Protocadherins mediate dendritic self-avoidance in
the mammalian nervous system
Julie L. Lefebvre1, Dimitar Kostadinov1, Weisheng V. Chen2, Tom Maniatis2& Joshua R. Sanes1
Dendritic arborizations of many neurons are patterned by a
process called self-avoidance, in which branches arising from a
single neuron repel each other1–7. By minimizing gaps and overlaps
ofa neuron’s territorybyits neurites1–3. Remarkably, some neurons
that display self-avoidance interact freely with other neurons of the
same subtype, implying that they discriminate self from non-self.
Herewedemonstrate rolesforthe clusteredprotocadherins (Pcdhs)
in dendritic self-avoidance and self/non-self discrimination. The
Pcdh locus encodes 58 related cadherin-like transmembrane
adhesion in heterologous cells and are expressed stochastically and
combinatoriallyinsingle neurons7–11. Deletion of all 22Pcdh genes
in the mouse c-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
and mediate both self-avoidance and self/non-self discrimina-
tion4–7,12–15. Thus, although insect Dscam and vertebrate Pcdh
strategies for endowing neurons with distinct molecular identities
and patterning their arborizations.
and c-subclusters, called Pcdha, Pcdhb and Pcdhg, which encode 14, 22
and 22 cadherin-like proteins, respectively8(Fig. 1a). In the Pcdha and
Pcdhg subclusters, single variable exons encoding extracellular, trans-
membrane and juxtamembrane domains are spliced to three constant
cellular domains8. The complexity of this locus is reminiscent of that of
Dscam1, which mediates self-avoidance in Drosophila4–7,15. Moreover,
Pcdh genes, like Dscam1, exhibit stochastic expression, and both Pcdhg
In contrast, the two vertebrate Dscams are not complex genes, so
although theymediate bothrepulsive and attractive interactionsamong
neurons16–19, they are unlikely to underlie self/non-self discrimination.
We therefore investigated roles of Pcdh genes in these processes.
Previous studies of mouse mutants lacking all 22 Pcdhg genes
revealed that they are required for survival of multiple neuronal
types20–23. To seek roles of Pcdhgs in self-avoidance, we focused on a
retinal interneuron, the SAC, which expresses Pcdhg genes22and
exhibits marked dendritic self-avoidance24. Radially symmetric SAC
plexiform (synaptic) layer; SACs have no axons. Dendrites of a single
SAC seldom cross one another, yet dendrites of neighbouring SACs
cross freely (Fig. 1b, c; Supplementary Fig. 1) and even form synapses
with each other24,25, suggesting that they can distinguish ‘self’ from
We used a conditional mutant (Pcdhgfcon3)22to bypass the neonatal
lethality of constitutive Pcdhg mutants and employed Cre drivers that
delete Pcdhg genes from all or subsets of retinal cells. We visualized
individual neurons by infection with recombinant adeno-associated
virus (rAAV) expressing a fluorescent protein (XFP; Fig. 1d, e),
biolistic delivery of DNA encoding XFP, or intracellular injection of
a fluorescent dye. We identified SACs, the sole cholinergic neurons in
retina, withantibodies to choline acetytransferase (ChAT),which also
demonstrated the association of XFP-positive SAC dendrites with
dendrites from other (XFP-negative) SACs (Supplementary Figs 1
SAC morphology was profoundly altered in Pcdhg mutant retinas
(Pcdhgfcon3/fcon3; retina-cre, called Pcdhgrko/rkohere; see Methods for
genotypes). Dendrites arising from a single SAC frequently crossed
each other and sometimes formed loose bundles (Fig. 1f–i and
Supplementary Fig. 1). Crossing frequency was increased several-fold
in both proximal and distalregions of the arborization(Fig. 1j). These
defectswerehighlyspecific,inthat thediameterof SACarborizations,
the number of dendritic termini, the laminar targeting of SAC
dendrites, and the mosaic arrangement of SAC bodies were all
unaffected in Pcdhgrko/rkomutants (Fig. 1k, l and Supplementary
Figs 1 and 2). Thus, Pcdhgs are dispensable for many aspects of
SAC morphogenesis but are required for their self-avoidance.
numbers during the period of naturally occurring cell death20–23.
Although SACs are largely spared in Pcdhg mutants22, their dendritic
defects might be secondary to loss of other neurites with which they
ordinarily interact. To test this possibility, we blocked apoptosis by
deleting the Bax gene, which is required for naturally occurring and
Pcdhg-dependent neuronal death22,23,26. SAC morphology was normal
in Bax2/2mice, but self-avoidance defects persisted in Bax2/2;
Pcdhgrko/rkodouble mutants (Supplementary Fig. 3).
