Journal: bioRxiv

Article Title: Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification

doi: 10.1101/2025.11.21.689716

Figure Lengend Snippet: Loss of trx in ISCs skews cell divisions toward the EE fate Flies carrying cell-specific Gal4 drivers were crossed either to UAS-trx-RNAi to deplete trx or to w1118 to generate the corresponding control genotype. Young mated female flies were dissected 7 days post temperature shift at 29°C and immunostainings were performed for the ISC marker Delta (Dl, cytoplasmic puncta) and the enteroendocrine (EE) cell marker Prospero (Pros, solid nuclear staining). Scale bar: 20 µm. (A, B) Immunostaining of posterior midguts from control and UAS-trxRNAi (BDSC#33703) flies carrying the ISC/EB/EEP driver esg ts > GFP. Number of midguts: n = 12 (control) and n = 12 (trxRNAi). Arrows mark enteroendocrine cells (EE, Pros⁺). (C, D) Immunostaining of UAS-trxRNAi expression (BDSC#33703) using the ISC-specific esg-Gal4 ts >GFP; Su(H)GBE-Gal80 driver. Number of midguts: n = 10 (control) and n = 8 (trxRNAi). (E, F) Immunostaining of RNAi-mediated knockdown of trx (v37715) using the EB-specific Su(H)GBE-Gal4 ts driver. Number of midguts: n = 8 (control) and n = 12 (trxRNAi). (G) Quantification of Pros+ cells using the different Gal4 drivers expressed as percentage of total DAPI-stained cells. For each genotype, two to four zoom-in images from each midgut were randomly selected and are represented as a single dot in the graphs. Statistical analysis was performed using a two-tailed unpaired t-test. Data are presented as mean ± standard deviation (SD). (H) Quantification of Dl-positive intestinal stem cells across the Gal4 drivers crossed with w1118 or UAS-trxRNAi (BDSC#33703) shown in the images, expressed as the percentage of total DAPI-stained cells. Each dot represents one zoom-in image. Statistical significance was determined using a two-tailed unpaired t-test. Data are shown as mean ± SD. (I) GFP⁺ cells were quantified in control and trxRNAi midguts using esg ts Gal4 (marking all progenitor cells) and Su(H) ts Gal4 (marking enteroblasts), expressed as a percentage of total DAPI⁺ cells. Each dot corresponds to one zoom-in image. Statistical significance was assessed using a two-tailed unpaired t-test. Data are presented as mean ± SD. For trx knockdown, BDSC#33703 was used for the esg ts Gal4 crosses, whereas v37715 was used for the Su(H) ts Gal4 crosses.

Article Snippet: The next day, samples were washed three times (10 min each) in PT buffer (1× PBS with 0.2% Triton X-100), incubated with secondary antibodies diluted in PBT for 1 hour at room temperature in the dark, and then washed again three times in PT before being mounted in Drop-n-Stain EverBriteTM Mounting Medium with DAPI (Biotium).

Techniques: Control, Marker, Staining, Immunostaining, Expressing, Knockdown, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification

doi: 10.1101/2025.11.21.689716

Figure Lengend Snippet: (A–C) Confocal images of midguts from flies expressing esg^ts>GFP crossed with two independent trxRNAi lines (v37715 and BDSC#31092) or control (w1118), showing the increased number of Pros+ enteroendocrine (EE) cells upon trx knockdown. (D) Quantification of Pros+ cells as a percentage of total DAPI-stained nuclei from the genotypes shown in (A–C). Each point represents an individual zoom-in image; Data indicate mean ± SD.

Article Snippet: The next day, samples were washed three times (10 min each) in PT buffer (1× PBS with 0.2% Triton X-100), incubated with secondary antibodies diluted in PBT for 1 hour at room temperature in the dark, and then washed again three times in PT before being mounted in Drop-n-Stain EverBriteTM Mounting Medium with DAPI (Biotium).

