Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Pax6 overexpression resulted in abnormalities of cortical patterning. ( A ) pCAGGS-EGFP (Control) or pCAGGS-Pax6-EGFP (Pax6 overexpression) constructs were transfected into E15.5 embryo cortex by in utero electroporation, and brain samples were collected three days after electroporation (E15.5 + 3d) for DAPI staining ( E , H , and I ), and thionine staining ( K and L ). ( B and C ) are the overview of the brain of the control and Pax6 overexpression group. ( D ) Statistical graph of brain size of the control and Pax6 overexpression. ( E ) Horizontal section of Pax6-overexpressing brain where the right side is Pax6-transfected. ( F ) is the amplification of the framed area of E. ( G ) Statistical graph of cortical thickness of the non-transfected and transfected side of horizontal Pax6-overexpressing brain section. ( H and I ) are the coronal section of non-transfected and transfected cortex of Pax6-overexpressing brain. ( J ) Statistical graph of cortical thickness of non-transfected and transfected side of coronal Pax6-overexpressing brain section. Thionine staining was performed on the coronal section of Pax6-overexpressing brain to label cortical layers; ( K and L ) show non-transfected and transfected cortex of coronal Pax6-overexpressing brain section, respectively. ( M ) Statistical graph of the thickness of each cortical layer in non-transfected and transfected cortex of coronal Pax6-overexpressing brain section. ( N ) Statistical graph of the percentage of each sublayer in cortical plate of non-transfected and transfected cortex of coronal Pax6-overexpressing brain section. Scale bars are 200 μm in E, 50 μm in ( F , K and L ), and 100 μm in ( H and I ). Abbreviations: OE, overexpression; VZ, ventricular zone; SVZ; subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone; ns, no significance. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: Over Expression, Control, Construct, Transfection, In Utero, Electroporation, Staining, Amplification

Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Neurons were detained in the intermediate zone of cortex after Pax6 overexpression. Three days after the transfection of pCAGGS-EGFP (Control), and pCAGGS-Pax6-EGFP (Pax6 overexpression) constructs into E15.5 embryo cortex, brain samples (E15.5 + 3d) were coronally sectioned for subsequent examinations ( A ). Immunostainings against Pax6 were performed to track Pax6-overexpressing cells: DAPI (Blue in B and C ), GFP (Green in D and E ), Pax6 (Red in F and G ), and Merge ( H and I ). ( J ) Statistical graph of percentage of GFP + cells in different cortical regions of the control and Pax6 overexpression group. ( K ) Pie chart of the proportion of GFP + /Pax6 + cells in Pax6-overexpressing cortex. L) Statistical graph of percentage of GFP + or GFP + /Pax6 + cells in different cortical regions of the control and Pax6 overexpression group. ( M ) Schematic diagrams of neuronal migration in Pax6-overexpressing cortex at E15.5 + 3d. GFP + /Pax6 + cells are pointed by white arrowheads in ( G and I ). Scale bar = 50 μm. Abbreviations: OE, overexpression; VZ, ventricular zone; SVZ, Subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: Over Expression, Transfection, Control, Construct, Migration

Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Pax6 overexpression temporally disrupted neuronal migration during corticogenesis. pCAGGS-EGFP (Control) or pCAGGS-Pax6-EGFP (Pax6 overexpression) constructs were transfected into E13.5 embryo cortex, and brain samples were collected respectively at 1 day (E13.5 + 1d, A - H ), 3 days (E13.5 + 3d, I - P ), 5 days (E13.5 + 5d, Q - X ), and 7 days (E13.5 + 7d, Y - f ) after electroporation for immunostainings against Pax6. Immunostaining shows DAPI (Blue in A , E , I , M , Q , U , Y , and c ), GFP (Green in B , F , J , N , R , V , Z , and d ), Pax6 (Red in C , G , K , O , S , W , a , and e ), and Merge ( D , H , L , P , T , X , b , and f ). White arrowheads highlight GFP + /Pax6 + cells in Pax6 overexpression group. ( g - j ) Statistical graph of percentage of GFP + or GFP + /Pax6 + cells in different cortical regions of the control and Pax6 overexpression at 1 day ( g ), 3 days ( h ), 5 days ( i ) and 7 days ( j ) after electroporation. ( k ) Schematic diagrams of neuronal migration in Pax6 overexpressing cortex at E13.5 + 1d, E13.5 + 3d, E13.5 + 5d and E13.5 + 7d. Scale bar = 50 μm. Abbreviations: OE, overexpression; VZ/SVZ, ventricular zone/subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone; ns, no significance. n = 3. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: Over Expression, Migration, Control, Construct, Transfection, Electroporation, Immunostaining

Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Axonal outgrowth was affected by Pax6 overexpression. Immunostaining against Pax6 was performed on E15.5 + 3d brain sections ( A - H ): DAPI (Blue in A and B ), GFP (Green in C and D ), Pax6 (Red in E and F ), and Merge ( G and H ). ( I ) Statistical graph of GFP + and GFP + /Pax6 + cells with vertical or lateral migratory morphology in the IZ of control and Pax6 overexpression group, respectively. ( J and K ) are the amplification of the framed area in G and H . White arrowheads in J and K indicate GFP + and GFP + /Pax6 + cells exhibiting distinct morphologies in the IZ of control and Pax6 overexpression group, respectively. ( L ) Statistical graph of neuronal cells of different morphologies in the IZ of the control and Pax6 overexpression group. GFP-SH-SY5Y and Pax6 OE-SH-SY5Y cell lines were generated by lentiviral transduction, followed by immunostaining against Pax6: DAPI in blue, GFP in green, and Pax6 in red ( M and N ). White arrowheads in M and N highlight GFP-SH-SY5Y and Pax6 OE-SH-SY5Y cells with different shapes, respectively. ( O ) Statistical graph of SH-SY5Y cells of different shapes in the control and Pax6 overexpression group. Scale bars are 50 μm in A-H, M, and N; 20 μm in J and K. Abbreviations: OE, overexpression; VZ, ventricular zone; SVZ, Subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. n = 3. * p < 0.05, ** p < 0.01.

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: Over Expression, Immunostaining, Control, Amplification, Generated, Transduction

Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Pax6 overexpression promoted the proliferation of neural stem cells. Immunostaining against SOX2 was performed on coronal sections of Pax6-overexpressing brain to test the change of neural stem cells: DAPI (Blue in A and E ), GFP (Green in B and F ), SOX2 (Red in C and G ), and Merge ( D and H ). ( I and J ) are the amplification of the framed area in ( D and H ), respectively. ( K ) Statistical graph of SOX2-postive cells in the cortex of control and Pax6 overexpression group. ( L ) Statistical graph of percentage of SOX2-positive cells in different layers of cortex. To further investigate whether Pax6 promoted cell proliferation, immunostaining against Ki67 was conducted on coronal Pax6-overexpressing brain sections ( M - T ): DAPI (Blue in M and Q ), GFP (Green in N and R ), Ki67 (Red in O and S ), and Merge ( P and T ). ( U and V ) are the amplification of the framed area in P and T , respectively. ( W ) Statistical graph of Ki67-positive cells in cortex of control and Pax6 overexpression group. We also conducted immunostaining against Ki67 on SH-SY5Y cells transfected with pCAGGS-EGFP (control) or pCAGGS-Pax6-EGFP constructs (Pax6 overexpression) ( X - e ): DAPI (Blue in X and b ), GFP (Green in Y and c ), Ki67 (Red in Z and d), Merge (a and e). ( f ) Statistical graph of percentage of Ki67 positive cells in control and Pax6 overexpression group. Scale bars are 100 μm in ( M - T ); 50 μm in ( A - H ); 25 μm in ( I , J , U , and V ); 20 μm in X-e. Abbreviations: OE, overexpression; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone; ns, no significance. n = 3. * p < 0.05, **** p < 0.0001.