We next asked whether Pcdhgs are required for the development
of SAC arborizations, or only for their maintenance. In wild-type
neonates, SACs extended dendrites that branched profusely and
contacted each other (Fig. 2a–c). By postnatal day (P)12, however,
excess neurites and isoneuronal contacts were eliminated, resulting
in a radial arborization with evenly spaced branches (Fig. 2d, and
of isoneuronal ‘sampling’. In Pcdhgrko/rkomice, SACs were clearly
aberrant by P3, exhibiting excessive crossing and tangling of neurites
(Fig. 2e–g). Excess branches were subsequently eliminated, but
1Center for Brain Science and Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts 02138, USA.2Department of Biochemistry and Molecular
Biophysics, Columbia University Medical Center, 701 West 168th Street, New York, New York 10032, USA.
2 3 A U G U S T 2 0 1 2 | V O L 4 8 8 | N A T U R E | 5 1 7
Macmillan Publishers Limited. All rights reserved
whereas most crossing branches were eliminated in controls, many
by mediating repulsive interactions that bias the rearrangement pro-
cess to selectively eliminate contacts among isoneuronal branches.
avoidance, we next asked whether they act cell-autonomously. We
selectively removed Pcdhg genes from SACs using a ChAT-Cre line.
In this case, Pcdhg-negative SACs were surrounded by Pcdhg-positive
neurons of other types. We also deleted Pcdhg genes from individual
SACs using a transgenic line that expressed tamoxifen-activated Cre
recombinase in SACs; we activated Cre with a low dose of tamoxifen
and introduced a Cre-dependent reporter to mark mutant SACs. In
this case, Pcdhg-negative SACs were surrounded by Pcdhg-positive
SACs. In both cases, SACs lacking Pcdhg genes exhibited striking
self-avoidance defects (Supplementary Fig. 4). To test whether
Pcdhgs can act in completely isolated SACs, we used fluorescence-
activated cell sorting to purify SACs from a transgenic line in which
0 5 10 15202530
Variable exons (22)
Dendritic field diameter
Terminal branch number
Dendrite self-crossings per SAC
Figure 1 | Pcdhgs are required for self-avoidance of SAC dendrites. a, Pcdh
locus comprisesPcdha, Pcdhb and Pcdhg subclusters. Pcdha and Pcdhg isoforms
transmembrane domains to three constant exons encoding the intracellular
ganglion cell layer (GCL) and extend dendrites that form radially symmetrical
arborizations confined to thin sublaminae in the inner plexiform layer (IPL).
a single SAC, labelled with membrane-Cherry, in the GCL in control 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 0.2mm x–y resolution in single 0.8-mm optical sections (e, g, i show
magnified views of boxed areas in d, f, h). Images with 0.2mm z resolution are
branches per SAC. Graph underestimates difference between genotypes because
the most severely affected mutant SACs could not be scored. **P,0.01.
from 5–6 animals per genotype. Scale bars, 50mm (d, f, h) and 10 mm (e, g, i).
1.44 1.48 1.52 1.56 1.60 1.64
Fractal dimension, Df
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. DIV, days in vitro.
(black) and 47 mutant (grey) SACs. Wild-type SAC in i has Dfof 1.61 and
mutant SAC in j has Dfof 1.53. l, Mean Dffor cultured SACs (n547 cells),
SACs in vivo at P5 (n56) and adult (n59). ***P,0.001. Error bars, s.e.m.
Scale bars, 50mm (a–d, also apply respectively to e–h) and 20mm (i, j).
5 1 8 | N A T U R E | V O L 4 8 8 | 2 3 A U G U S T 2 0 1 2
Macmillan Publishers Limited. All rights reserved
they are selectively labelled by an orange fluorescent protein (Thy1-
OFP3) and cultured them at low density. Isolated SACs extended
dendrites that formed radial, web-like arborizations (Fig. 2i),
reminiscent of those observed at ,P5 in vivo (Fig. 2b). In contrast,
SACs from Pcdhgrko/rko; Thy1-OFP3 mice exhibited less symmetrical
and unevenly spaced arborizations, reminiscent of those observed in
the space-filling capacity of dendritic arborizations2,27(see Methods)
We next assessed the requirement for isoform diversity in Pcdhg-
dependent self-avoidance. We used RT–PCR (PCR with reverse tran-
scription) to survey expression of Pcdhg isoforms in whole retina, in
amacrines generally and in SACs specifically. All 22 Pcdhg variants
were expressed in each preparation, with no indication of decreased
diversity in purified subpopulations (Supplementary Fig. 6). We
then analysed a targeted mouse mutant, Pcdhgtcko, in which three
contiguous Pcdhg variable exons, C3–C5, had been deleted.
Expression of the remaining 19 Pcdhg isoforms is unperturbed in
this allele28. Because Pcdhgtckohomozygous mice die at birth28, we
generated transheterozygous animals (Pcdhgtcko/fcon3;retina-cre) so
that only retina lacks both copies of Pcdhgc3-c5. In these retinas,
neuronal death was as prevalent as in those of Pcdhgrko/rkomice22,28,
yet SACs exhibited normal self-avoidance (Fig. 3a, e).