Techniques: Expressing, Control, Knockdown, Staining

Journal: bioRxiv

Article Title: Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification

doi: 10.1101/2025.11.21.689716

Figure Lengend Snippet: Trx loss does not affect ISC proliferation (A, B) Immunostaining of posterior midguts from control (w1118) and UAS-trxRNAi flies (BDSC#33703) using the ISC/EB/EEP driver esg ts GFP 7 days after temperature shift to 29°C. Mitotic cells were labeled with phospho-Histone H3 (PH3,nuclear red). Scale bars: 20 µm. (C) Quantification of pH3+ positive cells representing mitotic cells in control and trx-RNAi midguts. Cells were counted from whole midguts and each dot represents a whole midgut in the quantification graph. n = 17 for control flies and n = 16 for trx-RNAi flies. Data were pooled from three individual experiments. Data represented as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. (D–G) MARCM clonal analysis comparing control (D) and trx deficient (F) clones 12 days after heat-shock induction. Immunostaining was performed for the markers Delta (Dl) and Prospero (Pros) to label ISCs and EE cells, respectively. Clones were induced by heat shock at 37 °C for 1 h. Flies were then maintained at 25 °C throughout the clonal expansion period. Higher-magnification views from control and trx mutant clones are shown in (E) and (G), respectively. Scale bars: 50 µm (overview D, F), 20 µm (zoom-in E, G). (H) Quantification of GFP+ cells per clone, showing clonal growth in control and trx mutant clones. A two-tailed unpaired t-test was used to evaluate statistical significance. Values are shown as mean ± SD. (I) Quantification of Pros+ cells expressed as a percentage within GFP-marked clones to assess the EE numbers produced upon trx loss. Statistical analysis was conducted with a two-tailed unpaired t-test. Data represented as mean ± SD. (J-K) Representative immunofluorescence images of midguts from control (J-J’) and UAS-trxRNAi (K-K’) flies (BDSC#33703) using the G-Trace system 6 days after temperature shift to 29°C. Progenitor cells are marked by both RFP and nuclear EGFP. Newly formed ECs are identified as large polyploid EGFP⁺ cells and newly formed EEs as Pros⁺, EGFP⁺ cells. DAPI marks nuclei. Scale bars: 20 µm. (L) Newly produced cells marked by nuclear GFP were quantified and expressed as a percentage of total DAPI-stained cells 6 days after the temperature shift to 29 °C to assess ISC proliferation in control and trxRNAi midguts. Data represent mean ± SD from n = 5 midguts per genotype. Statistical significance was determined using unpaired two-tailed Student’s t-test. (M) Quantification of newly formed enteroendocrine cells (EEs), shown as the percentage of Pros⁺GFP⁺ cells relative to the total number of GFP⁺ cells in control and trx-RNAi midguts. Data represent mean ± SD from n = 5 midguts per genotype. Statistical significance was determined using unpaired two-tailed Student’s t-test.

Article Snippet: The next day, samples were washed three times (10 min each) in PT buffer (1× PBS with 0.2% Triton X-100), incubated with secondary antibodies diluted in PBT for 1 hour at room temperature in the dark, and then washed again three times in PT before being mounted in Drop-n-Stain EverBriteTM Mounting Medium with DAPI (Biotium).

Techniques: Immunostaining, Control, Labeling, Two Tailed Test, Clone Assay, Mutagenesis, Produced, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification

doi: 10.1101/2025.11.21.689716

Figure Lengend Snippet: Trx loss drives excess EE differentiation in stress and aging (A-C) Representative confocal images of posterior midguts from control (A) and UAS-trxRNAi (B) flies (BDSC#33703), using the esg-Gal4tsUAS-GFP system. Flies were maintained at 29 °C for a total of 7 days to induce RNAi expression, and were fed DSS during the final 48 hours to elicit an acute inflammatory response. Midguts were stained and quantified for phosphorylated histone H3 (PH3, nuclear red) to assess mitotic activity. Counts were obtained from entire midguts. Each dot represents a whole midgut measurement; Data were pooled from three independent experiments and correspond to n=14 for control and n=16 midguts for trxRNAi. Data are shown as mean ± SD. Statistical analysis was performed using a two-tailed unpaired t-test. Scale bars: 20 µm. (D-F) Midguts from control (D) and trxRNAi (E) flies (BDSC#33703) using the esg-Gal4 driver were stained for Delta (Dl, cytoplasmic puncta) and Prospero (Pros, red nuclear) to evaluate the numbers of intestinal stem cells and enteroendocrine cells, respectively. Flies were maintained at 29 °C for a total of 7 days, with DSS feeding during the final 48 hours to model an acute inflammatory condition. Quantification of Pros+ cells (F) is expressed as a percentage of total DAPI-stained cells. Statistical significance was performed using a two-tailed unpaired t-test. Data are presented as mean ± standard deviation (SD). Scale bars: 20 µm. (G-H) Representative immunofluorescence images of midguts from control (G–G’) and trx-RNAi (H–H’) flies (BDSC#33703) analyzed with the G-TRACE system after 6 days of RNAi induction at 29°C with DSS feeding during the final 24 hours to trigger ISC divisions. Progenitor cells are labeled by RFP and nuclear EGFP, while newly differentiated ECs appear as large polyploid EGFP⁺ cells and newly differentiated EEs as double Pros⁺ EGFP⁺ cells. Nuclei are visualized with DAPI. Scale bars: 20 µm. (I-J) Quantification of ISC proliferation (I) and EE differentiation (J) in control and trx-RNAi midguts after 6 days at 29 °C, with DSS administered during the final 24h. Proliferation was evaluated by measuring newly produced cells marked by nuclear GFP, expressed as a percentage of total DAPI-stained cells (I), while EE differentiation was assessed as the proportion of newly formed EEs (Pros⁺GFP⁺) relative to the total GFP⁺ population (J). Data represent mean ± SD; statistical significance was determined using an unpaired two-tailed Student’s t-test. (K-L) Confocal images of posterior midguts from aged control and trx-RNAi flies (BDSC#33703) maintained at 29 °C for 30 days, with flies transferred to fresh food every 3–4 days. Nuclei were stained with DAPI (blue). Pros was used to label enteroendocrine (EE) cells and recently produced EEs were identified as Pros⁺GFP⁺ (EEPs, indicated by arrows). Data are shown as mean ± SD. Scale bars: 20 µm. (M-N) Posterior midgut immunostainings from aged control and trxRNAi flies (BDSC#33703) subjected to DSS-induced epithelial damage. Flies were kept at 29 °C for 30 days and transferred to fresh food every 3–4 days. On day 28, flies were fed DSS for 48 h to stimulate ISC divisions in the aged epithelium. Nuclei were visualized with DAPI (blue). EE cells were marked with Pros, and newly generated EEs (EE progenitors) were identified as Pros⁺GFP⁺ cells (arrows). Data are presented as mean ± SD. Scale bars: 20 µm. (O) Percentage of Pros⁺ cells relative to total DAPI-stained nuclei in the aged midgut epithelium of control and trxRNAi flies under normal feeding (30d 29 °C) or following a 2-day DSS treatment (30d 29 °C + DSS). Data are mean ± SD and statistical significance was determined by unpaired two-tailed Student’s t-test.

