Journal: bioRxiv

Article Title: EBF1 limits the numbers of cochlear hair and supporting cells and forms the scala tympani and spiral limbus during the development of the inner ear

doi: 10.1101/2023.04.28.538789

Figure Lengend Snippet: A. Results of qRT-PCR analysis for Ebf1 in the inner ear of wild-type mice from E9.5 to P0. The value of each date is normalized to the value of E9.5. Box plot representing medians and interquartile ranges of relative mRNA expression of Ebf1 . B and C . in situ hybridization (ISH) for Ebf1 and Sox2 in cross-sections of the inner ear of wild-type mice at E13.5 (B) and E10.5 and E18.5 (C). Areas enclosed by dashed lines indicate the inner ear epithelium. Low-magnification images of the cochlear basal turn and vestibule are presented in the uppermost panels of B . High-magnification images of the apical turn of the cochlear duct and vestibules are presented in the middle and lower panels of B , respectively. Ebf1 was expressed in the prosensory domain of the cochlea (white arrows) and medial side of the cochlear floor, a part of Sox2 positive region within the vestibule (black arrows), spiral ganglion (white asterisks), and surrounding mesenchymal tissues (asterisks). Images of E10.5 otocysts, E18.5 cochleae, and E18.5 vestibules are presented in the uppermost, middle, and lowermost images of C , respectively. Ebf1 was expressed on the lateral and ventromedial sides of the otocyst at E10.5 (arrowheads). Ebf1 was also expressed in the cochleo-vestibular ganglion (white asterisk). In the inner ear of E18.5, Ebf1 was expressed in Kölliker’s organ, the organ of Corti, the macula, and the crista. Ebf1 was also expressed in the spiral ligament and tympanic border cells (white arrowhead). D . Immunohistochemical images of E14.5 Ebf1 +/+ and Ebf1 -/- mice cochlear ducts. In Ebf1 +/+ mice, EBF1 is expressed throughout Kölliker’s organ and in the medial part of prosensory domain, whereas SOX2 was expressed in the lateral part of Kölliker’s organ and in the prosensory domain. In Ebf1 -/- mice, EBF1 expression was absent in both the epithelia and mesenchyme, and the region of the SOX2-positive Kölliker’s organ was expanded (arrowhead). One-way analysis of variance (ANOVA) with Tukey–Kramer post hoc tests was performed. * p <0.05, ** p <0.01. D, dorsal; L, lateral. cd, cochlear duct; sg, spiral ganglion; Ut, utricle; Sa, saccule; Lc, lateral crista; SL, spiral ligament; Ko, Kölliker’s organ; oC, organ of Corti. Pd, prosensory domain. Scale bars: 100μm.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-EBF1 antibody (1:1000, Millipore, Darmstadt, Germany, AB10523), mouse anti-MYO7A antibody (1:1000, Developmental Studies Hybridoma Bank[DSHB], Iowa City, IA, USA, 138-1), rabbit anti-MYO7A antibody (1:1000, Proteus BioSciences, Waltham, MA, USA, 25-6790), goat anti-SOX2 antibody (1:250, R&D Systems, Minneapolis, MN, USA, AF2018), rabbit anti-SOX2 antibody (1:100, Proteintech, Manchester, UK, 11064-1-AP), rabbit anti-VGLUT3 antibody (1:500, Synaptic systems, Goettingen, Germany, 135 203), goat anti-JAG1 antibody (1:500, Santa cruz, Dallas, Texas, USA, sc-6011), mouse anti-p27Kip1 antibody (1:200, BD Transduction Laboratories, 610242), rabbit anti-Tubulin β 3 (TUJ1) antibody (1:1000, Biolegend, San Diego, CA, USA, PRB-435P), rabbit anti-PROX1 antibody (1:500, Millipore, AB5475), rabbit anti-p75 antibody (1:500, Millipore, AB1554), and rabbit anti Cleaved caspase3 (CC3) antibody (1:400, Cell Signaling Technology, Danvers, MA, USA, Asp175).