In a complementary approach, we generated a line in which the
single PcdhgC3 isoform, fused to a fluorescent protein (mCherry),
could be expressed in any cell in a Cre-dependent manner
Thus, in cC3-mCherry;Pcdhgrko/rkomice, Cre both deletes all 22
ΔPcdhgC3-C4-C5 Isoform diversity=19
Self-crossings branch order 1′-4′
22 19 1 0
PcdhgC3 Isoform diversity=1
ΔPcdhgA1-A2-A3 Isoform diversity=19 PcdhgA1 Isoform diversity=1
Figure 3 | NosinglePcdhgisoformisnecessaryandanyisoformissufficient
for dendrite self-avoidance. a, SACs lacking Pcdhgc3–c5
(pcdhgtcko/fcon3;retina-cre) exhibit self-avoidance. b, Replacement of all 22
Pcdhgs by the PcdhgC3 isoform, using the RC::cC3-cherry transgene, rescues
SAC dendrite self-avoidance. c, SACs lacking Pcdhga1-a3 (pcdhgtako/tako)
exhibit self-avoidance. d, Replacement of all 22 Pcdhgs by the PcdhgA1
isoform, using the RC::cA1-cherry transgene, rescues SAC dendrite self-
avoidance. e, Compared to mutants lacking all 22 isoforms, self-crossings of
SACs in retinas expressing 19 or 1 isoforms are restored to control levels.
***P,0.001; n.s., not significant. Data are mean6s.e.m., from 7 SACs from
pcdhgtcko/fcon3;retina-cre retinas, 3 SACs from pcdhgtako/tako, and 9 from
remaining genotypes. Scale bar in a, 50mm; applies to b–d also.
Real imageGreen cell flipped
Total overlap length (μm)
Real Flip Rotate
cA1 or cC3;
Length per overlap (μm)
R F R FR F
cA1 or cC3;
Figure 4 | Reducing Pcdhg diversity disrupts heteroneuronal SAC
interactions. a, Two nearby SACs from a wild-type mouse injected with
from the two panels in a. The green cell was rotated in 45u steps or flipped and
then rotated (manipulations indicated by symbols beneath graph); third and
fourth barsshow mean overlap6s.e.m. derivedfrom these images (n57). 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 (c), Pcdhgrko/rko(d) and cA1;Pcdhgrko/rko(e) mice. Overlap
shown in black. f, Overlap between neighbouring cells, expressed as ratio
for11,9 and8 pairsfromwild-type,Pcdhgrko/rkoandsingle isoform-expressing
(cA1;Pcdhgrko/rkoand cC3;Pcdhgrko/rko) animals. Expression of a single isoform
in neighbouring SACs decreases their interaction. g, Mean length of
overlapping segments between SAC pairs. R, real image; F, flipped image.
*P50.05; **P,0.05; ***P,0.01. Error bars, s.e.m.; n as in f. Scale bar in
a, 50mm; applies to c–e also.
2 3 A U G U S T 2 0 1 2 | V O L 4 8 8 | N A T U R E | 5 1 9
Macmillan Publishers Limited. All rights reserved
endogenous Pcdhg genes and activates the single PcdhgC3-mCherry
firmed Cre-dependent expression of the transgene in all retinal cells
and appropriate localization of the fusion protein to cell membranes
and synaptic layers (Supplementary Fig. 7). Expression of Pcdhgc3
alone rescued self-avoidance defects of Pcdhg mutants (Fig. 3b, e).
To test the possibility that only some isoforms are dispensable for
self-avoidance, we analysed a second set of isoforms. We generated
Pcdhgtako, which lacks the Pcdhga1-a3 variable exons28, and a line that
expresses Pcdhga1-mCherry in a Cre-dependent manner (cA1-
mCherry). Results were similar to those for the C3–C5 group: self-
avoidance persisted in the absence of PcdhgA1–A3 and was rescued
by replacement of all Pcdhg isoforms with PcdhgA1 alone (Fig. 3c–e
and Supplementary Fig. 7). From these results, we conclude that no
single Pcdhg isoform is necessary but any single isoform is sufficient
for dendritic self-avoidance.
Although Pcdhg isoform diversity is not required for isoneuronal
self-avoidance, it may be required to ensure that dendrites of adjacent
SACs do not avoid each other, which would prevent them from inter-
acting. The ability to generate a SAC population expressing a single
Pcdhg isoform (Pcdhga1 or Pcdhgc3) enabled us to test this idea. We
injected closely spaced pairs of SACs with different fluorophores
To determine whether this method reliably revealed interactions
among SACs, we rotated, flipped or rotated and flipped the image of
one of the cells, and recalculated overlap. Only the real image showed
an overlap greater than that of the manipulated images (Fig. 4b). We
then measured overlap for pairs of SACs from wild-type, mutant and
single isoform-expressing mice, normalizing for intercellular distance
by comparing overlap to the value calculated from the flipped image
(Fig. 4c–e and Supplementary Fig. 8). Overlap was equivalent in
wild-type and mutant retina, but significantly decreased in retinas
expressing a single isoform (Fig. 4f); values for Pcdhga1 and Pcdhgc3
were similar(1.01and 1.08).Likewise,themeanlengthofoverlapping
segments was greater than expected for random overlap in wild-type
and mutant but not in single isoform-expressing pairs (Fig. 4g). Thus,
when all SACs express the same Pcdhg isoform, heteroneuronal
dendrites avoid each other, just as isoneuronal dendrites do in control
isoneuronal from heteroneuronal dendrites.
