|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online November 21, 2008
doi: 10.1242/10.1242/dev.027912
-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina
1 Department of Molecular and Cellular Biology and Center for Brain Science,
Harvard University, Cambridge, MA 02138, USA.
2 Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern
University, Evanston, IL 60208, USA.
* Author for correspondence (e-mail: sanesj{at}mcb.harvard.edu)
Accepted 24 September 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
(Pcdh-) genes
encode transmembrane proteins with distinct cadherin-related extracellular
domains and a common intracellular domain. Genetic studies have implicated
Pcdh- genes in the regulation of neuronal survival and synapse
formation. Because mice lacking the Pcdh- cluster die perinatally, we
generated conditional mutants to analyze roles of Pcdh- genes in the
development and function of neural circuits. Retina-specific deletion of
Pcdh-s led to accentuation of naturally occurring death of interneurons
and retinal ganglion cells (RGCs) during the first two postnatal weeks.
Nonetheless, many neuronal subtypes formed lamina-specific arbors. Blocking
apoptosis by deletion of the pro-apoptotic gene Bax showed that even
neurons destined to die formed qualitatively and quantitatively appropriate
connections. Moreover, electrophysiological analysis indicated that processing
of visual information was largely normal in the absence of Pcdh- genes.
These results suggest that Pcdh- genes are dispensable for elaboration
of specific connections in retina, but play a primary role in sculpting
neuronal populations to appropriate sizes or proportions during the period of
naturally occurring cell death.
Key words: Apoptosis, Interneuron, Laminar specificity, Receptive field, Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
, -β and
-) arrayed in tandem on a single chromosome
(Kohmura et al., 1998
;
Obata et al., 1998;
Wu and Maniatis, 1999;
Wu and Maniatis, 2000).
Second, - and -protocadherins arise through the combination of
distinct extracellular domains with a common cytoplasmic domain, suggesting a
mechanism in which distinct recognition events promote a common cellular
response. Third, Pcdhs are members of the cadherin superfamily, other members
of which mediate selective intercellular interactions, including synapse
formation (Takeichi, 2007).
Fourth, Pcdhs are expressed predominantly in the nervous system, with
individual family members expressed in combinatorial patterns
(Esumi et al., 2005;
Frank et al., 2005;
Kohmura et al., 1998;
Zou et al., 2007). Fifth, Pcdh
proteins are associated at least in part with synaptic membranes
(Kohmura et al., 1998;
Phillips et al., 2003;
Wang et al., 2002). Finally,
clustered protocadherin orthologs are present in vertebrates but not in
invertebrates (Hill et al.,
2001; Hirayama and Yagi,
2006; Noonan et al.,
2004). Together, these features suggest that Pcdhs might underlie
complex patterns of selective neural connectivity in vertebrates.
The first genetic test of this hypothesis led to an unexpected result:
targeted mouse mutants lacking all 22 Pcdh- genes exhibited massive
apoptosis of spinal interneurons during late fetal life and died within hours
of birth (Wang et al., 2002).
Synapse number was also reduced in mutant spinal cords. This was not a trivial
consequence of decreased neuronal number, as synaptic defects and perinatal
lethality persisted when apoptosis was blocked
(Weiner et al., 2005). Thus,
neural connectivity may be defective in the absence of Pcdh- genes, and
apoptosis may be secondary to circuit defects. However, the associated
lethality and the complexity of spinal circuitry have made it difficult to
test these possibilities. In addition, it remains unknown whether Pcdh-
genes are required for neuronal survival and synaptogenesis in other regions
of the nervous system.
To address these issues, we generated conditional alleles of the
Pcdh- cluster, restricting inactivation to defined neuronal populations
and bypassing neonatal lethality. Here, we focus on the retina, which has
several advantages, including a stereotyped structure, markers for many
neuronal and synaptic subtypes, and a clear understanding of the tissue's
function (Masland, 2001;
Wässle, 2004). We used
Cre recombinase to delete Pcdh- genes from retinal neurons and glia,
and assessed the consequences for neuronal structure and function.
Surprisingly, lamina-specific arbors and complex functional circuits formed in
the absence of Pcdh- genes, suggesting that these genes play limited
roles in synaptic specificity. By contrast, loss of Pcdh- genes
accentuated naturally occurring death of multiple retinal cell types. These
results suggest a primary role for Pcdh- genes in neuronal population
matching during development.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
fusg and Pcdh-
del mutants have been described previously
(Wang et al., 2002). Mice in
which regulatory elements from the Chx10 gene drive expression of a
Cre-GFP fusion protein linked by an internal ribosome entry site (IRES) to
placental alkaline phosphatase (Chx10-Cre)
(Rowan and Cepko, 2004) were
provided by Constance Cepko (Harvard). Mice in which a short enhancer fragment
from the Pax6 gene drive expression of a Cre-IRES-GFP cassette
(Pax-Cre)
(Marquardt et al., 2001) were
provided by Peter Gruss (Göttingen, Germany). Mice in which regulatory
elements from the β-actin gene drive expression of Cre
(Actin-Cre) (Lewandoski et al.,
1997) were provided by Gail Martin (UCSF). Bax mutants
(Knudson et al., 1995) and
Z/EG reporter mice (Novak et al.,
2000) were obtained from Jackson Laboratories.
The Pcdh- fcon3 targeting vector was
modified from the Pcdh- fusg vector shown
in Fig. 2B of Wang et al.
(Wang et al., 2002) by
inserting a loxP sequence into an NheI site upstream of the final
coding exon. The Pcdh- fdel allele was
generated by re-targeting the ES cells used to generate Pcdh-
fusg with the vector that had been used to generate
Pcdh- del. This vector inserted a loxP
sequence directly upstream of variable exon A1. Homologous recombinants and
germ line chimeras were generated by standard methods. Mice were maintained on
a C57/B6J background.
