in RBS cells. Figure S5. mTORC2 show regulated genes do not show a coherent response to L-leucine. Figure S6. GTL2-regulated miRNAs are increased in RBS cells independent of L-Leu. Figure S7. Homeobox (HOX) gene expression is reduced in RBS cells. (ZIP 4.64 MB
A 2.8 kb fragment of the dpp enhancer is sufficient for activation and repression of <i>dpp</i> in the leg disc.
<p>A: Schematic representation of the <i>dpp</i> locus and the 6 enhancer fragments used in this study. The <i>dpp</i> transcription unit is centered around 86 kb (arrow). [Map coordinates (in kilobases) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000142#pone.0000142-Blackman1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000142#pone.0000142-Bergstrom1" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000142#pone.0000142-St.Johnston1" target="_blank">[53]</a>. The leg disc enhancer is located between 20–30 kb downstream of the <i>dpp</i> coding region. Filled stars represent dTCF-binding sites confirmed by footprinting, open stars are predicted sites and pentagons are BRK binding sites. Arrowheads indicate fusion to the ß galactosidase reporter gene. APRD refers to the 4 relevant domains A (region required for Activation), P (proximal TCF sites), R (repressor domain), D (distal TCF sites). B–E: 3rd instar leg imaginal discs with dorsal up and anterior to the left. B: Normal <i>dpp</i> mRNA expression detected by <i>in situ</i> hybridization. Bracket indicates ventral region, where <i>dpp</i> is repressed. C: A 10 kb <i>dpp</i> enhancer fragment (BS3.0) drives expression of lacZ in a stripe that recapitulates normal <i>dpp</i> expression including ventral repression (bracket). D: Expression driven by the 2.8 kb APRD <i>dpp</i> enhancer fragment mimics <i>dpp</i> mRNA and BS3.0 expression. Again, note ventral repression (bracket). E: Ventral repression is lost (bracket) in the 2.3 kb APR- fragment which has a 500 bp region of APRD that contains the distal cluster of dTCF binding sites (D) deleted. F: An 800 bp fragment (AP--, BS3.1) containing the proximal cluster of dTCF sites (P) is not sufficient for ventral repression (bracket). G: The AP-D fragment does not show ventral repression (bracket). Sequences in the 1.4 kb between the proximal and distal dTCF sites do not contain dTCF sites but are required for ventral repression.</p
The <i>dpp</i> enhancer responds to WG signaling
<p>A–D: 3rd instar leg imaginal discs. Dorsal is up, anterior is to the left. Expression of the 2.8 kb APRD reporter fragment is monitored by β-galactosidase activity. A: In wild type leg discs (mesothoracic shown), APRD>LacZ expression is repressed in the ventral region (bracket). B: WG signaling is required for ventral repression. In a pair of everting prothoracic leg discs from a <i>wg<sup>ts</sup></i> larva, ventral repression of APRD>LacZ is lost after shifting to restrictive temperature (brackets). C: Expression of the APRD reporter is repressed ventrally in Hs>Gal4; UAS>DNdTCF animals reared at 18° (bracket). DNdTCF is a dominant negative form of dTCF that cannot bind ARM. These animals and their discs are small compared to their non DNdTCF bearing sibs even when maintained continuously at low temperature, presumably due to low level expression of Hs>Gal4. However, these control animals maintained at low temperature do survive as viable, mophologically intact adults. D: When heat shocked in late third instar, repression is lost within 2.5 hours (bracket). At least 6 animals of each genotype were examined and all legs exhibited the same responses.</p
Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the <i>Drosophila</i> Ovary
<div><p>The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the <i>Drosophila</i> ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the <i>Drosophila</i> ovary to promote germ cell differentiation. <i>Piwi</i> knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of <i>dpp</i>, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress <i>dpp</i>. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the <i>piwi</i> knockdown somatic cells. Germ cell-specific knockdown of <i>piwi</i> surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the <i>Drosophila</i> ovary.</p></div
Piwi is required intrinsically to maintain PGCs or control GSC formation.
