Journal: Frontiers in Physiology

Article Title: FGF10 Is Required for Circumvallate Papilla Morphogenesis by Maintaining Lgr5 Activity

doi: 10.3389/fphys.2018.01192

Figure Lengend Snippet: Morphology of developing CVP and expression patterns of Lgr5 and FGF10 during CVP development. (A–E) HE staining shows the dynamic morphological changes during CVP development. (A) The epithelial invagination of CVP into the adjacent mesenchyme was observed at E13.5. (B) At 15.5, the epithelial invagination of both sides of CVP had deepened. (C) At 17.5, deep trenches were formed from the epithelium invagination of CVP. (D) At PN1, von Ebner’s glands were connected to the invaginated epithelium of CVPs. (E) At the adult stage, circular furrows and taste buds were observed in the well-formed epithelial trenches of CVP. (F–J, J’) The Lgr5 expression pattern was confirmed by in situ hybridization. (F) Lgr5 expression was observed in the CVP epithelium but not in the tongue epithelium or adjacent mesenchyme at E13.5. (G) At 15.5, Lgr5 was strongly expressed in the epithelial invagination region and dorsal epithelium of CVP. (H) At 17.5, strong Lgr5 expression was detected in the epithelial trenches of CVP but partially observed in the dorsal epithelium of CVP. (I) Expression of Lgr5 was detected in the taste bud-forming region but not in the circular furrow-forming region, while partial expression was detected in the dorsal epithelium of CVP at PN1. (J) In the adult stage, the expression was only detected around taste buds and bottom region of the epithelial trench. (J’) Higher magnification of the blue box in Panel J . (K,L) At 15.5 and 17.5, FGF10 was strongly localized in the mesenchyme of the CVP-forming region but not in the FFP-forming region. (M–P) Higher magnification results indicated that the localization of FGF10 was strongly detected in the CVP mesenchyme but not in the FFP mesenchyme at E15.5 and at 17.5. Scale bars are 100 μm in (A–J,J’,M–P) and 500 μm in (K,L) .

Article Snippet: Heparin formate-derived beads (Sigma-Aldrich, United States) were incubated in 50 μg/ml recombinant human FGF10 (R&D systems, United States) for 1 h at room temperature.

Techniques: Expressing, Staining, In Situ Hybridization

Journal: Frontiers in Physiology

Article Title: FGF10 Is Required for Circumvallate Papilla Morphogenesis by Maintaining Lgr5 Activity

doi: 10.3389/fphys.2018.01192

Figure Lengend Snippet: Morphological differences and rescue study after tongue epithelium recombination. (A–C) Schematic diagram indicating the three different experiments for in vitro culture. (A) In the control recombination, the CVP epithelium was separated, recombined in the same position and cultured for 72 h. (B) In the reverse recombination condition, the tongue epithelium was rotated 180 degrees in the anterior-posterior axis and then recombined with the tongue mesenchyme. To indicate the CVP region, Indian ink marks the dorsal surface of the CVP epithelium. (C) FGF10-soaked bead implantation was performed under the Indian ink-labeled CVP epithelium in the reverse recombination condition. (D) HE staining shows proper morphogenesis of the developing CVP in the control recombination condition ( n = 9/10). (E) In the reverse recombination tongue, FFP-like structure was observed in the CVP epithelium (20/20). (F,G) Implantation of FGF10-soaked beads rescued CVP epithelial invagination in the reverse recombination condition, while BSA-soaked beads could not rescue CVP morphology. (H,I) Epithelial cell marker Pan-CK was clearly localized in epithelium of control and reverse recombination. (J,K) Pan-CK was strongly localized both in BSA and FGF10 bead implanted group. (L–O) Lgr5 was expressed in control recombination and FGF10 bead implanted group. However, Lgr5 signal was negative in reverse recombination and BSA bead implanted group without epithelial invagination. ∗ , bead. Scale bars are 100 μm.

Article Snippet: Heparin formate-derived beads (Sigma-Aldrich, United States) were incubated in 50 μg/ml recombinant human FGF10 (R&D systems, United States) for 1 h at room temperature.