Techniques: Quantitative RT-PCR, Expressing, In Situ Hybridization, Immunohistochemical staining

Journal: bioRxiv

Article Title: EBF1 limits the numbers of cochlear hair and supporting cells and forms the scala tympani and spiral limbus during the development of the inner ear

doi: 10.1101/2023.04.28.538789

Figure Lengend Snippet: in situ hybridization (ISH) for Ebf1 and Sox2 on cross-sections of the inner ear of wild-type mice at E11.5, E12.5, E16.5, and P0. Areas enclosed by dashed lines indicate the inner ear epithelium. From E11.5 to P0, Ebf1 is expressed in the sensory epithelium of the cochlea (white arrows), the vestibular and semicircular canals (arrows), the spiral ganglion (white asterisks), and the surrounding mesenchymal tissues (asterisks). sg, spiral ganglion; Ut, utricle; Sa, saccule; Lc, lateral crista; SL, spiral ligament; Ko, Kölliker’s organ; oC, organ of Corti. Scale bars: 100μm.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-EBF1 antibody (1:1000, Millipore, Darmstadt, Germany, AB10523), mouse anti-MYO7A antibody (1:1000, Developmental Studies Hybridoma Bank[DSHB], Iowa City, IA, USA, 138-1), rabbit anti-MYO7A antibody (1:1000, Proteus BioSciences, Waltham, MA, USA, 25-6790), goat anti-SOX2 antibody (1:250, R&D Systems, Minneapolis, MN, USA, AF2018), rabbit anti-SOX2 antibody (1:100, Proteintech, Manchester, UK, 11064-1-AP), rabbit anti-VGLUT3 antibody (1:500, Synaptic systems, Goettingen, Germany, 135 203), goat anti-JAG1 antibody (1:500, Santa cruz, Dallas, Texas, USA, sc-6011), mouse anti-p27Kip1 antibody (1:200, BD Transduction Laboratories, 610242), rabbit anti-Tubulin β 3 (TUJ1) antibody (1:1000, Biolegend, San Diego, CA, USA, PRB-435P), rabbit anti-PROX1 antibody (1:500, Millipore, AB5475), rabbit anti-p75 antibody (1:500, Millipore, AB1554), and rabbit anti Cleaved caspase3 (CC3) antibody (1:400, Cell Signaling Technology, Danvers, MA, USA, Asp175).

Techniques: In Situ Hybridization

Journal: bioRxiv

Article Title: EBF1 limits the numbers of cochlear hair and supporting cells and forms the scala tympani and spiral limbus during the development of the inner ear

doi: 10.1101/2023.04.28.538789

Figure Lengend Snippet: A. Cross sections of basal and apical turns of the cochlea at E18.5 from Ebf1 +/+ and Ebf1 -/- mice labeled with MYO7A (green) and SOX2 (magenta). Magnified images of organ of Corti (oC) and Kölliker’s organ (Ko) are presented in the right four panels. MYO7A-and SOX2-positive cell numbers increase in Ebf1 -/- mice. The apical turn of Ebf1-/- mice had multiple layers of SOX2-positive cells (asterisk). B – E . Whole mount cochlear images at E18.5 from Ebf1 +/+ and Ebf1 -/- mice labeled with MYO7A (green, B and C ), phalloidin (white, C ), SOX2 (magenta, D ), and PROX1 (green, E ). In Ebf1 -/- mice, MYO7a-positive hair cells increased in number ( B ) and ectopic MYO7A-positive cells ( B , white arrows) were found in GER (medial region of the cochlear floor). Magnified images of ectopic MYO7A-positive cells ( C ) show stereocilia-like structures stained with phalloidin (arrows in C ), suggesting that these cells are hair-cell like cells. Scale bars: 50 μm in A , D , and E , 100 μm in B , 10 μm in C .

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-EBF1 antibody (1:1000, Millipore, Darmstadt, Germany, AB10523), mouse anti-MYO7A antibody (1:1000, Developmental Studies Hybridoma Bank[DSHB], Iowa City, IA, USA, 138-1), rabbit anti-MYO7A antibody (1:1000, Proteus BioSciences, Waltham, MA, USA, 25-6790), goat anti-SOX2 antibody (1:250, R&D Systems, Minneapolis, MN, USA, AF2018), rabbit anti-SOX2 antibody (1:100, Proteintech, Manchester, UK, 11064-1-AP), rabbit anti-VGLUT3 antibody (1:500, Synaptic systems, Goettingen, Germany, 135 203), goat anti-JAG1 antibody (1:500, Santa cruz, Dallas, Texas, USA, sc-6011), mouse anti-p27Kip1 antibody (1:200, BD Transduction Laboratories, 610242), rabbit anti-Tubulin β 3 (TUJ1) antibody (1:1000, Biolegend, San Diego, CA, USA, PRB-435P), rabbit anti-PROX1 antibody (1:500, Millipore, AB5475), rabbit anti-p75 antibody (1:500, Millipore, AB1554), and rabbit anti Cleaved caspase3 (CC3) antibody (1:400, Cell Signaling Technology, Danvers, MA, USA, Asp175).

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: EBF1 limits the numbers of cochlear hair and supporting cells and forms the scala tympani and spiral limbus during the development of the inner ear

doi: 10.1101/2023.04.28.538789

Figure Lengend Snippet: Cross sections of the E18.5 vestibular region immunostained for MYO7A (green) and SOX2 (magenta). Low-magnification (left four panels) and high-magnification (right panels) images of the saccule, lateral crista, and utricle are presented. Ut, utricle; Sa, saccule; Lc, lateral crista. Scale bars: 100μm.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-EBF1 antibody (1:1000, Millipore, Darmstadt, Germany, AB10523), mouse anti-MYO7A antibody (1:1000, Developmental Studies Hybridoma Bank[DSHB], Iowa City, IA, USA, 138-1), rabbit anti-MYO7A antibody (1:1000, Proteus BioSciences, Waltham, MA, USA, 25-6790), goat anti-SOX2 antibody (1:250, R&D Systems, Minneapolis, MN, USA, AF2018), rabbit anti-SOX2 antibody (1:100, Proteintech, Manchester, UK, 11064-1-AP), rabbit anti-VGLUT3 antibody (1:500, Synaptic systems, Goettingen, Germany, 135 203), goat anti-JAG1 antibody (1:500, Santa cruz, Dallas, Texas, USA, sc-6011), mouse anti-p27Kip1 antibody (1:200, BD Transduction Laboratories, 610242), rabbit anti-Tubulin β 3 (TUJ1) antibody (1:1000, Biolegend, San Diego, CA, USA, PRB-435P), rabbit anti-PROX1 antibody (1:500, Millipore, AB5475), rabbit anti-p75 antibody (1:500, Millipore, AB1554), and rabbit anti Cleaved caspase3 (CC3) antibody (1:400, Cell Signaling Technology, Danvers, MA, USA, Asp175).

Techniques:

Journal: bioRxiv

Article Title: EBF1 limits the numbers of cochlear hair and supporting cells and forms the scala tympani and spiral limbus during the development of the inner ear

doi: 10.1101/2023.04.28.538789

Figure Lengend Snippet: A . Schematic drawings of the cochlea duct showing the positions of basal, middle, and apical region of the cochlea. B . Total hair cell numbers per 200μm of E18.5 Ebf1 +/+ , Ebf1 +/- , and Ebf1 -/- mouse cochlear ducts at the basal, middle, and apical region. Ebf1 -/- mice showed significantly smaller numbers of hair cells through the whole cochlea. Even Ebf1 +/- mice showed a significantly higher number of cochlear hair cells than Ebf1 +/+ mice in the middle and apical regions C . The numbers of SOX2-positive cells per 100μm at the basal region of the cochlea. SOX2-positive cell numbers significantly increased in Ebf1 -/- mice. D . The numbers of PROX1-positive cells per 200μm at the basal, middle, and apical region of the cochlea. PROX1-positive cell numbers significantly increased in Ebf1 -/- mice at basal and middle region. two-way ANOVA test with Bonferroni post hoc tests ( B and D ) and Student’s t -test ( C ) were performed. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not significant. Error bars represent mean ± SD. n = 4.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-EBF1 antibody (1:1000, Millipore, Darmstadt, Germany, AB10523), mouse anti-MYO7A antibody (1:1000, Developmental Studies Hybridoma Bank[DSHB], Iowa City, IA, USA, 138-1), rabbit anti-MYO7A antibody (1:1000, Proteus BioSciences, Waltham, MA, USA, 25-6790), goat anti-SOX2 antibody (1:250, R&D Systems, Minneapolis, MN, USA, AF2018), rabbit anti-SOX2 antibody (1:100, Proteintech, Manchester, UK, 11064-1-AP), rabbit anti-VGLUT3 antibody (1:500, Synaptic systems, Goettingen, Germany, 135 203), goat anti-JAG1 antibody (1:500, Santa cruz, Dallas, Texas, USA, sc-6011), mouse anti-p27Kip1 antibody (1:200, BD Transduction Laboratories, 610242), rabbit anti-Tubulin β 3 (TUJ1) antibody (1:1000, Biolegend, San Diego, CA, USA, PRB-435P), rabbit anti-PROX1 antibody (1:500, Millipore, AB5475), rabbit anti-p75 antibody (1:500, Millipore, AB1554), and rabbit anti Cleaved caspase3 (CC3) antibody (1:400, Cell Signaling Technology, Danvers, MA, USA, Asp175).

