9 research outputs found

    Sox21a regulates intestinal regeneration.

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    <p>(<b>A-B</b> and <b>A’-B’</b>) Confocal images of unchallenged control (UN, A) and <i>Ecc15</i>-infected (B) midguts carrying <i>Sox21a-GFP</i> and <i>Su(H)-lacZ</i>. Representative ISCs (<i>Sox21a-GFP</i><sup><i>+</i></sup> <i>Su(H)-lacZ</i><sup>—</sup>) are labeled with white arrows, and EBs (<i>Su(H)-lacZ</i><sup><i>+</i></sup>) with yellow arrows. Some EBs are further indicated with stars of different colors in (B): <i>Sox21a-GFP</i><sup><i>high</i></sup> <i>Su(H)-lacZ</i><sup><i>high</i></sup> (orange), <i>Sox21a-GFP</i><sup><i>low</i></sup> <i>Su(H)-lacZ</i><sup><i>high</i></sup> (red) and <i>Sox21a-GFP</i><sup><i>negative</i></sup> <i>Su(H)-lacZ</i><sup><i>low</i></sup> (green). (<b>C</b>) Quantification of relative <i>Sox21a-GFP</i> intensity within individual ISC-EB pairs from intestines shown in A (n = 41 pairs, from 5 guts) and B (n = 54 pairs, from 7 guts). (<b>D-F</b> and <b>E’</b>) Representative intestines of wild-type flies (D) or flies over-expressing <i>Sox21a</i> in progenitors using <i>esg</i><sup><i>TS</i></sup> driver for 36 hours at 29°C (E, E’ and F). Note that Armadillo (Arm) is evenly distributed along the plasma membrane in (D) but is highly enriched at the interface between progenitor cells in (E) (arrows). Prospero (Pros, red, nuclear) marks EEs. Close-up view of a progenitor pair outlined in (E) is shown in (E’). An <i>esg>GFP</i><sup><i>+</i></sup> nest containing multiple cells is outlined in (F). (<b>G</b>-<b>H</b>) Quantification of the ratio of differentiating EBs out of all the <i>esg>GFP</i><sup><i>+</i></sup> cells (G) and the average number of cells in each <i>esg>GFP</i><sup><i>+</i></sup> nest (H) in intestines of wild-type flies (<i>esg</i><sup><i>TS</i></sup><i>>w</i>) or flies over-expressing <i>Sox21a</i> in progenitors (<i>esg</i><sup><i>TS</i></sup><i>>Sox21a</i>) for 36 hours. (<b>I-J</b>) Schematic progenitor contact categories (I) and the distribution of each contact category in intestines of wild-type flies (<i>esg</i><sup><i>TS</i></sup><i>>w</i>) or flies over-expressing <i>Sox21a</i> in progenitors (<i>esg</i><sup><i>TS</i></sup><i>>Sox21a</i>) for 36 hours (J). Nuclei are stained by DAPI. (<b>K</b>) Representative image of progenitors from <i>esg</i><sup><i>TS</i></sup><i>>Sox21a</i> flies carrying both ISC and EB markers after 36 hours of transgene expression. Progenitors are shown with <i>esg>mCherry</i> (green). Yellow arrow indicates the <i>Dl-GFP</i><sup><i>+</i></sup> cell with Notch activity. (<b>L</b>) Schematic representation of modes of ISC division and cell fate commitment during homeostasis and regeneration. The presence of strong junction between the two progenitors promotes Notch signaling allowing rapid differentiation. Green lines indicate Dl, and red fillings denote Notch activation. Each dot represents one gut.</p

    Sox21a functions downstream of JAK-STAT pathway for EB differentiation.

