Neurite arborization and mosaic spacing in the mouse retina require DSCAM.

Abstract

To establish functional circuitry, retinal neurons occupy spatial domains by arborizing their processes, which requires the self-avoidance of neurites from an individual cell, and by spacing their cell bodies, which requires positioning the soma and establishing a zone within which other cells of the same type are excluded. The mosaic patterns of distinct cell types form independently and overlap. The cues that direct these processes in the vertebrate retina are not known. Here we show that some types of retinal amacrine cells from mice with a spontaneous mutation in Down syndrome cell adhesion molecule (Dscam), a gene encoding an immunoglobulin-superfamily member adhesion molecule, have defects in the arborization of processes and in the spacing of cell bodies. In the mutant retina, cells that would normally express Dscam have hyperfasciculated processes, preventing them from creating an orderly arbor. Also, their cell bodies are randomly distributed or pulled into clumps rather than being regularly spaced mosaics. Our results indicate that mouse DSCAM mediates isoneuronal self-avoidance for arborization and heteroneuronal self-avoidance within specific cell types to prevent fasciculation and to preserve mosaic spacing. These functions are analogous to those of Drosophila DSCAM (ref. 6) and DSCAM2 (ref. 7). DSCAM may function similarly in other regions of the mammalian nervous system, and this role may extend to other members of the mammalian Dscam gene family.

Full text

Page 1

LETTERS
Neuritearborizationandmosaicspacinginthemouse
retina require DSCAM
Peter G. Fuerst1, Amane Koizumi2{, Richard H. Masland2& Robert W. Burgess1
To establish functional circuitry, retinal neurons occupy spatial
domains by arborizing their processes, which requires the self-
avoidance of neurites from anindividual cell, and by spacing their
cellbodies,whichrequirespositioningthesomaandestablishinga
zone within which other cells of the same type are excluded1. The
mosaic patterns of distinct cell types form independently and
overlap. The cues that direct these processes in the vertebrate
retina are not known2,3. Here we show that some types of retinal
amacrine cells from mice with a spontaneous mutation in Down
syndrome cell adhesion molecule (Dscam), a gene encoding an
immunoglobulin-superfamily member adhesion molecule4,5, have
defects in the arborization of processes and in the spacing of cell
bodies. In the mutant retina, cells that would normally express
Dscam have hyperfasciculated processes, preventing them from
creating an orderly arbor. Also, their cell bodies are randomly
distributed or pulled into clumps rather than being regularly
spacedmosaics.OurresultsindicatethatmouseDSCAMmediates
isoneuronal self-avoidance for arborization and heteroneuronal
self-avoidance within specific cell types to prevent fasciculation
and to preserve mosaic spacing. These functions are analogous to
thoseofDrosophilaDSCAM(ref.6)andDSCAM2(ref.7).DSCAM
mayfunctionsimilarlyinotherregionsofthemammaliannervous
system, and this role may extend to other members of the mam-
malian Dscam gene family.
We have identified a spontaneous mutation in mice that creates a
loss-of-functionalleleofDscam,theDrosophilahomologuesofwhich
function in both isoneuronal self-avoidance for dendrite arboriza-
tion and heteroneuronal self-avoidance for axon tiling7–12. The
recessive mutation arose in the BALB/cByJ genetic background and
caused an overt neurological phenotype. Mutant and wild-type mice
are indistinguishable at birth, but are severely uncoordinated by
postnatal day 3 (P3); as adults, the mutant mice have spontaneous
seizuresandkyphosis,butarefertileandlong-lived(.24months;see
http://www.jax.org/research/media/wild_type.html
Throughpositionalcloning(seeMethods),amutationwasidentified
in Dscam. Sequencing of genomic and complementary DNA from
affected mice revealed a 38-bp deletion in exon17, causing a frame
shift resulting in ten unique amino acids followed by a premature
stop codon (Supplementary Fig. 1). This mutation truncates the
protein in the second fibronectin repeat (Fig. 1a). Dscam messenger
RNA levels in the brain were reduced by 70% in affected mice, con-
sistent with nonsense-mediated decay (Fig. 1b).
forvideo).
1TheJacksonLaboratory,BarHarbor,Maine04609,USA.2MassachusettsGeneralHospital,Boston,Massachusetts02114,USA.{Presentaddress:NationalInstituteforPhysiological
Sciences, 38 Nishigonaka, Myodaiji, Okazaki, 444-8585, Japan.
b
+/+
+/+
ONL
RGL
IPL
INL
OPL
Dscam
Actb
DSCAM
+/+
RGL
IPL
INBL
ONBL
Dscam AS
k
+/+
g
fd
c
–/– –/–
–/–+/–
–/–
e
Ig 1–9
FN 1–4
Ig 10
FN 5–6
PID
THDSCAM
+/+
TH
DSCAM
h
+/+
i
DSCAM
+/+
j
S1
S2
S3
S4
S5
a
Figure 1 | Identification of a mouse Dscam mutation. a, A schematic of the
DSCAM protein domain structure. The extracellular portion of DSCAM
consists of ten immunoglobulin-like repeats (Ig) and six fibronectin
domains (FN). The Dscam deletion truncates the coding sequence in the
secondfibronectindomain(arrow),whichisbeforethetransmembraneand
PAK1-interacting domains (PID). b, Northern blotting of mRNA purified
from whole brains of wild-type (1/1), Dscam1/2and Dscam2/2mice
revealed a 70% reduction in Dscam mRNA in the mutant sample. b-actin
(Actb) was used as a loading control. c–f, Haematoxylin and eosin stained
sections of Dscam2/2and wild-type retinas from P0 and adult mice. The
Dscam2/2retina is indistinguishable from that of the wild type during
embryonic stages of development and at birth (c, d). INBL, innerneuroblast
layer; ONBL, outerneuroblast layer; ONL, outernuclear layer; OPL,
outerplexiform layer. e, f, In the adult Dscam2/2retina, the inner nuclear
(INL), inner plexiform (IPL) and retinal ganglion (RGL) layers are
disorganized compared to those of the wild-type retina, whereas other
retinal layers are indistinguishable from that of the wild type. g, In situ
hybridization with Dscam antisense probes (Dscam AS) revealed expression
in a subset of cells in the inner nuclear and ganglion cell layers. h, Whole
controlP15retinastainedwithantibodiestoDSCAMandTH.i,Asectionof
a control adult (6–10-week-old) retina labelled using antibodies to DSCAM
andTH.j,Asectionofwild-typeP15retinastainedwithDSCAMantibodies.
k, A section of the Dscam2/2retina stained with an antibody to DSCAM.
Scalebars:c,d,80mm;e,f,106mm;g,160mm;h,45mm;i,120mm;j,k,65mm.
Vol 451|24 January 2008|doi:10.1038/nature06514
470
Nature