Finally, we asked whether Pcdhgs mediate self-avoidance in areas
other than retina. We examined cerebellar Purkinje cells, which have
elaborate, planar dendritic arborizations known to exhibit self-
avoidance3(Fig. 5a–c). Importantly, stochastic and combinatorial
expression, which underlies the ability of Drosophila Dscam1 to
mediate self-avoidance4–6,12,14,15,29, has been documented for Pcdhg
genes in Purkinje cells10. We selectively deleted Pcdhg genes from
that expresses fluorescent proteins in a Cre-dependent manner, and
examined them at P15, P21 and at P35, after arborizations have
effect on their survival, shape, size or branching pattern (Fig. 5d, e, h, i
and Supplementary Fig. 9), but their arborizations were disorganized
and dendrites often crossed over each other (Fig. 5f, g). Use of a
Cre-dependent reporter revealed that deletion remained incomplete
at P8, at which time Purkinje dendrite growth was already advanced
(Supplementary Fig. 9). It is therefore possible that earlier deletion
of Pcdhg genes would lead to a more dramatic effect. Nonetheless,
these results demonstrate a role for Pcdhg genes in Purkinje cell
In summary, although vertebrate Pcdh genes and Drosophila
Dscam1 are structurally unrelated, they have remarkable parallels:
both encode numerous isoforms from a single locus, the isoforms
are expressed stochastically and combinatorially, and the encoded
proteins interact homophilically7,8,10–14. We have now shown that in
mammalian neurons, Pcdhgs, like Dscam1 (refs 4–6, 12), promote
In addition, for both Dscam1 and Pcdhg genes, diversity appears to
L7-Cre; Pcdhg+/fcon3 P35
L7-Cre; Pcdhgfcon3/fcon3 P35
PC density (number/area)
Dendrite arbor area (103 mm2)
Figure 5 | Purkinje cell dendrite self-avoidance requires Pcdhgs.
cre transgenic mouse. Self-avoidance is clear in high-magnification view in
c (shows area boxed in b). d–f, Purkinje cells lacking Pcdhgs and labelled as in
a–c have disorganized arborizations marked by frequent self-crossing defects.
Panel f shows area boxed in e. g, Self-crossings detected in single confocal
z-sections of 7,225mm2unit area from controls and mutants. **P,0.01;
***P,0.001; n58, 15 and 15 cells at P15, P21 and P35 respectively from $3
mice per genotype. h, i, Area of dendritic arborizations (n520 cells) and cell
Data show mean6s.e.m. Scale bars, 50mm (a, b, d, e) and 10mm (c, f).
5 2 0 | N A T U R E | V O L 4 8 8 | 2 3 A U G U S T 2 0 1 2
Macmillan Publishers Limited. All rights reserved
underlie self/non-self discrimination, presumably because neighbour-
free to interact7,12,14,15,29. Thus, two phyla appear to have recruited
different molecules to mediate similar, complex strategies for self-
recognition during formation of neuronal arborizations. These
parallels raise the question of why vertebrate and invertebrate
nervous systems have invested heavily in mechanisms that promote
self-avoidance. In principle, self-avoidance allows neurons to cover
their receptive or projective fields maximally while retaining the
ability to overlap those of neighbouring neurons1–3. However, to our
knowledge, the effect of perturbing self-avoidance on circuit function
has yet to be assessed in any system. We can now address this issue by
targets in Pcdhg mutant mice.
Transgenic, knockout and knock-in mouse lines used for this study, as well as
Dendrite self-crossings were quantified by numberof branchoverlaps detectedin
single confocal planes.
Full Methods and any associated references are available in the online version of
Received 19 December 2011; accepted 7 June 2012.
Published online 29 July 2012.
1.Kramer, A. P. & Kuwada, J. Y. Formation of the receptive fields of leech
mechanosensory neurons during embryonic development. J. Neurosci. 3,
Montague, P. R. & Friedlander, M. J. Expression of an intrinsic growth strategy by
mammalian retinal neurons. Proc. Natl Acad. Sci. USA 86, 7223–7227 (1989).
axon spacing. Cold Spring Harb. Perspect. Biol. 2, a001750 (2010).
Matthews, B. J. et al. Dendrite self-avoidance is controlled by Dscam. Cell 129,
Soba, P. et al. Drosophila sensory neurons require Dscam for dendritic self-
avoidance and proper dendritic field organization. Neuron 54, 403–416 (2007).
Hughes, M. E. et al. Homophilic Dscam interactions control complex dendrite
morphogenesis. Neuron 54, 417–427 (2007).
neural circuit assembly. Cell 143, 343–353 (2010).
Wu, Q. & Maniatis, T. A striking organization of a large family of human neural
cadherin-like cell adhesion genes. Cell 97, 779–790 (1999).