Article Snippet: The next day, samples were washed three times (10 min each) in PT buffer (1× PBS with 0.2% Triton X-100), incubated with secondary antibodies diluted in PBT for 1 hour at room temperature in the dark, and then washed again three times in PT before being mounted in Drop-n-Stain EverBriteTM Mounting Medium with DAPI (Biotium).

Techniques: Control, Expressing, Staining, Activity Assay, Two Tailed Test, Standard Deviation, Immunofluorescence, Labeling, Produced, Generated

Journal: bioRxiv

Article Title: Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification

doi: 10.1101/2025.11.21.689716

Figure Lengend Snippet: Trithorax functions upstream of proneural /Notch network (A) RT-qPCR analysis of mRNA expression levels of EE-related genes (scute, asense, phyllopod, prospero) in progenitor cells depleted of trx compared to controls. Virgin female flies carrying the esg-Gal4tsGFP driver were crossed to UAS-trx-RNAi (BDSC#33703) to induce trx knockdown, or to w1118 to generate the corresponding control genotype. Young mated females were dissected 7 days after the temperature shift to 29°C. Total RNA was extracted from two biological replicates of FACS-sorted GFP⁺ progenitors, isolated from 120 control and 120 trx-RNAi midguts per replicate. Expression levels were normalized to rpl32, as indicated. (B-M) Representative images of posterior midguts from young female flies expressing the indicated RNAi constructs under the control of esg -Gal4 ts GFP driver, following 7 days of total induction at 29 °C and 48 hours of DSS feeding to trigger ISC divisions. BDSC#33703 was used as the RNAi line to knock down trx . Flies carrying only the single-gene manipulations were obtained from the same parental crosses as those used to produce the combinatorial trxRNAi genotypes, ensuring a consistent genetic background. All genotypes were raised and handled under identical environmental conditions and DSS was administered uniformly prior to dissection. Enteroendocrine cells (EEs) were visualized by immunostaining for the EE marker Prospero (Pros, red) and nuclei were counterstained with DAPI (blue). Scale bar; 20 μm, applies to all images. (N) Quantification of EE content expressed as the percentage of Pros+ cells over total DAPI+ nuclei. Data represent mean ± SD. All experimental genotypes showed statistically significant differences compared to control (p < 0.001, two-tailed unpaired t-test).

Article Snippet: The next day, samples were washed three times (10 min each) in PT buffer (1× PBS with 0.2% Triton X-100), incubated with secondary antibodies diluted in PBT for 1 hour at room temperature in the dark, and then washed again three times in PT before being mounted in Drop-n-Stain EverBriteTM Mounting Medium with DAPI (Biotium).

