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MEGF10 and MEGF11 mediate homotypic interactions required for mosaic spacing of retinal neurons - PubMed

  • ️Sun Jan 01 2012

MEGF10 and MEGF11 mediate homotypic interactions required for mosaic spacing of retinal neurons

Jeremy N Kay et al. Nature. 2012.

Abstract

In many parts of the nervous system, neuronal somata display orderly spatial arrangements. In the retina, neurons of numerous individual subtypes form regular arrays called mosaics: they are less likely to be near neighbours of the same subtype than would occur by chance, resulting in 'exclusion zones' that separate them. Mosaic arrangements provide a mechanism to distribute each cell type evenly across the retina, ensuring that all parts of the visual field have access to a full set of processing elements. Remarkably, mosaics are independent of each other: although a neuron of one subtype is unlikely to be adjacent to another of the same subtype, there is no restriction on its spatial relationship to neighbouring neurons of other subtypes. This independence has led to the hypothesis that molecular cues expressed by specific subtypes pattern mosaics by mediating homotypic (within-subtype) short-range repulsive interactions. So far, however, no molecules have been identified that show such activity, so this hypothesis remains untested. Here we demonstrate in mouse that two related transmembrane proteins, MEGF10 and MEGF11, have critical roles in the formation of mosaics by two retinal interneuron subtypes, starburst amacrine cells and horizontal cells. MEGF10 and 11 and their invertebrate relatives Caenorhabditis elegans CED-1 and Drosophila Draper have hitherto been studied primarily as receptors necessary for engulfment of debris following apoptosis or axonal injury. Our results demonstrate that members of this gene family can also serve as subtype-specific ligands that pattern neuronal arrays.

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Figures

Figure 1
Figure 1

Expression of Megf10 and Megf11 in SACs and HCs. a. Relative expression level of Megf10, Megf11, and the known SAC markers Chat, Calb1 (encoding Calbindin), and Isl1 (Islet1) in thirteen amacrine (green), bipolar (blue) and retinal ganglion cell (black) subtypes analyzed with microarrays. Level in SACs set to 1 for each gene. Isl1 was also detected in ON bipolar cells as previously reported. Abbreviations for subtypes are defined in Supplementary Table S2. b. Schematic of retina. ONL, outer nuclear layer containing photoreceptors; OPL, outer plexiform layer with photoreceptor synapses; INL, inner nuclear layer with horizontal, bipolar, amacrine, and Müller glial cells; IPL, inner plexiform layer with synapses among bipolar, amacrine and ganglion cells; GCL, ganglion cell layer including displaced amacrine cells. SACs (red), HCs (green), and Müller cells (dark gray row) are indicated. c. In situ hybridization for Megf10 (left panel; red in right panel) combined with anti-Calbindin immunohistochemistry (green in right panel) at P5 shows expression in SACs (black arrows) and HCs (arrowheads). d. Megf10 (red) in E16 retina. Islet1 (green) marks SACs migrating through the outer neuroblast layer (ONbL) and in the INL. Megf10 (red) is expressed by migrating SACs as they arrive in the INL (arrowhead), but not at earlier stages of their migration through the ONbL (arrows). e. Megf10 expression appears in Müller glia (Mü) and is lost from SACs and HCs by P14. f. Double-label immunostaining for MEGF10 (green) and ChAT (red). MEGF10 protein localizes to somata and processes of developing SACs (also see Fig. S1). Scale bars, 20 μm (c-e) or 10 μm (f).