Techniques:

Journal: bioRxiv

Article Title: EBF1 limits the numbers of cochlear hair and supporting cells and forms the scala tympani and spiral limbus during the development of the inner ear

doi: 10.1101/2023.04.28.538789

Figure Lengend Snippet: A . Cross-sections of the cochlear basal regions at E12.5, E13.5, E14.5, and E16.5 from Ebf1 +/+ and Ebf1 -/- mice. Sections were immunostained with EdU (green) and SOX2 (magenta). E16.5 sections were counter-stained with DAPI (gray). Areas enclosed by dashed lines indicate the cochlear ducts and brackets indicate prosensory domains. Although the Ebf1 +/+ mice cochlear floor had EdU negative region (bracket), EdU was detected in the corresponding area of the Ebf1 -/- mice at E13.5 (bracket). ZNPC appeared in the Ebf1 -/- mice cochlear floor at E14.5 (bracket). Kölliker’s organ at E16.5 loses EdU-positive cells in the Ebf1 +/+ mice. In contrast, EdU positive cells were still present in Kölliker’s organ of the Ebf1 -/- mice at E16.5 (arrowhead). B – D . Quantitative assessment of the SOX2 positive region in the cochlea epithelia. The numbers of SOX2-positive cells ( B ) and EdU-and SOX2-double positive cells ( C ) were counted, and the percentage of EdU-positive cells among SOX2-positive cells ( D ) was calculated. In Ebf1 -/- mice, the number of SOX2-positive and EdU- and SOX2-double positive cells significantly increased in the cochlear duct at E13.5 and E14.5. The percentage of EdU-positive cells among SOX2-positive cells was significantly higher in Ebf1 -/- mice than in Ebf1 +/+ mice only at E13.5. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Error bars represent mean ± SD. n = 4.. E . Cross-sections of the cochlear basal regions at E13.5 and E14.5 from Ebf1 +/+ and Ebf1 -/- mice immunostained with p27Kip1 (green) and SOX2 (magenta). In Ebf1 -/- mice, p27Kip1 expression (brackets) was absent in the cochlear duct at E13.5 (arrows), although it was present at E14.5. Scale bars: 100μm.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-EBF1 antibody (1:1000, Millipore, Darmstadt, Germany, AB10523), mouse anti-MYO7A antibody (1:1000, Developmental Studies Hybridoma Bank[DSHB], Iowa City, IA, USA, 138-1), rabbit anti-MYO7A antibody (1:1000, Proteus BioSciences, Waltham, MA, USA, 25-6790), goat anti-SOX2 antibody (1:250, R&D Systems, Minneapolis, MN, USA, AF2018), rabbit anti-SOX2 antibody (1:100, Proteintech, Manchester, UK, 11064-1-AP), rabbit anti-VGLUT3 antibody (1:500, Synaptic systems, Goettingen, Germany, 135 203), goat anti-JAG1 antibody (1:500, Santa cruz, Dallas, Texas, USA, sc-6011), mouse anti-p27Kip1 antibody (1:200, BD Transduction Laboratories, 610242), rabbit anti-Tubulin β 3 (TUJ1) antibody (1:1000, Biolegend, San Diego, CA, USA, PRB-435P), rabbit anti-PROX1 antibody (1:500, Millipore, AB5475), rabbit anti-p75 antibody (1:500, Millipore, AB1554), and rabbit anti Cleaved caspase3 (CC3) antibody (1:400, Cell Signaling Technology, Danvers, MA, USA, Asp175).

Techniques: Staining, Expressing

Journal: Cell Cycle

Article Title: Mesenchymal traits are selected along with stem features in breast cancer cells grown as mammospheres

doi: 10.4161/cc.22543

Figure Lengend Snippet: Figure 2. ALDH1 expression in the breast cancer cell lines analyzed. ( A ) ALDH1A1 and ( B ) ALDH1A3 mRNA-relative expression determined by qRT-PCR. Data were normalized to three reference genes as described in Materials and Methods. Bars represent standard deviation. ( C ) SOX2 transcript determined by RT-PCR. GAPDH is reported as RNA control. NT2D1, NTera D1 cell line was used as a positive control for SOX2.