Histology
Mice were euthanized with intraperitoneal injection of Nembutal and eye
cups were fixed in 4% paraformaldehyde. Tissue was cryoprotected in sucrose,
frozen and sectioned at 20 µm in a cryostat. Slides were incubated
successively with blocking solution, primary antibodies (12-16 hours at
4°C) and Alexa Fluor-conjugated secondary antibodies (Invitrogen; 3 hours
at room temperature). Primary antibodies were: anti-GFP (Aves and Chemicon);
anti-calbindin (Swant); anti-choline acetyltransferase (Chemicon);
anti-protein kinase C (AbCam); anti-neurokinin receptor 3 (Calbiochem);
anti-synaptotagmin II (Zebrafish International Resource Center); anti-disabled
1 (a gift from T. Curran); anti-G13 (Santa Cruz); anti-Bassoon
(Stressgen); anti-synaptophysin (Zymed); anti-Chx10 (Exalpha Biologicals);
anti-Sox9 (Chemicon); anti-glutamine synthetase (BD Biosciences); anti-cleaved
caspase 3 (Cell Signaling Technology); anti-Brn3a (Chemicon); anti-VGlut3
(Chemicon); anti-syntaxin (Sigma); anti-Thy1.2 (BD Pharmingen); anti-GlyT1
(Santa Cruz); and anti-tyrosine hydroxylase (Chemicon). Peanut agglutinin was
from Invitrogen. Nuclei were labeled with DAPI, Po-pro1 or NeuroTrace Nissl
435/455 (Invitrogen).
For measurements of retinal layer thickness and cell number, areas were
chosen at equivalent retinal eccentricities from the optic nerve head or ora
serrata. Layer thickness was measured on single optical sections, adjacent to
the optic nerve head. Two to four areas were measured from each retina and two
sets of perpendicular measurements were made per area. Both
Chx10-Cre;Pcdh- fcon3/+ and
Pcdh- fcon3/+ littermates were used as
controls for Chx10-Cre;Pcdh- fcon3/fcon3
mutants, and similarly for Pcdh- fdel.
Immunolabeled cells were quantified from 0.13 mm2 (calbindin, ChAT,
Brn3a and Pax-GFP), 0.05 mm2 (Chx10), 0.02 mm2
(Sox9) or 1280 µm2 (photoreceptors) optical sections. Apoptotic
cells were counted on sections spanning the optic nerve head to the ora
serrata. Cells were classified as apoptotic if cleaved caspase 3
immunoreactivity partially or completely surrounded a nucleus. Means were
compared using ANOVA, Student's t-test on condition of equivalent
variances determined by F-test or with Mann-Whitney non-parametric test.
In situ hybridization of retinal sections was performed as described
previously (Wang et al.,
2002).
Retinas were dissociated with papain by a modification of the protocol
described by Meyer-Franke et al.
(Meyer-Franke et al., 1995).
Dissociated cells were plated onto poly-D-lysine coated eight-well Permanox
chamber slides (Nunc), then fixed with 4% paraformaldehyde/4% sucrose for 15
minutes, and immunostained. RGCs were enriched with CD90 magnetic Microbeads
(Miltenyi-Biotec).
Electrophysiology
Dark-adapted retinas were isolated under an infrared microscope into
Ringer's solution at room temperature. A piece of retina, 
3-4 mm in
diameter, was placed with RGCs facing down on a 61-electrode array superfused
with Ringer's (Kim et al.,
2008). Extracellular action potentials were recorded and single
units identified by spike-sorting methods as described previously
(Meister et al., 1994). White
light stimuli were delivered from a computer-driven display projected on the
retina.
To map spatio-temporal receptive fields, we projected gratings of adjacent
thin bars (8.3 or 16.6 µm width). Each bar flickered black or white
according to a pseudo-random binary sequence (16.6 millisecond frame
duration). For any given RGC, we computed the spike-triggered average of the
flickering bar stimulus (Kim et al.,
2008) using
where s(x,t) is the stimulus intensity at location x and time t, with the time-averaged intensity subtracted, and the neuron fired a total of n spikes at times {tj}. Examples are shown in Fig. S2 in the supplementary material.
We then approximated this function as the product of a spatial receptive
field b(x) and a temporal integration function
a(t):
 | (Eq.2) |
These are the spatial and temporal components analyzed in
Fig. 9. For analysis of
response threshold and gain, we fitted the time-dependent RGC firing rate
r(t) by a linear-nonlinear model
(Chichilnisky,
2001):
where Z is chosen so that y(t) has unit
variance, and
is a rectifying function with threshold 
and gain
G.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
genes in
retina. We used targeted mutant mice in which GFP is fused to the shared C
terminus, tagging all 22 Pcdh- isoforms (Pcdh-
fusg) (Wang et al.,
2002). Homozygous Pcdh-
fusg/fusg mutants are viable and fertile, and show none of
the defects documented previously in Pcdh- mutants
(Wang et al., 2002;
Weiner et al., 2005). We
therefore believe that GFP is a neutral reporter of endogenous Pcdh-
localization.
The retina consists of three cellular layers separated by two synaptic or
`plexiform' layers (Fig. 1A).
The cellular layers are the outer nuclear layer (ONL), containing
photoreceptors; the inner nuclear layer (INL), containing interneurons
(horizontal, bipolar and amacrine cells) and Müller glia; and the
ganglion cell layer (GCL), containing RGCs and displaced amacrine cells. The
outer plexiform layer (OPL) contains synapses of photoreceptors onto
horizontal and bipolar cells, and the inner plexiform layer (IPL) contains
synapses of bipolar and amacrine cells onto RGCs. As judged by localization of
GFP in Pcdh- fusg mice, Pcdh-s are
present in all five retinal layers (Fig.
1A). In the ONL, Pcdh-s are present in outer segments and
around photoreceptor somata (Fig.
1B); in the INL and GCL, Pcdh-s outline neuronal somata
(Fig. 1C,D). Pcdh-
levels are highest in the most membrane-rich layers: IPL, OPL and the optic
fiber layers that carry RGC axons to the brain
(Fig. 1A',D). In situ
hybridization confirmed Pcdh- expression by cells in the INL and GCL,
though this method did not reliably detect Pcdh- RNA in
photoreceptors (Fig. 1E).
To determine which retinal cell types express Pcdh- genes,
we dissociated Pcdh- fusg retinas and
immunostained cells with antibodies to cell-type-specific markers
(Haverkamp and Wässle,
2000; Wahlin et al.,
2004; Zhang et al.,
2004). This method circumvented the difficulty of determining
which of the cells abutting Pcdh--rich membranes are themselves
Pcdh- positive. Markers included Brn3a and Thy1 for RGCs, syntaxin 1
for amacrine cells, Chx10 for bipolar cells, calbindin for horizontal cells,
recoverin for photoreceptors and glial fibrillary acidic protein, Sox9 and
glutamine synthetase for Müller glia. All six cell types were
Pcdh- positive (Fig.
1F-K; data not shown). Thus, Pcdh-s are expressed in all
cell types of the neural retina.
