Human Molecular Genetics, 2003, Vol. 12, No. 24
DOI: 10.1093/hmg/ddg356
3269–3276
Abnormalities of the vitreoretinal interface
caused by dysregulated Hedgehog signaling
during retinal development
Graeme C.M. Black1,2,3,{, Chantal J. Mazerolle4,{, Yaping Wang4, Katrina D. Campsall4,
Dino Petrin5, Brian C. Leonard5, Karim F. Damji5, D. Gareth Evans1, David McLeod2 and
Valerie A. Wallace4,5,*
1
Received July 10, 2003; Revised and Accepted October 6, 2003
Mutations in Patched (PTCH), encoding the Hedgehog (Hh) receptor, underlie Basal Cell Naevus syndrome
(BCNS) and, in addition to tumor predisposition, are associated with a wide range of ‘patterning’ defects. The
basis for the underlying patterning problems in Hh-dependent tissues in BCNS and their long-term
consequences on tissue homeostasis are, however, not known. Hh signaling is required for normal growth
and organization of the mammalian retina and we show that PtchlacZ þ/ mice exhibit vitreoretinal
abnormalities resembling those found in BCNS patients. The retinas of PtchlacZ þ/ mice exhibit abnormal
cell cycle regulation, which culminates in photoreceptor dysplasia and Müller cell-derived gliosis. In BCNS,
the intraretinal glial response results in epiretinal membrane (ERM) formation, a proliferative and contractile
response on the retinal surface. ERMs are a cause of significant visual loss in the general, especially elderly,
population. We hypothesize that alteration of Müller cell Hh signaling may play a role in the pathogenesis of
such age-related ‘idiopathic’ ERMs.
INTRODUCTION
Basal Cell Naevus syndrome or Gorlin syndrome (BCNS,
MIM#109400) is an autosomal dominant disorder characterized by birth defects (dental, skeletal and radiographic
abnormalities including Falx calcification, bifid/fused ribs and
altered vertebral segmentation), as well as a predisposition to
tumor development [including early-onset basal cell carcinomata (BCCs), medulloblastoma (5%) and rhabdomyosarcoma]. BCNS results from mutations of the PTCH gene on
chromosome 9q23.1 (1,2). The human homologue of the
Drosophila patched gene, PTCH is a transmembrane protein
that functions as the receptor for members of the Hedgehog
(Hh) family of intercellular signaling molecules (3,4). Sonic
hedgehog (Shh), the most widely represented member of this
family, is required for many aspects of developmental
patterning in the vertebrate embryo (5).
Ptch normally functions to block Hh signaling by antagonizing
the activity of Smoothened, a seven domain transmembrane
protein that is required for transmission of the Hh signal to the
nucleus via cubitus interruptus/Gli transcription factors; this
results in the activation of target genes including Ptch and Gli
(6–11). The developmental abnormalities observed in BCNS
patients, and in mice that are heterozygous for the Ptch gene
(PtchlacZ þ/ mice) (12,13), indicate that Hh pathway activation
is sensitive to Ptch gene dosage. Mutations in PTCH have also
been documented in sporadic BCCs and medulloblastomas,
indicating that the gene also functions as a tumor suppressor (14).
Of the various developmental abnormalities described in
BCNS, the ocular features remain poorly characterized.
*To whom correspondence should be addressed at: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada.
Tel: þ1 6137378234; Fax: þ1 6137378803; Email: vwallace@ohri.ca
{
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Human Molecular Genetics, Vol. 12, No. 24 # Oxford University Press 2003; all rights reserved
Downloaded from http://hmg.oxfordjournals.org/ at Pennsylvania State University on February 23, 2013
Academic Unit of Medical Genetics and Regional Genetic Service, St Mary’s Hospital, Hathersage Road,
Manchester, UK, 2Academic Department of Ophthalmology, Manchester Royal Eye Hospital, Oxford Road,
Manchester, UK, 3Centre for Molecular Medicine, Stopford Building, Oxford Road, Manchester, UK,
4
Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario,
Canada and 5University of Ottawa Eye Institute, 501 Smyth Road, Ottawa, Ontario, Canada
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Human Molecular Genetics, 2003, Vol. 12, No. 24
Estimated to be present in between 15 and 25% of patients
(15,16), previous reports include defects of organogenesis
(microphthalmia, coloboma), as well as both anterior segment
(cataract) and posterior segment abnormalities, the latter
including inappropriate retinal myelination and retinoschisis
(abnormal splitting of the retina) (17–20). Hh pathway
activation is known to play a role in mammalian visual system
development. Shh is expressed in the retinal ganglion cells
(RGC), the first neurons to differentiate in the retina, and Ptch
and Gli are expressed in retinal neuroblasts, as well as astrocyte
precursor cells in the optic nerve (21–23 and summarized in
Fig. 1B). RGC-derived Shh expression is required for Hh target
gene induction in the retina and optic nerve and plays a role in
precursor cell proliferation, photoreceptor differentiation and
normal cellular organization in the rodent retina (21–25).