PublishingGroup
©2008

Page 2

Despite the overt neurological phenotype of Dscam2/2mice,
examination of the central nervous system of adults and developing
embryos didnot reveal any gross disorganization, withthe exception
of a caudal folium of the cerebellum (Supplementary Fig. 2), imply-
ing a subtle phenotype or a subpopulation of affected cells.
Wedidfinddisorganizationofpostnatalretinalanatomy(Supple-
mentary Fig. 3). At birth or before, the mutant retina was indistin-
guishable from that of control mice (Fig. 1c, d). However, by P4, the
ganglion cell layer and developing inner plexiform layer (IPL) were
disorganized (not shown). This disorganization persisted into adult-
hood in amacrine and ganglion cells (Fig. 1e, f), and was confirmed
withmarkeranalysis(SupplementaryFig.4).Withinthesecellpopu-
lations, many subtypes are present13,14, and Dscam was expressed in a
subset of cells in these layers (Fig. 1g). Double in situ hybridization
withmarkersofdefinedamacrine subtypesindicatedthatDscamwas
expressed in dopaminergic amacrine cells (tyrosine hydroxylase
(Th)-positive) and in bNOS-positive (Nos1-expressing) cells, but
not in choline acetyltransferase (Chat)-positive starburst amacrine
cells (SACs) or disabled 1-positive (Dab1) AII amacrine cells
(Supplementary Fig. 5). Immunofluorescence with an antibody
against the extracellular domain showed that DSCAM completely
overlapped dopaminergic amacrine cell staining in .400cells exam-
ined. In whole-mount retinas at P15, anti-DSCAM staining was dis-
tributed across the soma and processes of dopaminergic amacrine
cells, but redistributed to the cell body in the adult retina (Fig. 1h, i).
Additional immunoreactivity was detected in retinal cross-sections
in lower, more vitreal layers of the IPL, below the strata of TH-
positive processes (Fig. 1j). DSCAM immunoreactivity was absent
Density (cells per mm2)
Distance (µm)
Average density, 58 cells per mm2
DRP, TH WT
Average density,
80 cells per mm2
DRP, TH –/–
Average density,
92 cells per mm2
DRP, TH –/–
Average density,
41 cells per mm2
DRP, TH WT
100
80
60
40
50 100150200
Distance (µm)
50100150 200
Distance (µm)
50 100150 200
20
0
Density (cells per mm2)60
40
50
20
30
10
0
Density (cells per mm2)
100
120
140
80
60
40
20
0
Distance (µm)
50100150200
Density (cells per mm2)
150
200
100
50
0
TH
TH
TH
b
d
ac
+/+–/–
TH
k
m
TH
l
n
o
+/+
–/–
0
400
800
1,200
1,600
WT
–/––/–
–/––/–
1.6
1.2
WT
0.8
0.4
0
1.2
0.8
0.4
0
WT
1.2
0.8
0.4
0
WT
*
*
__
__
__
__
__
__
__
__
gh
i
j
+/+
TH
–/–
TH
e
f
Length of
neurites (µm)
Crosses per
100-µm neurite
Branches per
100-µm neurite
Crosses per
branches
Figure 2 | Arborization and mosaic patterning of dopaminergic amacrine
cellsin controland Dscam2/2retinas. a, A wild-type P6 retinastained with
anti-TH. Dopaminergic amacrine cell processes have little proximal contact
and rarely self-cross. b, DRP analysis of cell-body spacing identified an
exclusion zone surrounding dopaminergic neurons in wild-type retinas.
c, A P6 Dscam2/2retina stained with anti-TH. Individual dopaminergic
amacrine cells frequently self-cross. Error bars in all figures represent s.d.
d, DRP analysis of Dscam2/2dopaminergic amacrine cells indicates normal
soma spacing at P6. e, f, Representative isolated P6 wild-type (e) and
Dscam2/2(f) dopaminergic amacrine cells are shown; insets contain traces
of the neurites. g–j, Analysis of 20 isolated dopaminergic amacrine neurons
fromeitherwild-typeorDscam2/2retinas.g,Nosignificantdifferenceinthe
totalneurite lengthwasobservedbetweenwild-typeandDscam2/2neurons
(t-test, P50.55). h, Similarly, no significant difference in neurite branches
per unit length was observed (P50.38). i, j, A significant increase in the
number of self-crossingsper length of neurite (i) or per number of branches
(j) was observed for the Dscam2/2neurons over controls (P50.003 and
0.015, respectively). k–n, Whole adult (6–8weeks) wild-type (k, l) or
Dscam2/2(m, n) retinas stained with anti-TH. TH-positive neurite fascicles
run throughout the Dscam2/2retina (m) in contrast to the wild-type retina
(k), in which neurites arborize evenly. l, DRP analysis indicates that
dopaminergic amacrine cells in the adult wild-type retina maintain and
expand their mosaic spacing and exclusion zones. n, DRP analysis of adult
Dscam2/2retinas indicates aggregationof dopaminergic amacrineneurons.
o, Schematic of arborization and mosaic formation in wild-type (left) and
Dscam2/2(right) retinas. At P6, both wild-type and Dscam2/2
dopaminergic amacrine cells are organized in a mosaic pattern; however,
wild-type neurites arborize, whereas Dscam2/2neurites self-cross. By P10,
wild-type dopaminergic amacrine cells have an increased exclusion zone,
whereas Dscam2/2dopaminergic amacrine neurons aggregate along
bundled fascicles. Scale bars: a, c, e, f, 133mm; k, m, 388mm.
NATURE|Vol 451|24 January 2008
LETTERS
471
Nature

