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:

Journal: Scientific Reports

Article Title: Effects of neuroactive agents on axonal growth and pathfinding of retinal ganglion cells generated from human stem cells

doi: 10.1038/s41598-017-16727-1

Figure Lengend Snippet: Effect of SLIT1 supplementation on axonal growth of hESC- and hiPSC-derived RGCs. ( a ) Axonal growth of hESC- and hiPSC-derived RGCs is observed by immunostaining of NFL. In the control, hESC- and hiPSC-derived RGCs axons grow radially and straight (left panels). In contrast, the growth pattern of axons after SLIT1 supplementation is complex. Axonal growth of hESC- and hiPSC-derived RGCs is not inhibited by addition of 0.2 and 1.0 µg of SLIT1 supplementation from D27−30 (centre and right panels, respectively). However, axonal paths are disturbed (centre and right panels). The assessment at D27 is performed in RMM supplemented with 1.0% FBS and 100 ng/ml BDNF. ( b ) Real-time PCR analysis of mRNA expression of axonal markers, TAU , NFL , and TUJ1 . Expression levels of axonal markers are not influenced by SLIT1 supplementation at any of the concentrations tested. Scale bar, 100 μm. Error bars indicate ± SD. Each column shows an average value for the studied samples. The sample size for all mRNA data is five (n = 5). NS, not significant.

Article Snippet: Recombinant human NGF, diluted in reduced Matrigel (BD Bioscience), was used at 10 ng/ml, and SEMA3A, diluted in reduced Matrigel (BD Bioscience), was used at a concentration of 200 ng/ml, while recombinant human SLIT1 (R&D Systems), diluted in Matrigel, was used at concentrations of 5 μg/ml at a temperature of approximately 4 °C.

Techniques: Derivative Assay, Immunostaining, Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Effects of neuroactive agents on axonal growth and pathfinding of retinal ganglion cells generated from human stem cells

doi: 10.1038/s41598-017-16727-1

Figure Lengend Snippet: Effect of locally sustained release of SLIT1 (5μg/ml) from hydrogels placed in front of the attached OVs. Locally sustained release of SLIT1 using hydrogel does not affect axonal growth of hESC- and hiPSC-derived RGCs (( a ) and ( d ), respectively, magnification 40x). Only a subset of axons is repelled by SLIT1 release (( b ) and ( e ), magnification 100x: squares in ( a ) and ( d ), respectively), while other axons remain unaffected. Filopodia of hESC- and hiPSC-derived RGCs (( c ) and ( f ), respectively) have collapsed in response to SLIT1 release, as evident from phalloidin and GAP43 staining. The assessment from D27 is performed under RMM supplemented with 1.0% FBS and 100 ng/ml BDNF. Each experiment was repeated at least five times. Squares in ( a ) and ( d ) correspond to ( b ) and ( e ), respectively. Scale bars in ( a ) and ( d ), 100 μm. Scale bars in ( b ) and ( e ), 40 μm. Scale bars in ( c ) and ( f ), 10 μm.

Article Snippet: Recombinant human NGF, diluted in reduced Matrigel (BD Bioscience), was used at 10 ng/ml, and SEMA3A, diluted in reduced Matrigel (BD Bioscience), was used at a concentration of 200 ng/ml, while recombinant human SLIT1 (R&D Systems), diluted in Matrigel, was used at concentrations of 5 μg/ml at a temperature of approximately 4 °C.

Techniques: Derivative Assay, Staining

Journal: Scientific Reports

Article Title: Effects of neuroactive agents on axonal growth and pathfinding of retinal ganglion cells generated from human stem cells

doi: 10.1038/s41598-017-16727-1

Figure Lengend Snippet: The effect of focally sustained release of SLIT1 on pathfinding of axons derived from hESC- and hiPSC-derived RGCs. Phase-contrast images showing SLIT1-releasing beads (blue-coloured) placed next to the bottom of attached OV, and corresponding immunohistochemistry for the axons of the RGCs by NFL staining. ( a,c ) The paths of the axons of hESC- and hiPSCs- derived RGCs (( a ) and ( c ), respectively) stained by NFL radiate straight from the attached OV, even though the bead is located close by. ( b , d ) Compared with the control, the axons of hESC- and hiPSC-derived RGCs (( b ) and ( d ), respectively) are repelled by SLIT1 release from the bead, and axonal paths are not straight, but winding, and avoid the beads. The assessment from D27 is performed in RMM supplemented with 1.0% FBS and 100 ng/ml BDNF. Each experiment was repeated at least five times. Scale bars, 100 μm.

Article Snippet: Recombinant human NGF, diluted in reduced Matrigel (BD Bioscience), was used at 10 ng/ml, and SEMA3A, diluted in reduced Matrigel (BD Bioscience), was used at a concentration of 200 ng/ml, while recombinant human SLIT1 (R&D Systems), diluted in Matrigel, was used at concentrations of 5 μg/ml at a temperature of approximately 4 °C.

Techniques: Derivative Assay, Immunohistochemistry, Staining, Control