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    <p>(<b>A-D</b>) Representative intestines of flies with the indicated genotype observed 14 days after transgene expression using EB-specific driver <i>Su(H)GBE</i><sup><i>TS</i></sup>. <i>Su(H)GBE>GFP</i> (green) labels EBs. (<b>E</b>) Quantification of mitotic index in the midgut of flies after knocking down respective factors using EB-specific driver <i>Su(H)GBE</i><sup><i>TS</i></sup> for 14 days. (<b>F</b>) Quantification of tumor formation capacity in the midgut of flies shown in (A-D). The tumor-grading system is based on the degree of EB accumulation and examples are color-coded and denoted in (A-D). (<b>G-H</b>) Representative images of intestines with indicated genotype observed 14 days after transgene expression using the progenitor-specific driver <i>esg</i><sup><i>TS</i></sup> (G) and quantification of progenitor tumor frequency (H). The tumor-grading standard is shown in (G). <i>esg>GFP</i> (green) labels ISC/EB. (<b>I-J</b>) Representative intestines of flies with the indicated genotype observed 7 days after transfer to 29°C allowing transgene expression using <i>esg</i><sup><i>TS</i></sup>. (<b>K</b>) Quantification of mitotic index of midgut from flies with the indicated genotype. (<b>L</b>) Quantification of the frequency of intestinal progenitor tumors in <i>esg</i><sup><i>TS</i></sup><i>>Stat-IR</i> flies and <i>esg</i><sup><i>TS</i></sup><i>>Stat-IR</i> flies co-expressing <i>Sox21a</i> to induce differentiation for 14 days. Each dot represents one gut.</p

    GATAe is required in EBs for <i>Sox21a</i>-induced differentiation.

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    <p>(<b>A-B</b>) Representative intestines of wild-type flies (A) or flies over-expressing <i>GATAe</i> (B) in progenitors using <i>esg</i><sup><i>TS</i></sup> driver for 5 days. EC marker Pdm1 is shown in red. Note many differentiating progenitors already express Pdm1 (indicated by arrows). (<b>C</b>) The percentage of of <i>Pdm1</i><sup><i>+</i></sup> <i>esg>GFP</i><sup><i>+</i></sup> cells over all the <i>esg>GFP</i><sup><i>+</i></sup> progenitors in both control (<i>esg</i><sup><i>TS</i></sup><i>>w</i>) and <i>GATAe</i> overexpressing intestines (<i>esg</i><sup><i>TS</i></sup><i>>GATAe</i>). (<b>D-E</b>) Intestines of <i>Su(H)GBE</i><sup><i>TS</i></sup><i>>w</i> (control, D) and <i>Su(H)GBE</i><sup><i>TS</i></sup><i>>GATAe-IR</i> (E) flies which were shifted to 29°C to induce transgene expression for 3 days, then orally infected with <i>Ecc15</i> for 2 days and recovered for 3 additional days. <i>Su(H)GBE>GFP</i> (green) labels EBs. (<b>F-G</b>) Representative intestines of flies with the indicated genotype observed 14 days after transfer to 29°C allowing transgene expression using <i>esg</i><sup><i>TS</i></sup>. (<b>H</b>) Quantification of mitotic index of midgut from flies with the indicated genotype. (<b>I</b>) Quantification of the frequency of intestinal progenitor tumors in <i>esg</i><sup><i>TS</i></sup><i>>Stat-IR</i> flies and <i>esg</i><sup><i>TS</i></sup><i>>Stat-IR</i> flies co-expressing <i>GATAe</i> for 14 days. (<b>J-K</b>) Intestines of <i>Sox21a</i> mutant fly and <i>Sox21a</i> mutant fly carrying <i>esg</i><sup><i>TS</i></sup><i>>GATAe</i> transgenes, kept at 22°C for 20 days to allow the formation of progenitor tumors and then shifted to 29°C for 2 days to induce transgene expression. (<b>L</b>) Tumor frequency in intestines of <i>Sox21a</i> mutant and <i>Sox21a</i> mutant expressing <i>GATAe</i> in progenitors using <i>esg</i><sup><i>TS</i></sup> for 14 days at 29°C. <i>esg>GFP</i> (green) labels midgut progenitors. Each dot represents one gut.</p

    Sox21a promotes differentiation by enhancing the Dl/Notch signaling.