<p>(<b>A–D</b>) <i>nos-gal4</i>-driven <i>piwiKD</i> (<b>B</b>, <b>C</b>) leads to a reduction in PGC number in the third-instar larval gonads in comparison with the control (<b>A</b>). PGCs (arrows, <b>A</b>–<b>C</b>) are positive for Vasa (red) and also carry a spectrosome). <b>D</b> quantifies normal (<b>A</b>), moderate (<b>C</b>) and severe (<b>B</b>) phenotypes based on PGC numbers. (<b>E</b>, <b>F</b>) <i>nos-gal4</i>-driven <i>piwiKD</i> leads to complete germ cell loss in the germaria of the newly eclosed females, leaving empty germaria (arrows). (<b>G–I</b>) <i>nos-gal4</i>-driven <i>armi</i> (<b>H</b>) or <i>aub</i> (<b>I</b>) knockdown decreases nuclear Piwi expression in germ cells, but does not affect GSCs because the germaria still contain two or three GSCs (broken lines) as the control germarium (<b>G</b>). Nuclear Piwi expression remains in ECs (arrowheads) of the knockdown germaria (<b>H</b>, <b>I</b>). (<b>J–L</b>) <i>armi</i> (<b>K</b>) or <i>aub</i> (<b>L</b>) mutant germaria decrease nuclear Piwi expression in germ cells, but still have 2 or 3 GSCs as the control germarium (<b>J</b>). Nuclear Piwi expression remains in mutant ECs (arrowheads; <b>K</b>, <b>L</b>). Scale bars: 75 µm (<b>E</b> and <b>F</b>); 25 µm (<b>A–C</b> and <b>G–L</b>).</p
Piwi is required intrinsically to maintain GSCs and promote germ cell differentiation.
<p>(<b>A</b>) A flip-out strategy for <i>nos-gal4</i>-driven <i>piwi</i> knockdown specifically in adult GSCs and their progeny, which are also labeled by GFP expression. (<b>B</b>, <b>C</b>) GFP-marked control GSCs (circles) detected 1 d AHS (<b>B</b>) are still maintained 3 w AHS (<b>C</b>). (<b>D–H</b>) GFP-marked <i>piwiKD</i> GSCs (circles) detected 1 d AHS (<b>D</b>, <b>E</b>) are lost 3 w AHS (<b>F</b>, <b>G</b>). Consequently, the <i>piwiKD</i> germaria (arrowheads) completely lose their germ cells, and some marked GSCs have developed into GFP-positive egg chambers (arrows). <b>H</b> represents the quantitative results on the germaria containing no germ cells. (<b>I</b>, <b>J</b>) <i>piwiKD</i> germaria accumulate excess SGCs (arrow), which are negative for Piwi protein though GFP-negative, outside the GSC niche 3w AHS. As expected, all somatic cells are still positive for Piwi. (<b>K</b>) A working model for the roles of Piwi in TF/cap cells, ECs and germ cells. Scale bars: 25 µm.</p
Piwi is required in ECs to promote germ cell differentiation and maintain EC survival.
<p>Ovals indicate cap cells, whereas brackets denote the germarial region covered by ECs. (<b>A</b>) <i>c587</i> drives GFP expression specifically in ECs (arrow). (<b>B</b>) <i>c587</i>-mediated <i>piwi</i> knockdown (<i>piwiKD</i>) leads to an accumulation of many single germ cells (SGCs) carrying a spectrosome (arrowhead) mixed with differentiated cysts containing a branched fusome (arrow) in the germarium and its associated egg chamber. (<b>C</b>) Piwi protein is expressed in <i>PZ1444</i>-positive cap cells and ECs (arrow) as well as in follicle cells and germ cells. (<b>D</b>–<b>F</b>) <i>c587</i>-mediated <i>piwiKD</i> by three independent RNAi lines efficiently eliminates Piwi protein expression in <i>PZ1444</i>-positive cap cells and ECs (arrows), whereas Piwi protein expression in germ cells remains normal. (<b>G</b>–<b>I</b>) <i>c587</i>-mediated <i>piwiKD</i> by THU and HMS lines leads to the accumulation of SGCs (arrowhead) mixed with differentiated cysts (arrow). <b>I</b> shows the quantitative results on the numbers of SGCs and agametic germaria (n indicates total germaria examined). (<b>J</b>–<b>L</b>) <i>c587</i>-mediated <i>piwiKD</i> causes the formation of the germaria (arrows) containing no germ cells. (<b>M</b>–<b>P</b>) <i>c587</i>-mediated <i>piwiKD</i> (<b>O</b>, <b>P</b>) results in a significant reduction in EC numbers in comparison with the control (<b>M</b>). <b>N</b> shows the quantitative results on EC numbers (n indicates total germaria examined). Scale bars: 25 µm.</p
Piwi is required in ECs to repress transposon activity and thus prevent DNA damage.