Given the importance of the Hh signaling pathway in eye
morphogenesis and retinal development, we reasoned that
dysregulation of this pathway caused by haploinsufficiency for a
key regulatory component, the Ptch receptor, could cause ocular
defects. We studied 30 patients with BCNS and documented a
wide range of ocular abnormalities. Amongst these were defects
of retinogenesis including fibroglial epiretinal membrane
(ERM) formation (Fig. 1B) and abnormal ganglion-cell axon
myelination. To understand the basis for these retinal abnormalities in BCNS patients, we undertook a histological analysis of
the retinas of PtchlacZ þ/ mice (12). Approximately 50% of
adult PtchlacZ þ/ mice exhibited dysplastic foci in the retina
that were associated with an abnormal Müller cell-derived
gliotic response. Analysis of perinatal PtchlacZ þ/ mice
revealed ectopic proliferation and delayed differentiation. Our
findings confirm a role for Ptch/Shh signaling in retinal
histogenesis and implicate this pathway in glial cell homeostasis. Furthermore, our findings indicate that there is an
underlying developmental basis for ERM formation.
RESULTS
Abnormal retinal phenotypes associated
with Basal Cell Naevus syndrome
Thirty BCNS patients were examined and, in keeping with
previous reports, we documented a wide range of ocular
abnormalities. The frequency and range of non-ocular
manifestations, as previously described, was in keeping with
those expected for BCNS (15). Since BCCs around the eye are
a general feature of BCNS they were not included in this study.
The ocular abnormalities included: squint (9/30), microphthalmos (1/30) and defects of both anterior segment development
(Peters’ anomaly 1/30, cataract 7/30) and posterior segment
development. In total, 9/30 patients had early-onset unilateral
visual reduction as a direct result of a structural ocular
abnormality that could be attributed to BCNS. There were no
cases of bilateral visual reduction.
Examination of the posterior segment revealed retinal and
vitreoretinal abnormalities in 11 patients. The range of retinal
and vitreous abnormalities is shown in Figure 2 and may be
classified into two groups:
Inner retinal abnormalities. ERM formation, which is manifest as a cellular proliferation in the surface of the retina, was
observed in eight eyes and in six eyes there was evidence of
retinal myelination (Fig. 2B). In a single eye the abnormalities
at the interface between the retina and vitreous were associated
with a discrete opaque nodule that was consistent with astrocytic proliferation (Fig. 2C). In other eyes there were focal retinal
abnormalities located close to retinal arteries or at sites where
retinal arteries and veins cross. Finally in one eye there was
an extensive fibroglial ERM encompassing the optic nerve head
associated with abnormal retinal vessels (Fig. 2D).
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Figure 1. General eye anatomy and summary of Hh pathway gene expression in the retina. (A) Adult mouse eye stained with hematoxylin and photographed at low
magnification. RPE, retinal pigment epithelium; NR, neural retina; ON, optic nerve; per and cen refer to the peripheral and central retina, respectively. (B) Diagram
of the developing and adult mouse retina. At late stages of embryogenesis, the retina consists of two layers, the retinal ganglion cell (RGC) layer and the neuroblast
(NB) layer, which contains proliferating precursor cells. RGC axons are located on the surface of the retina and exit the eye at the optic disc to form the optic nerve.
The adult retina is organized into three cellular layers: RGC, inner nuclear layer (INL), and the rod and cone-containing outer nuclear layer (ONL). The nuclear
layers are separated by the inner and outer plexiform layers (IPL, OPL), which contain neuronal processes. Müller cells, radial-type glial cells, have processes that
span the width of the retina from the RGC to the ONL and cell bodies that are located in the middle of the INL. In the embryonic and adult retina Shh is expressed
in RGCs and Ptch is expressed by dividing precursor cells in the neuroblast layer (embryonic) and Müller cells (adult). The diagram on the far right depicts a
photoreceptor rosette in the ONL, and an epiretinal membrane (ERM) at the vitreoretinal interface where the invasion of Müller cell processes into the vitreous
is associated with the recruitment of contractile cells, which in some instances can pull on the retina causing distortion of the blood vessels and retinal detachments.
