Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells

Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). We demonstrate here that loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero‐endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type‐specific transcriptome analysis combined with Dam‐ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU‐domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation‐promoting factors, such as Pdm1

Regional Cell-Specific Transcriptome Mapping Reveals Regulatory Complexity in the Adult Drosophila Midgut

SummaryDeciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1–R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, we generated RNA-sequencing transcriptomes of all five cell types isolated by FACS from each of the five regions, R1–R5. In doing so, we identify transcriptional diversities among cell types and document regional differences within each cell type that define further specialization. We validate cell-specific and regional Gal4 drivers; demonstrate roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation, and study the transcriptional response of midgut cells upon infection. The resulting transcriptome database (http://flygutseq.buchonlab.com) will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions

Robustness of glycolysis in yeast to internal and external noise

Gehrmann E, Gläßer C, Jin Y, Sendhoff B, Drossel B, Hamacher K. Robustness of glycolysis in yeast to internal and external noise. Physical Review E. 2011;84(2): 021913.Glycolysis is one of the most essential intracellular networks, found in a wide range of organisms. Due to its importance and due to its wide industrial applications, many experimental studies on all details of this process have been performed. Until now, however, to the best of our knowledge, there has been no comprehensive investigation of the robustness of this important process with respect to internal and external noise. To close this gap, we applied two complementary and mutually supporting approaches to a full-scale model of glycolysis in yeast: (a) a linear stability analysis based on a generalized modeling that deals only with those effective parameters of the system that are relevant for its stability, and (b) a numerical integration of the rate equations in the presence of noise, which accounts for imperfect mixing. The results suggest that the occurrence of metabolite oscillations in part of the parameter space is a side effect of the optimization of the system for maintaining a constant adenosine triphosphate level in the face of a varying energy demand and of fluctuations in the parameters and metabolite concentrations

Robustness of glycolysis in yeast to internal and external noise

Glycolysis is one of the most essential intracellular networks, found in a wide range of organisms. Due to its importance and due to its wide industrial applications, many experimental studies on all details of this process have been performed. Until now, however, to the best of our knowledge, there has been no comprehensive investigation of the robustness of this important process with respect to internal and external noise. To close this gap, we applied two complementary and mutually supporting approaches to a full-scale model of glycolysis in yeast: (a) a linear stability analysis based on a generalized modeling that deals only with those effective parameters of the system that are relevant for its stability, and (b) a numerical integration of the rate equations in the presence of noise, which accounts for imperfect mixing. The results suggest that the occurrence of metabolite oscillations in part of the parameter space is a side effect of the optimization of the system for maintaining a constant adenosine triphosphate level in the face of a varying energy demand and of fluctuations in the parameters and metabolite concentrations

Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long-Term Response of Arabidopsis to Light Quality Shifts

International audienceNatural illumination conditions are highly variable and because of their sessile life style plantsare forced to acclimate to them at cellular and molecular level. Changes in light intensity orquality induce changes in the reduction/oxidation (redox) state of the photosynthetic electronchain that act as trigger for compensatory acclimation responses comprising functional andstructural adjustments of photosynthesis and metabolism. Such responses include redoxcontrolledchanges in plant gene expression in nucleus and organelles. Here we describe astrategy for the identification of early redox-regulated genes (ERGs) in the nucleus of themodel organism Arabidopsis thaliana which significantly respond 30 or 60 min after thegeneration of a reduction signal in the photosynthetic electron transport chain. By comparingthe response of wild-type plants with that of the acclimation mutant stn7 we could specificallyidentify ERGs. The results reveal a significant impact of chloroplast redox signals on distinctnuclear gene groups including genes for the mitochondrial electron transport chain,tetrapyrrole biosynthesis, carbohydrate metabolism and signalling lipid synthesis. Theseexpression profiles are clearly different from that observed in response to reduction of thephotosynthetic electron transport (PET) by high light treatments. The identified ERGs, thus,are unique to redox imbalances in PET and were used for the analysis of potential redoxresponsivecis-elements, trans-factors and chromosomal regulatory hot spots. The dataidentify a novel redox-responsive element and indicate extensive redox control attranscriptional and chromosomal levels that point to an unprecedented impact of redox signalson epigenetic processes

Cic targets genes in ISCs found by DamID-Seq.