Techniques: In Vitro, Control, Cell Culture, Labeling, Staining, Marker

Journal: Frontiers in Physiology

Article Title: FGF10 Is Required for Circumvallate Papilla Morphogenesis by Maintaining Lgr5 Activity

doi: 10.3389/fphys.2018.01192

Figure Lengend Snippet: Morphogenesis and cellular events analysis after BIO treatment. (A–T) E15.5 mouse tongues were treated with BIO (1 ng/ml) or SU5402 (20 μm) and cultured for 72 h. (A) In the control group, normal CVP morphology was observed. (C) In the BIO-treated group, significantly disrupted CVP structure was observed with a reduced epithelial invagination depth. (B,D) To confirm the efficiency of BIO treatment, RT-qPCR was performed. Fgf10 and Lgr5 expression levels were significantly reduced after BIO treatment compared to those in the control group. (E,G) CVP morphology was disrupted by SU5402 treatment. (F) RT-qPCR result indicated that Lgr5 expression level was reduced after SU5402 treatment. (H) No Caspase3-positive CVP epithelial cells were detected in the control group. (I,J) In the BIO and SU5402 treatment group, apoptotic cells were detected at the crypt region of disrupted CVP epithelium where stem cells are normally located. (K) Numerous Ki67-positive proliferating cells were observed in not only the CVP epithelium but also the tongue epithelium. (L,M) Proliferating cells were reduced after BIO or SU5402 treatment both in the CVP epithelium and tongue epithelium. (N) Lgr5 was expressed in DMSO control group. (O,P) Positive signal of Lgr5 was not observed after BIO and SU5402 treatment. (Q–T) Apoptotic and proliferating cell number was counted after BIO and SU5402 treatment. Number of apoptotic cell was induced and proliferating cell was reduced by BIO and SU5402 treatment. Scale bars are 100 μm. ∗ p < 0.05.

Article Snippet: Heparin formate-derived beads (Sigma-Aldrich, United States) were incubated in 50 μg/ml recombinant human FGF10 (R&D systems, United States) for 1 h at room temperature.

Techniques: Cell Culture, Control, Quantitative RT-PCR, Expressing

Journal: Frontiers in Physiology

Article Title: FGF10 Is Required for Circumvallate Papilla Morphogenesis by Maintaining Lgr5 Activity

doi: 10.3389/fphys.2018.01192

Figure Lengend Snippet: Schematic representation of CVP development. The molecular interactions between Fgf10 and Lgr5 modulate cellular events including apoptosis and cell proliferation to ensure correct CVP development.

Article Snippet: Heparin formate-derived beads (Sigma-Aldrich, United States) were incubated in 50 μg/ml recombinant human FGF10 (R&D systems, United States) for 1 h at room temperature.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Lacrimal Gland Development and Fgf10-Fgfr2b Signaling Are Controlled by 2-O- and 6-O-sulfated Heparan Sulfate

doi: 10.1074/jbc.m111.225003

Figure Lengend Snippet: FIGURE 3. Genetic interaction between Hs6st and Fgf10. A–D, lacrimal gland bud was present in both Fgf10/ and Hs6stCKO mutants at E14.5 but lost in the Fgf10/;Hs6stCKO compound mutants (arrow). E, quantification of the lacrimal gland phenotype is shown.

Article Snippet: Briefly, 80–120- m diameter heparin acrylic beads (Sigma) were washed in PBS and incubated with 250 g/ml recombinant FGF10 (R&D Systems) or 5 mg/ml BSA in PBS at 4 °C overnight.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Lacrimal Gland Development and Fgf10-Fgfr2b Signaling Are Controlled by 2-O- and 6-O-sulfated Heparan Sulfate

doi: 10.1074/jbc.m111.225003

Figure Lengend Snippet: FIGURE 4. Hs2st/Hs6st mutants disrupted Fgf10/Fgfr2b signaling in lacrimal gland development. A–D, in situ binding (LACE assay) of Fgf10/Fgfr2b on lacrimal gland sections were reduced in the Hs2stCKO and Hs6stCKO single mutants and abolished in the Hs2stCKO;Hs6stCKO double mutants (arrow). E–L, downstream of FGF signaling, phospho-ERK and Erm expression was also reduced in the Hs2stCKO and Hs6stCKO single mutants and lost in the Hs2stCKO;Hs6stCKO double mutants (arrow).

Article Snippet: Briefly, 80–120- m diameter heparin acrylic beads (Sigma) were washed in PBS and incubated with 250 g/ml recombinant FGF10 (R&D Systems) or 5 mg/ml BSA in PBS at 4 °C overnight.