We asked whether Pcdh- genes are present in the retina during early
postnatal life, when neural circuits form. At postnatal day (P) 0, the retina
contains ganglion cell and neuroblast layers, separated by a nascent IPL. All
RGCs have been born by this time, while neurogenesis and migration of newborn
interneurons and photoreceptors continue in the neuroblast layer. At this
time, Pcdh- is present on cells in the neuroblast layer, in the IPL,
and on RGC axons (Fig. 2A). At
P3, Pcdh- is apparent in the layer of horizontal cells that prefigures
the OPL (Fig. 2B). By P7,
Pcdh- appears in the OPL, as it divides the neuroblast layer into INL
and ONL (Fig. 2C). By P14, the
adult pattern described above is established
(Fig. 2D).
|
|
proteins in the retina proteins, we
focused on the OPL, because its synapses are larger than those in the IPL. We
labeled photoreceptor terminals with antibodies to bassoon, which is present
in both rod terminals (spherules) and cone terminals (pedicles), and labeled
spherules and pedicles selectively with anti-PSD-95 and peanut agglutinin,
respectively (Blanks et al.,
1987; Koulen et al.,
1998; tom Dieck and
Brandstätter, 2006). Pcdh- was associated with both
spherules and pedicles (Fig.
3A-C). We labeled bipolar cell dendrites with antibodies to
protein kinase C and neurokinin receptor 3, which mark rod and cone
bipolars, respectively, and to G protein 13 (G13), which is
present in subsets of both rod and cone bipolars
(Haverkamp et al., 2003;
Huang et al., 2003).
Pcdh- was associated with both rod and cone bipolar dendrites
(Fig. 3D; data not shown).
Thus, Pcdh- genes were present in pre- and postsynaptic compartments of
rod and cone synapses. By contrast, although horizontal cell processes labeled
with anti-calbindin (Sharma et al.,
2003) were Pcdh- positive, little Pcdh- was present
in their synaptic varicosities (Fig.
3E).
Pcdh- was also present throughout the IPL. GFP-positive puncta often
overlapped with bassoon-positive presynaptic ribbons in bipolar cells,
glutamate decarboxylase- and GlyT1-positive terminals of inhibitory amacrines,
and PSD-95-positive postsynaptic membranes of excitatory synapses
(Fig. 3F; data not shown).
Taken together, these results suggest that Pcdh-s are present at many
synapses in the retina, although they are not confined to synapses.
Inactivation of Pcdh-s in the retina leads to neuronal and synaptic loss
Pcdh- null and hypomorphic mice die shortly after birth
(Wang et al., 2002;
Weiner et al., 2005). We
examined retinas of Pcdh- null mutants at late embryonic
stages [embryonic day (E) 17-18; birth is at E19] but found no obvious defects
in retinal structure (see below). However, as the development of retinal
circuitry occurs largely during postnatal life, roles of Pcdh- in
circuit formation and function could not be studied in these mutants. We
therefore generated two conditional inactivation alleles to bypass neonatal
lethality (Fig. 4A). In
Pcdh- fdel, loxP sites flank the entire
Pcdh- locus, such that Cre-mediated recombination generates a
null allele. In Pcdh- fcon3, the
C-terminal exon shared by all isoforms is flanked by loxP sites, such that Cre
truncates all Pcdh- genes. In both alleles, GFP is fused to this
C-terminal exon, allowing us to use loss of GFP as an indicator of
Cre-mediated Pcdh- excision. The Pcdh--GFP fusion protein was
identical to that in the Pcdh- fusg allele
described above.
|
fdel and Pcdh-
fcon3 alleles in the germline by mating them to transgenic
mice in which Cre is expressed ubiquitously (Actin-Cre)
(Lewandoski et al., 1997).
Homozygotes generated from these animals died at birth and exhibited spinal
cord phenotypes similar to those described previously in null mutants
(Wang et al., 2002),
indicating that recombination inactivates the Pcdh- gene (data
not shown). Western blotting reported by Prasad et al.
(Prasad et al., 2008) failed
to detect Pcdh- protein in Actin-Cre;Pcdh-
fcon3/fcon3 mice, indicating that this allele is
effectively a protein null. The truncation in Pcdh-
fcon3 is more extensive than the hypomorphic allele
described previously (Pcdh- tr), in which
Pcdh- levels were decreased several-fold
(Weiner et al., 2005). We
speculate that increased truncation of Pcdh-
fcon3 and lack of a polyadenylation signal led to greater
destabilization of Pcdh- protein and mRNA, respectively.
To selectively inactivate Pcdh-s in retina, we crossed
Pcdh- fdel and Pcdh-
fcon3 mutants with mice in which a GFP-Cre recombinase
fusion protein is expressed under the control of regulatory elements from the
Chx10 gene (Chx10-Cre)
(Rowan and Cepko, 2004). These
elements drive expression of GFP-Cre transiently in embryonic retinal
progenitors and postnatally in bipolar cells. To assay recombination in
retinas of Chx10-Cre mice, we crossed them to a reporter line in
which β-galactosidase (lacZ) and GFP label non-recombined and
recombined cells, respectively (Z/EG)
(Novak et al., 2000).
Recombination was extensive (>90%) in the INL and ONL, but occasional
columns of cells were spared (see Fig. S1A in the supplementary material). By
contrast, approximately half of the cells in the GCL were GFP-negative and
lacZ positive (see Fig. S1B in the supplementary material). This
pattern may reflect the fact that many RGCs are born by E12
(Farah and Easter, 2005),
before Cre accumulates in progenitors. We then used loss of GFP to
assay Chx10-Cre-mediated loss of Pcdh--GFP from the
Pcdh- alleles. This method did not allow us to assess excision
in bipolars, in which GFP was expressed from the Chx10 transgene (see
Materials and methods). Nonetheless, Chx10-Cre excised Pcdh-
fdel, Pcdh- fcon3 and Z/EG
genes in similar patterns and to a similar extent (see Fig. S1C-E in the
supplementary material). The efficacious excision of the Pcdh-
fdel allele is surprising given the length of the floxed
segment.
Chx10-Cre;Pcdh- fdel/fdel mice are
healthy and outwardly normal. We first examined these mutants at P18, by which
time the retinal architecture is well developed. Labeling of nuclear layers
with DAPI and plexiform layers with antibodies to the synaptic vesicle protein
synaptophysin revealed that mutant retinas were properly laminated
(Fig. 4B-G). However, mutant
retinas were 25% thinner than those of wild-type mice or heterozygote
littermates. The difference resulted from a selective reduction of 40% in
the thickness of the INL and the IPL (Fig.