Article Snippet: Western blotting was performed on cell protein lysates (50 mM Tris, pH 7.6, 250 mM NaCl, 0,2% Triton X-100, 0,3% NP40, 2 nM EGTA, 2 nM EDTA, 0.1% P-40, complete protease inhibitor cocktail Roche Molecular Biochemicals) using mouse antibodies reacting to human SOX2 (R&D Systems), actin (Santacruz) and rabbit antibodies to human SNAI1 (Cell Signaling) and β-tubulin (Santacruz).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Control, Positive Control

Journal: Cell Cycle

Article Title: Mesenchymal traits are selected along with stem features in breast cancer cells grown as mammospheres

doi: 10.4161/cc.22543

Figure Lengend Snippet: Figure 3. Stem properties were modulated by MS-proficient conditions. ( A ) ALDH1A1 and ( B ) ALDH1A3 transcript levels in cells grown as primary (M1), secondary (M2) and tertiary (M3) mammospheres (gray columns) or in adherence (ctr, black columns) in the same-stem medium. Data, obtained by qRT-PCR, were normalized to three reference genes as described in Materials and Methods. Bars represent standard deviation; asterisks p < 0.05 in one-way Anova analysis. ( C ) SOX2 protein levels, determined by western blotting, in MCF7 cells grown as primary (M1), secondary (M2) and tertiary (M3) mammospheres or in adherence (ctr) in the same-stem medium. NT2D1 is reported as a positive control. The increment of SOX2, determined by the ratio of SOX2 signal over tubulin signal (used as a loading control), is reported.

Article Snippet: Western blotting was performed on cell protein lysates (50 mM Tris, pH 7.6, 250 mM NaCl, 0,2% Triton X-100, 0,3% NP40, 2 nM EGTA, 2 nM EDTA, 0.1% P-40, complete protease inhibitor cocktail Roche Molecular Biochemicals) using mouse antibodies reacting to human SOX2 (R&D Systems), actin (Santacruz) and rabbit antibodies to human SNAI1 (Cell Signaling) and β-tubulin (Santacruz).

Techniques: Quantitative RT-PCR, Standard Deviation, Western Blot, Positive Control, Control

Journal: Biology Open

Article Title: In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

doi: 10.1242/bio.20123160

Figure Lengend Snippet: In utero electroporation was performed at E37, and sections were prepared at E40. The sections were immunostained with anti-Sox2, anti-Pax6 and anti-Tbr2 antibodies. The cerebral cortex is shown in A . The areas within the white boxes are magnified and are shown in B . CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 200 µm (A) and 100 µm (B).

Article Snippet: Sections were made using a cryostat, permeabilized with 0.1–0.5% Triton X-100/PBS, and incubated overnight with primary antibodies, which included anti-Sox2 antibody (R&D Systems), anti-Pax6 antibody (Covance), anti-Tbr2 antibody (Abcam) and anti-neurofilament-M antibody (Chemicon).

Techniques: In Utero, Electroporation

Journal: Biology Open

Article Title: In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

doi: 10.1242/bio.20123160

Figure Lengend Snippet: In utero electroporation was performed at E35, and coronal sections were prepared at P0 and were stained with Hoechst 33342. Many GFP-positive cells were distributed throughout the cortex ( A ). The areas within the white boxes in A are magnified and are shown in B . The areas within the white boxes in B are shown in C . Note that the morphology of GFP-positive cells was clearly visible even without immunostaining. ( D , E ) The sections were immunostained with anti-Sox2 antibody (D) and anti-Pax6 antibody (E), and high magnification images of the OSVZ are shown. The GFP-positive cells (arrowheads) expressed Sox2 (D) and Pax6 (E), and had basal fibers but not apical fibers, suggesting that these cell are oRG cells. GFP-positive fibers running tangentially in the inner OSVZ were also visible (arrow). *Lateral ventricle. CP, cortical plate; OSVZ, outer subventricular zone; ISVZ, inner subventricular zone; VZ, ventricular zone. Scale bars: 1 mm (A), 500 µm (B), 100 µm (C) and 20 µm (D,E).

Article Snippet: Sections were made using a cryostat, permeabilized with 0.1–0.5% Triton X-100/PBS, and incubated overnight with primary antibodies, which included anti-Sox2 antibody (R&D Systems), anti-Pax6 antibody (Covance), anti-Tbr2 antibody (Abcam) and anti-neurofilament-M antibody (Chemicon).

Techniques: In Utero, Electroporation, Staining, Immunostaining