<p>Ovals highlight cap cells. (<b>A</b>–<b>C</b>) Somatic <i>piwiKD</i> (<b>B</b>) causes an increase in γ-H2Av-positive and <i>PZ1444</i>-positive ECs in comparison with the control (<b>A</b>) in which <i>PZ1444</i>-positive ECs are negative for γ-H2Av. <b>A′</b> and <b>B′</b> highlight <i>PZ1444</i>-positive ECs in <b>A</b> and <b>B</b>, respectively. <b>C</b> represents quantitative results on γ-H2Av-positive ECs. (<b>D</b>–<b>F</b>) Quantitative RT-PCR results show that the transcripts for <i>gyspy</i> (<b>D</b>) and <i>zam</i> (<b>E</b>), but not <i>tart</i>, increase significantly in the <i>piwiKD</i> ECs in comparison with the control. (<b>G</b>–<b>J</b>) The <i>piwiKD</i> ECs (arrows, <b>H</b>–<b>J</b>) elevate <i>gypsy-lacZ</i> expression in comparison with the control ECs (arrow, <b>G</b>). (<b>K</b>–<b>M</b>) The <i>YbKD</i> ECs (arrows, <b>L</b> and <b>M</b>) lose Yb protein expression in comparison with the control ECs (arrow, <b>K</b>). (<b>N</b>, <b>O</b>) The <i>YbKD</i> ECs (arrows) elevate <i>gypsy-lacZ</i> expression. Scale bars: 25 µm.</p
Piwi knockdown in ECs results in an elevation of BMP signaling in SGCs outside the GSC niche.
<p>Asterisks indicate the GSC niche. (<b>A</b>–<b>C</b>) Some SGCs (arrows) outside the GSC niche are positive for pMad in the <i>piwiKD</i> germaria (<b>B</b>, <b>C</b>) in addition to GSCs (arrowhead) in contrast with the control germarium in which only GSCs (arrowhead) are positive (<b>A</b>). (<b>D</b>–<b>G</b>) Some SGCs (arrows) outside the GSC niche are positive for <i>Dad-lacZ</i> in the <i>piwiKD</i> germaria (<b>E</b>, <b>F</b>) in contrast with the control germarium in which only GSCs (arrowhead) are positive (<b>D</b>). <b>G</b> shows quantification results on <i>Dad-lacZ</i> expression in GSCs. (<b>H</b>, <b>I</b>) RNA sequencing (<b>H</b>) and qRT-PCR (<b>I</b>) results show that mRNA expression levels for <i>dpp</i>, but not for <i>gbb</i> and <i>dally</i>, are significantly upregulated in the <i>piwi</i> knockdown ECs compared to the control ECs (FKPM stands for fragments per kilobase of exon per millions of reads). (<b>J</b>–<b>L</b>) <i>c587</i>-mediated <i>dpp</i> knockdown can partially rescue the germ cell differentiation defects caused by <i>piwi</i> knockdown. <b>L</b> shows quantification results on percentages of germaria carrying three or more SGCs among the <i>piwiKD</i> and <i>piwi dppKD</i> germaria, which still contain at least one GSC. Scale bars: 25 µm.</p