Human Molecular Genetics, 2003, Vol. 12, No. 24
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Developmental vitreous abnormalities. Three eyes showed
persistence of the fetal hyaloid system. In a further three eyes
isolated vitreous cysts were present. In one, this was free floating in the vitreous humor. In two cases (Fig. 2E and F) the cysts
were close to the macular region of the retina. In one case there
was an associated full-thickness retinal (macular) hole (Fig. 2F).
The site and appearance of the surface retinal pathology
strongly suggested recruitment of contractile (fibroglial) elements. Furthermore they were focal and indicative of developmental disturbances.
Retinal dysplasia and gliosis associated with
Ptch mutation in mice
The basis for these retinal abnormalities in BCNS patients is
unclear, therefore we examined the retinas of mice that are
heterozygous for the Ptch gene, PtchlacZ þ/ mice (12).
External ocular examination, fundoscopy and electroretinography detected no gross abnormalities in 8–12-week-old
PtchlacZ þ/ mice compared with wildtype littermates and
age matched C57Bl/6 mice (data not shown). Histological
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Figure 2. Surface retinal anomalies in BCNS patients. Retinal photographs of normal (A) and BCNS patients (B–F). (A) Normal retina indicating the position of
the optic disc (OD), the exit point for RGC axons and the entry point for the major retinal blood vessels (BV). The macula, the region of the retina required for high
acuity visual tasks, is outlined by the dashed circle. (B) Retinal myelination is visible as an arcuate opaque region above the macula. Surface retinal (epiretinal)
membrane formation results in wrinkling of the retinal surface and is visible as a fan of lines radiating from its epicenter (arrowed). (C) Discrete opaque nodule,
consistent with astrocytic proliferation, embedded in the retina (arrowed n). As in several other cases, an associated epiretinal membrane was located close to retinal
arteries (arrowed e). (D) Abnormal glial proliferation around optic disc. (E, F) Premacular isolated vitreous cysts. In (E) the cyst is situated close to the macula,
while in (F), in a second patient the cyst was associated with a full-thickness retinal detachment at the macula (macular hole) (arrowed).
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analysis of retinas from adult (3–6 months) PtchlacZ þ/ mice
revealed that 50% (n ¼ 7/14) exhibited foci of dysplasia
(including rosetting or clustering of photoreceptor nuclei
around a central lumen as illustrated in Fig. 1B) involving
photoreceptors in the outer nuclear layer (ONL) compared with
10% of controls (n ¼ 1/9) (Fig. 3B and C). Since these types of
abnormalities are usually associated with gliosis or activation
of the Müller glial cells (Fig. 1B), we stained companion
sections with antibodies against glial fibrillary acidic protein
(GFAP), which is normally not expressed by Müller cells, but is
induced when they are activated. Our analysis revealed that the
dysplastic regions contained reactive Müller cells, as indicated
by exaggerated GFAP and glutamine synthetase (a Müller cell
marker) staining (Figs 3B, C and 4A, B). In some cases the
Müller cell gliotic response was confined to the inner and outer
plexiform layers and was not associated with retinal dysplasia
(Fig. 4A and B and data not shown). Dysplastic lesions were
observed as early as postnatal day 21 (P21) in 4/7 of PtchlacZ þ/
mice and in none of their littermates (n ¼ 6) (Fig. 4C and D);
these dysplastic regions were more numerous and larger than
those in adult PtchlacZ þ/ mice, suggesting that they arose
early and often resolved before adulthood. At P21 they were
sometimes associated with an accumulation of photoreceptor
cell bodies outside the outer limiting membrane (Fig. 4C and
D); this was never observed in adult PtchlacZ þ/ mice. GFAP
staining associated with the dysplastic foci was weak or absent
at P21, suggesting that the induction of Müller cell-derived
gliosis that we observed in adult PtchlacZ þ/ mice occurs as a
consecutive or secondary response to the outer retinal dysplasia
(Fig. 4C and D).