PublishingGroup
©2008

Page 3

from the mutant retinas, consistent with a protein-null mutation
(Fig. 1k).
Wild-type dopaminergic amacrine cells were first labelled by anti-
TH staining at around P6, when the neurons are still extending
processes(Fig.2a).Thespacingofdopaminergicamacrinecellbodies
was analysed by density recovery profiling (DRP)—a statistical mea-
sureofthecells’spatialautocorrelation, measuringtheprobabilityof
encountering a homotypic cell body at varied distances from the
reference cell. Regularly spaced cells have a zone of exclusion for
other homotypic cells, indicated by a below-average cell density at
short distances15(Fig. 2b). Although their spacing was normal, the
Dscam2/2dopaminergic amacrine cells showed abnormal morpho-
logy at P6 (Fig. 2c, d). Examining individual cell morphologies in
20 isolated dopaminergic amacrine cells in control and Dscam2/2
retinas revealed defects in arborization in the mutant retina
(Fig. 2e, f). Quantification indicated no differences in total length
ofprocessesorinthenumberofbranchesperunitlength,butmutant
cells had a significantly larger number of processes that self-crossed
bNOS
ac
bNOS
f
DAB1
ChAT
e
g
ChAT
Dab1
i
k
Dab1
d
f
h
Density (cells per mm2)1,500
j
l
AII
bNOS+
SAC
DAC
S1
S2
S5
S4
S3
m
Dscam-positive cell type
Wild-type retina Dscam–/– retina
f
DAB1
Dscam-negative cell type
Wild-type retina Dscam–/– retina
n
j
f
+/+
+/+
+/+
–/–
–/–
–/–
Density (cells per mm2)
Distance (µm)
DRP SAC, WT
Average density,
63 cells per mm2
DRP bNOS, WT
Average density,
212 cells per mm2
DRP bNOS, –/–
Average density,
1,020 cells per mm2
DRP SAC, –/–
Average density,
1,200 cells per mm2
DRP, All, –/–
Average density,
1,560 cells per mm2
DRP, All, WT
Average density, 1,120 cells per mm2
100
80
60
40
50100150200
Distance (µm)
20406080100
20
0
Density (cells per mm2)
Distance (µm)
300
50
100
150
200
250
50 100150 200
0
Density (cells per mm2)1,500
1,000
500
0
Distance (µm)
20 4060 80 100
1,000
500
0
Distance (µm)
20 40305010
Density (cells per mm2)
1,500
2,000
1,000
500
0
Distance (µm)
20 40305010
Density (cells per mm2)
1,500
2,000
1,000
500
0
b
Figure 3 | Morphology and cell body spacing of other amacrine cell types.
a, b, Cell bodies ofbNOS-positivecells, whichalso expressDscam incontrol
retinas, are spaced and arborized in a manner similar to dopaminergic
amacrineneurons.c, d,The bNOS-positiveamacrinecellsshowfasciculated
processes and randomly spaced cell bodies in Dscam2/2retinas. Starburst
amacrine cells have an exclusion zone around the cell body and a meshwork
of arborized processes in both control (e, f) and Dscam2/2(g, h) retinas.
(Cellbodiesare pseudo-colored red to distinguishthemfrom themeshwork
of processes in S2 of the IPL (green).) AII amacrine cells are also evenly
spaced withvery shortprocessesincontrol(i, j) andDscam2/2(k, l)retinas.
m, Schematic depicting the stratification of bNOS-positive, SAC, DAC and
AII neurites in the inner plexiform layer. Dopaminergic amacrine neurites
arborize predominantly in S1, AII amacrine neurites arborize in between S1
and S2, bNOS-positive amacrine neurites arborize in S3, and starburst
amacrine neurites arborize in S2 (soma in INL) or S4 (soma in RGL).
n, Schematic summarizing mosaic formation in the Dscam2/2retina.
DSCAM-positive cell types arborize and maintain mosaics in the wild-type
retina, but fasciculate and fail to maintain mosaics in the Dscam2/2retina.
Cell types in which DSCAM expression was not detected arborize and
maintain mosaics in both the wild type and the Dscam2/2retina. Scale bars:
a, c, 388mm; e, g, 62mm; i, k, 123mm. Different scales are used on the
ordinates of DRP graphs for each cell type.
LETTERS
NATURE|Vol 451|24 January 2008
472
Nature