Kohmura, N. et al. Diversity revealed by a novel family of cadherins expressed in
neurons at a synaptic complex. Neuron 20, 1137–1151 (1998).
monoallelic and biallelic expression in single Purkinje cells. J. Biol. Chem. 281,
11. Schreiner, D. & Weiner, J. A. Combinatorial homophilic interaction between
c-protocadherin multimers greatly expands the molecular diversity of cell
adhesion. Proc. Natl Acad. Sci. USA 107, 14893–14898 (2010).
12. Zhan, X. L. et al. Analysis of Dscam diversity in regulating axon guidance in
Drosophila mushroom bodies. Neuron 43, 673–686 (2004).
13. Wojtowicz, W. M., Flanagan, J. J., Millard, S. S., Zipursky, S. L. & Clemens, J. C.
Alternative splicing of Drosophila Dscam generates axon guidance receptors that
exhibit isoform-specific homophilic binding. Cell 118, 619–633 (2004).
14. Neves, G., Zucker, J., Daly, M. & Chess, A. Stochastic yet biased expression of
multiple Dscam splice variants by individual cells. Nature Genet. 36, 240–246
thousands of Dscam1 isoforms. Nature 461, 644–648 (2009).
16. Yamagata, M. & Sanes, J. R. Dscam and Sidekick proteins direct lamina-specific
synaptic connections in vertebrate retina. Nature 451, 465–469 (2008).
17. Fuerst, P. G., Koizumi, A., Masland, R. H. & Burgess, R. W. Neurite arborization
and mosaic spacing in the mouse retina require DSCAM. Nature 451, 470–474
types in the developing mouse retina. Neuron 64, 484–497 (2009).
19. Sanes, J. R. & Zipursky, S. L. Design principles of insect and vertebrate visual
systems. Neuron 66, 15–36 (2010).
20. Wang, X. et al. Gamma protocadherins are required for survival of spinal
interneurons. Neuron 36, 843–854 (2002).
21. Prasad,T.,Wang,X.,Gray,P.A.& Weiner,J.A.Adifferentialdevelopmental pattern
of spinal interneuron apoptosis during synaptogenesis: insights from genetic
analyses of the protocadherin-c gene cluster. Development 135, 4153–4164
22. Lefebvre, J. L., Zhang, Y., Meister, M., Wang, X. & Sanes, J. R. c-Protocadherins
regulate neuronal survival but are dispensable for circuit formation in retina.
Development 135, 4141–4151 (2008).
23. Weiner, J. A., Wang, X., Tapia, J. C. & Sanes, J. R. Gamma protocadherins are
24. Stacy, R. C. & Wong, R. O. Developmental relationship between cholinergic
amacrine cell processes and ganglion cell dendrites of the mouse retina. J. Comp.
Neurol. 456, 154–166 (2003).
25. Lee, S. & Zhou, Z. J. The synaptic mechanism of direction selectivity in distal
processes of starburst amacrine cells. Neuron 51, 787–799 (2006).
26. White, F. A., Keller-Peck, C. R., Knudson, C. M., Korsmeyer, S. J. & Snider, W. D.
Widespread elimination of naturally occurring neuronal death in Bax-deficient
mice. J. Neurosci. 18, 1428–1439 (1998).
27. Jelinek, H. F. & Fernandez, E. Neurons and fractals: how reliable and useful are
calculations of fractal dimensions? J. Neurosci. Methods 81, 9–18 (1998).
28. Chen, W. V. et al. Functional significance of isoform diversification in the
protocadherin gamma gene cluster. Neuron. (in the press).
29. Wang, J. et al. Transmembrane/juxtamembrane domain-dependent Dscam
distribution and function during mushroom body neuronal morphogenesis.
Neuron 43, 663–672 (2004).
30. Kaneko, M. et al. Remodeling of monoplanar Purkinje cell dendrites during
cerebellar circuit formation. PLoS ONE 6, e20108 (2011).
Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank members of our laboratory for providing advice and
reagents, including D. Cai and K. Cohen (rAAV), I.-J. Kim (fstl4-line 1 mice) and
M. Yamagata for modified Rosa-CAG targeting vector. We also thank B. Stevens
NARSAD Young Investigator Award to J.L.L.
Author Contributions J.L.L., D.K. and J.R.S. designed experiments and prepared the
the project. W.V.C. and T.M. generated Pcdhgtakoand Pcdhgtckomice. All authors
commented on the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to J.R.S. (firstname.lastname@example.org).
2 3 A U G U S T 2 0 1 2 | V O L 4 8 8 | N A T U R E | 5 2 1
Macmillan Publishers Limited. All rights reserved
Mouse strains. The Pcdhgfcon3conditional mutant allele, in which the third con-
stant exon is flanked by loxP sequences and which generates a functionally null
allele following Cre recombination, was described previously21,22. Retina-
specific Chx10-cre31and Six3-cre transgenic mice32were provided by C. Cepko
(Harvard) and W. Klein (M.D. Anderson Cancer Center), respectively. Bax2/2
mutants33, Purkinje-specific L7Bac-cre transgenic mice34, Chat-cre, in which the
Cre recombinase gene was targeted to the endogenous ChAT gene35, and Rosa-
CAG-LoxP-STOP-LoxP-tdTomato-WPRE reporter mice36were obtained from
Inthisline, calledline1todistinguishitfromthe linecalled‘BD’inref. 37,CreER
was expressed in SACs, as well as sparse other amacrine cells. We believe that
expression reflects influences at the site of transgene integration rather than
expression of fstl4. Thy1-OFP3 transgenic mice, in which Thy1 promoter and
regulatory elements direct expression of Kusabira Orange (OFP) in SACs and
subsets of retinal ganglion cells (RGCs), were described previously37. Pcdhgtcko
and Pcdhgtakomice were generated using standard gene targeting techniques28.
in accordance with protocols approved by the Harvard University Standing
Committee on the Use of Animals in Research and Teaching.