Techniques: In Situ, Binding Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Lacrimal Gland Development and Fgf10-Fgfr2b Signaling Are Controlled by 2-O- and 6-O-sulfated Heparan Sulfate

doi: 10.1074/jbc.m111.225003

Figure Lengend Snippet: FIGURE 6. Disruption of Fgf10 signaling in the Hs6st/Hs2st knock-out MEF cells. A, immortalized MEF cells were clonally selected to generate the Hs2st and Hs6st null (Hs2st/, Hs6st/, and Hs2st/;Hs6st/) cells. The disaccharide composition of heparan sulfate derived from the cells was determined by enzymatic depolymerization and liquid chromatography/mass spectrometry. Each disaccharide is defined by a structure code: D0H0, UA-GlcNH2; D0H6, UA-GlcNH2-6S; D2H0, UA2S-GlcNH2; D0A0, UA-GlcNAc; D0S0, UA-GlcNS; D2H6, UA2S-GlcNH26S; D0A6, UA-GlcNAc6S; D0S6, UA-GlcNS6S; D2A0, UA2S-GlcNAc; D2S0, UA2S-GlcNS; D2A6, UA2S-GlcNAc6S; D2S6, UA2S-GlcNS6S, where UA 4,5-unsaturated uronic acid generated during the heparin lyase reaction (47). *, one-way analysis of variance: p 0.05 for the comparison with the wild type. The data reflected three independent experiments for each cell line and were confirmed in two independently cloned cells for each genotype. B, the amount of N-acetyl groups and sulfate groups at various positions was calculated from the data in panel A. *, one-way analysis of variance: p 0.05 for the comparison with the wild type; N.S., not significant. C, the Hs2st and Hs6st knock-out MEF cells were stimulated with 10 ng/ml Fgf10, and the time course of ERK phosphorylation was determined by fluorescent Western blot. D, the relative level of phospho-ERK was significantly reduced in the Hs2s/;Hs6st/ cells. Three independent experiments were performed for each cell line, and the results were confirmed in two independently cloned cells for each genotype. *, p 0.01 for the comparison with the wild type.

Article Snippet: Briefly, 80–120- m diameter heparin acrylic beads (Sigma) were washed in PBS and incubated with 250 g/ml recombinant FGF10 (R&D Systems) or 5 mg/ml BSA in PBS at 4 °C overnight.

Techniques: Disruption, Knock-Out, Derivative Assay, Liquid Chromatography, Mass Spectrometry, Generated, Comparison, Clone Assay, Phospho-proteomics, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Lacrimal Gland Development and Fgf10-Fgfr2b Signaling Are Controlled by 2-O- and 6-O-sulfated Heparan Sulfate

doi: 10.1074/jbc.m111.225003

Figure Lengend Snippet: FIGURE7.Fgf10requiresHs6st/Hs2sttoinducelacrimalglandbuddingex vivo. A–H, although implantation of BSA beads did not affect endogenous lacrimal gland budding (arrows), Fgf10-soaked beads induced ectopic lacri- mal gland buds in wild type control, Hs2stCKO, and Hs6stCKO explants (arrow- heads). However, no lacrimal gland buds were observed in the Hs2stCKO; Hs6stCKO mutant explants. * denotes implanted beads. I, the lacrimal gland budding rate for each Hs6st/Hs2st mutant is shown.

Article Snippet: Briefly, 80–120- m diameter heparin acrylic beads (Sigma) were washed in PBS and incubated with 250 g/ml recombinant FGF10 (R&D Systems) or 5 mg/ml BSA in PBS at 4 °C overnight.

Techniques: Control, Mutagenesis

Journal: Cell stem cell

Article Title: Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells.