<p>(A) Graph showing the location of Cic binding relative to annotated transcript TSSs. The distance is from the summit of the Cic peaks to the nearest TSS. Dashed red line showed the summit of the graph is 500bp away from TSS. (B) Box plot showing fold change of peaks in CicDamID and <i>P</i>.<i>e</i>. infected CicDam. (C) Heatmap showing fold enrichment of Cic peaks from Cic DamID-Seq without or with <i>P</i>.<i>e</i>. infection. Y axis represents genes associated with the Cic binding peaks. (D) Expression heatmap of cell cycle regulators and DNA replication related genes from RNA-Seq data from <i>cic-RNAi</i> expressing FACS sorted progenitor cells. The names of the genes that had Cic binding sites by DamID are written in green. (E) Venn diagram showing the overlap between genes upregulated > 1.5 fold upon <i>cic-RNAi</i> (left) and genes associated with Cic binding peaks (right) in ISC/EBs. (F) Graph showing correlation between genes upregulated in Cic-depleted progenitor cells, and the Cic-DamID peaks that changed significantly upon <i>P</i>.<i>e</i>. infection (upper panel). Lower panel show genes ranked by absolute expression change, and then plotted for expression fold change (bottom). (G, H) Cic binding sites in the <i>CycE</i> and <i>stg</i> loci, as determined by Cic DamID-Seq in ISC/EBs from control (above) and <i>P</i>.<i>e</i>. infected (below) midguts. Vertical bars represent the log2 ratio of the Dam-fusion signal to the Dam-only signal. Red arrows indicate TGAATG(G/A)A motifs. (I-K) mRNA level fold changes of <i>stg</i> and <i>CycE</i> analyzed by qRT-PCR and normalized to <i>β-Tub</i> and <i>Rp49</i>. (I) <i>stg</i> and <i>CycE</i> fold enrichment from whole midguts after knocking down or over expressing <i>cic</i> in all cells using the <i>tub</i>^<i>ts</i><i>(tubGal4; tubGal80ts)</i> driver. Transcription of both <i>stg</i> and <i>CycE</i> was induced in <i>cic</i> knock-down midguts and inhibitied in <i>cic</i> over-expressing midgut. (J) <i>stg</i> and <i>CycE</i> expression is upregulated in <i>cic</i>-depleted, FACS-sorted progenitor cells (ISC &EB) and ISCs. (K) <i>stg</i> and <i>CycE</i> expression fold change in <i>cic</i> over expressing midguts after <i>P</i>.<i>e</i>. infection. The induction of <i>stg</i> and <i>CycE</i> by <i>P</i>.<i>e</i>. infection was suppressed by <i>cic</i>^<i>ΔC2</i> overexpression. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Error bars in each graph represent standard deviations.</p

Cic controls ISC proliferation by regulating <i>pnt</i> transcription.

<p>(A–E) Effects caused by <i>pntP1</i> overexpression and RNAi’s. Transgenes were induced using the <i>esg</i>^<i>ts</i> system at 29°C for 4 days, and samples were stained for GFP (green), DNA (blue) and mitoses (PH3, red). White arrows pointing out PH3 signals. (A) Control adult midgut. (B) <i>pntP1</i> overexpressing midgut after 4 days induction at 29°C. (C) Control midgut after 12 hours <i>P</i>.<i>e</i>. infection. (D) <i>pnt</i> knockdown midgut after 12 hours <i>P</i>.<i>e</i>. infection. Fewer GFP+ and PH3+ cells are observed. (E) <i>Ets21C</i> overexpressing midgut, showing more PH3+ ISCs (arrows) and GFP+ ISCs and EBs (green). (F-H) Ectopic expression or loss of <i>pnt</i> bypasses ISC phenotypes caused by <i>cic</i> overexpression or depletion. (F) <i>pnt</i> and <i>cic</i>^<i>ΔC2</i> co-over-expressing midgut after 4 days induction at 29°C. GFP+ progenitor cells were still able to proliferate. (G) <i>cic</i> knockdown adult midgut and (H) <i>pnt</i>, <i>cic</i> double knockdown midgut. The increased number of progenitor cells marked by GFP upon <i>cic</i> knockdown was decreased by also knocking down <i>pnt</i>. (I-L) Quantification of PH3+ cells in adult midguts of the indicated genotypes. (I) <i>pntP1</i>, <i>pntP2</i> or <i>Ets21C</i> overexpression driven by <i>esg</i>^<i>ts</i> or <i>Dl</i>^<i>ts</i>. All the <i>pntP1</i>, <i>pntP2</i> and <i>Ets21C</i> overexpressing midguts contained more dividing ISCs. (J) <i>pnt</i> or <i>Ets21C</i> knockdown midguts after <i>P</i>.<i>e</i>. infection. ISC mitoses caused by <i>P</i>.<i>e</i>. infection were reduced in <i>pnt</i> or <i>Ets21C</i> knockdown midguts. (K) <i>pnt</i> and <i>cic</i> knock down using <i>Dl</i>^<i>ts</i> system. Fewer mitotic ISCs were observed in the <i>pnt</i> and <i>cic</i> double knockdown midgut than the <i>cic</i> knockdown midgut. (L) <i>pnt</i> and <i>cic</i>^<i>ΔC2</i> co-overexpressing midguts. <i>cic</i>^<i>ΔC2</i> overexpression could not inhibit ISC mitoses caused by <i>pnt</i> overexpression. (M) Quantification of PH3+ cells from adult midguts following <i>P</i>.<i>e</i>. infection. <i>MEK-RNAi</i> completely blocked infection-driven ISC mitoses, but could not inhibit ISC proliferation driven by overexpressed Ets21c. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Error bars in each graph represent standard deviation. Scale bars represent 50μm.</p

Cic regulates ISC proliferation as a downstream effector of EGFR signaling.

