EMBO/soda-nel huggingface dataset:
Biomechanical stress regulates mammalian tooth replacement via the integrin β1-RUNX2-Wnt pathway (2019)
Figure 4

Figure 4. Biomechanical stress regulates the integrin β1-ERK1-RUNX2 pathway in mesenchyme between DC and PC.

A-L IF of integrin β1, ERK1, and RUNX2 in miniature pig canine frontal sections at embryonic day 60 (E60), E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days; (A'-L') are magnifications of boxed regions in the corresponding figure panels.

M Relative IF expression levels of integrin β1, ERK1, and RUNX2 during E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days.

N Morphology of the human PC at weeks 18-19 (H&E staining); (N') is magnification of the boxed region in (N). DC, deciduous canine; PC, permanent canine; IEE, inner enamel epithelium; OEE, outer enamel epithelium. Mesenchyme between DC and PC is indicated with a green arrow.

O-Q ISH expression patterns of ITGB1, ERK1, and RUNX2 in human PCs. Green arrows indicate the mesenchyme between DC and PC.

R IF of RUNX2 in human PC. Mesenchyme between DC and PC is indicated with a red arrow. Red arrow indicates the mesenchyme between DC and PC.

S PC epithelium areas in E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days.

T Illustration of force exertion on cultured dental follicle cells (DFCs).

U IF of RUNX2 and phospho-ERK1/2 (p-ERK1/2) upon force loading with 0 or 1.0 g/cm2 for 2 h or after force was removed and the cells cultured for an additional 1, 2, and 4 hours.

V Relative IF expression levels of RUNX2 and p-ERK1/2 between groups in (U).

W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126).

X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W).

Data information: Data represent the means ± SEM. n = 3. Scale bars = 25 µm (A', G') and 50 µm (other panels). Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for V and X, *P < .05, **P < .01, ***P < .001; NS, not significant.
Biomechanical stress regulates mammalian tooth replacement via the integrin β1-RUNX2-Wnt pathway (2019)
Figure 5

Figure 5. RUNX2 overexpression inhibits initiation of PC germs.

A-C H&E staining of miniature pig canine frontal sections from embryonic day 60 (E60 group) (A) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag (overexpression group) (B) or overexpression control lentiviral vector with a Myc tag (O-control group) (C); (A'-C') are magnifications of boxed regions in the corresponding figure panels.

D-F Immunohistochemical (IHC) staining of Myc expression in mandible slices from the E60, overexpression, and O-control groups.

G-I IF staining of RUNX2 expression (yellow arrows) in mandible slices of the E60, overexpression, and O-control groups.

J, K Relative expression levels of Myc and RUNX2 in the E60, O-control, and overexpression groups.

L, M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector (knockdown control, K-control) (L) or RUNX2 shRNA lentiviral vector (knockdown) (M); (L'-M') are magnifications of boxed regions in the corresponding figure panels.

N Relative RUNX2 expression levels between K-control and knockdown groups.

O-Q H&E staining of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (O'-Q') are magnifications of boxed regions in the corresponding figure panels.

R-T IF staining of RUNX2 expression (yellow arrows) of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (R'-T') are magnifications of boxed regions in the corresponding figure panels.

U Relative RUNX2 expression levels among the three groups in (R-T).

V PC epithelium areas in the overexpression, O-control, knockdown, knockdown + force, K-control, and K-control + force groups.

Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 100 µm (D-F), 25 µm (R'-T'), and 50 µm (other panels). Dashed lines mark the outlines of PCs. Unpaired t-tests for N and V; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, K, and U, *P < .05, **P < .01, ***P < .001.
Biomechanical stress regulates mammalian tooth replacement via the integrin β1-RUNX2-Wnt pathway (2019)
Figure 6

Figure 6. Wnt modulation between the mesenchyme and epithelium regulates PC initiation.

A, B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 (E60) and E90. (A', B') are magnifications of boxed regions in the corresponding figure panels.

C Relative ISH expression levels of RUNX2 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90.

D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels.

F Relative ISH expression levels of β-catenin in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90.

G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels.

I Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90.

J FISH of β-catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope. Boxed regions of mesenchyme are magnified in the right panels. Epithelium is indicated with a yellow arrow.

K Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60, E90, and E60 cultured for 12 and 24 h.

L, M IF of β-catenin at E60 and E90; The yellow arrows indicate the localization of β-catenin in the nucleus.

N Relative IF expression levels of β-catenin in the nucleus of epithelium and mesenchyme at E60 and E90.

