Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Cadherin expression in Cdh3-GFP-RGCs. (A) We propose that DSCAM masks inappropriate adhesion, allowing indifference between homotypic neurites. Without DSCAM, unmasked adhesion drives clustering and fasciculation, predicting that reducing adhesion will partially rescue fasciculation, and that ectopic overexpressing of a CAM will make random cells fasciculate together as if they were homotypic. (B–E) In situ hybridization (RNAscope), with probes against GFP (green), Cdh3 (red), and Cdh6 (cyan), performed on P0 Cdh3-GFP retinas. Forty-eight of 50 GFP-positive cells were positive for both Cdh3 and Cdh6 (dotted lines). Many GFP-negative cells were Cdh6 positive (filled arrowheads in B and C), while occasional cells that were GFP-negative but Cdh3/Cdh6 double positive were also observed (open arrowheads in D and E, n = 6 retinas). (Scale bar: 50 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Expressing, In Situ Hybridization

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Cadherin-mediated adhesion contributes to Cdh3-GFP-RGC fasciculation. Confocal image projections through Cdh3-GFP-RGC dendrites in whole-mount retinas from wild-type (A), Cdh3−/− (B), Cdh6−/− (C), and Ctnna2CDF/+ (D) mice and from all genotypes in combination with Dscam−/− (E–H) demonstrate that dendrite fascicles are less severe in double mutants than in Dscam−/− alone (arrowheads). Images were quantified using the FS (I) and the Elo score (J). n = 4 to 12 retinas per genotype (actual n values are noted in J) over one to four microscope fields of view (median, 3 fields per retina). Box plots represent the median, first and third quartiles, range, and outliers. Blue diamonds in I represent the means. *P < 0.05, **P < 0.01, and ***P < 0.001 by pairwise Wilcoxon rank sum test compared with Dscam−/−. (Scale bar: 100 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Cdh3 overexpression can drive fasciculation between nonhomotypic cells. Retinas of Vgat-Cre mice were electroporated in vivo at P0 with the Cre-inducible expression construct pCALNL-DsRed, alone or along with pCALNL-Cdh3. After 2 wk, these were fixed, stained for DsRed, and imaged en face. (A and B) Mice wild-type for Dscam (A) and DscamcKO/cKO mutants with conditional mutation in GABAergic amacrine cells (B) were electroporated with DsRed alone (Upper). There was no appreciable fasciculation (depicted in Lower) between electroporated cells. (C) Likewise, when retinas wild-type for Dscam were coelectroporated with DsRed and Cdh3 (Upper), no fasciculation (depicted in Lower) was observed. (D) In contrast, when DsRed and Cdh3 were together introduced into DscamcKO/cKO mutant retinas (Upper), significant fasciculation (depicted in Lower) was observed (arrowheads). (E) Images were compared for fasciculation using the Elo score. P = 0.025 by pairwise Wilcoxon rank sum test compared with each other condition. n = 6 mice per condition. (Scale bar: 100 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Over Expression, In Vivo, Expressing, Construct, Staining, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members

doi: 10.1073/pnas.1809430115

Figure Lengend Snippet: Trans DSCAM interactions mask the CDH3 adhesive response. Cortical neurons from wild-type (A and D) and Dscam−/− (B and E) mice were transfected with constructs encoding CDH3 with a C-terminal FLAG tag (green) and then incubated for 1 h with beads coated with CDH3 and DSCAM ectodomains (A and B, magenta) or CDH3-EC alone (D and E, magenta). The accumulation of CDH3-FLAG at sites of contact between beads and neurons was quantified (C and F). In all four conditions, CDH3 was present both in the neuron and on the bead. While neuronal CDH3-FLAG was largely indifferent to the beads when DSCAM was both in the neuron and on the bead (A), CDH3 accumulated at these sites when DSCAM was present only on the bead (B), only in the neuron (D), or completely absent (E), demonstrating that trans DSCAM interactions masked this accumulation. Means ± SEM are presented in C and F. n = 20 to 31 neurons per condition, from cultures separately prepared from three different mice per genotype (actual n values are noted in C and F). ***P < 0.001 by two-tailed Student’s t test. (Scale bar: 10 μm.)

Article Snippet: An expression construct containing Myc-DDK–tagged cadherin-3 (pLenti-Cdh3) was obtained from Origene (MR227582L1).

Techniques: Transfection, Construct, FLAG-tag, Incubation, Two Tailed Test