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: Over Expression, Immunostaining, Amplification, Control, Transfection, Construct

Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Pax6 overexpression induced the expression of Wnt3a. ( A ) Table of 5 Pax6 potential binding sites within the regulatory sequence of Wnt3a. Immunostaining against Wnt3a was conducted on coronal sections of Pax6-overexpressing brain ( B - I ): DAPI (Blue in B and F ), GFP (Green in C and G ), Wnt3a (Red in D and H ), and Merge ( E and I ). J and K are the amplification of the framed area in E and I , respectively. ( L ) Quantitative PCR was performed to detect the alteration of Pax6 and Wnt3a at the mRNA level in Pax6-overexpressing N2a cells. The level of Pax6/Wnt3a protein in Pax6-overexpressing N2a cells ( M ) and their medium ( O ) was examined by Western blot. ( N and P ) are the statistical graph of ( M and O ). Scale bars are 50 μm in ( B - I ), 25 μm in ( J and K ). Abbreviations: Con, control; OE, overexpression; VZ, ventricular zone; SVZ, Subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. n = 3 in B-I and M-P; n = 4 in L. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: Over Expression, Expressing, Binding Assay, Sequencing, Immunostaining, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Wnt3a participates in the Pax6-overexpression-causing defects. pCAGGS-EGFP (Control), or pCAGGS-EGFP + pECMV-Wnt3a-Flag (Wnt3a overexpression) constructs were transfected into E13.5 embryo cortex, and brain samples were collected three days after electroporation (E13.5 + 3d) for DAPI staining ( B - D , I and J ) or Thionine staining ( F - G ). ( A ) Schematic graph of coronal section of Wnt3a-overexpressing brain. ( B ) Coronal overview of Wnt3a-overexpressing coronal brain section. ( C and D ) are the amplification of non-transfected and transfected cortex of Wnt3a-overexpressing brain. ( E ) Statistical graph of cortical thickness of non-transfected and transfected cortex in Wnt3a-overexpressing brain. Thionine staining was performed on the coronal brain sections of control ( F ) and Wnt3a overexpression ( G ) to label cortical layers. ( H ) Statistical graph of the thickness of each layer in the control and Wnt3a overexpression. ( I and J ) show DAPI staining of control and Wnt3a-overexpressing cortex: DAPI in Blue, and GFP in Green. ( K ) Statistical graph of percentage of GFP + cells in each layer of the control and Wnt3a-overexpressing cortex. To investigate whether Wnt3a participates in the Pax6-overexpression-causing defects, Wnt3a was co-suppressed with Pax6 overexpression in E15.5 embryo cortex, and brain samples were collected in 3 days (E15.5 + 3d) for immunostaining against Pax6. ( L ) Schematic graph of in utero electroporation and coronal brain section. Immunostaining against Pax6 was performed in the control ( M , P , S , and V ), Pax6 overexpression ( N , Q , T , and W ) and Pax6 overexpression + Wnt3a knockdown ( O , R , U , and X ): DAPI (Blue in M - O ), GFP (Green in P - R ), Pax6 (Red in S - U ), and Merge ( V - X ). White arrowheads highlight GFP + /Pax6 + cells in Pax6 overexpression and Pax6 overexpression + Wnt3a knockdown group. ( Y ) Statistical graph of percentage of GFP + or GFP + /Pax6 + cells in different cortical regions of the control, Pax6 overexpression and Pax6 overexpression + Wnt3a knockdown. Scar bars are 200 μm in ( B ); 50 μm in ( C , D , I , J , and M - X ); 30 μm in ( F and G ). Abbreviations: A, anterior; P, Posterior; OE, overexpression; KD, knockdown; VZ, ventricular zone; SVZ; subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone; ns, no significance. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: Over Expression, Control, Construct, Transfection, Electroporation, Staining, Amplification, Immunostaining, In Utero, Knockdown

Journal: Scientific Reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet:

Article Snippet: Pax6 or Wnt3a proteins were detected using Mouse anti-Pax6 primary antibody (1:5000, ab197768, Abcam, UK) or Rabbit anti-Wnt3a primary antibody (1:5000, ab28472, Abcam) and HRP-conjugated anti-Mouse secondary antibody (1:5000, ZDR-5307, ZSGB-BIO, China) or HRP-conjugated anti-Rabbit secondary antibody (1:5000, ZDR-5306, ZSGB).

Techniques: