Journal: Cell

Article Title: Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels

doi: 10.1016/j.cell.2018.06.007

Figure Lengend Snippet: Functional Validation of Genetic Reagents and Test of Interaction between Robo and Notch Signaling, Related to , , and (A and B) Validation of dnRobo and myrRobo as dominant-negative and constitutively active for Robo signaling, respectively. In (A), growth cone collapse assay of growing axons from explants of embryonic mouse retinas, electroporated to express Gfp or dnRobo and exposed to recombinant Slit protein or vehicle solution. Failure of response to Slit upon dnRobo-overexpression demonstrates its dominant-negative effect (n = 44-58 growth cones per group, 3 independent experiments). In (B), branching assay of growing axons from single neurons of embryonic rat dorsal root ganglion, overexpressing Gfp alone or with myrRobo constructs as indicated. Exuberant axonal branching typically elicited by Slit-Robo signaling occurs in myrRobo-expressing neurons in the absence of Slit, demonstrating constitutive activation of Robo signaling (n = 5-10 neurons per group). (C–F) Validation of crispr constructs for disruption of Dll1 in mouse and human. (C) Top, sequence of the gRNA targeting mouse Dll1 , and schematic of the orientation and location of the targeting site (black arrow) within the mDll1 coding sequence (gray bar). Bottom, validation of Crispr-mediated editing of the mDll1 locus upon electroporation with gDll1 plus Cas9, but not with Cas9 alone. Different lanes correspond to independent electroporated embryos. M, molecular weight marker. Arrow indicates 1,025 bp amplicon, arrowheads indicate the products of PCR amplicon digestion by Syrveyor Nuclease (656 + 368 bp), absent in the Cas9-alone lanes. (D) Left, sequence of the gRNA targeting human Dll1 , and schematic of the orientation and location of the targeting site (black arrow) within the hDll1 coding sequence (gray bar). Right, chromograms for genome sequence validation of Crispr-mediated editing of the hDll1 locus upon electroporation of cerebral organoid with gDll1 plus Cas9. A 270bp fragment was inserted at position 54 of the coding sequence, introducing a STOP codon in position 76. (E and F) Effect of electroporating crDll1 in NCx (green cells) on the abundance of Dll1 protein (red). Details are examples VZ cells loosing Dll1 protein (arrowheads) from the cell surface upon crDll1 (n = 3 embryos per group). (G–K) Antibody stain for GFP and Robo1 or Dll1 in NCx at E13.5 upon electroporation of the indicated plasmid combinations at E12.5, and quantifications (paired t test). Arrowheads indicate area of increased Robo (n = 3 embryos per group). Values are mean + SEM; paired or independent samples t tests; ∗ p < 0.05; ns, not significant. Scale bars: 30 μm (E), 50 μm (G and H).

Article Snippet: After 24h of incubation, recombinant mouse Slit protein (R&D Systems) was added to the medium (250ng/μl) and the explants were fixed 1h later with PFA 2% during 15 min.

Techniques: Functional Assay, Biomarker Discovery, Dominant Negative Mutation, Recombinant, Over Expression, Construct, Expressing, Activation Assay, CRISPR, Disruption, Sequencing, Electroporation, Molecular Weight, Marker, Amplification, Staining, Plasmid Preparation

Journal: Current biology : CB

Article Title: FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons.

doi: 10.1016/j.cub.2014.01.042

Figure Lengend Snippet: Figure 2. Slit1 Enables Netrin-1 Attraction by Activating the Robo1 Receptor

Article Snippet: Recombinant Slit1 (5 nM; R&D Systems), FLRT3-ECD [15] (1 mg/ml), a mouse anti-DCC antibody (AF5 clone; Abcam), or control mouse IgG was (J and K) Robo1+/+ rTCAs grow rostrally as a compact bundle (J), whereas the (L) Quantification of the data represented in (J) and (K). rTCA dispersion at the v tailed Student’s t test. (M) Scheme representing the topographic guidance of rTCAs and iTCAs in t Slit1 and Netrin-1 [21].

Techniques: