EMBO/soda-nel huggingface dataset:
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 6

Enhanced induction of p53‐dependent apoptosis in response to irradiation in LGR5hi cells compared to LGR5lo cells

A mRNA gene expression analysis by microarray. The figure shows the number of differentially expressed genes (#dg) and the median absolute fold change (med. Abs. FC) for genes that are regulated in both LGR5hi cells and LGR5lo cells in response to IR (n = 4–5 samples per group). NIR, non‐irradiated; IR, irradiated. Wilcoxon test.
B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (B) qPCR analysis of relative mRNA expression of apoptosis‐related genes compared to GAPDH. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. (C) Representative Western blot analysis of cell lysates for the expression of phospho‐p53 and cleaved caspase‐3.
D, E Three‐month‐old mTerc+/+ mice were γ‐irradiated with 12 Gy. Small intestinal tissue was collected at 3 h after IR (n = 3 mice per group). (D) Representative picture of TUNEL staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (E) Percentage of TUNEL‐positive ISPCs at indicated positions in basal crypts. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
F–P Two‐month‐old LGR5‐GFPki, p53+/+ mice and LGR5‐GFPki, p53−/− mice were exposed to 12 Gy γ‐irradiation (n = 4 mice per group). Basal crypts were isolated at 24 h after IR. (F–H) Flow cytometry analysis of the survival rate of LGR5+ cells (F), LGR5hi and LGR5lo cells (G), and LGR5hi‐high and LGR5hi‐low cells (H) of irradiated mice (24 h after IR) compared to non‐irradiated mice (NIR). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. (I–P) Representative FACS plots of LGR5+ cells in freshly isolated basal crypts from irradiated and non‐irradiated mice of indicated genotypes. Note the stronger rescue of survival rate of LGR5hi‐high than the LGR5hi‐low cells upon p53 deletion. NIR, non‐irradiated; IR, irradiated.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 7

Chemical modification of canonical Wnt/β‐catenin signaling activity changes the radio‐sensitivity of cultured ISPCs

Crypts of 2‐ to 3‐month‐old LGR5ki mice were cultured and γ‐irradiated with 6 Gy on day 10 of culture. Sixteen hours before irradiation, cells were exposed to the indicated treatment (n = 4 independent experiments per group).

A–J Representative FACS plots of indicated treatments are given. NIR, non‐irradiated; IR, irradiated.
K–P Flow cytometry analysis of the percentage of LGR5+, LGR5hi, and LGR5lo cells of irradiated cultures (24 h after IR) compared to the percentage in non‐irradiated controls (NIR) set to 100%. (K–M) Note that inhibition of Wnt signaling by DKK1 treatment or R‐spondin reduction (50% Rspo) partially rescued the decrease in LGR5+ cells in irradiated cultures. (N–P) Note that activation of Wnt signaling by 6‐BIO treatment aggravated the decrease in LGR5+ cells in irradiated cultures. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 8

Transient inhibition of canonical Wnt/β‐catenin signaling activity decreases radio‐sensitivity of ISPCs in vivo

A–G Two‐month‐old LGR5‐GFPki, mTerc+/+ mice were i.v. injected with LRP6 neutralization antibody, and IgG serves as a negative control. Thirty‐six hours after injection, small intestinal tissue was collected for GFP immunofluorescence staining. (A–F) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm. (G) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, fifty positively stained cells were measured per mouse). Note the GFP staining intensity was substantially reduced in anti‐LRP6 antibody‐injected group with intact maintenance of ISPCs at the crypt bottom as shown by DAPI staining.
H–L Two‐month‐old mTerc+/+ mice were i.v. injected with anti‐LRP6 antibody 12 h before γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3–4 mice per group). IgG serves as a negative control. (H–K) Representative pictures of PCNA staining of small intestine of non‐irradiated mice (H, I) or 6 Gy‐irradiated mice (J, K). Note the anti‐LRP6 antibody injection did not induce loss of CBC cells at position 1 and 2 in non‐irradiated mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (L) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR with indicated doses or non‐irradiated mice.
M–R Two‐month‐old LGR5‐GFPki, mTerc+/+ mice were i.v. injected with LRP6 neutralization antibody 12 h before 12 Gy γ‐irradiation. IgG serves as a negative control. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (M–P) Representative FACS plots of indicated treatments. (Q) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (R) Quantification of the number of LGR5+, LGR5hi, and LGR5lo cells of mice with indicated treatment by flow cytometry. Note that the total LGR5+ cell frequency was not significantly different between anti‐LRP6 antibody‐injected and control IgG‐injected mice, though the LGR5hi fraction was reduced in the LRP6 antibody‐injected group.