4H). Thus, Pcdh-s are required for development or
maintenance of retinal interneurons and the layer in which they form synapses.
The INL and IPL were thinned to the same extent in
Chx10-Cre;Pcdh- fcon3/fcon3 and
Chx10-Cre;Pcdh- fdel/fdel mice
(Fig. 4C,D,F,G), consistent
with the idea that Pcdh- fcon3 is
functionally a null. In subsequent studies, we used the two alleles
interchangeably, but most of the results reported here are from
Pcdh- fcon3 mice.
|
-Cre, in which Cre is expressed
under the control of retina-specific sequences from the Pax6 gene
(Marquardt et al., 2001). The
Pax6-Cre transgene drives expression of Cre
transiently in embryonic retinal progenitors, leading to essentially complete
(>99%) inactivation in peripheral retina; a sector in central retina is
spared, as described below. Postnatally, Pax6-Cre is
expressed in a subset of amacrines, which can be identified by a GFP reporter
within the transgene.
We used cell type-specific markers (see above) to quantify cell loss from
peripheral regions of Pax6-Cre;Pcdh-
fcon3/fcon3 retinas at P18. Numbers of bipolar, amacrine
and retinal ganglion cells were reduced by 45-65%
(Fig. 4I). Müller glia
were also decreased, but only by 20%. By contrast, numbers of horizontal
cells and photoreceptors differed little between mutants and controls
(Fig. 4I). Together, these
results demonstrate that Pcdh- genes are essential for the survival of
many but not all retinal cell types.
Increased postnatal apoptosis in the absence of Pcdh- genes
We next asked when retinal defects arise in Pcdh- mutants,
and whether they are progressive. We detected no differences in laminar
arrangement or thickness between mutant and control retinas perinatally
(E17.5-P3) (Fig. 5A,B,G,H and
data not shown). By P7, however, shortly after the ONL and INL form, mutant
retinas were thinner than those of controls
(Fig. 5C,D). We used
Chx10-Cre; Pcdh- fcon3 mice for
quantification of these defects. Both layers were 40% thinner in mutants
than in controls by P14, then changed little over the following months
(Fig. 5E-I). Thus, the
difference between mutant and control retinas appears during the first
postnatal week, is maximal by the end of the second postnatal week, and
neither abates nor worsens substantially thereafter.
A process of naturally occurring programmed cell death eliminates many
retinal neurons during the first two postnatal weeks
(Pequignot et al., 2003;
Young, 1984). Apoptosis
followed a similar time course in Pcdh- deficient retinas, but levels
were significantly higher in mutants than in controls
(Fig. 5J-L). Although increased
apoptosis was seen in both neuroblast and ganglion cell layers at P0, it was
confined to the INL at P7 (Fig.
5M). This pattern is consistent with the finding that naturally
occurring cell death in the GCL is complete several days before that in the
INL (Farah and Easter, 2005;
Pequignot et al., 2003;
Young, 1984). These results
suggest that Pcdh- genes regulate neuronal survival during the period
of naturally occurring programmed cell death.
Cell autonomy of Pcdh--dependent cell survival in retina
Are Pcdh- genes required for cell survival in cells that express
them, in neighboring cells, or in both? As a first step to distinguish between
these possibilities, we capitalized on the recombination pattern in
Pax6-Cre transgenic mice. As noted above, Cre is
expressed in all progenitors in peripheral retina as well as in a subset of
amacrine cells marked by the GFP in the transgene. In a large dorsoventrally
oriented swath of central retina, however, Cre is expressed in amacrines but
not progenitors (Marquardt et al.,
2001; Stacy et al.,
2005). Thus, in central retina of
Pax6-Cre;Pcdh- fcon3/fcon3
mice, GFP-positive amacrine cells lack Pcdh- genes, whereas all other
cells, including Müller glia, are Pcdh- positive
(Fig. 6A-E).
We asked whether Pax6-positive (that is, GFP-positive) amacrines
were lost from central retina of
Pax6-Cre;Pcdh- fcon3/fcon3
mice, despite being surrounded by Pcdh--positive cells. The number of
Pax6-amacrines in central retina was 58% lower in mutants than in
controls (Fig. 6B,C,F). Because
multiple, neighboring amacrines are Pcdh- deficient in the central
region, this result does not demonstrate cell autonomy sensu strictu, but does
indicate that loss of Pcdh- genes from a single cell type impairs its
survival, even when the majority of its synaptic inputs (bipolar cells) and
targets (RGCs) are wild type. This result also rules out the possibility that
neuronal apoptosis in the absence of Pcdh- is secondary to a defect in
surrounding glial cells. Furthermore, loss of Pax6-positive amacrines
is equivalent in peripheral and central retina
(Fig. 6F), indicating that
Pcdh--negative cells are not protected from apoptosis when surrounded
by Pcdh--positive cells.
|
s from Pax6-amacrines was
detrimental to survival of neighboring cells
(Fig. 6F). Loss of
Pcdh-s from the Pax6-positive amacrines had no detectable effect
on the survival of bipolar cells, horizontal cells or Müller glia.
Likewise, survival of a distinct, intermingled subpopulation of amacrines -
the cholinergic starburst cells - was unaffected in central retina. By
contrast, we detected a small (15%) but significant loss of
Brn3a-positive RGCs from central retina of
Pax6-Cre;Pcdh fcon3/fcon3
mice. We do not know whether this loss reflects absence of Pcdh-s per
se, or death of amacrines, which regulate at least some aspects of RGC
development (Goldberg et al.,
2002).
Pcdh- genes are dispensable for laminar targeting of retinal neurons
Although the width of the IPL is dramatically reduced in
Pcdh--deficient retina, it nonetheless contains synapses, as judged by
the presence of pre- and postsynaptic markers such as synaptophysin and
bassoon (Fig. 4E-G;
Fig. 8A,B). Are they
appropriate synapses? The retina is well-suited to test specificity, because
discrete subsets of bipolar and amacrine cells arborize and synapse in just
one or a few of at least 10 closely spaced parallel sublaminae within the IPL
(Pang et al., 2002;
Roska and Werblin, 2001;
Wässle, 2004).
We used markers of 10 lamina-specified amacrine and bipolar subtypes to
assess lamina-specific arborization and connectivity in the absence of
Pcdh-s. We follow a scheme in which five sublaminae of equal width are
numbered, from the border of the INL (S1) to the border of the ganglion cell
layer (Ghosh et al., 2004;
Yamagata and Sanes, 2008).