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    <p>(<b>A-B</b>) ISC-EB pairs in control (<i>esg</i><sup><i>TS</i></sup><i>>w</i>) and <i>Sox21a</i>-expressing (<i>esg</i><sup><i>TS</i></sup><i>>Sox21a</i>) flies. ISCs are detected with <i>Dl-GFP</i>, EBs with <i>Su(H)-lacZ</i> and progenitors with <i>esg>mCherry</i> (green). Arrows indicate ISCs in (A) and highlight the ISC-EB contact in (B). (<b>C</b>) Relative <i>Dl</i> mRNA levels in control and <i>Sox21a</i>-expressing progenitors from an RNA-seq experiment using sorted <i>esg>GFP</i><sup><i>+</i></sup> cells (n = 50 midguts/group, in two biological replicates, Robinson and Smyth Exact test, ***p < 0.001). <i>esg</i><sup><i>TS</i></sup><i>>Sox21a</i> flies were shifted to 29°C for 12 or 24 hours to induce Sox21a expression. (<b>D-E</b>) Confocal images of intestines expressing <i>Sox21a</i> alone (D) or simultaneously with a <i>Dl-RNAi</i> construct (E) using <i>esg</i><sup><i>TS</i></sup> for 36 hours. Arrows in (D) indicate some differentiating EBs, which are not present upon depleting <i>Dl</i> (E).</p

    Invasive tumor growth induced by Ct depletion is due to changes in adhesive cell properties.

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    <p>(A) Changes in expression of cell adhesion genes in 3<sup>rd</sup> instar eye-antennal imaginal discs of <i>ey</i>::<i>Dl</i>;<i>2xct<sup>RNAi</sup></i> versus <i>ey</i>::<i>Dl</i> animals identified by expression profiling experiments. Red arrows indicate reduced expression, green arrow induced expression of the respective genes in <i>ey</i>::<i>Dl</i>;<i>2xct<sup>RNAi</sup></i> animals. (B) Top: Representative pictures of tumor growth in <i>ey</i>::<i>Dl</i>;<i>βPSintegrin<sup>RNAi</sup></i> and <i>ey</i>::<i>Dl</i>;<i>αPS4integrin<sup>RNAi</sup></i> flies. Green arrowhead marks secondary tumor growth in the abdomen. Bottom: Quantification of primary and secondary tumor growth in <i>ey</i>::<i>Dl</i>;<i>αPS4integrin<sup>RNAi</sup></i>, <i>ey</i>::<i>Dl</i>;<i>βPSintegrin<sup>RNAi</sup></i>, <i>ey</i>::<i>Dl</i>;<i>αPS2integrin<sup>RNAi</sup></i> and <i>ey</i>::<i>Dl</i>;<i>Timp<sup>RNAi</sup></i> flies. (C) Relative transcript levels of <i>DE-Cad</i>, <i>Cad86C</i> and <i>Cad99C</i> in eye-antennal discs of 3<sup>rd</sup> instar larvae of control animals (<i>Dcr2; ey::lacZ</i>) and in animals with reduced Ct activity (<i>Dcr2; ey::2xct<sup>RNAi</sup></i>). (D) Quantification of secondary tumor growth rates in different genetic backgrounds. Co-expression of E-Cad strongly reduces invasive tumor growth rates in <i>eyeful</i>+<i>ct<sup>RNAi</sup>;p35</i> flies. (E) Schematic drawing of a 3<sup>rd</sup> instar larva expressing GFP in eye-imaginal discs (either <i>ey</i>::<i>GFP</i> or <i>eyeful+GFP;ct<sup>RNAi</sup>;p35</i>). Locations of GFP-labeled eye-imaginal discs and the insect circulatory fluid, the hemolymph, are indicated by arrows. For analysis of the hemolymph, the insect circulatory fluid is extracted by bleeding out the larvae after cutting at the posterior end (indicated by dashed, blue line). (F) Left: Quantification of GFP-positive cells in the hemolymph of wild-type, <i>ey</i>::<i>GFP</i> and <i>eyeful</i>+<i>GFP</i>;<i>ct<sup>RNAi</sup></i>;<i>p35</i> 3<sup>rd</sup> instar larvae. Right: Relative <i>GFP</i> transcript levels in the hemolymph of <i>ey</i>::<i>GFP</i> and <i>eyeful</i>+<i>GFP</i>;<i>ct<sup>RNAi</sup></i>;<i>p35</i> 3<sup>rd</sup> instar larvae.</p

    The Ct switch function represents a cancer prevention mechanism.