Data information: Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. NIR, non‐irradiated; IR, irradiated; con, control IgG antibody injected; LRP6, anti‐LRP6 antibody injected.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage (2014)
Figure 9

Genetically activating canonical Wnt/β‐catenin signaling activity by APC mutation increases the radio‐sensitivity of ISPCs in vivo

A–C GFP immunofluorescence staining of small intestine from 2‐month‐old LGR5‐GFPki, APC+/+ mice and LGR5‐GFPki, APCmin/+ mice. (A) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, 50 positively stained cells were measured per mouse). Note the GFP staining intensity was increased in APCmin/+ mice, particularly in position 4 and 5 cells. (B, C) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm.
D–F Two‐month‐old APC+/+ mice and APCmin/+ mice were exposed to 6 Gy γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3 mice per group). (D) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR. IR, irradiated. (E, F) Representative pictures of PCNA staining are given. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm.
G–K Two‐month‐old LGR5‐GFPki, APC+/+ mice and LGR5‐GFPki, APCmin/+ mice were exposed to 6 Gy γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (G) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (H–K) Representative FACS plots of indicated groups are given.

Data information: Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. NIR, non‐irradiated; IR, irradiated.
Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut (2022)
Figure 1

Figure 1. Bacterial infection can induce EB mitosis.

(A) ISC lineages in Drosophila adult midguts. Dl marks ISCs. Su(H)-Gal4>UAS-CD8:GFP marks EBs. Pdm1 and Prospero (Pros) are markers for EC and EE, respectively.

(B-K') The Drosophila posterior midguts containing Su(H)-Gal4>UAS-CD8:GFP (Su(H)>GFP) with sucrose (Suc, control) (B-C'), P.e for 36 h (D-F'), Ecc15 for 36 h (G-I') and DSS for 36 h (J-K') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (C-C', E-E', H-H' and K-K') Magnification of selected areas containing PH3+GFP─ cells from B, D, G and J. The EB cell close to the PH3+ cell (white arrow) in C-C' is out of focus. (F-F' and I-I') Magnification of selected areas containing PH3+ EBs (GFP+) from D and G. White arrowheads and red arrows indicate PH3 in GFP─ and GFP+ cells, respectively.

(L and M) Quantification of PH3+GFP─ ISCs (L) and PH3+GFP+ EBs (M) with given treatments. n = 21 guts for each treatment.

Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (L, M).
Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut (2022)
Figure 2

Figure 2. Dividing EBs do not gain Dl expression.

(A-C'') The midguts from the adult female flies fed with Suc (control) (A-A''), P. e (B-B'') or DSS (C-C'') were immunostained with anti-PH3 and anti-Dl antibodies. The white arrows indicate that GFP─ cells with PH3 have Dl staining and the red arrows indicate that EBs with GFP and PH3 (GFP+PH3+) do not have Dl expression. The yellow arrows in B show that two ISCs with PH3 are out of focus.

(D-F'') When the ISCs were marked by esgGal4, su(H)-Gal80>UAS-GFP, PH3 expression was found in GFP+ ISCs with feeding of Suc (control) (D-D''), P.e (E-E'') and Dss (F-F''). However, PH3 expression is present in EBs with GFP─Pros─ and small nuclei upon P.e infection (E-E'', red arrows). The white arrows indicate GFP+ cells with PH3 expression.

(G) Quantification of PH3+GFP─Pros─ EBs with given treatments. n = 11 guts for each treatment. Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (G).
Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut (2022)
Figure 4

Figure 4. Activation of EGFR-Ras signaling in EBs is sufficient and required to induces cell-autonomous EB mitosis.

(A) P. e infection up-regulates the expression of EGFR ligands (vn and Krn).

(B-B'') In the control, the dpErk level is high in ISCs (yellow arrowheads), but low in EBs (white arrows).

(C-C'') The level of dpErk is greatly up-regulated in both EBs (white arrows) and ISCs (yellow arrowheads) upon bacterial infection.

(D-N') The Drosophila midguts expressing Su(H)ts>GFP with control (D-E'), UAS-RasV12 for 6 days (F-H'), EGFRA887T for 4 days (I-K') and UAS−λTop for 5 days (L-N') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (E-E', G-G', J-J' and M-M') Magnification of selected areas containing PH3+GFP─ cells from D, F, I and L. (H-H', K-K' and N-N') Magnification of selected areas containing PH3+GFP+ cells from F, I and L. White arrowheads and red arrows indicate PH3 in GFP─ ISCs and GFP+ EBs, respectively.

(O, P) Quantification of PH3+GFP─ (P) and PH3+GFP+ (O) in midguts with indicated genotypes. n = 13 guts for each genotype.

(Q) Quantification of PH3+ EBs (GFP+) with the given genotypes in response to P. e infection. n = 11 guts for each genotype.

Data information: Three independent experiments were performed, and error bars are ± SEM. **, P < 0.01 (Student's t-test) (A). ***, P < 0.001 (Student's t-test) (O─Q).
Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut (2022)
Figure 6

Figure 6. Dl+ ISC-like cells are generated from EBs upon P.e infection.