To determine whether the Müller cell activation in the
dysplastic regions of PtchLacZ þ/ mice was associated with
changes in Hh pathway activation, we also examined b-gal
activity in these regions. The Ptch locus in PtchlacZ þ/ mice is
disrupted by the insertion of the lacZ gene, thus b-gal activity
is a convenient readout for Ptch gene expression in this mouse
strain. In the apparently normal regions of the PtchlacZ þ/
retina (i.e. those areas where dysplasia and gliosis were absent)
b-galþ cells were localized to the INL (i.e. in Müller cell
nuclei) and in a subset of astrocytes in the nerve fiber layer
(Fig. 3D and E). In dysplastic regions, however, the density of
b-galþ cells was reduced (Fig. 3F), indicating that these regions
contain fewer Müller cells or that those cells that normally
express the gene do so at a reduced level.
Since the retinal dysplasia that we observed in PtclacZ þ/
mice is similar to that resulting from dysregulated expression
of cell cycle components in the retina (26–30), we sought to
determine whether defective cell cycle regulation could
underlie the retinal abnormalities in PtchlacZ þ/ mice.
Retinal maturation proceeds from center to periphery such
that, by P5, proliferation (as assessed by BrdU incorporation)
has ceased in the central retina and cell division is confined to
the retinal periphery (Fig. 5C and D). In all four PtclacZ þ/
mice examined, however, we observed BrdUþ cells in the
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Figure 3. Extensive dysplasia and gliosis in the retinas of adult PtchlacZ þ/ mice. GFAP (A, B), glutamine synthetase (GS) staining, (C) and b-galactosidase
activity (blue stain) (D–F) in the retinas of adult PtchlacZ þ/ mice. Retinal cross sections were stained with the indicated antibodies (brown) and nuclei were
counterstained blue with hematoxylin. GFAP staining is normally confined to astrocytes located in the nerve fibre layer, the layer of RGC axons on the retinal
surface (A), however gliosis is indicated by the extension of GFAPþ processes into the retina (B). GS staining, which marks Müller cells, of a companion section
to (B) reveals that Müller cells are involved in the gliotic response. Nuclear staining with hematoxylin reveals foci of dysplasia of cells in the outer nuclear layer of
the retina, note rosettes in (B) and (C). X-gal staining reveals that b-galactosidase activity is reduced in dysplastic, red box in (D), compared with normal, green box
in (D), regions of the retina. (E) and (F) represent higher magnification views of the areas in (D) indicated by the green and red boxes, respectively. Please see
Figure 1 for a definition of the abbreviations.
Human Molecular Genetics, 2003, Vol. 12, No. 24
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central retina and a greater extent and intensity of BrdU
labeling in the peripheral retina compared with in littermate
controls (Fig. 5A and B). There were associated abnormalities
of photoreceptor and horizontal cell maturation; the intensity
of both rhodopsin staining in the ONL (Fig. 5 compare I and
K, J and L) and syntaxin staining at the border of the
developing outer plexiform layer (Fig. 5 compare E and G and
F and H) being reduced in PtclacZ þ/ mice compared with
littermate controls. Retinal maturation in PtclacZ þ/ mice is
likely to have been delayed rather than permanently disrupted
since we did not observe differences in adult PtclacZ þ/ mice
in staining for a variety of retinal markers (data not shown)
and, aside from the dysplastic foci, normal lamination was
established.
DISCUSSION
Our analysis of the retinas of BCNS patients and PtchlacZ þ/
mice revealed defects in retinal histogenesis and glial cell
function that were developmental in origin and focal in nature.
The similarities in the retinal abnormalities between BCNS
patients and PtchlacZ þ/ mice also suggest that the latter
represent a true model of the human disorder. Our findings
indicate that retinal histogenesis is sensitive to Ptch gene dosage
and support the hypothesis for a developmental basis for ERM
formation.