Generation of single Pcdhg isoform conditional knock-in mice. Pcdhga1 and
pcdhgc3 full-lengthcDNAs were amplifiedfromRNAisolated from P21 C57/BL6
mouse brain, and cloned in frame into pCMV-mCherry-N1 (Clontech). Linker
sequence residing between the third constant exon and gfp in Pcdhgfusgknock-in
mice and shown to produce functional Pcdhg-GFP fusion proteins in vivo20was
subcloned into pCMV-pcdhga1/c3-mCherry-N1. Targeting vector pRosa26-
PAS38was modified as described in ref. 39 to include a CAG cassette (chicken
b-actin promoter and CMV immediate-early enhancer), a Gateway RfA destina-
11925). LoxP-STOP-loxP-Pcdhga1/c3-mCherry wasrecombinedinto pROSA26-
Pcdhga1/c3-mCherry-WPRE-FNF-iSceI targeting vectors. The iSceI-linearized
vectors were electroporated into 129/B6 F1 hybrid ES cell line V6.5. G418-
resistant, targeted ES clones were identified by PCR: 1.7kb fragment amplified
by 59-Rosa-F: GGCGGACTGGCGGGACTA and 59-CAG-R: CCAGGCGGGCC
ATTTACCGTAAG; and 8.2kb fragment amplified by 39-CherryF: CTCCCA
TACAAC. ES cell transfections and blastocyst injections were performed by the
Genome Modification Facility, Harvard University. Following germ-line trans-
mission, the FRT-neo-FRT cassette was excised by crossing to mice that express
Labelling of neurons. Plasmid encoding pAAV2/2-CAG-palmitoylation tag-
mCherry-WPRE was used to generate recombinant AAV2/2 expressing
membrane-tagged Cherry. To label SACs in retina expressing cC3-mCherry or
cA1-mCherry, we used rAAV2/2-CBA-YC3.6-WPRE expressing a calcium
sensor that includes cytosolic YFP and used here for visualization of neuronal
morphology41. Recombinant AAV2/2-CAG-memb-mCherry and rAAV2/2-
YC3.6 were prepared at the Harvard Gene Therapy Institute ((1–2)31012
genome copies per ml). Optimal titres of (1–2)3109viral genome particles
per ml for AAV2/2-CAG-memb-mCherry and 231010viral genome particles
per ml for rAAV2/2- YC3.6 were prepared in phosphate-buffered saline (PBS,
pH57.4). rAAV2/9 expressing GFP and mCherry were generated and provided
by D. Cai and K. Cohen in our laboratory; high titre virus was produced at the
University of Pennsylvania Vector Core.
To injectvirus into eyes,adult mice were anaesthetized with ketamine/xylazine
by intraperitonealinjection.A 30KG needle wasused to make a small hole in the
temporal eye, below the cornea, and 1.5ml of rAAV virus was injected into the
vitreous humour with a Hamilton syringe and 33G blunt-ended needle. Animals
virus infection, P1–P2 mice were anaesthetized with ice and a small puncture was
Mice were analysed 12–35days after infection.
For biolistic transfection of SACs, gold particles (1.0mm diameter, Bio-Rad)
were coated with plasmids encoding tdTomato driven by CMV promoter24. Live
retinas were dissected, transected with four radial incisions, flattened with
photoreceptor side down, and mounted onto a nitrocellulose filter (Millipore).
Gold particles were deliveredusinga Biolistics HeliosGene gundevice(Bio-Rad),
and retinas were cultured in Ames medium (Sigma) in an oxygenated incubator
heated to 37uC for 12–16h.
To assess interactions between dendrites of neighbouring SACs, we injected
pairs of cells with fluorescent dyes. Retinas from mice expressing OFP in SACs
(Thy1-OFP3) were mounted RGC side up and perfused with Ames medium
bubbled with 95%O2/5%CO2at 25uC. OFP1SACs were visualized with epifluor-
escence, and impaled with high resistance electrodes (50MV) filled with a K1
based intracellular recording solution supplemented with 50mM Alexa Fluor 568
(for targeting) and 200mM of Alexa Fluor 488 or 647 (for filling, Invitrogen).
Square voltage pulses of ,3V were applied to SACs at 50Hz using a BK
Precision Model 3011B function generator. After filling one SAC, the electrode
filled.Images of labelled SAC pairs in live retinas were acquired at 403 on a Zeiss
LSM 510 confocal microscope.
perfused with Ringer’s solution followed by 4% paraformaldehyde (PFA) in PBS.
