Replacing the PDZ-interacting C-termini of DSCAM and DSCAML1 with epitope tags causes different phenotypic severity in different cell populations
- The Jackson Laboratory, United States
- University of Idaho, United States
Figures
Figure 1 with 2 supplements
The C-terminus of DSCAM is not required for protein stability or localization.
(A) In the Dscam∆C allele, the sequence encoding the final ten amino acids was replaced with a Myc tag by homologous recombination. (B) Western blots of protein immunoprecipitated from HEK293T cells …
Figure 1—figure supplement 1
DSCAM’s C-terminus interacts with PDZ domains.
(A,B) Both MAGI-2 and MAGI-3 were found to interact with DSCAM by yeast two-hybrid. The C-terminal 20 amino acids of DSCAM (PDZ) were used as bait and the Gal4 binding domain alone (GBD) was used as …
Figure 1—figure supplement 2
DSCAM protein localization is grossly unchanged in Dscam∆C/∆C retinas.
(A–F) Confocal images of P9 retinas immunolabeled for DSCAM and melanopsin show normal colocalization between the proteins in control (A–C) Dscam∆C/∆C animals (D–F). The colocalization is quantified …
Figure 2 with 1 supplement
DSCAM-mediated self-avoidance requires C-terminal interactions in only some amacrine cell types.
(A–C) Dopaminergic amacrine cells (stained for tyrosine hydroxylase, TH) are non-randomly spaced in wild type retinas (A), but lose mosaic spacing and form neurite fascicles in two-week old Dscam∆C/∆…
Figure 2—figure supplement 1
Examples of Voronoi tessellation domains in Dscam mutants.
Representative Voronoi tessellation domains of TH-positive DA cells (A–C) and bNOS-positive amacrine cells (D–F) show the differential effect of the C-terminal deletion.
Figure 3 with 1 supplement
RGCs also display differential dependence on DSCAM C-terminal interactions for self-avoidance.
(A–C) Melanopsin-positive intrinsically photoresponsive retinal ganglion cells are found in a mosaic pattern in wild type retinas (A), but at two weeks of age, ipRGC cell bodies in Dscam∆C/∆C (B) …
Figure 3—figure supplement 1
Examples of Voronoi tessellation domains in Dscam mutants.
Representative Voronoi tessellation domains of melanopsin-positive ipRGCs (A–C) and Cdh3-GFP RGCs (D–F) show the differential effect of the C-terminal deletion.
Figure 4 with 2 supplements
DSCAML1-mediated self-avoidance requires C-terminal interactions in only some cell types.
(A) Dscaml1∆C/∆Cmutant mice were generated by replacing the sequence encoding the final ten amino acids with an HA tag by homologous recombination. See also Figure 4—figure supplement 1. (B–D) …
Figure 4—figure supplement 1
DSCAML1-∆C has a normal membrane topology, but does not interact with MAGI-3.
Live staining of HEK293T cells transfected with full-length DSCAML1 with an N-terminal HA tag (A) and DSCAML1-∆C with a C-terminal HA tag (B) show that, for both proteins as expected, the N-terminus …
Figure 4—figure supplement 2
Examples of Voronoi tessellation domains in Dscaml1 mutants.
Representative Voronoi tessellation domains of AII amacrine cells (A–C, Dab1) and VGLUT3-positive amacrine cells (D–F) show the differential effect of the C-terminal deletion.
Figure 5
Increased cell density is not sufficient to explain spacing defects.
(A) ipRGC cell density was similar in Dscam∆C/∆C and Bax-/- retinas. Despite this, clustering was more severe in Dscam∆C/∆C as measured by DRP (B) and Voronoi (C), but not by nearest neighbor …
Figure 6 with 1 supplement
Neurite fasciculation is separable from density-dependent cell body clustering.
(A) DA cell neurites evenly fill their receptive fields in control mice, but form fascicles in Dscam∆C/∆C (B) and Dscam-/- (C) animals. (D) DA fascicles are rarely observed in Bax-/- mutants. (E) M2 …
Figure 6—figure supplement 1
Fasciculation of Cdh3-GFP RGCs and bNOS-positive amacrine cells.
Fasciculation of Cdh3-GFP RGC dendrites (A–C) and bNOS-positive neurites (D–G) were compared between genotypes at two weeks of age. (H) Elo ranking of fasciculation severity demonstrates that …
Figure 7
Laminar specificity in ∆C mutants.
Neurite stratification in the IPL was analyzed in immunolabeled cryosections. (A,B) bNOS-positive amacrine cells stratified normally in Dscam∆C/∆C retinas, as did ipRGCs and DA cells (C,D), which …
Tables
Table 1
Phenotypes by cell type in ∆C mutants. The phenotypes assessed in Dscam∆C/∆C and Dscaml1∆C/∆C are summarized. n.a. is not applicable, n.s. is not shown.
| Cell type | Cell spacing | Cell density | Fasciculation | Neurite stratification |
|---|---|---|---|---|
| DA | nearly null | nearly null | nearly null | normal |
| bNOS | nearly wild type | nearly wild type | nearly wild type | normal |
| Cdh3-GFP | nearly wild type | nearly wild type | nearly wild type | normal (n.s.) |
| ipRGC | Intermediate | nearly wild type | Intermediate | normal |
| VGLUT3 | nearly null | nearly null | n.a. | misprojection |
| AII | Intermediate | nearly wild type | n.a. | normal |
Additional files
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Supplementary file 1
p-values from all pairwise comparisons.
p-values were calculated from each pairwise comparison across genotypes using the indicated measurement and test. NN = nearest neighbor analysis; PF = packing factor derived from DRP (density recovery profiling).
- https://doi.org/10.7554/eLife.16144.018