(A) The schematic drawing of the Flip-out lineage tracing system.

(B-C''') Flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ were fed with Suc (B-B''') or P.e (C-C''') for 36h and their midguts were dissected and immunostained with antibodies against GFP, β-gal and Dl. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─).

(D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control, P. e infected midguts, n=50.

(E) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control with Suc or P. e, or knockdown of EGFR with Suc or P. e infection, n=50.

Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (D, E).
Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut (2022)
Figure 7

Figure 7. Activation of EGFR-Ras signaling in EBs induces EBs to produce Dl+ ISC-like cells.

(A-C''') The midguts from adult female flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ with (B-B''') or without (A-A''') UAS-RasV12 or with UAS-RasV12 and UAS-stg-RNAi (C-C''') were dissected and immunostained with antibodies against GFP, β-gal and Dl. The bigger GFP─ nucleus with LacZ expression (only red arrowheads) indicate differentiating or mature ECs. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─).

(D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the indicated genotypes, n=50.

Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (D).
Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut (2022)
Figure 8

Figure 8. Activating EGFR-Ras signaling in EBs or P.e infection induces EB division to generate two ISCs.

(A) Schematic drawing of the cell division that produces differentially labeled twin-spot cells (RFP+ GFP− and RFP−GFP+) through FRT-mediated mitotic recombination. UAS-Flp and transgenic overexpression is driven by Su(H)ts.

(B) Schematic drawings of differentially labeled twin-spot clones generated by FLP/FRT-mediated mitotic recombination of dividing cells in EBs.

(C-E'') The representative twin clones from midguts when EGFRA887T is overexpressed in EBs (D-D'', red and green arrows) or adult flies were fed with P.e for 36h (E-E'', red and green arrows), but no twin clone is found in the control (C-C'').

(F) Quantification of different types of twin clones in the control midguts, or in the midguts with EGFRA887T overexpression or P.e infection. n = 10 guts for each genotype.

Data information: Three independent experiments were performed (F).
Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut (2022)
Figure 9

Figure 9. The regenerative ISCs from EBs can differentiate into mature cells.

(A-C'') ISCs from EBs upon RasV12 expression or P. e infection could differentiate to generate EE or pre-EE cells (B-C'', LacZ+Pros+GFP─, blue arrowheads), but no EE or pre-EE cells from EBs are found in the control (A-A'', white arrowheads).

(D) Quantification of EE cells from progenies of EBs with indicated genotypes or treatments. n = 9 guts for each genotype or treatment.

(E-F'') ECs which were marked by Pdm1 expression were found in pairs of single-color clones (white arrows) in midguts with EGFRA887T expression or upon P.e infection.

(G) A model for bacterial infection and activation of EGFR-Ras signaling to induce EB mitosis and ISC regeneration.

Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (D).

Supplementary Figure legends:
Receptor guanylyl cyclase‐G is a novel thermosensory protein activated by cool temperatures (2014)
Figure 1

Guanylyl cyclase (GC) subtype GC‐G activity is stimulated by cool temperatures, and the hinge (H) region and cyclase (CYC) domain are essential for its activation by coolness

Coolness stimulates intracellular cyclic guanosine monophosphate (cGMP) accumulation in HEK‐293T cells expressing GC‐G. Two days after transfection of empty vector or GC‐G‐encoding plasmid, cells were exposed to ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured.
Temperature dependence of GC‐G activity. Membrane preparations from HEK‐293T cells transfected with empty vector or GC‐G‐encoding plasmid were used to determine cyclase activity at the indicated temperatures for 20 min (asterisks denote values significantly elevated compared to 37°C).
GC‐G is the only murine transmembrane GC that can be stimulated by cool temperatures. Expression plasmids encoding FLAG‐tagged GC subtypes GC‐A to GC‐G were transfected into HEK‐293T cells. Two days after transfection, membrane protein fractions were prepared and used for cyclase activity assays at 37 or 15°C. Protein expression of each receptor GC was confirmed by Western blot (WB) analysis with anti‐FLAG antibody.
The H+CYC domain is critical for stimulation by coolness. The domain structure of full‐length (FL) GC‐G and its mutant variants is shown in the upper panel. A FLAG tag was added at the N‐terminus of each protein. The ΔECD+KLD mutant protein lacks amino acids 73–455 and 556–833; the ΔCYC variant lacks amino acids 834–1,003. The H+CYC mutant protein contains amino acids 806–1,100. ECD, extracellular domain; KLD, kinase‐like domain; SP, signal peptide; TM, transmembrane region. HEK‐293T cells expressing the indicated GC‐G constructs were incubated at 37 or 15°C for 20 min, then intracellular cGMP concentration was measured (lower panel).