<p>(A–C) Results of the <i>λtop</i> and <i>cic</i> epistasis tests, carried out using the <i>esg</i>^<i>ts</i><i>F/O</i> system, to co-express the indicated transgenes with GFP for 2 days at 29°C. (A) Control of adult midgut (B) <i>λtop</i> overexpresssing midgut (C) <i>λtop</i> and <i>cic</i>^<i>ΔC2</i> co-overexpresssing midgut. GFP+ clones (green) expressing <i>λtop</i> were much smaller when <i>cic</i>^<i>ΔC2</i> was co-overexpressed. Samples stained with anti-PH3 (red) and DAPI (blue). (D-F) Results of the epistasis test between <i>cic</i> and <i>egfr</i>, carried out using the <i>esg</i>^<i>ts</i> system to express the indicated transgenes for 4 days at 29°C. (D) Control adult midgut, (E) <i>Egfr-RNAi</i> expressing midgut, (F) <i>Egfr-RNAi</i> and <i>cic-RNAi</i> co-expressing midgut. The number of GFP+ cells (green) still promoted by depleting <i>cic</i> in EGFR/Ras inactivated background. Samples were stained with anti-PH3 (red) and DAPI to visualize nuclei. (G-H) Results of epistasis tests between <i>Ras</i>^{<i>V12S35</i>} and <i>cic</i>, carried out using the <i>esg</i>^<i>ts</i><i>F/O</i> system. The transgenes were induced for 2 days at 29°C (G) <i>Ras</i>^{<i>V12S35</i>} over-expressing midgut (H) <i>Ras</i>^{<i>V12S35</i>} and <i>cic</i>^<i>ΔC2</i> co-over expressed midgut. Size of GFP+ clones (green) in <i>Ras</i>^{<i>V12S35</i>} and <i>cic</i>^<i>ΔC2</i> co-overexpressing midgut was significantly reduced. Samples were stained with anti-PH3 (red) and DAPI to visualize nuclei. (I-K) ISC mitoses as quantified by scoring PH3+ cells. (I) Quantification of ISCs mitoses for the <i>λtop</i> and <i>cic</i> epistasis test. The increase in mitoses induced by <i>λtop</i> was completely suppressed by <i>cic or cic</i>^<i>ΔC2</i> over expression. (J) Quantification of ISC mitoses from <i>Ras</i>^{<i>V12S35</i>}/<i>cic</i> epistasis tests. The increase in mitosis induced by <i>Ras</i>^{<i>V12S35</i>} was partially suppressed by <i>cic or cic</i>^<i>ΔC2</i> over expression. (K) Quantification of ISCs mitosis in <i>cic</i> and either <i>Egfr or Ras</i> double knock down midguts. The increase in ISC mitoses induced by <i>cic-RNAi</i> is still observed when either <i>Egfr</i> or <i>Ras RNAi</i> is also expressed. Error bars represent standard deviations. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Scale bars represent 50μm (A-H).</p

<i>cic</i> inactivation promotes ISC proliferation and hyper-activation inhibits ISC proliferation.

<p>(A,B) Knock down of Cic in ISCs using the <i>esg</i>^<i>ts</i><i>; Su(H)-Gal80</i> system. ISCs were marked by YFP (green). Samples were stained with anti-PH3 (red) for mitosis and DAPI (blue) for DNA. (A) Control adult midgut (B) Cic knock down midgut after 4 days induction 29°C. Increases in the number of YFP+ cells are observed in <i>cic</i> depleted midguts as was a large increase in mitotic cells. (C) Midguts were scored for PH3+ cells after 4 days of induction of <i>cic-RNAi</i>. A strong increase in numbers of ISC mitosis was observed in <i>cic</i> knockdown midguts. (D) Clone areas of cic mutant and control WT clones 10, 20, and 30 days after clone induction. Mutant ISCs divided faster and generated bigger clones. (E) Increased number of cells per clone was detected in <i>cic</i> mutant clones. Data was quantified 10 days after <i>cic</i> mutant clones were generated with the MARCM system. (F) Quantification of pH3-positive cells per adult midgut of the indicated genotype. <i>cic</i> transheterozygotes contained significantly more mitotic cells than controls. (G) Quantification of ISC proliferation after 12 hours <i>P</i>.<i>e</i>. infection. A decreased number of PH3+ cells, representing dividing ISCs, was observed in midguts overexpressing either <i>cic</i> or <i>cic</i>^<i>ΔC2</i> after <i>P</i>.<i>e</i>. infection. (H-J) Clones generated by the <i>esg</i>^<i>ts</i><i>F/O</i> system are marked with GFP (green), Cic over-expression was confirmed by anti-Cic (red) staining, and nuclei were visualized by DAPI (blue) staining. (E) Control adult midgut 12 days after clone induction (F) midgut overexpressing Cic (G) midgut overexpressing Cic^ΔC2 12 days after clone induction. The size of clones marked by GFP was reduced after Cic or Cic^ΔC2 overexpression. Statistical significance was determined by Student’s t test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Error bars represent standard deviations. Scale bars represent 20 μm in A-B and 50 μm in E-G.</p