O, P FISH of β-catenin in mandible slices infected with RUNX2 shRNA lentivirus (knockdown) or scrambled shRNA lentivirus (scramsh) via RNAscope.

Q Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups.

Data information: Scale bars = 50 µm. Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for C, F, I, N, and Q; One-way ANOVA for K, *P < .05, **P < .01, ***P < .001.
Biomechanical stress regulates mammalian tooth replacement via the integrin β1-RUNX2-Wnt pathway (2019)
Figure 7

Figure 7. RUNX2 regulates Wnt signaling in DFCs.

A, B IF of Lef1 in mandible slices at embryonic day 60 (E60) infected with RUNX2 overexpression lentiviral vector (overexpression) or overexpression control lentiviral vector (O-control). (A'-B') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC.

C Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) of the overexpression and O-control groups.

D, E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days. (D', E') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC.

F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure.

G Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector. (G') Relative expression levels between control vector and RUNX2 overexpression groups.

H Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with scrambled shRNA (scramsh) or RUNX2 knockdown shRNA (knockdown) lentiviral vectors. (H') Relative expression levels between scramsh and knockdown groups.

I Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs of RUNX2 overexpression (O), control vector (V), RUNX2 knockdown (K), scramsh (S), compressed force (F; 1.0 g/cm2), and control (C; 0 g/cm2) groups. Sample were loaded in a gradient (7.5, 15, 30, and 60 μg) showing a linear relationship for each group.

J Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs treated with IWR-1-endo. Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR-1-endo treatment.

K Diagram illustrating the biomechanical stress regulation of Wnt/β-catenin signaling in the mesenchyme between the deciduous (DT) and permanent tooth (PT) via the integrin β1-ERK1-RUNX2-Wnt/β-catenin pathway.

L Diagram illustrating the biomechanical stress-associated downregulation of RUNX2-Wnt/β-catenin pathway in the mesenchyme, inducing upregulation of Wnt signaling in the epithelium, which triggers PT development.

Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for C, F, G', and H'; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, *P < .05, **P < .01, ***P < .001; NS. not significant.

Expanded View Figure legends
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 1

Figure 1 – Tau protein is not involved in regulating the rate of adult hippocampal neurogenesis under basal conditions.

A-B Representative image of a proliferative cell in the DG labeled with an anti-PH3 antibody (green) (A) and quantification of the number of PH3+ cells in the DG of WT and Tau-/- mice (B) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).

C-D Representative image of an apoptotic cell in the DG labeled with an anti-fractin antibody (red) (C) and quantification of the number of fractin+ cells in the DG of WT and Tau-/- mice (D) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).

E-F Schematic diagram of the cell survival experimental design using thymidine analogs (F) and quantification of the percentage of surviving cells in Tau-/- mice as compared to WT mice at each cell age (1-, 2-, 4-, 6- and 8-week-old cells) (F) (mean ± SEM (normalized data); Student´s t-test).

G Schematic diagram of the cell markers representative of each maturational stage during adult hippocampal neurogenesis.

H Quantification of the percentage of 1-, 4- and 8-week-old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau-/- mice (χ2 test).

I-J Representative images of progenitor cells in the DG labeled with anti-Sox2 (red) and anti-BLBP (green) antibodies (I), and neuroblasts and immature neurons labeled with anti-DCX (red) and anti-calretinin (green) antibodies, respectively (J).

K-N Quantification of the number of Sox2+ (K), BLBP+ (L), DCX+ (M) and calretinin+ (N) cells in the DG of WT and Tau-/- mice (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).

Data information: the number of animals used for the quantification with thymidine analogs (F, H) is indicated in (E). In representative images cell nuclei were labeled with DAPI (blue). White scale bars 10 µm. Red scale bars 20 µm. GL: granular layer; SGL: subgranular layer; H: hilus. Brightness and contrast of representative confocal microscopy images shown in the Figure were minimally adjusted in order to improve visualization.
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 2

Figure 2 – Tau protein is necessary for the dendritic and synaptic maturation of newborn granule neurons.

A Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus. A schematic representation of Sholl´s analysis is shown in the WT image.

B-C Quantification of total dendritic length (B) and Sholl´s analysis (C) of 4-week-old newborn granule neurons in WT and Tau-/- mice.

D Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus.

E-F Quantification of total dendritic length (E) and Sholl´s analysis (F) of 8-week-old newborn granule neurons in WT and Tau-/- mice.

G Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).

H-I Quantification of the number of PSDs/µm (H) and PSD area (I) in each dendritic branching order of 4-week-old newborn granule neurons in WT and Tau-/- mice.

J Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).

K-L Quantification of the number of PSDs/µm (K) and PSD area (L) in each dendritic branching order of 8-week-old newborn granule neurons in WT and Tau-/- mice.

Data information: in (B, C, E, F, H, I, K, L) data are presented as mean ± SEM; n= 3 mice per genotype; *0.05 > p ≥ 0.01, **0.01 > p ≥ 0.001, ***p < 0.001 (Student´s t test or Mann-Whitney U test). In representative images cell nuclei were labeled with DAPI (blue). White scale bars 50 µm. Red scale bars 5 µm. Brightness and contrast of representative confocal microscopy images shown in the Figure were minimally adjusted in order to improve visualization.
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 3

Figure 3 – Tau protein deficiency affects basal synaptic activity in granule neurons.

A Representative traces of mEPSCs recorded at a holding potential of −65 mV from WT (black traces) and Tau-/- (red traces) mice.

B Exemplary traces of mEPSCs from one cell from WT (black trace) and Tau-/- (red trace) mice.

C Resting membrane potential of WT and Tau-/- mice (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; *0.05 > p ≥ 0.01, Student´s t-test).

D-E mEPSC amplitude (D) and mEPSC frequency (E) of WT and Tau-/- animals (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; Student´s t test or Mann-Whitney´s U test).

F Cumulative probability plot of the total mEPSC amplitudes, small mEPSCs (i) and big mEPSCs (ii) from both WT and Tau-/- mice (black and red traces, respectively) (***p < 0.001; Kolmogorov-Smirnov Z test).
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 4

Figure 4 – Tau protein is necessary for the stress-induced death of newborn granule neurons.

A Representative image of a mouse subjected to forced swimming in the Porsolt test.

B Schematic diagram of the experimental design.

C Representative images of 1-week-old newborn granule neurons labeled with CldU (green) belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice. Cell nuclei were labeled with DAPI (blue). Scale bar 50 µm.

D-E Quantification of the number of 1-week-old CldU+ cells (D) and apoptotic fractin+ cells (E) in each experimental group.

Data information: the number of animals used is indicated in (B). Data are presented as mean ± SEM. In (D) data are normalized with respect to WT CNP. Black asterisks (*) indicate differences between CNP and P groups: ***p < 0.001 (Two-way ANOVA). Colored crosses (+) indicate differences between the group represented by the same color and the WT P group: +++p < 0.001 (One-way ANOVA + LSD post hoc multiple comparisons). GL, granular layer; SGL, subgranular layer; H, hilus. Brightness and contrast of representative confocal microscopy images shown in the Figure were minimally adjusted in order to improve visualization.
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 5

Figure 5 – Tau protein mediates the deleterious effect of stress on the dendritic morphology and PSDs of newborn granule neuron.

A Schematic diagram of the experimental design.

B Representative images of 8-week-old newborn granule neurons of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice infected with a PSD95-GFP-expressing retrovirus. In the WT CNP image a schematic representation of Sholl´s analysis is shown.

C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).

E-F Representative images of an 8-week-old newborn granule neuron, in which proximal dendrites are highlighted (E), and of PSDs (green) in proximal dendrites belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (F).

G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).

I-J Representative Western Blot (I) and quantification (J) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau-/- mice subjected or not to the Porsolt test (n= 6 mice per experimental condition).

K Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).

L-M Representative images (L) and quantification of the area (M) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (n= 3 mice per experimental condition). Arrows indicate MFTs.

Data information: Data are presented as mean ± SEM. In (J) data are normalized with respect to WT CNP. Black asterisks (*) indicate differences between CNP and P groups: ***p < 0.001 (Two-way ANOVA). Colored asterisks (*) indicate differences between the group represented by the same color and the WT CNP group: *0.05 > p ≥ 0.001, **0.01 > p ≥ 0.001, ***p < 0.001; colored crosses (+) indicate differences between the group represented by the same color and the WT P group: +0.05 > p ≥ 0.001, +++p < 0.001; colored pads (#) indicate differences between the group represented by the same color and the Tau-/- CNP group: ##0.01 > p ≥ 0.001 (One-way ANOVA + LSD post hoc multiple comparisons). In representative images cell nuclei were labeled with DAPI (blue). White scale bars 50 µm. Red scale bar 5 µm. Orange scale bar 300 µm. Green scale bar 20 µm. Brightness and contrast of representative confocal microscopy images shown in the Figure were minimally adjusted in order to improve visualization.
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 6

Figure 6 – Tau protein mediates the proneurogenic effects of environmental enrichment.