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    <p>(A–M) Adult compound eyes of the respective genotypes are shown. (L) <i>eyeful::ct<sup>RNAi</sup>; p35</i> flies show high frequency of long range metastasis (marked by yellow arrowhead), a close-up of which is shown in (M). Eyes of such <i>eyeful::ct<sup>RNAi</sup>; p35</i> flies show undifferentiated and overproliferated eye tissue (marked by light blue arrowhead). (N) Quantification of primary and secondary tumor formation in different genetic backgrounds. (O) Relative transcript levels of selected genes involved in cell cycle control, DNA damage response, growth control and epigenetic regulation in eye-antennal discs of 3<sup>rd</sup> instar larvae of pre-oncogenic control animals (<i>ey::Dl</i>) and animals with reduced Ct activity (<i>ey::Dl;2xct<sup>RNAi</sup></i>). (P) Expression of the apoptosis marker Caspase-3 (Casp-3) and the proliferation marker Phosphorylated histone H3 (PH3) in representative 3<sup>rd</sup> instar eye-antennal discs of <i>ey::Dl</i> and <i>ey::Dl;2xct<sup>RNAi</sup></i> animals. An increase in Casp-3 and PH3 positive cells is seen in the area below the dashed, yellow line highlighting the morphogenetic furrow. (Q) Top panel: representative pictures of eyes from <i>ey::Dl;PI3K<sup>RNAi</sup></i> and <i>ey::Dl;2xct<sup>RNAi</sup>;PI3K<sup>RNAi</sup></i> animals. Bottom panel: quantification of tumorous eye growth, secondary tumor growth and “small eye” phenotype in <i>ey::Dl;2xct<sup>RNAi</sup></i> and <i>ey::Dl;2xct<sup>RNAi</sup>;PI3K<sup>RNAi</sup></i> and <i>ey::Dl;PI3K<sup>RNAi</sup></i> animals.</p

    Model of cancer prevention mechanism by cell fate specifying transcription factors like Cut.

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    <p>(A) During normal development, cell-type specification factors like Cut ensure the survival of cells by repressing apoptosis while at the same time these factors also induce a specific differentiation program, which generates cells with a specific terminal cell fate. (B) In the case of a mutation in a cell-type specification factor those cells unable to differentiate, which are potentially harmful to the organism, are removed by releasing apoptosis repression conferred by the same cell-type specification factor. Thus, the transcriptional coupling of differentiation and apoptosis regulation represents a very fast and efficient cancer prevention mechanism. (C) Together with other mutations creating a sensitized background, like the over-activation of the Notch (N) signaling pathway, cells that acquire the inability to differentiate and a resistance to apoptosis activation, two important hallmarks of cancer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002582#pgen.1002582-Hanahan1" target="_blank">[1]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002582#pgen.1002582-Harris1" target="_blank">[2]</a>, very easily develop into cancer cells.</p

    General function of Ct in apoptosis repression and induction of differentiation.

