Notch-Mediated Suppression of TSC2 Expression Regulates Cell Differentiation in the <em>Drosophila</em> Intestinal Stem Cell Lineage

<div><p>Epithelial homeostasis in the posterior midgut of <em>Drosophila</em> is maintained by multipotent intestinal stem cells (ISCs). ISCs self-renew and produce enteroblasts (EBs) that differentiate into either enterocytes (ECs) or enteroendocrine cells (EEs) in response to differential Notch (N) activation. Various environmental and growth signals dynamically regulate ISC activity, but their integration with differentiation cues in the ISC lineage remains unclear. Here we identify Notch-mediated repression of Tuberous Sclerosis Complex 2 (TSC2) in EBs as a required step in the commitment of EBs into the EC fate. The TSC1/2 complex inhibits TOR signaling, acting as a tumor suppressor in vertebrates and regulating cell growth. We find that TSC2 is expressed highly in ISCs, where it maintains stem cell identity, and that N-mediated repression of TSC2 in EBs is required and sufficient to promote EC differentiation. Regulation of TSC/TOR activity by N signaling thus emerges as critical for maintenance and differentiation in somatic stem cell lineages.</p> </div

TSC repression in EBs is sufficient for ISC differentiation.

<p>A. Loss of TSC1 (<i>Tsc1^Q87X</i>) rescues ISC tumor formation in a N^RNAi expressing background. MARCM clones; cell sizes in N, TSC1 double-mutant lineages are almost identical to wild-type clones, and ECs differentiate that acquire a brush border. GFP (MARCM clones), green; Phall (Phalloidin, highlighting the brush border), red; DAPI, blue. B. Loss of TSC1 induces polyploidy in N^RNAi loss-of-function background. Cells in MARCM clones deficient for TSC1 (<i>Tsc1^Q87X</i>) and N^RNAi have DNA content similar to wild-type ECs, compared to N^RNAI alone in which DNA content remains similar to wild-type ISCs. DNA content was analyzed by integrating DAPI intensity values across all pixels for individual nuclei using TCSNT software from Leica (arbitrary units [AU] are listed). ECs (outside of clone) and small Dl+/GFP+ cells (ISCs, within clones) were measured for WT, while EC (outside of clone) and GFP+ cells within clones were measured for N^RNAi and N^RNAi, <i>Tsc1^q87x</i>. Bar represent averages and standard deviations (N = 5–10 nuclei, from 3 independent guts), Student's Ttest. Nuclear sizes of WT/Dl+ cells and of N^RNAi/GFP+ cells show little variability, allowing precise quantification using small numbers of representative nuclei. See also quantification in C and images in E. C. The fraction of polyploid cells (defined as any cell with a nucleus that is larger than the smallest Dl+ cell) in ISC clones decreases dramatically in N loss of function conditions, and is rescued when <i>Tsc1</i> is also lost. Bar represent averages and standard deviations (N shown in each bar), Student's Ttest. D, E. Expression of Dl is reduced, while expression of <i>pdm1</i> is restored to wild-type levels in N, <i>Tsc1^q87x</i> double-mutant lineages, confirming the loss of ISC identity and the acquisition of EC identity. Quantification of the fraction of Pdm1+ cells in clones is shown in D (Averages and standard deviations, N shown in each bar; Student's Ttest), confocal micrographs of Dl (red, panels a) and Pdm1 antibody staining (red and white, panels b and c) in E. GFP green, DAPI blue. White arrowheads point to individual clones, highlighting the absence of Pdm1+ cells in N loss of function conditions and the restoration of Pdm1+ ECs when <i>Tsc1</i> is lost. While most N^RNAi/<i>Tsc1^Q87X</i> cells become polyploid, have a brush border and are Dl−, occasionally large Dl+ cells can be observed (as seen in one clone in a). F. Intestines expressing N^RNAi and TSC1 or 2^RNAi under the control of esg>Gal80^ts for 7 days. Note that loss of TSC1 or 2 reduces Dl expression in N^RNAi-expressing ISCs and EBs, indicating differentiation of ISCs. GFP (ISCs and EBs), green; Dl, red; DAPI, blue. Quantification of the sizes of individual stem cell clusters is shown on the right. Averages and SEM; Student's Ttest. N = 7 guts each, 15–40 clusters/gut.</p

Model for TSC–mediated regulation of cell differentiation in the ISC lineage.

<p>Our results identify transcriptional repression of TSC2 as a required step in the induction of EC differentiation by the Notch signaling pathway. This regulation integrates with Insulin-mediated repression of TSC2 function to promote EC growth and endoreplication. Under conditions of low Notch activation, TSC2 expression in EBs remains high and prevents growth of cells differentiating into EE cells. Similarly, in ISCs, high TSC2 expression serves as a ‘buffer’ from nutritional changes that increase IIS activity or can otherwise activate the TOR pathway, thus promoting ISC maintenance.</p

Nutrient-dependent dysfunction of TSC mutant ISCs.

<p>A. Timecourse analysis of MARCM clone growth. <i>Tsc1^q87x</i> mutant and wild type clones were induced at 3 days of age by a 45 min heat shock. The number of GFP labeled cells in each clone was determined for 5–10 guts and 30–50 clones for each timepoint. Averages and STDEV are shown. P values from Student's T-test. B. TSC1 mutant clones are progressively lost from the intestinal epithelium. Number of MARCM clones observed in each midgut at 7 and 15 d ahs is shown (Averages and SEM; Student's T-test). C, D. TSC1 mutant ISCs become dysfunctional in high-calorie food. TSC1 (<i>Tsc1^Q87X</i>) mutant ISCs from flies fed regular food (RF, 2% yeast) form larger clones (more cells per clone) compared to TSC1 mutant clones from flies fed high calorie food (HY, 15% yeast). High-yeast food also reduces clone sizes of wild-type ISCs somewhat, but these are not significantly different from wild type (compare p values determined between high-yeast and regular food conditions for each genotype). Averages and SEM are shown in this quantification. E. In most TSC mutant clones maintained on HY, Dl+ ISCs become large (right panels), while mutant clones maintained on RF retain small diploid ISCs. In wild-type clones, Dl+ cells are small and diploid independently of the food conditions. Dl red, GFP, green, DAPI, blue. F, G. MARCM clones deficient for TSC1 (<i>Tsc1^Q87X</i>) or TSC2 (<i>TSC2^RNAi</i>), or over-expressing Rheb (UAS-Rheb) contain EE cells significantly more rarely than wild-type clones. Example clones are shown in F. EE cells identified by anti-pros staining (arrowheads; Armadillo and prospero red, GFP green, and DAPI blue). Quantification in G (Averages and SEM. Student's T-test. The number of guts in which the percentage of EE containing clones was quantified is shown in each bar.</p

TSC repression in EBs is required for ISC differentiation.

<p>A. Flp-out-mediated over-expression of N^ICD promotes ISC differentiation (small clones with large, Dl− cells are formed). Co-expression of TSC1 and 2 rescues this phenotype. RFP (clone), green; Dl, red; DAPI, blue. The percentage of clones containing at least 1 Dl+ cell is quantified on the right (Averages and SEM; Student's Ttest; N shown in each bar). B. The formation of large, Dl− cells in intestines of flies expressing N^ICD in ISCs and EBs (esg::Gal4, tub::Gal80^ts) is also rescued by over-expressing TSC1 and 2. GFP (ISC and EB), green; Dl, red; DAPI, blue. Quantification shows percentage of GFP+ cells expressing Dl (Averages and SEM; Student's Ttest; N shown in each bar).</p