Populations examined were starburst amacrines, labeled by choline
acetyltransferase; glutamatergic amacrines (anti-vGlut3); GABAergic amacrines
(anti-GAD65/67); dopaminergic amacrines (anti-tyrosine hydroxylase); type AII
amacrines (anti-disabled); calbindin-positive amacrines and RGCs; OFF bipolar
cells (anti-synaptotagmin 2); ON bipolar cells (anti-G13); OFF bipolars
(anti-NK3R); and rod bipolars (anti-protein kinase C)
(Fig. 7E). In all 10 cases,
processes were arrayed in appropriate sublaminae in mutant retinas at P18,
although disruptions or gaps were sometimes present
(Fig. 7A-D; data not shown). We
also observed proper laminar targeting of amacrine subsets at P7, and of
bipolar subsets at P14, in each case soon after these synapses formed in
controls (data not shown).
|
-deficient neurons destined to dies are dispensable
for formation of neural circuits in retina. Alternatively, however, some INL
interneurons might fail to target appropriate sublaminae and then die. In this
case, neuronal apoptosis in Pcdh- mutants might be secondary to circuit
defects. To test this possibility, we blocked apoptosis in
Pcdh- fcon3/fcon3 mice by deleting the
pro-apoptotic gene, Bax. Naturally occurring death in many regions of
the nervous system, including retina, is dramatically reduced in
Bax-/- mice (Mosinger
Ogilvie et al., 1998;
Pequignot et al., 2003;
White et al., 1998), and
deletion of Bax preserves spinal interneurons that would otherwise
die in Pcdh- null spinal cord
(Weiner et al., 2005).
Likewise, Bax deletion rescued neurons destined to die in
Pcdh- mutant retina: the thickness of the INL and GCL and the
number of Chx10-positive bipolar cells were indistinguishable in
Chx10-Cre;Pcdh-
+/fcon3;Bax-/-and
Chx10-Cre;Pcdh-
fcon3/fcon3;Bax-/- retinas
(Fig. 8A-D; data not
shown).
Deletion of Bax also resulted in expansion of the IPL. The IPL in
Chx10-Cre;Pcdh-
fcon3/fcon3;Bax-/- mice was thicker than that
in Pcdh- mutants and indistinguishable from that in
Pcdh--positive Bax-/- mutants
(Fig. 8A-D). The density of
synapses in the IPL and OPL, as judged by staining for PNA or bassoon, did not
differ significantly between Pcdh--positive and Pcdh--negative
Bax-/- retinas (Fig.
8E-L; see Fig. S2 in the supplementary material). Thus, loss of
Pcdh- had little effect on synapse number in the IPL when apoptosis was
prevented. This result is consistent with the idea that much of the synapse
loss in the IPL of Pcdh--deficient retina is a consequence of
decreased neuron number.
To assess the laminar targeting of interneurons that would have died in the
presence of Bax, we stained Chx10-Cre;Pcdh-
fcon3/fcon3; Bax-/- retinae with the panel of
markers listed above. In all cases, targeting of processes to appropriate
laminae was as specific in double mutants as in Pcdh- single
mutants (Fig. 8M,N; data not
shown). Moreover, thinning and disruptions of layers observed in
Pcdh- single mutants were rescued in Pcdh-
-/-;Bax-/- double mutants (compare
Fig. 7B,D with
Fig. 8M,N). We therefore
conclude that the gaps observed in Pcdh- deficient retina are secondary
to the loss of cells rather than a manifestation of improper laminar
targeting.
Functional visual circuits form in the absence of Pcdh-s
To test whether circuits that form in Pcdh--deficient retina are
functional, we recorded light responses from RGCs. These cells integrate
signals from amacrine and bipolar interneurons and send the resulting spike
trains to the brain. RGCs differ in their responses to visual stimuli,
depending on the synaptic inputs they receive. Thus, ON RGCs, which respond
primarily to light onset, receive synapses from ON bipolar cells in the inner
half of the IPL (nearest the GCL); OFF RGCs receive synapses from OFF bipolars
in the outer IPL; and ON-OFF RGCs receive both types of synapses. Further
specializations, such as responses that are transient, sustained or selective
for moving objects, result from innervation by specific subsets of bipolar and
amacrine cells (Masland, 2001;
Wässle, 2004).
Accordingly, the presence of diverse, specific responses from RGCs is a
sensitive indicator of precisely patterned synaptic connectivity. We therefore
monitored action potentials simultaneously from large populations of RGCs in
control and Pcdh- mutant retinas, using a multi-electrode array
(Meister et al., 1994).
Results from Chx10-Cre;Pcdh- fcon3/fcon3
retinas and peripheral regions of
Pax6-Cre;Pcdh- fcon3/fcon3
retinas were similar, so they are combined here. We did not use
Pcdh-;Bax double mutants for this study, because
visual responses are compromised in Bax single mutants
(Pequignot et al., 2003).
|
|
mutant and control retinas showed a similar variety of
responses to small flashing spots, including sustained and transient ON, OFF
and ON-OFF responses (Fig. 9A).
Proportions of ON- and OFF-dominated responses were identical in mutants and
controls (Fig. 9C). Mutant RGCs
responded to very dim flashes, which excite only rods, and also to bright
flashes, which predominantly excite cones (data not shown), indicating that
both rod- and cone-activated pathways were functional. We also probed the
retina with moving bars and gratings to elicit direction-selective responses,
which are known to depend on specific patterns of connectivity in the IPL
(Masland, 2001). Both ON and
ON-OFF direction-selective cells were encountered in mutant retinas
(Fig. 9B; data not shown). Some
control and mutant RGCs had receptive field surrounds, where light has the
opposite effect of the center (see Fig. S3 in the supplementary material),
indicating that lateral inhibitory connections are functional in these
circuits.
|
;
Meister et al., 1994). This
measures spatio-temporal receptive fields, revealing how RGCs respond to light
intensity at different points on the retina and at different times in the past
(see Fig. S3 in the supplementary material). The size of the receptive field
center varied greatly among RGCs, but the distribution was similar in control
and mutant retinas (Fig. 9D).
However, the time course of the light response was significantly slower in
mutant retina (Fig. 9E).