doi: 10.1016/j.stem.2021.10.010

Figure Lengend Snippet: Figure 5. ENS cells promote in vitro growth and patterning of gastric mesenchyme (A) Schematic illustrating the method of recombining hAGOs with ENCCs at day 6 and day 9 of the hAGO protocol. (B) Representative images of 4-week in vitro hAGOs with (bottom) and without (top) ENCCs recombined on day 6 of the hAGO protocol, stained with TUJ1 neurons (green) and FOXF1 mesenchyme (red) and epithelial ECAD (white). Higher-magnification images are shown on the right. (C) Representative images of 4-week in vitro hAGOs with (bottom) and without (top) ENCCs recombined on day 6 (left) or day 9 (right) of the hAGO protocol, demonstrating an increase in FOXF1+ mesenchyme (red). (D) Quantification of FOXF1+ mesenchyme contribution (n = 16–24 fields from at least 3 organoids from one differentiation; the same trend is seen across at least two individually seeded differentiations; *p < 0.05, ***p < 0.001, Student’s t test). Scale bars represent S.D. (E) Relative expression of key gastric mesenchymal genes (BARX1, NKX3-2, FGF10, ISL1, and SIX2) in hAGOs+ENCCs compared with control hAGOs without ENCCs (n = 4–12 wells with a minimum of 3 organoids per well from 5 individual differentiations, *p < 0.05, Student’s t test). Scale bars represent S.D. See also Figure S4, Video S2, and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Human Stomach and Duodenum Samples Wells Lab CCHMC GI-1B-9952 A B Human Antral and Fundic Samples Helmrath Lab CCHMC 14-241, 14-242, 14-243, 14-244 Human Stomach and Duodenum Samples CCHMC Pathology Core N/A Chemicals, peptides, and recombinant proteins 1x DMEM Invitrogen Cat# 11965-084 50x B27 supplement Thermo Fisher Scientific Cat# 17504044 50x B27 supplement w/o Vitamin A Thermo Fisher Scientific Cat# 12587-010 A8301 Torcis Cat# A83-01 Activin A Cell Guidance Systems Cat# GFH6 Accutase Thermo Fisher Scientific Cat# A11105-01 Advanced DMEM:F12 Thermo Fisher Scientfic Cat# 12634-010 Atropine sulfate salt monohydrate Sigma-Aldrich Cat# A0257 Bethanechol Sigma-Aldrich Cat# 1071009 C59 Cellagen Technology Cat# C7641-2s CHIR99021 ReproCell Cat# 04-0004-10 Collagen Type IV Thermo Fisher Scientific Cat# 17104019 Defined fetal bovine serum (dFBS) Hyclone Cat# SH30070.02 Dispase Thermo Fisher Scientific Cat# 17105-041 DMEM/F12 Thermo Fisher Scientific Cat# 11320033 DMEM/F-12, GlutaMAX Thermo Fisher Scientific Cat# 10565018 EGF Sigma-Aldrich Cat# 1257-0.1MG F12 Media Invitrogen Cat# 11765-054 Fetal Bovine Serum (FBS) HyClone Cat# SH30071.03 Fibronectin Corning Cat# 354008 Geltrex Thermo Fisher Scientific Cat# A1413201 HEPES Buffer Thermo Fisher Scientific Cat# 15630080 hESC-qualified Matrigel BD Biosciences Cat# 354277 Insulin from bovine pancreas Sigma-Aldrich Cat# I4011-50 L-glutamine (100x) Thermo Fisher Scientific Cat# 25030-081 L-NAME Sigma-Aldrich Cat# N5751 Matrigel Matrix Basement Membrane BD Biosciences Cat# 354234 mTeSR1 media Stem Cell Technologies Cat# 5850 N2 Supplement Thermo Fisher Scientific Cat# 17502-048 Non-essential Amino Acids (100x) Thermo Fisher Scientific Cat# 11140050 Normal donkey serum Jackson Immunoresearch Labs Cat# 017-000-121 Neurobasal Medium Thermo Fisher Scientific Cat# 21103049 PD0325901 Stem Cell Technologies Cat# 72184 Pen/Strep (100x) Thermo Fisher Scientific Cat# 15140-122 PIK90 EMD Millipore Cat# 528117 PMA Torcis Cat# 1201 recombinant human BMP4 R&D Systems Cat# 314-BP recombinant human EGF R&D Systems Cat# 236-EGF recombinant human FGF2 R&D Systems Cat# 233-FB recombinant human FGF4 R&D Systems Cat# 235-F4 recombinant human FGF10 R&D Systems Cat# 345-FG recombinant human Noggin R&D Systems Cat# 6057-NG recombinant human WNT3A R&D Systems Cat# 5036-WN (Continued on next page) Cell Stem Cell 29, 36–51.e1–e6, January 6, 2022 e2

Techniques: In Vitro, Staining, Expressing, Control