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Control, Isolation, Construct, Dissection, Immunostaining, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification

doi: 10.1101/2025.11.21.689716

Figure Lengend Snippet: (A-A’) Expression of the cad-GFP reporter reveals cad expression throughout the R4 region of the intestinal epithelium, detected in all intestinal cell types. Prospero marks enteroendocrine (EE) cells, and nuclei are counterstained with DAPI. Scale bars: 50 µm (overview) and 20 µm (zoom-in). (B-B’) Expression of the sens-2 reporter line is observed throughout the R4 region of the intestinal epithelium, encompassing all intestinal cell types, including small diploid cells. Nuclei were visualized with DAPI. Scale bars: 50 µm (overview) and 20 µm (zoom-in). (C) Quantification of Pros⁺ cells (expressed as percentage of total DAPI-stained cells) in midguts after RNAi knockdown of candidate EE fate repressors (CG34376, sens-2, and caudal), compared to control (UAS-mcherryRNAi). For the double knockdown of caudal and trithorax, the RNAi lines BDSC#33703 (trithorax) and BDSC#57546 (caudal) were used. This analysis tests whether depletion of the candidates in esg+ cells produces a Pros⁺ cell expansion reminiscent of the phenotype observed in trx-depleted progenitors. Statistical comparisons between each RNAi line and the control are shown above the corresponding bars. Data are shown as mean ± SD; significance was determined by a two-tailed unpaired t-test. (D-D’’) Expression pattern of the Ptx1-GFP reporter line indicates strong expression of Ptx1 in the R3 region of the midgut epithelium and a weaker but still detectable expression in the R4 region of the posterior midgut. Prospero has been used as an EE cell marker and nuclei have been counterstained with DAPI. Representative area of middle and posterior midgut is shown. Scale bar; 150μΜ (Α-A’) and 20μΜ (Α’’).

Article Snippet: The next day, samples were washed three times (10 min each) in PT buffer (1× PBS with 0.2% Triton X-100), incubated with secondary antibodies diluted in PBT for 1 hour at room temperature in the dark, and then washed again three times in PT before being mounted in Drop-n-Stain EverBriteTM Mounting Medium with DAPI (Biotium).

Techniques: Expressing, Staining, Knockdown, Control, Two Tailed Test, Marker

Journal: bioRxiv

Article Title: Trithorax balances ISC fate decisions via Ptx1-mediated repression of EE specification

doi: 10.1101/2025.11.21.689716

Figure Lengend Snippet: Genetic validation of Ptx1 as a Trx-dependent repressor of EE fate (A-B) Confocal images of esgts>GFP midguts crossed to control (mcherryRNAi) or UAS-Ptx1RNAi (BDSC#57494), showing a significant increase in the proportion of Pros+ enteroendocrine (EE) cells under homeostatic conditions 7 days upon RNAi induction at 29°C. Scale bars: 20 µm. (C) Quantification of Pros+ cells (% of DAPI-stained nuclei) for the genotypes shown in (A-B), using two independent Ptx1RNAi lines (BDSC#57494 and v19831) and UAS-trxRNAi (BDSC#33703). To generate the corresponding control genotype, mcherryRNAi was used as an additional control to ensure that the observed trxRNAi phenotype was not specific to the standard w1118 line. All measurements are representative of at least two independent experiments. Statistical analysis was performed using t-test; Data are shown as mean ± SD. (D-G) Immunofluorescence images from posterior midguts of young female flies carrying the esg-Gal4tsGFP driver and the indicated RNAi constructs. RNAi-mediated knockdown of trx and Ptx1 was performed using BDSC#33703 and BDSC#57494, respectively. Expression was induced at 29 °C for 7 days, with flies being exposed to DSS during the last 48 hours to enhance ISC activity. Single and double genotypes were generated from the same crosses using the identical RNAi lines. All genotypes were raised and handled under the same environmental conditions, and DSS treatment was applied uniformly before dissection. Enteroendocrine cells were identified by Prospero (red), with nuclei counterstained using DAPI (blue). All panels show a 20 µm scale bar. (H) Quantification of Pros⁺ cells (panels D–G) expressed as a percentage of total DAPI-stained cells. Data points correspond to individual zoom-in images. Statistical analysis was performed using a two-tailed unpaired t-test. Data are presented as mean ± SD.

Article Snippet: The next day, samples were washed three times (10 min each) in PT buffer (1× PBS with 0.2% Triton X-100), incubated with secondary antibodies diluted in PBT for 1 hour at room temperature in the dark, and then washed again three times in PT before being mounted in Drop-n-Stain EverBriteTM Mounting Medium with DAPI (Biotium).

Techniques: Biomarker Discovery, Control, Staining, Immunofluorescence, Construct, Knockdown, Expressing, Activity Assay, Generated, Dissection, Two Tailed Test