Moreover, mutant RGCs fired at much lower rates in response to flicker stimuli
(Fig. 9F). In principle, this
could result from an elevated response threshold; alternatively, the gain of
the response might be lower once the threshold is crossed. Based on fitting
with a linear-nonlinear model
(Chichilnisky, 2001), we found
that the threshold is unaltered, but the gain is reduced in mutants
(Fig. 9G-H). |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Hamada and Yagi,
2001; Hirayama and Yagi,
2006; Kohmura et al.,
1998; Morishita and Yagi,
2007; Serafini,
1999; Shapiro and Colman,
1999; Washbourne et al.,
2004; Wu and Maniatis,
1999; Yagi and Takeichi,
2000). Several observations that led to this notion are summarized
in the Introduction. Moreover, Hasegawa et al.
(Hasegawa et al., 2008)
recently showed that olfactory axons bearing a single type of odorant receptor
fail to coalesce properly onto glomeruli in olfactory bulbs of mice lacking
Pcdh- genes.
These considerations, coupled with the finding that most retinal cells
express Pcdh- genes, led us to expect that retinal circuitry might be
grossly defective in their absence. Surprisingly, it was not. Synaptic
specializations were present in the OPL and IPL of Pcdh-
mutants, and the light-responsiveness of RGCs indicates that synapses in both
laminae were functional. Moreover, synapses in the IPL were sublamina specific
as judged by distribution of arbors. This distribution provides a stringent
test of targeting, in that 10 or more IPL sublaminae are separated by only a
few tens of micrometers (Roska and
Werblin, 2001; Wässle,
2004).
The loss of neurons in Pcdh--deficient retinas potentially
complicates this interpretation: neurons making improper arbors or connections
could be selectively eliminated, so only neurons that wired up properly would
be retained. The ability to block apoptosis in Pcdh- mutant retinas by
deleting the Bax gene allowed us to test this possibility.
Lamina-specific targeting was, if anything, more precise in the absence of
Bax than in its presence, in that disruptions and irregularities seen
in Pcdh- -/- laminae were absent in double
mutants. Therefore, IPL disruptions observed in Pcdh-
-/- retinas presumably reflected loss of cells rather than
mistargeting of neurites. Moreover, with apoptosis prevented by Bax
deletion, loss of Pcdh had no detectable effect on the number of synapses in
either the IPL or the OPL.
The ability of mutant retinas to process visual information was also
remarkably preserved. RGCs exhibited a wide range of complex responses, and
their receptive field sizes were normal. Because the spatial extent of RGC
receptive field centers are largely determined by their dendritic fields,
which collect input from bipolar cells
(Wässle, 2004), this
result suggests that mutant RGC arbors are normal in size. As bipolar cells
provide the main excitation to RGCs, their decreased number could account for
the lower firing rate and response gain in mutant RGCs. Most likely to result
from lack of Pcdh- rather than from decreased cell number are the
defects in response dynamics, which are controlled by synaptic properties in
the OPL (DeVries, 2000) and IPL
(Nirenberg and Meister, 1997).
One way to distinguish which defects are due to loss of interneurons and
altered ratios of cell types and which to loss of Pcdh- genes per se
will be to record from retinas lacking both Pcdh- genes and Bax. This
work is under way, but is complicated by the fact that naturally occurring
cell death is also blocked by Bax deletion, and that visual responses
are compromised in these mutants
(Pequignot et al., 2003).
Synaptic circuitry and neuronal survival
Patterns of apoptosis in Pcdh--deficient retina are similar to those
in spinal cord (Wang et al.,
2002; Weiner et al.,
2005) (see also Prasad et al.,
2008) in several respects. First, approximately half of the
interneurons in each region are lost in the absence of Pcdh- genes.
Second, some interneuronal subtypes and primary sensory neurons (dorsal root
ganglion cells and photoreceptors) are spared in both regions, even though
they express Pcdh- genes. Third, the loss of neurons in
Pcdh- mutants occurs during the period of naturally occurring cell
death. One apparent difference is that the output neurons of the spinal cord,
motoneurons, are spared in Pcdh--deficient mice, whereas those of
retina, RGCs, are affected. However, at least some apoptosis of RGCs is
cell-nonautonomous, reflecting either loss of Pcdh- from presynaptic
cells or loss of input cells themselves. It is possible that in the mutants
analyzed to date, motoneurons retain a larger fraction of their inputs than do
RGCs, and that this contributes to their survival.
Retina and spinal cord phenotypes are also similar in that loss of
Pcdh- genes leads to decreased numbers of synapses in both tissues. In
spinal cord, synapse loss does not result simply from neuron loss, as shown by
analysis of Pcdh--deficient mice in which apoptosis was blocked:
neuronal number was normal in these animals, but synapse number was still
reduced (Weiner et al., 2005).
This result is consistent with the idea that failure of synapse formation or
function impairs neuronal survival (see also
Prasad et al., 2008). In fact,
complete blockade of synaptic function in embryonic brain leads to increased
apoptosis (Verhage et al.,
2000). By contrast, deletion of Pcdh- in a
Bax-/- background does not decrease synapse density in
retina. In addition, given the electrophysiological evidence for maintained
synaptic function, it seems unlikely that any synaptic defect is sufficient in
magnitude to explain the massive apoptosis we observe. Likewise, synaptic
patterns in the IPL of Pcdh- mutants are at least as well preserved in
the absence of Bax as in its presence, ruling out the possibility that
apoptosis reflects selective elimination of inappropriate synapses.
Thus, synapses can be lost in the absence of neuronal loss in the spinal
cord, and neurons can be lost in the absence of major synaptic defects in
retina. These results suggest that Pcdh- regulates neuronal survival
and synaptic maturation by distinct mechanisms, and that effects on these two
processes differ in severity among brain regions. The combinatorial diversity
provided by the Pcdh- genes may therefore be useful for selectively
controlling the size of diverse neuronal populations.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/4141/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Benson, D. L., Colman, D. R. and Huntley, G. W.
(2001). Molecules, maps and synapse specificity. Nat.
Rev. Neurosci. 2,899
-909.[CrossRef][Medline]
Blanks, J. C., Hageman, G. S. and Johnson, L. V.
(1987). Appearance of PNA-binding cells within the outer nuclear
layer coinciding with photoreceptor degeneration in rd mice. Prog.
Clin. Biol. Res. 247,229
-242.[Medline]
Chichilnisky, E. J. (2001). A simple white
noise analysis of neuronal light responses. Network
12,199
-213.[Medline]
DeVries, S. H. (2000). Bipolar cells use
kainate and AMPA receptors to filter visual information into separate
channels. Neuron 28,847
-856.[CrossRef][Medline]
Esumi, S., Kakazu, N., Taguchi, Y., Hirayama, T., Sasaki, A.,
Hirabayashi, T., Koide, T., Kitsukawa, T., Hamada, S. and Yagi, T.