PublishingGroup
©2008

Page 4

(Fig. 2g–j). This phenotype was more pronounced at P10 (not
shown), and eventually large fascicles of TH-positive processes from
many cells form in the mutant retina (Supplementary Fig. 6).
Dopaminergic processes in the wild-type adult retina form a broad,
uniform plexus, and the cell bodies are evenly spaced (Fig. 2k, l). The
fasciculated adult Dscam2/2dopaminergic amacrine cells had
abnormal positioning of their cell bodies, which associated with
the fascicles and clumped significantly (Fig. 2m, n and Supple-
mentary Figs 6 and 7).
In the mutant retina, bNOS-positive amacrine cells, which nor-
mally express Dscam, also had marked differences in the cell mor-
phology, with short bundled processes and randomly spaced cell
bodies (Fig. 3a–d). Starburst and AII amacrine populations that do
not express Dscam were normal in their cell-body distribution
(Fig. 3e–l), maintaining an exclusion zone in the control and
Dscam2/2retinas. The meshwork of processes in the IPL from star-
burst cells in the inner nuclear layer was preserved in the Dscam2/2
retinas, with no abnormal fasciculation seen, and the short processes
of AII cells were also normal. Cross-sections of control and
Dscam2/2retinaswerealsoexamined,andallcelltypesreachedtheir
appropriate vertical layer in the retina (Supplementary Fig. 8).
In normal retinas, the mosaic spacing of each cell type, such as AII
and starburst cells, is independent, allowing dendritic overlap of
functionally different cells16. If DSCAM-mediated recognition was
required for this process, then cell types that express Dscam might
influence the spacing of each other. We therefore examined the dis-
tribution of dopaminergic amacrine and bNOS-positive amacrine
cells, relative to each other, in control and mutant retinas (Fig. 4
and Supplementary Fig. 9). These cell bodies were randomly spaced
with respect to one another in both genotypes, indicating that
DSCAM is necessary for maintaining mosaics in homotypic cells
but is not sufficient to confer identifying signals between cell types.
Also, despite the fasciculation of dopaminergic and bNOS-positive
processesintheDscam2/2retinas,noassociationsofprocessesacross
cell types were seen.
Mammalian DSCAM is therefore required for isoneuronal self-
avoidance and for heteroneuronal recognition within a cell type. In
the absence of DSCAM, the processes of an individual dopaminergic
or bNOS-positive amacrine cell fail to form proper arbors, and
instead fasciculate with processes of other cells of the same type,
secondarily pulling the cell bodies out of their mosaic position.
However, differentcelltypesexpressing Dscamdonotinfluenceeach
other’s spacing. A neurodevelopmental role for vertebrate DSCAM
wasnotpreviouslyestablishedbeyondanearly,non-neuron-specific,
developmental phenotype seen in zebrafish17.
Our results are consistent with previous studies showing that ret-
inal mosaics form very early, before neurons have extensive arbors,
and that arborization and mosaic formation are largely indepen-
dent18,19. The normal spacing of cells in the P6 Dscam2/2retina
indicates that the early stages of mosaic formation are intact.
However, DSCAM-mediated heteroneuronal self-avoidance is
needed to prevent fasciculation of homotypic cells, which destroys
their mosaic pattern.
Decreased cell death may also indirectly contribute to the disrup-
tionofmosaics,basedontheincreaseindopaminergicandbNOScell
densities in the mutant retina. The absolute density of cells that
would normally express Dscam was increased by ,250% for dopa-
minergic amacrine cells, and by ,300% for bNOS-positive cells in
themutant retina (DRPsin Figs 2and3). Incontrast, the densities of
the ChAT-positive (starburst) and DAB1-positive (AII) cells were
slightly decreased. Dopaminergic amacrine cells rely in part on cell
death to sculpt their mosaic pattern20. We saw decreased TdT-
mediated dUTP nick end labelling (TUNEL)-positive cells in the
developing Dscam2/2retina, but saw no change in the number of
proliferating cells (Supplementary Fig. 10); we also identified that
clumped dopaminergic amacrine cells were not clonal, indicating
that they are not the result of overproliferation from a progenitor,
but consistent with lateral migration of cell bodies (Supplementary
Fig. 11). DSCAM-mediated self-recognition may therefore be
upstream of cell death in the wild-type retina, whereas non-Dscam-
expressing cells may use other mechanisms for spacing21.
The fact that DSCAM mediates both isoneuronal and heteroneur-
onal self-avoidance in given amacrine cell types is interesting given
recent work in Drosophila. In flies, Dscam is alternatively spliced to
generate tens of thousands of protein isoforms6. The homophilic
interaction of DSCAM is highly isoform-specific, and this specificity
is determined by the variable immunoglobulin domains8,22,23. There-
fore, Drosophila neurons use DSCAM for isoneuronal self-avoidance
and arborization because each cell recognizes only its own processes,
which express the same set of isoforms8,9,24. Reducing or eliminating
the diversity of DSCAM isoforms perturbs Drosophila circuit
organization25,26. Dscam2 in Drosophila is not extensively alterna-
tively spliced and mediates heteroneuronal cell avoidance in tiling
bNOS+
DAC
Wild type
Dscam–/–
a
c
TH
TH
bNOS
bNOS
–/–
+/+
Density (cells per mm2)
Distance (µm)
Cross DRP, TH to bNOS, –/–
Average density,
63 cells per mm2
Cross DRP, TH to bNOS, WT
Average density,
211 cells per mm2
100
80
60
40
50100150200
Distance (µm)
50 100150200
20
0
b
Density (cells per mm2)
300
200
250
150
100
50
0
d
fe
Figure 4 | Co-existence of dopaminergic and bNOS-positive amacrine
cells. a, In wild-type retinas double-labelled for two Dscam-expressing cell
populations (dopaminergic amacrine and bNOS-positive), the cell bodies
and processes appear independent. b, This was confirmed by cross-DRP
analyses comparing dopaminergic to bNOS-positive cells, which indicated
no difference from average density (random spacing). c, In Dscam2/2
mutant retinas, the fasciculation and cell-body-spacing defects are evident,
but the spacing of cells remains independent and they do not aggregate, as
indicated by cross-DRP analyses (d). In addition, the fascicles of processes
alsoremainindependent.e,Schematicdepictingtheindependenceofretinal
neuron mosaic formation. Retinal neurons form organized mosaics that are
independent of the spacing of other cell types. f, Schematic depicting
dopaminergicamacrineandbNOS-positiveneuronarborizationinthewild-
type and Dscam2/2retina. Wild-type dopaminergic amacrine neurons
arborize in S1, whereas bNOS-positive neurons arborize in S3. In the
Dscam2/2retina, the arborization of dopaminergic amacrine and bNOS-
positive neurites becomes diffuse but, although neurites of both cell types
fasciculate with homotypic neurites (dopaminergic amacrine with
dopaminergicamacrineorbNOS-positivewithbNOS-positive),theydo not
fasciculate with heterotypic neurites (dopaminergic amacrine with bNOS-
positive). Scale bars in a and c represent 240mm.
NATURE|Vol 451|24 January 2008
LETTERS
473
Nature