Data information: All data are mean ± SD from three experiments in triplicate. *P 0.05; **P 0.01.
Receptor guanylyl cyclase‐G is a novel thermosensory protein activated by cool temperatures (2014)
Figure 2

Chimeric GC‐A/G receptor containing the H+CYC domain of GC‐G acquires coolness‐evoked activity

In the chimeric GC‐A/G protein, the H+CYC domain of GC‐A was replaced by that of GC‐G (upper panel). HEK‐293T cells expressing the indicated proteins were exposed to ambient temperature of 37 or 15°C for 20 min; then, intracellular cGMP concentration was measured. Data are mean ± SD from three experiments in triplicate. **P 0.01.
Receptor guanylyl cyclase‐G is a novel thermosensory protein activated by cool temperatures (2014)
Figure 3

The hinge region is critical for activation of GC‐G by cool temperatures

A Representation of the domain structure from different C‐terminal fragments of GC‐G analyzed for coolness‐induced activity (amino acids of GC‐G encoded in each expression plasmid are indicated).
B, C Protein expression and coolness‐stimulated cGMP synthesis of the above shown divergent C‐terminal fragments of GC‐G with intact or ablated hinge (H) region. The expression plasmids encoding the H+CYC domain or a deletion construct (Δ1–Δ2) tagged with a FLAG epitope were transfected into HEK‐293T cells. Two days after transfection, cells were exposed to the ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured (C). Protein expression of each construct was confirmed by Western blot (WB) analysis with anti‐FLAG antibody (B).
D A recombinant protein containing the H+CYC domain of GC‐G acquires coolness‐evoked activity. Diagram of recombinant GST.GC‐G‐CYC protein containing the H+CYC domain of GC‐G (amino acids 849–1,078) compared to the domain structure of full‐length GC‐G (upper panel). The purified GST.GC‐G‐CYC protein was exposed to the indicated temperatures for 20 min, and GST.GC‐G‐CYC cyclase activity was measured (lower panel; asterisks denote values significantly elevated compared to 37°C).

Data information: All data are mean ± SD from three experiments in triplicate. **P 0.01.
Receptor guanylyl cyclase‐G is a novel thermosensory protein activated by cool temperatures (2014)
Figure 4

Cool temperatures enhance dimerization/oligomerization of GC‐G but not GC‐A

HEK‐293T cells were co‐transfected with plasmids encoding two differential epitope‐tagged (FLAG or Myc) versions of GC‐G or GC‐A, respectively. After 2 days, transfected cells were lysed with detergent and lysates were subjected to immunoprecipitation (IP) for 2 h with anti‐Myc antibody at two different temperatures (15 or 25°C), followed by Western blotting (WB) using anti‐FLAG antibody to determine the amount of dimerized/oligomerized GC proteins (upper panel). These approaches demonstrated that in contrast to GC‐A, homomeric protein/protein association of GC‐G seems to be enhanced by cool temperatures (upper panel). Cell lysates werealso precipitated and immunoblotted with the anti‐Myc antibody, demonstrating that cooling does not interfere with immunoprecipitation of Myc‐tagged GC‐G via the anti‐Myc antibody (middle panel). Likewise, precipitation and immunoblotting with the anti‐FLAG antibody yielded bands of similar intensity for 15 and 25°C, respectively (lower panel).
Relative quantification of homomeric protein/protein association of GC‐G and GC‐A was performed by densitometric scanning. Each intensity value from the upper panel was subsequently normalized to the total protein levels as determined in the middle and the lower panel of Fig A (intensity of the immunoreactive bands obtained with the anti‐Myc or anti‐FLAG antibody, respectively). Relative dimerization level at 15°C was further calculated by normalizing to values obtained at 25°C; the latter were set as 1. Results are means ± SD of three experiments, **P 0.01 (compared to 25°C).
Receptor guanylyl cyclase‐G is a novel thermosensory protein activated by cool temperatures (2014)
Figure 5

Increase in intracellular calcium [Ca2+]i concentration in HEK‐293T cells co‐expressing GC‐G and CNGA3 in response to cool temperatures

HEK‐293T cells expressing CNGA3 alone (upper panel) or together with GC‐G (full‐length in the middle panel and H+CYC domain in the lower panel) were loaded with the Ca2+ indicator Fura‐2 to monitor intracellular [Ca2+]i concentration. Cells co‐expressing GC‐G and CNGA3 showed a rapid increase in [Ca2+]i when the temperature was lowered from 37 to 15°C (middle and lower panel); cells only expressing CNGA3 are not responsive to cool temperatures (top panel). Removal of extracellular Ca2+ (−Ca2+; middle panel) completely suppressed the response to cooling. ΔF represents changes in the ratio of the fluorescence intensity of Fura‐2 at 340/380 nm excitation. The ratiometric Ca2+ responses are representative of 30 cells recorded from three experiments.