A Representative images of control housing (CH) and environmental enrichment (EE) cages.

B Schematic diagram of the experimental design.

C-D Representative images of DCX+ cells in the DG of CH and EE animals (C), and quantification of the number of DCX+ cells of WT and Tau-/- animals in CH or EE conditions (D).

E Representative images of 8-week-old newborn granule neurons labeled with IdU (red) belonging to WT CH, WT EE, Tau-/- CH and Tau-/- EE mice.

F-G Quantification of the number of 8-week-old IdU+ cells (F) and apoptotic fractin+ cells (G).

Data information: the number of animals used is indicated in (B). Data are presented as mean ± SEM. In (F) data are normalized with respect to WT CH. Black asterisks (*) indicate differences between CH and EE groups: ***p < 0.001 (Two-way ANOVA). Colored asterisks indicate differences between the group represented by the same color and the WT CH group: **0.01 > p ≥ 0.001, ***p < 0.001; colored crosses (+) indicate differences between the group represented by the same color and the WT EE group: +0.05 > p ≥ 0.01, ++0.01 > p ≥ 0.001 (One-way ANOVA + LSD post hoc multiple comparisons). GL, granular layer; SGL, subgranular layer; H, hilus; ML, molecular layer. In representative images cell nuclei were labeled with DAPI (blue). White scale bar 20 µm. Red scale bar 50 µm. Brightness and contrast of representative confocal microscopy images shown in the Figure were minimally adjusted in order to improve visualization.
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 7

Figure 7 – Tau protein is involved in the effect of EE on GABAergic innervation.

A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD-65 antibody (red), and high-power magnifications of the DG molecular layer showing GAD-65+ terminals in WT CH, Tau-/- CH, WT EE and Tau-/- EE mice. Cell nuclei were labeled with DAPI (blue). White scale bar 300 µm. Red scale bar 50 µm. Green scale bar 5 µm.

B-C Quantification of the density (B) and area (C) of GAD-65+ terminals in the molecular layer of mice in each experimental condition (n= 10 mice WT CH, n= 8 mice Tau-/- CH, n= 6 mice WT EE, n= 10 mice Tau-/- EE).

Data information: in (B-C) data are presented as mean ± SEM (normalized with respect to WT CH). Colored asterisks indicate differences between the group represented by the same color and the WT CH group: **0.01 > p ≥ 0.001, ***p < 0.001; colored crosses (+) indicate differences between the group represented by the same color and the WT EE group: +0.05 > p ≥ 0.01; colored pads (#) indicate differences between the group represented by the same color and the Tau-/- CH group: #0.05 > p ≥ 0.01, ###p < 0.001 (One-way ANOVA + LSD post hoc multiple comparisons). DG, dentate gyrus; ML, molecular layer.
Novel function of Tau in regulating the effects of external stimuli on adult neurogenesis (2016)
Figure 8

Figure 8 – Tau protein mediates the stimulatory effects of EE on the dendritic morphology, PSDs and MFTs of newborn neurons.

A Schematic diagram of the experimental design.

B Representative images of 8-week-old newborn granule neurons of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice infected with a PSD95-GFP-expressing retrovirus. In the WT CH image a schematic representation of Sholl´s analysis is shown.

C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).

E-F Representative images of an 8-week-old newborn granule neuron in which proximal and distal dendrites are highlighted (E) and of PSDs (green) in distal and proximal dendrites of WT and Tau-/- mice housed under either CH or EE conditions (F).

G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).

I Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).

J-K Representative images (J) and quantification of the area (K) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice (n= 3 mice per experimental condition). Arrows indicate MFTs.

Data information: Data are presented as mean ± SEM. Black asterisks (*) indicate differences between CH and EE groups: ***p < 0.001 (Two-way ANOVA). Colored asterisks indicate differences between the group represented by the same color and the WT CH group: *0.05 > p ≥ 0.001, **0.01 > p ≥ 0.001, ***p < 0.001; colored crosses (+) indicate differences between the group represented by the same color and the WT EE group: +++p < 0.001; colored pads (#) indicate differences between the group represented by the same color and the Tau -/- CH group: ##0.01 > p ≥ 0.001, ### p < 0.001 (One-way ANOVA + LSD post hoc multiple comparisons). In representative images cell nuclei were labeled with DAPI (blue). White scale bars 50 µm. Red scale bar 5 µm. Orange scale bar 300 µm. Green scale bar 20 µm. Brightness and contrast of representative confocal microscopy images shown in the Figure were minimally adjusted in order to improve visualization.