PublishingGroup
©2008

Page 5

the axons of L1 neurons in the medulla of the Drosophila visual
system7. Mouse DSCAM functions in a highly analogous way, pro-
moting self-avoidance for both arborization (isoneuronal) and the
prevention of fasciculation (heteroneuronal). Although the verte-
brate genes are not extensively alternatively spliced, they do undergo
homophilic and paralogue-specific binding5,27,28. The coexistence of
Dscam-expressing amacrine cell populations suggests that stratifica-
tion in different IPL layers may allow reuse of DSCAM as a recog-
nition signal. Alternatively, additional co-signals may distinguish
distinct cell populations, consistent with the continued lack of
interaction between bNOS and dopaminergic cells in the
Dscam2/2retina. Additional complexity for self-recognition could
also be introduced by other Dscam gene family members, such as
Dscam-like 1 (Dscaml1), which is expressed in a similar but discrete
population of retinal neurons27,28(P.G.F. and R.W.B., unpublished)
or the highly homologous Sidekick proteins29.
METHODS SUMMARY
Geneticsand Dscamanalysis.TheDscammutationwas mappedby breeding to
C57BL6/JandestablishinglinkageinF2micewithpolymorphicmarkers,andthe
mutationwasidentifiedby positionalcloningapproaches.Mice weregenotyped
by PCR from genomic DNA spanning the 38-bp deletion (Dscam forward,
CTTTGCGCGTTATGATCCT; Dscam reverse, GTGGTGTCGATACTGATG).
Control mice were littermates of the Dscam2/2animals to control for genetic
background effects and age.
Immunocytochemistry. Whole-mount dissected retinas or cryostat sections
were stained using standard immunofluorescence protocols. Antibody sources
are listed in the Methods. Fluorescence images were collected using a Leica
SP5 confocal microscope or a Nikon epifluorescence microscope with a digital
camera.
In situ hybridization. In situ hybridization was performed using digoxygenin-
and fluorescein isothiocyanate (FITC)-labelled riboprobes, detected by HRP-
conjugated secondary antibodies and TSA-Plus fluorescent substrates (Perkin
Elmer).
Analysis of mosaics. The spacing of cell bodies was analysed using DRP as
described previously, with the modification that the closest bin in the analysis
was corrected to account for the diameter of the cell body15,16.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 2 October; accepted 21 November 2007.
1.Wassle, H. & Boycott, B. B. Functional architecture of the mammalian retina.
Physiol. Rev. 71, 447–480 (1991).
Novelli, E., Resta, V. & Galli-Resta, L. Mechanisms controlling the formation of
retinal mosaics. Prog. Brain Res. 147, 141–153 (2005).
Galli-Resta,L.Local,possiblycontact-mediatedsignallingrestrictedtohomotypic
neurons controls the regular spacing of cells within the cholinergic arrays in the
developing rodent retina. Development 127, 1509–1516 (2000).
Yamakawa, K.et al.DSCAM: anovel member oftheimmunoglobulin superfamily
maps in a Down syndrome region and is involved in the development of the
nervous system. Hum. Mol. Genet. 7, 227–237 (1998).
Agarwala, K. L., Nakamura, S., Tsutsumi, Y. & Yamakawa, K. Down syndrome cell
adhesionmoleculeDSCAMmediateshomophilicintercellularadhesion.BrainRes.
Mol. Brain Res. 79, 118–126 (2000).
Schmucker, D. et al. Drosophila Dscam is an axon guidance receptor exhibiting
extraordinary molecular diversity. Cell 101, 671–684 (2000).
Millard, S. S., Flanagan, J. J., Pappu, K. S., Wu, W. & Zipursky, S. L. Dscam2
mediates axonal tiling in the Drosophila visual system. Nature 447, 720–724
(2007).
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).
2.
3.
4.
5.
6.
7.
8.
9.Zhan, X. L. et al. Analysis of Dscam diversity in regulating axon guidance in
Drosophila mushroom bodies. Neuron 43, 673–686 (2004).
10. Soba, P. et al. Drosophila sensory neurons require Dscam for dendritic self-
avoidance and proper dendritic field organization. Neuron 54, 403–416 (2007).
11.Matthews, B. J. et al. Dendrite self-avoidance is controlled by Dscam. Cell 129,
593–604 (2007).
12. Hughes, M. E. et al. Homophilic Dscam interactions control complex dendrite
morphogenesis. Neuron 54, 417–427 (2007).
13. Jeon, C. J., Strettoi, E. & Masland, R. H. The major cell populations of the mouse
retina. J. Neurosci. 18, 8936–8946 (1998).
14. Kong, J. H., Fish, D. R., Rockhill, R. L. & Masland, R. H. Diversity of ganglion cells in
the mouse retina: unsupervised morphological classification and its limits. J.
Comp. Neurol. 489, 293–310 (2005).
15. 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).
16. Rockhill,R.L.,Euler,T.&Masland,R.H.Spatialorderwithinbutnotbetweentypes
of retinal neurons. Proc. Natl Acad. Sci. USA 97, 2303–2307 (2000).
17. Yimlamai, D., Konnikova, L., Moss, L. G. & Jay, D. G. The zebrafish Down
syndrome cell adhesion molecule is involved in cell movement during
embryogenesis. Dev. Biol. 279, 44–57 (2005).
18. Galli-Resta, L., Resta, G., Tan, S. S. & Reese, B. E. Mosaics of islet-1-expressing
amacrine cells assembled by short-range cellular interactions. J. Neurosci. 17,
7831–7838 (1997).
19. Lin, B., Wang, S. W. & Masland, R. H. Retinal ganglion cell type, size, and spacing
can be specified independent of homotypic dendritic contacts. Neuron 43,
475–485 (2004).
20. Raven, M. A., Eglen, S. J., Ohab, J. J. & Reese, B. E. Determinants of the exclusion
zone in dopaminergic amacrine cell mosaics. J. Comp. Neurol. 461, 123–136
(2003).
21. Resta, V., Novelli, E., Di Virgilio, F. & Galli-Resta, L. Neuronal death induced by
endogenous extracellular ATP in retinal cholinergic neuron density control.
Development 132, 2873–2882 (2005).
22. Wojtowicz, W. M. et al. A vast repertoire of Dscam binding specificities arises
from modular interactions of variable Ig domains. Cell 130, 1134–1145 (2007).
23. Meijers, R. et al. Structural basis of Dscam isoform specificity. Nature 449,
487–491 (2007).
24. 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
(2004).
25. Hattori, D. et al. Dscam diversity is essential for neuronal wiring and self-
recognition. Nature 449, 223–227 (2007).
26. Chen, B. E. et al. The molecular diversity of Dscam is functionally required for
neuronal wiring specificity in Drosophila. Cell 125, 607–620 (2006).
27. Agarwala,K.L.etal.CloningandfunctionalcharacterizationofDSCAML1,anovel
DSCAM-like cell adhesion molecule that mediates homophilic intercellular
adhesion. Biochem. Biophys. Res. Commun. 285, 760–772 (2001).
28. Yamagata, M. & Sanes, J. R. Dscam and Sidekick proteins direct lamina-specific
synaptic connections in vertebrate retina. Nature doi:10.1038/nature06469 (this
issue).
29. Yamagata, M.,Weiner, J. A. &Sanes, J. R.Sidekicks: synaptic adhesion molecules
that promote lamina-specific connectivity in the retina. Cell 110, 649–660
(2002).
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank G. Cox and S. Ackerman for comments on the
manuscript, and M. Yamagata and J. Sanes for sharing unpublished results. The
authorsaresupportedbytheNEI,theKnightsTemplarEyeFoundation(P.G.F.)and
Research to Prevent Blindness (R.H.M.). We also thank the scientific services at
The Jackson Laboratory (supported by NCI Cancer Center).
Author Contributions P.G.F. identified the Dscam mutation and performed all
tissue staining and microscopy presented. A.K. carried out the quantitative
analyses of retinal cell mosaics and microinjected TH cells. R.H.M. helped to
interprettheanatomicalimagesanddesignedthemosaicanalysis.R.W.B.assisted
in experimental design and interpretation. All authors shared in writing the paper.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. Correspondence and requests for materials should be
addressed to R.W.B. (robert.burgess@jax.org).
LETTERS
NATURE|Vol 451|24 January 2008
474
Nature