Eyecups were removed andfixed in4% PFA onicefor 1h, followedby dissection
andpost-fixationofretinasforanadditional30min, thenrinsedwith PBS.Brains
were post-fixed in 4% PFA at 4uC overnight. Animal procedures were in compli-
ance with the US National Institutes of Health Guide for the Care and Use of
Laboratory Animals and approved by the Animal and Care and Use Program at
Whole-mount preparations and cryosections of retinas were performed as
described22,42. Briefly, whole retinas were incubated for 1–2h in blocking buffer
(0.4% Triton-X, 4% normal donkey serum in PBS), then incubated for 6days at
3h at room temperature with Alexa-conjugated secondary antibodies (Invitrogen
or Jackson ImmunoResearch). Whole retinas were flattened with photoreceptor
side down onto nitrocellulose filters. Retina flat-mounts and brain sections were
mounted onto glass slides, covered with Vectashield (Vector) or Fluoromount G
scope. Antibodies used were as follows: chick and rabbit anti-GFP (Aves and
Millipore); rabbit anti-DsRed (Clontech); goat anti-choline acetyltransferase
(Millipore); guinea pig anti-vGluT3 (Millipore); rabbit anti-Calbindin (Swant);
mouse anti-syntaxin HPC1 clone (Sigma); rabbit anti-cleaved caspase3 (Cell
Signaling Technology). Nuclei were labelled with DAPI, Po-pro1, or NeuroTrace
Nissl 435/455 (Invitrogen).
SACs in vitro, we crossed the Thy1-OFP3 transgene, which selectively directs
expression of Kusabira Orange (OFP) in SACs and subset of RGCs37, into
Pcdhgfcon3; Six3-cre mice. Retinas from genotyped Pcdhgfcon3/fcon3; Six3-cre;
Thy1-OFP3 mutant and control P2 mice were dissociated using papain22. OFP1
SACs were isolated by fluorescence activated cell sorting (FACS, MoFlo), plated
onto poly-L-lysine-coated glass coverslips (Warner) and cultured for 7–9days in
RGC growth media modified from Meyer-Franke43in the following ways: (1)
substitution of NS2144for B27, (2) substitution of N2 (Invitrogen) for Sato stock,
(3)additionofTGF-b1andTGF-b2 (2.5ngml21; Peprotech), and(4)addition of
mouse glia-conditioned medium (15%). One-third of media was exchanged with
fresh media every three days. Cells were fixed with cold 4%PFA/4% sucrose for
15min, and immunostained for syntaxin and calbindin to confirm SAC identity,
SACs due to variegated Six3-Cre activity in retina.
Image analysis. For best reproduction and clarity of SAC arborizations,
maximized projections of confocal images were inverted and contrast-enhanced
using Photoshop (Adobe Systems). For morphometric analysis of SACs, we used
Fiji software and selected confocal image series of wild-type and Pcdhg mutant
SACs situated in comparable retinal eccentricities. Self-crossings per dendritic
branch order were quantified as number of branch overlaps detected in single
confocal planes; crossings occurring distal to fifth branch order could not be
quantified accurately owing to severity of defects in mutants. Dendritic field
diameter was measured as the longest axis of arborization. In some cases, arbor-
izations were re-imaged by oversampling using a 603 1.45NA objective at x,y,z
resolution of 473473131mm and then subjected to deconvolution using
Huygens software (http://www.svi.nl/HuygensProfessional).
For analysis of SAC density and mosaic regularity, confocal z-stacks of ChAT-
labelled SACs through the GCL and INL were acquired at similar locations in
central retina. Sample sizes were 4–5 areas (0.099mm2) per animal, 2–4 animals
per genotype. For each field, x–y coordinates of SAC arrays were obtained by
manually marking centres of cells using Fiji and used to compute SAC density
(number per mm2), packing factor45, and density recovery profiles (DRP)46with
WinDRP software (http://www.mpimf-heidelberg.mpg.de/,teuler/WinDRP/
Macmillan Publishers Limited. All rights reserved
Tocomparethespace-fillingandcomplexityofcontrolandmutantSACarbor- Download full-text
izations, we computed fractal dimensions, Df, which provide a measure of how
completely dendrites fill its area2,27,47,48. To calculate Df, we applied the box-
counting method as implemented in the FracLac 2.5 plug-in for ImageJ software
images of cultured mutant and control SACs were obtained at equivalent laser
scanning parameters with a 603 oil immersion lens, and maximum projections
and thresholded, binaryimages were processed using ImageJ. Box counts using a
series of progressively smaller box sizes (d) were scanned in a region of interest
covering the SAC arborization, and the number of boxes intersected by pixels
[k(d)] were analysed; this computes Df, which represents an inverse linear regres-
sion between log[k(d)] and log(d). Dfranges from 1.0 (straight line with a dimen-
sion of 1) to 2.0 (plane with a dimension of 2); a difference of 0.1 represents a
doubling of complexity27.
