Protocadherins mediate dendritic self-avoidance in the mammalian nervous system

Abstract

Dendritic arborizations of many neurons are patterned by a process called self-avoidance, in which branches arising from a single neuron repel each other. By minimizing gaps and overlaps within the arborization, self-avoidance facilitates complete coverage of a neuron’s territory by its neurites. Remarkably, some neurons that display self-avoidance interact freely with other neurons of the same subtype, implying that they discriminate self from non-self. Here we demonstrate roles for the clustered protocadherins (Pcdhs) in dendritic self-avoidance and self/non-self discrimination. The Pcdh locus encodes 58 related cadherin-like transmembrane proteins, at least some of which exhibit isoform-specific homophilic adhesion in heterologous cells and are expressed stochastically and combinatorially in single neurons. Deletion of all 22 Pcdh genes in the mouse γ-subcluster (Pcdhg genes) disrupts self-avoidance of dendrites in retinal starburst amacrine cells (SACs) and cerebellar Purkinje cells. Further genetic analysis of SACs showed that Pcdhg proteins act cell-autonomously during development, and that replacement of the 22 Pcdhg proteins with a single isoform restores self-avoidance. Moreover, expression of the same single isoform in all SACs decreases interactions among dendrites of neighbouring SACs (heteroneuronal interactions). These results suggest that homophilic Pcdhg interactions between sibling neurites (isoneuronal interactions) generate a repulsive signal that leads to self-avoidance. In this model, heteroneuronal interactions are normally permitted because dendrites seldom encounter a matched set of Pcdhg proteins unless they emanate from the same soma. In many respects, our results mirror those reported for Dscam1 (Down syndrome cell adhesion molecule) in Drosophila: this complex gene encodes thousands of recognition molecules that exhibit stochastic expression and isoform-specific interactions, and mediate both self-avoidance and self/non-self discrimination. Thus, although insect Dscam and vertebrate Pcdh proteins share no sequence homology, they seem to underlie similar strategies for endowing neurons with distinct molecular identities and patterning their arborizations.

Figure 1. Pcdhgs are required for self-avoidance of SAC dendrites

a. Pcdh locus comprises Pcdha, Pcdhb and Pcdhg subclusters. Pcdha and Pcdhg isoforms are assembled by splicing of 1 variable exon to 3 constant exons. b. SACs are present in both inner nuclear and ganglion cell layers (INL, GCL) and extend dendrites that form radially symmetrical arbors confined to thin sublaminae in the inner plexiform layer (IPL). c. SAC dendrites avoid isoneuronal dendrites but form synapses with dendrites of other SACs. d-i. Morphology of single SAC, labeled with membrane-Cherry, in the GCL in wild-type and Pcdhg mutant retinas. Wild-type SAC dendrites self-avoid. In Pcdhg mutants, self-avoidance defects include self-crossing and bundling of dendrites. Crossings are detected at 0.2 μm x-y resolution in single 0.8 μm optical sections (g,i magnified inset in f,h). Images with 0.2 μm z resolution are shown in Supplementary Fig. 1. j. SAC dendritic self-crossings in 1^st-5^th order branches per SAC. Graph underestimates difference between genotypes because the most severely affected mutant SACs could not be scored. ** P<0.01 k,l. Number of terminal branches (i) and dendritic field diameter (j) do not differ between wild-type and mutant SACs. i-l show means ±s.e.m; n=8 cells from 5-6 animals per genotype. Scale bars, 50 μm (d,f) or 10 μm (e,g).

Figure 2. Pcdhgs pattern developing SAC dendrites in a cell-autonomous manner

a-h. SACs in developing wild-type and Pcdhg mutant retinas. Wild-type SACs extend fine, exuberant branches (P3, P5) that make transient intradendritic contacts (P5, P8); by P12, excess branches and isoneuronal contacts are eliminated. Dendrites of mutant SACs display excessive self-crossing and bundling by P3; by P12, excess branches are eliminated, but crossing dendrites remain. i,j. Cultured Pcdhg mutant SACs exhibit loss of symmetric growth and uneven distribution of neurites. k. Histogram of fractal dimensions (Df, metric for space-filling) for 47 wild-type (black) and 47 mutant (grey) SACs. Wild-type SAC in i has Df of 1.61 and mutant SAC in j has Df of 1.53. l. Mean Df for cultured SACs (n=47 cells), SACs in vivo at P5 (n=6) and adult (n=9). *** P<0.001. Error bars, s.e.m. Scale bars, 50 μm except 20 μm in i,j.

Figure 3. No single Pcdhg isoform is necessary and any isoform is sufficient for dendrite self-avoidance

a. SACs lacking Pcdhgc3-c5 (pcdhg^tcko/fcon3;retina-cre) exhibit self-avoidance. b. Replacement of all 22 Pcdhgs by the PcdhgC3 isoform rescues SAC dendrite self-avoidance. c. SACs lacking Pcdhga1-a3 (pcdhg^tako/tako) exhibit self-avoidance. d. Replacement of all 22 Pcdhgs by the PcdhgA1 isoform rescues SAC dendrite self-avoidance. e. Compared to mutants lacking all 22 isoforms, self-crossings in SACs expressing 19 or 1 isoform are restored to control levels. *** P<0.001; n.s., not significant. Bars are mean ± s.e.m, from 7 SACs from pcdhg^tcko/fcon3;retina-cre retinas, 3 SACs from pcdhg^tako/tako, and 9 from remaining genotypes. Scale bars, 50 μm.

Figure 4. Reducing Pcdhg diversity disrupts heteroneuronal SAC interactions

a. Two nearby SACs from a wild-type mouse injected with contrasting fluorescent dyes. Right panel shows image of the green SAC flipped vertically. b. Overlap between red and green cells in a. First two bars are derived from the two panels in a. The green cell was rotated in 45° steps or flipped and then rotated; third and fourth bars show mean overlap ± s.e.m. derived from these images (n=7). All inversions and rotations decrease overlap, indicating that overlap in the real image is non-random. c-e. Tracings of SAC pairs, and versions flipped as in a, from wild-type, Pcdhg^rko/rko and cA1;Pcdhg^rko/rko mice. Overlap shown in black. f. Overlap between neighboring cells, expressed as ratio between overlap measured in real and flipped images. Bars show mean ± s.e.m. for 11, 9 and 8 pairs from wild-type, Pcdhg^rko/rko and single isoform-expressing (cA1;Pcdhg^rko/rko and cC3;Pcdhg^rko/rko) animals. Expression of a single isoform in neighboring SACs decreases their interaction. g. Mean length of overlapping segments between SAC pairs. R, real image; F, flipped image. *P=0.05; **P<0.05; ***P<0.01. Error bars, s.e.m. N as in f. Scale bar, 50 μm.

Figure 5. Purkinje cell dendrite self-avoidance requires Pcdhgs

a-c. Control Purkinje cells labeled with Cre-dependent AAV-XFP in L7-cre transgenic mouse. Self-avoidance is clear in high magnification view in c (inset in b). d-f. Purkinje cells lacking Pcdhgs and labeled as in a-c have disorganized arbors marked by frequent self-crossing defects. f shows area boxed in e. g. Self-crossings detected in single confocal z-sections of 7225 μm² unit area. *** P <0.001; n=8, 15 and 15 cells at P15, P21 and P35 respectively from ≥ 3 mice per genotype. h,i. Area of dendritic arbors (n=20 cells) and cell density (>40 regions) do not differ between control and mutant Purkinje cells. Bars show mean ±s.e.m. Scale bars, 50 μm in a, b, d, e; 10 μm in c, f.