Journal: Cell reports

Article Title: Embryogenic stem cell-derived intestinal crypt fission directs de novo crypt genesis.

doi: 10.1016/j.celrep.2022.111796

Figure Lengend Snippet: Figure 1. Characterization of Setd4-expressing intestinal cells (A) smFISH for Setd4 and Lgr5 expression in crypt (as indicated with dotted lines). Scale bar, 30 mm. (B) CD45EpCAM+CD44+ cells were sorted for sequencing using a 10X Genomics platform (n = 6 mice, age of 8 weeks). (C) Left, unsupervised clustering of scRNA-seq data from isolated crypt cells (n = 6,653 single-cell transcriptomes as classified in Figure S1D). Right, expression of Setd4 gene. (D) Cell proportions of the indicated genes that distinguish CBCs or RSCs in crypt cells. (E) Cellular composition of Setd4+ cells. (F) Left, violin plot of Mki67 expression. Middle, expression of proliferation markers (black outline) is overlaid on the t-SNE graph. Gene-expression values are shown as median of log10(CPM). Right, analysis of co-expression of Setd4+ cells with these proliferation markers with proportions shown (n = 237 Setd4+ cells). (G) Illustration of lineage tracing strategy in Setd4cre-ERT2/+; Rosa26lsl-tdTomato mice. (H) Detection of tdTomato and Setd4 mRNA transcripts (white transition point outline) by smFISH. Scale bar, 30 mm. (I) Left, image showing examples of tdT+ cells produced at +4 positions relative to the crypt base, with dotted lines indicating crypt boundaries. Right, position of tdT+ cells at 1 dpt (n = 371 cells from five mice). Scale bar, 30 mm. (J) Schematic of a 3D confocal imaging strategy of crypt and villus. Representative z stack images of villus and crypt, with sections at different depth of z axis. Scale bar, 50 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glycerol Sangon Biotech Cat# A600232-0500 ProLongTM glass antifade mountant Thermo Fisher Cat# P36980 Isoflurane RWD Cat# R510-22 Critical commercial assays Click-iT EdU Alexa Fluor 647 Imaging Kit Thermo Fisher Cat# C10640 Advanced Cell Diagnostics RNAscope 2.0 HD Detection Kit Advanced Cell Diagnostics Cat# 323100 Deposited data Raw RNA-seq data This manuscript SRA: PRJNA741073 Experimental models: Organisms/strains Mouse: C57BL/6J Shanghai Model Organisms Center N/A Mouse: Rosa26lsl-tdTomato Jackson Laboratory JAX No. 007905 Mouse: Rosa26mT/mG Jackson Laboratory JAX No. 007676 Mouse: Setd4cre/+ Shanghai Model Organisms Center This manuscript Mouse: Setd4cre-ERT2/+ Shanghai Model Organisms Center This manuscript Oligonucleotides Primers for Setd4, see Table S1 This manuscript N/A Primers for mT/mG, see Table S1 This manuscript N/A Primers for Foxg1, see Table S1 This manuscript N/A Primers for Globin, see Table S1 This manuscript N/A Primers for tdTomato, see Table S1 This manuscript N/A Mm-Lgr5 probe Advanced Cell Diagnostics Cat# 312171-C2 Mm-Clu probe Advanced Cell Diagnostics Cat# 427891-C3 TdTomato probe Advanced Cell Diagnostics Cat# 317041-C2 Mm-Setd4 probe Advanced Cell Diagnostics Cat# 874641-C1 Bacterial dapB probe Advanced Cell Diagnostics Cat# 320871 Software and algorithms FlowJo (V10) Treestar https://www.flowjo.com/solutions/ flowjo/downloads CytExpert software (V2.3) Beckman https://www.beckman.com/flow-cytometry/ research-flow-cytometers/cytoflex/software CellRanger (V3.1.0) 10X Genomics https://support.10xgenomics.com/single- cell-gene-expression/software/pipelines/ latest/installation Seurat (V3.1.0) Satija Lab http://seurat.r-forge.r-project.org/ ZEN Blue (V2.6) Zeiss N/A Olympus FV31-SW software (V2.3) Olympus N/A Leica Application Suite X (V3.3.0.16799) Leica N/A GraphPad Prism (V8.0.1) GraphPad https://www.graphpad.com/scientificsoftware/ prism/ ImageJ (V1.52p).