(2005). Monoallelic yet combinatorial expression of variable
exons of the protocadherin-alpha gene cluster in single neurons.
Nat. Genet. 37,171
-176.[CrossRef][Medline]
Farah, M. H. and Easter, S. S., Jr (2005). Cell
birth and death in the mouse retinal ganglion cell layer. J. Comp.
Neurol. 489,120
-134.[CrossRef][Medline]
Frank, M., Ebert, M., Shan, W., Phillips, G. R., Arndt, K.,
Colman, D. R. and Kemler, R. (2005). Differential expression
of individual gamma-protocadherins during mouse brain development.
Mol. Cell. Neurosci. 29,603
-616.[CrossRef][Medline]
Ghosh, K. K., Bujan, S., Haverkamp, S., Feigenspan, A. and
Wässle, H. (2004). Types of bipolar cells in the mouse
retina. J. Comp. Neurol.
469, 70-82.[CrossRef][Medline]
Goldberg, J. L., Klassen, M. P., Hua, Y. and Barres, B. A.
(2002). Amacrine-signaled loss of intrinsic axon growth ability
by retinal ganglion cells. Science
296,1860
-1864.
Hamada, S. and Yagi, T. (2001). The
cadherin-related neuronal receptor family: a novel diversified cadherin family
at the synapse. Neurosci. Res.
41,207
-215.[CrossRef][Medline]
Hasegawa, S., Hamada, S., Kumode, Y., Esumi, S., Katori, S.,
Fukuda, E., Uchiyama, Y., Hirabayashi, T., Mombaerts, P. and Yagi, T.
(2008). The protocadherin-alpha family is involved in axonal
coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb
in mouse. Mol. Cell. Neurosci.
38, 66-79.[CrossRef][Medline]
Haverkamp, S. and Wässle, H. (2000).
Immunocytochemical analysis of the mouse retina. J. Comp.
Neurol. 424,1
-23.[CrossRef][Medline]
Haverkamp, S., Ghosh, K. K., Hirano, A. A. and Wässle,
H. (2003). Immunocytochemical description of five bipolar
cell types of the mouse retina. J. Comp. Neurol.
455,463
-476.[CrossRef][Medline]
Hill, E., Broadbent, I. D., Chothia, C. and Pettitt, J.
(2001). Cadherin superfamily proteins in Caenorhabditis elegans
and Drosophila melanogaster. J. Mol. Biol.
305,1011
-1024.[CrossRef][Medline]
Hirayama, T. and Yagi, T. (2006). The role and
expression of the protocadherin-alpha clusters in the CNS. Curr.
Opin. Neurobiol. 16,336
-342.[CrossRef][Medline]
Huang, L., Max, M., Margolskee, R. F., Su, H., Masland, R. H.
and Euler, T. (2003). G protein subunit G gamma 13 is
coexpressed with G alpha o, G beta 3, and G beta 4 in retinal ON bipolar
cells. J. Comp. Neurol.
455, 1-10.[CrossRef][Medline]
Kim, I. J., Zhang, Y., Yamagata, M., Meister, M. and Sanes, J.
R. (2008). Molecular identification of a retinal cell type
that responds to upward motion. Nature
452,478
-482.[CrossRef][Medline]
Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A.
and Korsmeyer, S. J. (1995). Bax-deficient mice with lymphoid
hyperplasia and male germ cell death. Science
270, 96-99.
Kohmura, N., Senzaki, K., Hamada, S., Kai, N., Yasuda, R.,
Watanabe, M., Ishii, H., Yasuda, M., Mishina, M. and Yagi, T.
(1998). Diversity revealed by a novel family of cadherins
expressed in neurons at a synaptic complex. Neuron
20,1137
-1151.[CrossRef][Medline]
Koulen, P., Fletcher, E. L., Craven, S. E., Bredt, D. S. and
Wässle, H. (1998). Immunocytochemical localization of
the postsynaptic density protein PSD-95 in the mammalian retina. J.
Neurosci. 18,10136
-10149.
Lewandoski, M., Meyers, E. N. and Martin, G. R.
(1997). Analysis of Fgf8 gene function in vertebrate development.
Cold Spring Harb. Symp. Quant. Biol.
62,159
-168.
Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R.,
Guillemot, F. and Gruss, P. (2001). Pax6 is required for the
multipotent state of retinal progenitor cells. Cell
105, 43-55.[CrossRef][Medline]
Masland, R. H. (2001). The fundamental plan of
the retina. Nat. Neurosci.
4, 877-886.[CrossRef][Medline]
Meister, M., Pine, J. and Baylor, D. A. (1994).
Multi-neuronal signals from the retina: acquisition and analysis.
J. Neurosci. Methods 51,95
-106.[CrossRef][Medline]
Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W. and Barres, B.
A. (1995). Characterization of the signaling interactions
that promote the survival and growth of developing retinal ganglion cells in
culture. Neuron 15,805
-819.[CrossRef][Medline]
Morishita, H. and Yagi, T. (2007).
Protocadherin family: diversity, structure, and function. Curr.
Opin. Cell Biol. 19,584
-592.[CrossRef][Medline]
Mosinger Ogilvie, J., Deckwerth, T. L., Knudson, C. M. and
Korsmeyer, S. J. (1998). Suppression of developmental retinal
cell death but not of photoreceptor degeneration in Bax-deficient mice.
Invest. Ophthalmol. Vis. Sci.
39,1713
-1720.
Nirenberg, S. and Meister, M. (1997). The light
response of retinal ganglion cells is truncated by a displaced amacrine
circuit. Neuron 18,637
-650.[CrossRef][Medline]
Noonan, J. P., Grimwood, J., Schmutz, J., Dickson, M. and Myers,
R. M. (2004). Gene conversion and the evolution of
protocadherin gene cluster diversity. Genome Res.
14,354
-366.
Novak, A., Guo, C., Yang, W., Nagy, A. and Lobe, C. G.
(2000). Z/EG, a double reporter mouse line that expresses
enhanced green fluorescent protein upon Cre-mediated excision.
Genesis 28,147
-155.[CrossRef][Medline]
Obata, S., Sago, H., Mori, N., Davidson, M., St John, T. and
Suzuki, S. T. (1998). A common protocadherin tail: multiple
protocadherins share the same sequence in their cytoplasmic domains and are
expressed in different regions of brain. Cell Adhes.
Commun. 6,323
-333.[Medline]
Pang, J. J., Gao, F. and Wu, S. M. (2002).