For analysis of dendrite overlap between arborizations of neighbouring SACs,
pairs with somata separated by 80–160mm were selected because their dendrites
are known to interact25. Images were processed using Fiji or Photoshop software.
To estimate the amount of dendritic overlap that would occur by chance if two
SAC arborizations occupy the same territory, we flipped or rotated the image of
one SAC, realigned cell body position, and merged images. This method was
inspired by work on tiling of RGC dendrites49. We measured total overlapping
pixels in real and flipped images, interpreting ratios of .1 (real/flipped) as indi-
cating non-random interactions between SACs.
Purkinje cell dendrite self-crossings detected in single confocal planes were
counted in a 7,225mm2region of interest assigned to middle of arborization.
Purkinje arborization areas were measured using the convex-hull selection in
Fiji. Calbindin-labelled Purkinje somata residing along a 635mm segment in
lobules III–VI in single confocal planes were counted to measure Purkinje cell
Means were compared using the two-tailed Student’s t test on condition of
equivalent variances determined by F-test, or with the Mann–Whitney non-
parametric test. Means of multiple samples were compared using ANOVA and
posthoc Tukey test.
RT–PCR of dissociated retina cells. We used FACS to sort live cells from dis-
sociated P7 whole retina, VC1.11amacrine cells, and OFP1;Thy1.22SACs cells,
as describedpreviously37,50. Amacrine cellswere sortedfroma live cell suspension
and an anti-IgM secondary conjugated to phycoerythrin-Cy7 (Southern). OFP1
SACs were sorted from OFP1RGCs by negative selection of Thy1.2-PE-Cy7
labelled RGCs. In each condition, 2,000 cells were sorted directly into RNA lysis
buffer (Qiagen); RNA was purified and first strand cDNAs were generated with
exon-constant exon spliced transcripts were adapted from ref. 21, with modifica-
of the sorted population, are listed in Supplementary Table 1. PCR program used
is:94uCfor2min; 30cyclesof94uC for 20s,56uCfor30s, 72uCfor1min; 72uC
Dev. Biol. 271, 388–402 (2004).
32. Furuta, Y., Lagutin, O., Hogan, B. L. & Oliver, G. C. Retina- and ventral forebrain-
33. Knudson,C.M., Tung, K.S.,Tourtellotte, W.G., Brown,G.A.& Korsmeyer,S. J.Bax-
34. Zhang, X. M. et al. Highly restricted expression of Cre recombinase in cerebellar
Purkinje cells. Genesis 40, 45–51 (2004).
energy balance and glucose homeostasis. Cell Metab. 13, 195–204 (2011).
system for the whole mouse brain. Nature Neurosci. 13, 133–140 (2010).
37. Kay, J. N. et al. Retinal ganglion cells with distinct directional preferences differ in
molecular identity, structure, andcentral projections. J.Neurosci. 31, 7753–7762
38. Srinivas, S. et al. Expression of green fluorescent protein in the ureteric bud of
transgenic mice: a new tool for the analysis of ureteric bud morphogenesis. Dev.
Genet. 24, 241–251 (1999).
39. Yamagata, M&. Sanes, J. R. Transgenic strategy for identifying synaptic
connections in mice by fluorescence complementation (GRASP). Front. Mol.
Neurosci. 5, 18 (2012).
40. Farley, F. W., Soriano, P., Steffen, L. S. & Dymecki, S. M. Widespread recombinase
expression using FLPeR (flipper) mice. Genesis 28, 106–110 (2000).
41. Kuchibhotla, K. V. et al. Ab plaques lead to aberrant regulation of calcium
homeostasis in vivo resulting in structural and functional disruption of neuronal
networks. Neuron 59, 214–225 (2008).
subsets in the mouse superior colliculus. J. Comp. Neurol. 519, 1691–1711
signaling interactions that promote the survival and growth of developing retinal
ganglion cells in culture. Neuron 15, 805–819 (1995).
44. Chen, Y. et al. NS21: re-defined and modified supplement B27 for neuronal
cultures. J. Neurosci. Methods 171, 239–247 (2008).
45. Whitney, I. E., Keeley, P. W., Raven, M. A. & Reese, B. E. Spatial patterning of
46. Rodieck, R. W. The density recovery profile: a method for the analysis of points in
the plane applicable to retinal studies. Vis. Neurosci. 6, 95–111 (1991).
47. Montague, P. R. & Friedlander, M. J. Morphogenesis and territorial coverage by
isolated mammalian retinal ganglion cells. J. Neurosci. 11, 1440–1457 (1991).
48. Smith, T. G. Jr, Lange, G. D. & Marks, W. B. Fractal methods and results in cellular
morphology—dimensions, lacunarity and multifractals. J. Neurosci. Methods 69,
49. Wa ¨ssle, H., Peichl, L. & Boycott, B. B. Dendritic territories of cat retinal ganglion
cells. Nature 292, 344–345 (1981).
retinal amacrine cell subtypes and regulates their fate. Nature Neurosci. 14,
Macmillan Publishers Limited. All rights reserved