Figure 2
Figure 2

Loss of SAC mosaic spacing in Megf10 mutant mice a. SAC mosaic in inner nuclear layer (INL) of wild-type (left) and Megf10−/− (center) retina, revealed by whole-mount staining with anti-ChAT. Wild-type mice have evenly spaced SAC somata, whereas mutants exhibit clumps and gaps similar to those seen in a simulation of a random cellular array (right). See Figure S3 for similar results in ganglion cell layer (GCL) SACs. b. ChAT-stained retinal sections from wild-type and Megf10 mutant animals. Laminar positions of SAC somata and processes are normal in mutants, even in regions where somata are clumped. c. Density of SACs and Vglut3+ amacrines is similar in wild-type (+/+) and Megf10 mutant (-/-) retina. d. Density recovery profiles (DRPs) for the SAC (INL) and Vglut3+ amacrine arrays. Graphs show the density of cells in a ring of radius x, relative to the density of cells in the image as a whole. Dashed line, DRP of random point array. The exclusion zone characteristic of mosaic spacing is measured as a dip below this line. e: Exclusion zone radius measured from (d). Dashed line, expected result for an array of cells distributed randomly, i.e. the diameter of a single cell. Increases above this minimum indicate spatial order. The mutant SAC exclusion zone radius was similar in size to a SAC cell diameter, and was significantly smaller than wild-type (*p < 0.0001). Vglut3+ amacrine exclusion zones were unaffected. f, g. SAC packing factor (f) and Voronoi domain regularity index (g) were significantly lower in Megf10−/− mice than in wild-type littermates (*p < 0.0001). Dashed line, mean for arrays of cells distributed randomly. Wild-type SACs, and Vglut3+ cells of both genotypes, were non-randomly arrayed. The SAC array in mutants was not significantly different from random arrays (f, p = 0.16; g, p = 0.48). h. Morphology of single GCL SACs, labeled with adeno-associated virus driving membrane-targeted Cherry fluorescent protein, showed no gross abnormalities in Megf10 mutants (n ≥ 8 cells each genotype). Data from P15 (a-g) or P80 (h) mice. Scale bars, 50 μm. Error bars, s.e.m.

Figure 3
Figure 3

Horizontal cell mosaic spacing requires Megf10 and Megf11. a-d. In situ hybridization for Megf11 at ages indicated. Megf11 (red) was not expressed at E16 (a). Calbindin immunostaining (green) labels SACs and HCs, which co-express Megf11 at P5 (b), P7 (c), and P14 (d). See Fig. 1 for abbreviations. e-i. Retinal whole-mounts stained for Calbindin to reveal the HC array. In wild-type mice (e), HCs are distributed evenly. Megf10−/− mutants (f) and Megf11−/− mutants (g) show subtle changes in the regular spacing of HCs, while double Megf10−/−; Megf11−/− mutants (h) show striking HC disorganization similar to a simulation of a random HC array (i). j-m. Quantification of HC spacing regularity in Megf10−/− (red) or Megf11−/− (green) single mutants; Megf10−/−; Megf11−/− double mutants (blue); and wild-type siblings (gray). In all genotypes, HCs were present at normal density (j) but were less regularly spaced relative to wild-type based on exclusion zone radius (k), packing factor (l), and Voronoi domain regularity (m) measurements as in Fig. 2. Double mutants showed significantly less order than single Megf10−/− or Megf11−/− mutants and approach random arrangement indicated by dashed lines (k, mean HC soma diameter; l,m, computed values for random arrays. P-values: *p < 0.01, **p <0.001, ***p< 0.0001. n.s. = not significant. Error bars give s.e.m. Data in e-m from P15 animals. Scale bars, 20 μm (a-d; b,c share scale) or 50 μm (e-i).

Figure 4
Figure 4

MEGF10 acts as both ligand and receptor to trigger SAC repulsion. a: A retinal patch transfected by electroporation with plasmid encoding MEGF10-FP fusion protein, viewed in flatmount. SAC somata in INL, stained with anti-ChAT, are excluded from a swath at the patch edge. SACs are evenly spaced elsewhere, except where the retina was pierced to inject DNA (inj. site). b: Higher-magnification views of patch edges. FP misexpression (left) did not affect SAC spacing, but MEGF10-FP (right) produced a SAC-free zone just inside the transfected region and induced apparent crowding of SACs immediately outside it (arrows). Dashed line, patch edge. See Fig. S11 for quantification of cell distribution at patch edges. c. Hypothesis for MEGF10 function based on a,b. In wild-type retina (top), SACs use MEGF10 as a ligand to signal their location to neighboring SACs. Cell 3 positions itself at the point where repulsive signals on either side (from Cell 1 and Cell 2) are equal. In MEGF10-FP overexpression (bottom), that location is now outside the patch. d. MEGF10-FP transfected whole-mount immunostained for ChAT and Vglut3. Dashed lines mark SAC-free zone (see Fig. S10). Vglut3+ ACs are present in this zone. e. MEGF10-FP is incapable of generating a SAC-free zone when misexpressed in Megf10 mutant retina, indicating that MEGF10 is needed for SAC responses to MEGF10. See Fig. S11 for quantification. Scale bars, 100 μm (a); 50 μm (b,d). All retinas from P10-P15 animals.

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