PublishingGroup
©2008

Page 6

METHODS
Animal husbandry. Mice were used in accordance with protocols approved by
the Animal Care and Use Committee at the Jackson Laboratory. The Jackson
LaboratoryisaccreditedbytheAssociationfortheAssessmentandAccreditation
orLaboratoryAnimal Care(AAALAC). Animalswerehoused inPIVcagingand
given food and water ad libitum.
Genetic mapping. The Dscamdel17mutation arose spontaneously in the Balb/cJ
mapping partner of an unrelated F1 mapping cross. The mutation was initially
localized to the distal region of chromosome 16 by typing 140 strain-specific
markers,spacedapproximatelyevery10Mb,onatotalof10mutantand10wild-
type mice, the phenotypes of which were confirmed by retinal histology. The
mutation was fine-mapped against DBA/2J, C57BL/6J and CAST/EiJ strains to
maximize the number of polymorphic markers available. In total, more than
1,800meiosesweretypedattheflankingmarkersD16Mit106andD16Mit94.The
intervalwasnarrowedto1.71Mb(0.2cM),betweenD16Mit219andD16Mit205.
Thisintervalcontained14annotatedgenesincludingmouseDscam.Foranalysis
of the Dscam gene, mRNA from affected and control brain was prepared using
Trizol(Invitrogen),reversetranscribedwithSuperscriptIII(Invitrogen)forPCR
and sequencing, or used for northern blot analysis.
Mouse strains. The official strain designation of the Dscam mutation is
Dscamdel17, reflecting the molecular lesion. It is referred to in this paper as
Dscam2. In segregating genetic backgrounds containing BALB/c or 129 loci,
these mice survive to adulthood and will breed as homozygotes. However, as
the background is made increasingly C57BL/6, the affected mice die neonatally.
The experiments described were therefore carried out on a mixed, but predo-
minantly BALB/cByJ, background. Littermates of experimental mutants were
used as controls whenever possible to account for age and genetic background.
The yellow fluorescent protein (YFP) transgenic strain used in Supplementary
Fig. 6 is the MitoZ strain.
Genotyping. The primers Dscam17F and Dscam17R (CTTTGCGCGTT-
ATGATCCT and GTGGTGTCGATACTGATG, respectively), which flank the
Dscamdel17deletion, were used to genotype mice. Tail biopsies were digested
overnight in 100ml of 1mgml21proteinase-K in buffer containing 100mM
Tris, pH8.5, 10mM EDTA and 200mM NaCl. Samples were then boiled for
15min to denature the DNA and to inactivate the proteinase-K. PCR was per-
formedusingEppendorfmastertaqwiththefollowingprogram:94uCfor1min,
followed by 35cycles of 94uC for 20sec, 53uC for 30sec and 72uC for 30sec,
followed by a single 72uC extension for 2min. PCR of DNA from homozygous
wild-type mice amplifies a single 170bp band, whereas PCR of DNA from
homozygous mutant mice have a single 133bp band, with both bands apparent
in PCR samples run using DNA from heterozygous mice.
AnalysisofmRNA.ForRNAisolation,totalretinalorbrainRNAwasisolatedby
dissecting the retina or brain of wild-type and Dscam2/2mice, which were then
homogenized in 500ml or 2ml Trizol reagent, respectively, according to the
manufacturer’s protocol. RNA samples were resuspended in diethyl pyrocarbo-
nate (DEPC)-treated H2O. Reverse transcription was performed using
SuperScript First-Strand Synthesis kit according to manufacturer’s instructions
using Superscript III reverse transcriptase and a mix of random hexamer and
oligo-dTprimers(Invitrogen).Fornorthernblotanalysis,aprobetotheamino-
terminal region of the Dscam transcript was generated by PCR with reverse
transcription (RT–PCR) with primers Dscam1 forward and Dscam1 reverse
(TCCTTCAAGCTTCAGCAC and CATTTCCTGTCACACTGC, respectively).
This fragment was labelled with
Random labelling kit according to the manufacturer’s instructions (GE
Healthcare). Total RNA (25mg) from each sample was heated at 75uC for
5min and loaded onto a 2% formaldehyde gel. Electrophoresis of samples was
conducted for 5h at 50volts (V). RNA was transferred to a charged nylon
membrane for 16–18hours using the standard sandwich technique. RNA was
crosslinkedtothemembranewithultravioletlightandprehybridizedat50uCin
10ml hybridization buffer for 1–2hours (53 SSC, 13 Denhardt’s solution, 1%
salmon sperm DNA (100mgml21), 50% formamide and 0.5% SDS). Labelled
probe (10mCi) was boiled in TE (10mM Tris, pH7.5, and 1mM EDTA) for
5min to denature and was added to 5ml of hybridization buffer. Blots were
hybridized at 65uC for 16–18hours. Blots were washed two times for 15min in
23 SSC and 0.5% SDS followed by two 15-min washes in 0.23 SSC and 0.1%
SDS at 65uC. Transcript levels were compared in three Dscam2/2mice, three
Dscam1/2mice and three Dscam1/1mice at tendays of age.
RNA in situ hybridization. Probe templates were prepared by PCR from first-
strand brain cDNA. The primers contained T7 (anti-sense) or SP6 (sense) tran-
scription initiation sequences at the 39 and 59 ends, respectively, of the PCR
products. RNA probes labelled with digoxygenin or fluorescein dUTP were
prepared using the following reaction mixture: 1mg DNA, 2ml 103 T7 or SP6
buffer (Roche), 1.5ml T7 or SP6 RNA polymerase (Roche), 0.5ml RNase out
32P using the Amersham Rediprime II
(Invitrogen), 2ml digoxyigenin or fluorescein dNTP mixture (Roche), and H2O