Techniques: Expressing, Sequencing, Isolation, Gene Expression, Produced, Imaging

Journal: Cell reports

Article Title: Embryogenic stem cell-derived intestinal crypt fission directs de novo crypt genesis.

doi: 10.1016/j.celrep.2022.111796

Figure Lengend Snippet: Figure 3. Setd4+ cells are resistant to irradiation and produce Sca-1+ cells for tissue repair (A) Experimental overview. Setd4cre-ERT2/+; Rosa26lsl-tdTomato mice (age of 8–12 weeks) were pulsed with a single TAM injection, followed by irradiation and tissue analyses (at least three mice for each time point). (B and C) Whole-mount imaging (B) and section imaging (C) of gut tube from non-irradiated (non-IR) or irradiated (IR) mice at 1 day post irradiation (1 dpi) showing the resistance of Setd4+ cells to X-ray. Boxed areas were magnified on the right with dotted lines indicating the crypt boundaries. Scale bar, 100 mm. (D) Absolute numbers of tdT+ cells per ten crypts at 1 dpi was quantified (n = 3 mice each; two-tailed unpaired Student’s t test; data are mean ± SEM). (E) Left, detection of Olfm4 in sections of the small intestine from non-IR or IR mice at 1 dpi. Scale bar, 100 mm. Right, western blot analysis of Olfm4 in non-IR and IR groups (n = 4 mice analyzed for each; two-tailed unpaired Student’s t test; data are mean ± SEM). (F) Left, detection of tdTomato and EdU in sections from non-IR mice or IR mice with 1-h EdU incorporation at 3 dpi. Each crypt (circled with dotted lines) is magnified to the right. Right, the percentage of tdTomato+EdU+ cells was quantified (n = 3 mice analyzed for each; two-tailed unpaired Student’s t test; data are mean ± SEM). Scale bar, 50 mm. (G) Detection of tdTomato and Sca-1 with dotted lines indicating crypt boundaries. Scale bar, 50 mm. (H) Flow cytometry of Sca-1 and tdTomato in crypt cells (gating strategy plots in Figure S3B; n = 3 mice at non-IR group and n = 7 mice at IR group analyzed; two- tailed unpaired Student’s t test; data are mean ± SEM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glycerol Sangon Biotech Cat# A600232-0500 ProLongTM glass antifade mountant Thermo Fisher Cat# P36980 Isoflurane RWD Cat# R510-22 Critical commercial assays Click-iT EdU Alexa Fluor 647 Imaging Kit Thermo Fisher Cat# C10640 Advanced Cell Diagnostics RNAscope 2.0 HD Detection Kit Advanced Cell Diagnostics Cat# 323100 Deposited data Raw RNA-seq data This manuscript SRA: PRJNA741073 Experimental models: Organisms/strains Mouse: C57BL/6J Shanghai Model Organisms Center N/A Mouse: Rosa26lsl-tdTomato Jackson Laboratory JAX No. 007905 Mouse: Rosa26mT/mG Jackson Laboratory JAX No. 007676 Mouse: Setd4cre/+ Shanghai Model Organisms Center This manuscript Mouse: Setd4cre-ERT2/+ Shanghai Model Organisms Center This manuscript Oligonucleotides Primers for Setd4, see Table S1 This manuscript N/A Primers for mT/mG, see Table S1 This manuscript N/A Primers for Foxg1, see Table S1 This manuscript N/A Primers for Globin, see Table S1 This manuscript N/A Primers for tdTomato, see Table S1 This manuscript N/A Mm-Lgr5 probe Advanced Cell Diagnostics Cat# 312171-C2 Mm-Clu probe Advanced Cell Diagnostics Cat# 427891-C3 TdTomato probe Advanced Cell Diagnostics Cat# 317041-C2 Mm-Setd4 probe Advanced Cell Diagnostics Cat# 874641-C1 Bacterial dapB probe Advanced Cell Diagnostics Cat# 320871 Software and algorithms FlowJo (V10) Treestar https://www.flowjo.com/solutions/ flowjo/downloads CytExpert software (V2.3) Beckman https://www.beckman.com/flow-cytometry/ research-flow-cytometers/cytoflex/software CellRanger (V3.1.0) 10X Genomics https://support.10xgenomics.com/single- cell-gene-expression/software/pipelines/ latest/installation Seurat (V3.1.0) Satija Lab http://seurat.r-forge.r-project.org/ ZEN Blue (V2.6) Zeiss N/A Olympus FV31-SW software (V2.3) Olympus N/A Leica Application Suite X (V3.3.0.16799) Leica N/A GraphPad Prism (V8.0.1) GraphPad https://www.graphpad.com/scientificsoftware/ prism/ ImageJ (V1.52p).

Techniques: Irradiation, Injection, Imaging, Two Tailed Test, Western Blot, Flow Cytometry

Journal: Cell reports

Article Title: Embryogenic stem cell-derived intestinal crypt fission directs de novo crypt genesis.

doi: 10.1016/j.celrep.2022.111796

Figure Lengend Snippet: Figure 7. Sedt4+ cells contribute to the construction of new crypts after birth (A) Experimental overview. (B) Representative images of tdT+ clones in whole mounts of crypt. The white transition points outlined are magnified in the corresponding boxed areas (n = 4 mice each point). Scale bar, 100 mm. (C) A representative whole-mount image of the crypt fission. Sections show differing z axis depths. Yellow arrows indicate a base-to-top (from Z = 1 to Z = 55) change of crypt lumens within a crypt undergoing fission. Scale bar, 20 mm. (D) Representative images of tdT+ clones in section of intestinal crypts at P6 and P15. Dotted lines indicate crypt boundaries. Scale bar, 50 mm. (E) Top, experimental overview. Bottom, representative whole-mount images of tdT+ clones at P35 and P55. Boxed areas are magnified to the right. Yellow arrows indicate the n R 2 tdT+ crypts adjacent to each other. Scale bar, 1 mm. (F) Gross morphology of the small intestine and colon at P55 (50 dpt) showing the distribution of tdT+ clones. Scale bar, 3 mm. (G) Representative images of continuous crypts organized by tdT+ cells. Scale bar, 50 mm. (H) The percentage of continuous tdT+ crypt clones in total crypt clones labeled by tdTomato from the proximal (pro) small intestine quantified at indicated chase times (n = 3 mice respectively, two-tailed unpaired Student’s t test; data are mean ± SEM). (I) Left, model summarizing two possible fluxes of tdT+ cells based on crypt fission. Right, a representative image of ribbons with continuous tdT+ crypts. Scale bar, 50 mm. (J) Experimental overview. Setd4cre-ERT2/+; Rosa26lsl-tdTomato mice were pulsed with a single TAM injection at P25 as control and chased at P75 (50 dpt). The P5 TAM-induced group was chased at P55 (50 dpt). (K) Left, representative whole-mount images of tdT+ clones (crypt-villus). Boxed areas (left) are magnified to the right. Far right, tdTomato-infiltrate crypts identified as in the left image and the percentage of continuous tdT+ crypt clones in total crypt clones labeled by tdTomato and quantified (n = 3 mice respectively analyzed, two-tailed unpaired Student’s t test; data are mean ± SEM). Scale bar, 1 mm. (L) The number of tdT+ crypts per 1-mm2 area of the proximal (Pro) or distal (Dis) small intestinal epithelium from P5 and P25 TAM-induced groups was quantified at 50 dpt (n = 90 images as K analyzed; n = 3 mice each; two-tailed Mann-Whitney test; box edges show 25th and 75th percentile, with the central point as the median and minimum and maximum values also shown). See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Glycerol Sangon Biotech Cat# A600232-0500 ProLongTM glass antifade mountant Thermo Fisher Cat# P36980 Isoflurane RWD Cat# R510-22 Critical commercial assays Click-iT EdU Alexa Fluor 647 Imaging Kit Thermo Fisher Cat# C10640 Advanced Cell Diagnostics RNAscope 2.0 HD Detection Kit Advanced Cell Diagnostics Cat# 323100 Deposited data Raw RNA-seq data This manuscript SRA: PRJNA741073 Experimental models: Organisms/strains Mouse: C57BL/6J Shanghai Model Organisms Center N/A Mouse: Rosa26lsl-tdTomato Jackson Laboratory JAX No. 007905 Mouse: Rosa26mT/mG Jackson Laboratory JAX No. 007676 Mouse: Setd4cre/+ Shanghai Model Organisms Center This manuscript Mouse: Setd4cre-ERT2/+ Shanghai Model Organisms Center This manuscript Oligonucleotides Primers for Setd4, see Table S1 This manuscript N/A Primers for mT/mG, see Table S1 This manuscript N/A Primers for Foxg1, see Table S1 This manuscript N/A Primers for Globin, see Table S1 This manuscript N/A Primers for tdTomato, see Table S1 This manuscript N/A Mm-Lgr5 probe Advanced Cell Diagnostics Cat# 312171-C2 Mm-Clu probe Advanced Cell Diagnostics Cat# 427891-C3 TdTomato probe Advanced Cell Diagnostics Cat# 317041-C2 Mm-Setd4 probe Advanced Cell Diagnostics Cat# 874641-C1 Bacterial dapB probe Advanced Cell Diagnostics Cat# 320871 Software and algorithms FlowJo (V10) Treestar https://www.flowjo.com/solutions/ flowjo/downloads CytExpert software (V2.3) Beckman https://www.beckman.com/flow-cytometry/ research-flow-cytometers/cytoflex/software CellRanger (V3.1.0) 10X Genomics https://support.10xgenomics.com/single- cell-gene-expression/software/pipelines/ latest/installation Seurat (V3.1.0) Satija Lab http://seurat.r-forge.r-project.org/ ZEN Blue (V2.6) Zeiss N/A Olympus FV31-SW software (V2.3) Olympus N/A Leica Application Suite X (V3.3.0.16799) Leica N/A GraphPad Prism (V8.0.1) GraphPad https://www.graphpad.com/scientificsoftware/ prism/ ImageJ (V1.52p).

Techniques: Clone Assay, Labeling, Two Tailed Test, Injection, Control, MANN-WHITNEY