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    <p>(A, E, I) Scanning electron micrographs of individual ommatidia of adult <i>Drosophila</i> fly eyes with indicated genotypes are shown. The closed, red arrowheads in (A) mark interommatidial bristles, the open, red arrowheads in (E) mark the absence of these structures. The closed, light red arrowheads in (I) indicate the presence of tissue that would normally develop into interommatidial bristles. (B, F, J) Projections of consecutive confocal sections of one ommatidium of 50 h pupal retinas labeled with DE-Cadherin. Interommatidial bristles are marked by red, closed arrowheads in (B). Open arrowheads in (F) mark absence of DE-Cad, light-red arrowheads in (J) mark reduced DE-Cadherin levels in shaft cells of interommatidial bristles. (C, G) Projections of consecutive confocal sections of one ommatidium of 50 h pupal retinas of <i>GMR::lacZ</i> control (C) and <i>GMR::ct<sup>RNAi</sup></i> flies (G). (D, H) Expression of the apoptosis marker Caspase-3 (Casp-3) in 3<sup>rd</sup> instar eye-antennal discs of control <i>Dcr2; ey::lacZ</i> (D) and <i>Dcr2; ey::2xct<sup>RNAi</sup></i> (H) animals. Yellow asterisks in (H) mark Casp-3 positive cells in <i>Dcr2; ey::2xct<sup>RNAi</sup></i> eye imaginal discs. (K) Relative mRNA expression levels of <i>rpr</i>, <i>grim</i>, <i>Wrinkled</i> (<i>W</i>) and <i>sickle</i> (<i>skl</i>) in 3<sup>rd</sup> instar eye-antennal discs of control <i>Dcr2; ey::lacZ</i> and <i>Dcr2; ey::2xct<sup>RNAi</sup></i> animals.</p

    Cut directly represses <i>rpr</i> and apoptosis in the PS primordium.

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    <p>(A) Posterior spiracle (PS) of a 1<sup>st</sup> instar wild-type <i>Drosophila</i> larva. The filzkörper is highlighted by red asterisks. (B, C) <i>rpr</i> mRNA (green) expression in stage 11 wild-type (B) and <i>ct</i> mutant (C) embryos. Spalt (Sal) protein (blue) labels stigmatophore precursor cells, Cut (Ct) protein (red, nuclear) marks spiracular chamber and filzkörper precursor cells and the apical membrane marker Crb (red) outlines the cells. Small, green arrows in (C) mark <i>rpr</i> positive spiracular chamber and filzkörper precursor cells in the eighth abdominal segment (A8) of <i>ct</i> mutant embryos. (D, E) Over-expression of the apoptosis sensor UAS-<i>Apoliner</i> using the <i>arm</i>-GAL4 driver in stage 11 wild-type (D) and <i>ct</i> mutant (E) embryos. Small, green arrows in (E) mark apoptotic cells in PS precursor cells (A8) of <i>ct</i> mutant embryos. (F, G) TUNEL stainings in wild-type (F) and <i>ct</i> mutant (G) embryos. Closed arrowhead in (G) marks TUNEL-positive cells in <i>ct</i> mutants, which are absent in wild-type embryos (F). (H, I) Co-localization of GFP protein and <i>rpr</i> mRNA (H) or Cut protein (I) in stage 15 <i>rpr</i>-HRE-571 embryos. White circles mark the PS primordium. (J) Top: conservation blot of <i>rpr</i>-HRE-571 genomic region obtained from the UCSC genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). Species used for generating blot are also shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002582#pgen.1002582.s002" target="_blank">Figure S2A</a>. Bottom: diagram of the <i>rpr</i>-HRE-571 deletion constructs tested. (K) EMSA using S2 sub-fragment with Ct binding sites either in wild-type (wt probe) or mutated (mut. probe) version and no protein (−), purified MBP protein (M), and purified Cut-MBP fusion protein consisting of the Cut repeat 3 and the Cut homeodomain (C). The black arrowheads indicate the specific DNA-protein complexes. Loading of equal amounts of labeled wild-type and mutated oligonucleotides is illustrated by formation of comparable amounts of unspecific DNA-protein complexes (black arrow). (L–O) Reporter gene expression in the PS of stage 15 embryos driven by the fragments described above. In the S2-Ctbs-GFP, line Ct binding sites within the <i>rpr</i>-HRE-571-S2 fragment are mutated. Spalt (Sal) and Cut (Ct) proteins label stigmatophore (blue) or spiracular chamber and filzkörper cells (red). Closed, yellow arrowheads in (N) and (M) mark reporter gene expression in filzkörper cells, whereas open, yellow arrowheads in (L) and (M) mark missing GFP expression.</p
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