to 20 ml. Samples were incubated at 37uC for 2h, after which 1ml of RNase-free
DNase I (Roche) was added. Samples were incubated for an additional 15min,
after which 1ml 0.5M EDTA was added to stop the reaction. The RNA was
precipitatedbyadding2.5mlof4MNH4Cland75mlofchilled(220uC)ethanol,
and resuspended in 40ml of DEPC-treated H2O. Typical yields were 300–
400ngml21of labelled riboprobe.
For hybridization, eyes were enucleated and fixed in 4% paraformaldehyde at
4uC for 4h and were frozen in cyro-embedding media. Sections were cut at 15–
20mm with a cryostat and incubated at 65uC for 10min. Sections were again
fixed with 4% paraformaldehyde for 10min at room temperature (20uC), fol-
lowed by three washes with DEPC-treated PBS. Sections were digested with
proteinase-K for 5min at room temperature (1mgml21proteinase-K in
50mMTris,pH7.5,and5mMEDTA).Afterdigestion,sampleswerefixedagain
in 4% paraformaldehyde for 5min at room temperature. Sections were washed
three times in DEPC-treated PBS. Sections were then acetylated for 10min at
room temperature (59ml H2O, 0.8ml triethanolamine, 0.105ml HCl and
0.15ml acetic anhydride). After acetylation, sections were permeabilized in 1%
Triton X-100/PBS for 30min at room temperature, followed by three 10-min
rinses in DEPC-treated PBS. Slides were prehybridized for one hour at room
temperature in hybridization solution (50% formamide, 53 SSC, 53 Denhart’s
solution, 250mgml21yeast tRNA, 500mg salmon sperm DNA and 50mgml21
heparin). While sections were prehybridizing, probes were prepared at a con-
centration of 200ngml21in hybridization solution. Probe solution (50ml) was
prepared per slide and incubated at 80uC for 5–10min to denature.
Prehybridization solution was replaced with hybridization solution, and the
slideswerecoverslippedwith50-mmglasscoverslipsandincubatedinahumidi-
fied chamber (50% formamide 53SSC) at 65–70uC overnight.
Forprobedetection,afterhybridization,sectionswerewashedtwicefor5min
with TBS at room temperature. Peroxidase-conjugated antibodies to digoxy-
genin or fluorescein (Roche) were diluted 1:2,000 in TNB (TSA plus system)
and incubated on sections at 4uC overnight. Sections were washed in TBST
(0.05% Tween) three times for 10min at room temperature. Probes were
detectedusingtheTSAplussystem(Perkin-Elmer).Fordouble-labellingexperi-
ments, POD-antibodies against FITC and digoxygenin (DIG) were applied
sequentially, and the peroxidase conjugate of the first detection antibody was
inactivated by H2O2treatment before applying the second antibody. Different
tyramide conjugates were used when developing the signals to achieve different
colours, and detectionreactions were allowedto proceed for 30min. Dscamand
amacrine-marker expression was examined at P12. At least four independent
retinas were examined. Sense-strand controls were used in all experiments to
confirm specificity.
Histology and immunocytochemistry. Tissues were fixed in Bouin’s fixative
overnight. Sections (4mm) were cut with a microtome and stained using a
standard haemotoxylin and eosin stain. For immunocytochemistry, embryos
or enucleated eyes were fixed in 4% paraformaldehyde overnight. Tissue was
equilibrated in 30% sucrose/PBS and frozen in OCT media. Thicker sections
(8mm) were cut using a cryostat and used immediately or stored for up to two
weeks at 220uC. Sections were blocked in 3% normal horse serum in 13 PBS
and 0.1% Triton X-100. Primary antibodies (indicated in the Methods
Summary) were incubated overnight at 4uC and subsequently washed twice
for10mininPBS.Sectionswereincubatedatroomtemperature withsecondary
antibodies for one hour, and then washed four times in PBS for 10min. The last
wash contained 2ml of 1mgml21DAPI per 40ml PBS.
Whole-mounted retinas. Mice were perfused transcardially with PBS and 4%
paraformaldehyde. Eyes were enucleated and placed in a dish containing PBS. A
small hole was made at the junction between the cilliary body and retina with a
30-gauge needle. The eye was hemisected and the retina was gently teased away
from the sclera. Retinas were fixed a second time in 4% paraformaldehyde for
onehour, followed by a rinse in PBS. Retinas were incubated with primary
antibodies in PBS, supplemented with 3% normal horse serum and 0.5%
Triton X-100, for 3–5 days at 4uC with gentle rocking. Primary antibodies were
washed off overnight in PBS, and the retinas were incubated with secondary
antibodies in PBS, 3% normal horse serum and 0.5% Triton. Retinas were
washed for 2–4h in PBS and were flat-mounted. Whole-mount images were
collected from mice between six and tenweeks of age, and at least six mutant
and control animals were included in each analysis.
Antibodies used. The following primary antibodies were used in standard
immunofluorescence and western blotting protocols. Goat anti-human
DSCAM(R&DSystems),rabbit
Biologicals), goat anti-choline acetyltransferase (Chemicon), rabbit anti-
Disabled1 (gift from B. Howell), anti-CHX10 (Santa Cruz Biotechnology),
anti-BRN3b (Santa Cruz Biotechnology), mouse anti-neurofilament (2H3,
Developmental Studies Hybridoma Bank), mouse anti-PAX6 (Developmental
anti-tyrosinehydroxylase(Novus
doi:10.1038/nature06514
Nature

PublishingGroup
©2008

Page 7

Studies Hybridoma Bank), rabbit anti-bNOS (Sigma-Aldrich), mouse anti-TH
(Novocastro), and mouse anti-Ki67 (Dako).
Westernblotanalysis.AP0retinawasdissectedandhomogenizedinhypertonic
sucrose lysis buffer (0.32M sucrose, 1mM NaHCO3, 1mM MgCl2and 0.5mM
CaCl2) containing protease inhibitors with a glass dounce homogenizer. Total
protein (40mg) was loaded onto 6% acrylamide gels, run and transferred.
DSCAM antibodies were used at a concentration of 1:500. The specificity of
the DSCAM antibody in western blotting was established using COS-7 cells
transfected with c-Myc-tagged Dscam cDNA. A single band of approximately
220kD was observed when using an antibody to either c-Myc or DSCAM in the
transfected sample only. Western blots from tissue were performed using the
DSCAM antibody after pre-absorption against mutant tissue to reduce back-
ground staining. As a result, immunoreactivity to truncated protein products
specific to the mutant could have been missed. The serum used in Fig. 1 for
immunocytochemistry was not pre-absorbed.
Cell morphologies. P6 retinas were collected and stained with anti-TH as
described previously. TH-positive amacrine cells that were sufficiently isolated
fromneighbouringcellstoallowtheirprocessestobedefinitivelyidentifiedwere
imagedusinga363objectivelensonaLeicaSP5confocalmicroscope.Thetotal
length of 20 wild-type and 20 Dscam2/2dendrites was traced, and measured
using ImageJ.
DRP and nearest neighbour analysis analysis. DRP and nearest neighbour
analysis (NNA) were performed as described previously23,24. The entire retinas
were imaged and reconstructed for analysis of TH and bNOS amacrine cells.
Four 775-mm fields for each wild type and Dscam2/2retina were imaged and
usedtoanalysestarburstandAIIamacrinecells.Cholinergicamacrinecellswere
imagedintheinnernuclearlayerforanalysis.Independentsamplesfromatleast
three animals, ages P6 and six to tenweeks, were included in each analysis.
TUNEL staining and analysis. TUNEL cell death detection kit was used to
perform TUNEL analysis according to the manufacturer’s instructions for dif-
ficult tissues (Roche). Eyes were enucleated and immediately frozen and cut in
10-mm sections using a cryostat. After finishing the TUNEL reaction, the slides
were washed in PBS for 5min, and immunocytochemistry was performed using
a PAX6 antibody as detailed previously. TUNEL-positive cells were counted
accordingtotheirlocation:retinalganglionlayer(includingtheinnerplexiform
layer), the inner neuroblast layer and the outer neuroblast layer. The inner and
outer neuroblast layers were distinguished by PAX6 immunoreactivity. At least
threeslideswitheightsectionsoneachslideforeachofthreemicewererecorded
at each time point for each genotype.
Mitotic index. The mitotic index in the wild-type and Dscam2/2retina was
determined at three time points, E14, E17 and P1. BrDU injections were admi-
nistered at a dosage of 10 mgkg2120min before tissue collections. Embryo
headsorenucleatedeyeswerefixedovernightin4%paraformaldehydeandwere
imbedded in paraffin blocks. Sections were cut at 5mm and stained with anti-
bodiestoBrDUandKi67afterboilingincitratebufferfor10min.Thenumberof
BrDU-positive/Ki67-positive cells (S-phase) were divided by the number of
Ki67-positive cells (G2/M phase). Cells that did not label for either marker were
alsocountedandnosignificantdifferencewasobservedintheirnumber.Atleast
threeslideswitheightsectionsoneachslideforeachofthreemicewererecorded
at each time point for each genotype.
doi:10.1038/nature06514
Nature

PublishingGroup
©2008