Fig 1: Genetic knockdown of Wnt7a and Decorin blocks CA*β1-mediated neurogenesis.(a) Immunostaining for fluorescein to show uptake of the morpholinos (MO) in a control E4 midbrain neuroepithelium electroporated with the DCN-MO, DCN-MM, Wnt7a-MO or Wnt7a-MM morpholinos. (b,c) Quantification of Decorin and Wnt7a staining intensity in E4 midbrain neuroepithelium after electroporation of morpholinos. n>3. (d) Illustration by immunohistochemistry for fluorescein, RFPnls and Tuj1 in E4 midbrain neuroepithelium co-electroporated with CA*β1 and a morpholino (either DCN-MO, DCN-MM, Wnt7a-MO or Wnt7a-MM). (e) Quantification of the number of Tuj1+ cells in E4 midbrain neuroepithelium electroporated only with the morpholinos. Note the loss of neurogenesis with the Wnt7a-MO. n>5. (f) Quantification of the number of Tuj1+ cells in E4 midbrain neuroepithelium co-electroporated with CA*β1 and a morpholino. Note the loss of the significant increase in neurogenesis resulting from CA*β1 expression with both the Wnt7a-MO and the DCN-MO. n>8. (g) Quantification of the thickness of the neuroepithelium in E4 midbrain neuroepithelium co-electroporated with CA*β1 and a morpholino. n>4. Scale bars, 20 μm. All graphs: mean and s.d., *P<0.05, **P<0.01, ***P<0.001, one-way analysis of variance.
Fig 2: Pharmacological inhibition of integrin, Wnt7a and Decorin activity in explants.(a) Immunostaining for Tuj1 in explants expressing CA*β1 alone or treated with Wnt inhibitors C-59, Dkk-1, IQ-1, DCN-blocking antibody CB-1, TGF-βR inhibitor or FAK inhibitor. Scale bar, 50 μm. (b) Quantification of the number of Tuj1+ cells in explants expressing CA*β1 alone or treated with the inhibitors. Note that all the inhibitors prevent the significant increase in neurogenesis resulting from CA*β1 expression. n>4. (c) Immunostaining for Tuj1 in non-electroporated explants alone and with the addition of Chiron. (d) Quantification of the number of Tuj1+ cells in explants treated with Chiron. n>4. (e) Immunostaining for GFP and Tuj1 in an explant expressing CA*β1 treated with a FAK inhibitor. White box outlines the area of the image in e'. Scale bar, 50 μm. (e') Enlarged area of e, white filled arrowheads label GFP+Tuj1+ cell bodies. (f) Quantification of GFP+Tuj1+ cells in explants expressing CA*β1 and treated with inhibitors. n>5. Mean and s.d. *P<0.05. n>4, one-way analysis of variance.
Fig 3: Wnt7a and Decorin promote neurogenesis.(a) Immunostaining for Tuj1 in explants—a non-electroporated control, one expressing CA*β1 and non-electroporated explants with recombinant Wnt7a or Decorin (DCN) added. Scale bar, 50 μm. (b) Quantification of the number of Tuj1+ cell bodies in explants electroporated with the different itgβ1 constructs. n>7. (c) Quantification of the number of Tuj1+ cell bodies in non-electroporated explants with recombinant Wnt7a or DCN added. n>5. All graphs: mean and s.d., *P<0.05, **P<0.01, one-way analysis of variance.
Fig 4: Transcriptome analysis of CA*β1-positive and -negative cells reveals a role for Wnt signalling.(a) Heat map showing fold change of gene expression between the CA*β1/GFP+ versus CA*β1/GFP− cells. (b) Validation of transcriptome results via quantitative PCR. Genes highlighted in red are the genes of interest. (c) Immunostaining for GFP and Decorin (DCN) in E4 midbrain neuroepithelium electroporated with hWTβ1 or CA*β1. Scale bar, 20 μm. (d) Immunostaining for GFP and Wnt7a in E4 midbrain neuroepithelium electroporated with hWTβ1 or CA*β1. Scale bar, 20 μm.
Fig 5: Model of the integrin-β1–Wnt7a–Decorin pathway.Schematic showing the proposed model of integrin-β1–Wnt7a–Decorin signalling pathway. The green cell represents the CA*β1-expressing cell, which undergoes proliferation and self-renewal with upregulation of Wnt7a. Wnt7a is secreted, promoting Wnt7a signalling within the CA*β1-expressing cell via the CBP branch of the Wnt pathway, leading to proliferation and self-renewal. Wnt7a also acts on the neighbouring, negative cell (blue/red cell) via the LRP5/6 receptor, signalling via the p300 branch of the Wnt pathway. This leads to the upregulation of Decorin expression. Decorin is secreted and acts in an autocrine manner on the negative cell via the TGF-β receptor, promoting differentiation.
Supplier Page from Abcam for Recombinant human Wnt7a protein