Segregation and integration of visual channels: layer-by-layer computation of
ON-OFF signals by amacrine cell dendrites. J.
Neurosci. 22,4693
-4701.
Pequignot, M. O., Provost, A. C., Salle, S., Taupin, P.,
Sainton, K. M., Marchant, D., Martinou, J. C., Ameisen, J. C., Jais, J. P. and
Abitbol, M. (2003). Major role of BAX in apoptosis during
retinal development and in establishment of a functional postnatal retina.
Dev. Dyn. 228,231
-238.[CrossRef][Medline]
Phillips, G. R., Tanaka, H., Frank, M., Elste, A., Fidler, L.,
Benson, D. L. and Colman, D. R. (2003). Gamma-protocadherins
are targeted to subsets of synapses and intracellular organelles in neurons.
J. Neurosci. 23,5096
-5104.
Prasad, T., Wang, X., Gray, P. A. and Weiner, J. A.
(2008). A differential developmental pattern of spinal
interneuron apoptosis during synaptogenesis: insights from genetic analyses of
the protocadherin- gene cluster. Development
135,4153
-4164.
Roska, B. and Werblin, F. (2001). Vertical
interactions across ten parallel, stacked representations in the mammalian
retina. Nature 410,583
-587.[CrossRef][Medline]
Rowan, S. and Cepko, C. L. (2004). Genetic
analysis of the homeodomain transcription factor Chx10 in the retina using a
novel multifunctional BAC transgenic mouse reporter. Dev.
Biol. 271,388
-402.[CrossRef][Medline]
Serafini, T. (1999). Finding a partner in a
crowd: neuronal diversity and synaptogenesis. Cell
98,133
-136.[CrossRef][Medline]
Shapiro, L. and Colman, D. R. (1999). The
diversity of cadherins and implications for a synaptic adhesive code in the
CNS. Neuron 23,427
-430.[CrossRef][Medline]
Sharma, R. K., O'Leary, T. E., Fields, C. M. and Johnson, D.
A. (2003). Development of the outer retina in the mouse.
Brain Res. Dev. Brain Res.
145,93
-105.[Medline]
Stacy, R. C., Demas, J., Burgess, R. W., Sanes, J. R. and Wong,
R. O. (2005). Disruption and recovery of patterned retinal
activity in the absence of acetylcholine. J. Neurosci.
25,9347
-9357.
Takeichi, M. (2007). The cadherin superfamily
in neuronal connections and interactions. Nat. Rev.
Neurosci. 8,11
-20.[CrossRef][Medline]
tom Dieck, S. and Brandstätter, J. H.
(2006). Ribbon synapses of the retina. Cell Tissue
Res. 326,339
-346.[CrossRef][Medline]
Verhage, M., Maia, A. S., Plomp, J. J., Brussaard, A. B.,
Heeroma, J. H., Vermeer, H., Toonen, R. F., Hammer, R. E., van den Berg, T.
K., Missler, M. et al. (2000). Synaptic assembly of the brain
in the absence of neurotransmitter secretion. Science
287,864
-869.
Wahlin, K. J., Lim, L., Grice, E. A., Campochiaro, P. A., Zack,
D. J. and Adler, R. (2004). A method for analysis of gene
expression in isolated mouse photoreceptor and Muller cells. Mol.
Vis. 10,366
-375.[Medline]
Wang, X., Weiner, J. A., Levi, S., Craig, A. M., Bradley, A. and
Sanes, J. R. (2002). Gamma protocadherins are required for
survival of spinal interneurons. Neuron
36,843
-854.[CrossRef][Medline]
Washbourne, P., Dityatev, A., Scheiffele, P., Biederer, T.,
Weiner, J. A., Christopherson, K. S. and El-Husseini, A.
(2004). Cell adhesion molecules in synapse formation.
J. Neurosci. 24,9244
-9249.
Wässle, H. (2004). Parallel processing in
the mammalian retina. Nat. Rev. Neurosci.
5, 747-757.[CrossRef][Medline]
Weiner, J. A., Wang, X., Tapia, J. C. and Sanes, J. R.
(2005). Gamma protocadherins are required for synaptic
development in the spinal cord. Proc. Natl. Acad. Sci.
USA 102,8
-14.
White, F. A., Keller-Peck, C. R., Knudson, C. M., Korsmeyer, S.
J. and Snider, W. D. (1998). Widespread elimination of
naturally occurring neuronal death in Bax-deficient mice. J.
Neurosci. 18,1428
-1439.
Wu, Q. and Maniatis, T. (1999). A striking
organization of a large family of human neural cadherin-like cell adhesion
genes. Cell 97,779
-790.[CrossRef][Medline]
Wu, Q. and Maniatis, T. (2000). Large exons
encoding multiple ectodomains are a characteristic feature of protocadherin
genes. Proc. Natl. Acad. Sci. USA
97,3124
-3129.
Yagi, T. and Takeichi, M. (2000). Cadherin
superfamily genes: functions, genomic organization, and neurologic diversity.
Genes Dev. 14,1169
-1180.
Yamagata, M. and Sanes, J. R. (2008). Dscam and
Sidekick proteins direct lamina-specific synaptic connections in vertebrate
retina. Nature 451,465
-469.[CrossRef][Medline]
Young, R. W. (1984). Cell death during
differentiation of the retina in the mouse. J. Comp.
Neurol. 229,362
-373.[CrossRef][Medline]
Zhang, J., Gray, J., Wu, L., Leone, G., Rowan, S., Cepko, C. L.,
Zhu, X., Craft, C. M. and Dyer, M. A. (2004). Rb regulates
proliferation and rod photoreceptor development in the mouse retina.
Nat. Genet. 36,351
-360.[CrossRef][Medline]
Zou, C., Huang, W., Ying, G. and Wu, Q. (2007).
Sequence analysis and expression mapping of the rat clustered protocadherin
gene repertoires. Neuroscience
144,579
-603.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
-protocadherins
This article has been cited by other articles:
|
|
 |
|
  A. M. Garrett and J. A. Weiner Control of CNS Synapse Development by {gamma}-Protocadherin-Mediated Astrocyte-Neuron Contact J. Neurosci., September 23, 2009; 29(38): 11723 - 11731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Prasad, X. Wang, P. A. Gray, and J. A. Weiner A differential developmental pattern of spinal interneuron apoptosis during synaptogenesis: insights from genetic analyses of the protocadherin-{gamma} gene cluster Development, December 15, 2008; 135(24): 4153 - 4164. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