We have shown previously that the Hh pathway is involved in
precursor cell proliferation in the retina (21,22). Our observation that proliferation is extended in the central retina of
PtchlacZ þ/ mice is consistent with previous reports showing
that other Hh-dependent processes are sensitive to Ptch gene
dosage (13,31). Hh pathway activation has been directly linked
to transcriptional activation of cell cycle genes (32–35), which
raises the possibility that the reduction in Ptch expression
levels in Ptch-LacZ þ/ mice could result in an increase in the
expression of cell cycle genes, thus predisposing cells to higher
rates of proliferation. However, our preliminary analyses
indicate that retinal precursor cells from Ptch-LacZ þ/ mice
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Figure 4. Dysplasia is an early feature in retinal histogenesis in PtchlacZ þ/ mice. GFAP (A, C, D) and GS (B) staining in the retinas of postnatal day 21 (P21)
PtchlacZ þ/ mice. Retinal cross sections were stained with the indicated antibodies (brown) and nuclei were counterstained blue with hematoxylin. Staining of serial
sections for GFAP and GS reveals the Müller cell contribution to gliosis in the inner nuclear layer [the region between the arrows in (A) and (B)]. (C, D) Examples of
dysplastic lesions involving photoreceptor nuclei located in the outer nuclear layer. Note in (C) the extension of photoreceptor nuclei past the outer limiting membrane at P21, which was never observed in adult PtchlacZ þ/ mice. The difference in the severity of the dysplasia in young versus old PtchlacZ þ/ mice is consistent
with the possibility that some of these abnormalities are corrected by cell death. The dysplasia at P21 was not always associated with gliosis, as indicated by the lack
of GFAP staining in (C), suggesting that gliosis occurs as a secondary response to an initial dysplastic lesion in the PtchlacZ þ/ retina.
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Human Molecular Genetics, 2003, Vol. 12, No. 24
do not appear to have overall a higher rate of proliferation in
response to recombinant Shh (data not shown).
Our results are consistent with the possibility that focal changes
in retinal precursor cell proliferation account for the localized
dysplasias that we observed in Ptch-LacZ þ/ mice and that we
infer to have occurred in BCNS patients. That cell cycle
dysregulation in the retina can result in a delay in differentiation
and retinal dysplasia is supported by the phenotypes of p27
mutant mice or transgenic mice expressing cell cycle promoting
genes (26–30). It is unclear, however, whether the retinal
dysplasia that we observed in the PtchlacZ þ/ mice is associated
with loss of expression of the wild type Ptch allele, as is the case
in medulloblastomas derived from these mice (36). In contrast to
the cerebellum, ectopic proliferation in Ptc-LacZ þ/ mouse retina
does not result in tumorigenesis, perhaps because of the increased
propensity of retinal cells to undergo apoptosis (29,30,37).
In both murine and human cases we have demonstrated focal
areas of dysplasia involving cells in the outer nuclear layer, which
in adults was associated with reactive Müller cells (gliosis). Hh
responsiveness in the adult mouse retina, as defined by Ptch
expression, is largely confined to Müller cells (23). Müller cells
play a key role in retinogenesis and, in vitro promote the
establishment of retinal lamination and counteract rosette
formation (38). Thus, it may be possible that some of the retinal
abnormalities that we observed in BCNS patients and PtchlacZ þ/
mice result, at least in part, from a signaling defect at the level of
the Müller cell. However, such a defect is likely to be a late event
in the disease process, as our analysis of 3-week-old PtchlacZ þ/
mice indicates that the gliosis likely occurs after the development
of retinal dysplasia and the gliotic lesions in adult mice were not
associated with an increase in b-galactosidase activity, an
indicator of Hh pathway activation in PtchlacZ þ/ mice.
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Figure 5. Ectopic proliferation and delayed differentiation in the retinas of perinatal PtchlacZ þ/ mice. Cross sections of the retinas of PtchlacZ þ/ (A, B, E, F, I,
J) and wildtype (C, D, G, H, K, L) littermates at P5 stained with (A–D) BrdU, (E–H) anti-syntaxin and (I–L) anti-rhodopsin (all brown signals). Comparison of
BrdU incorporation in the retinas of PtchlacZ þ/ and wildtype mice in a (A, C) low magnification view of the peripheral retina and a (B, D) high magnification
view of the central retina at the optic disc (asterisk) reveals ectopic BrdU incorporation, as indicated by the extension of BrdUþ cells beyond the bracket in the
peripheral retina [compare (A) versus (C)], and the presence of BrdUþ cells in the central retina in PtchlacZ þ/ mice [compare (B) and (D)]. Immunostaining with
anti-syntaxin antibodies, which identify amacrine neurons in the inner nuclear layer [arrows in (F)] and horizontal cells at the border of the outer plexiform layer
(OPL) [arrowheads in (F)], reveals that horizontal cell differentiation and the establishment of the OPL are delayed in PtchlacZ þ/ mice (compare straight line of
syntaxinþ cells that extends from the central to the peripheral retina in wildtype mice [(G) top and bottom, (H)] with the disorganized line of syntaxinþ cells that is
restricted to the central retina of PtchlacZ þ/ mice [(E) compare top versus bottom, (F)]. Similarly, staining with anti-rhodopsin antibodies reveals that rod photoreceptor differentiation, as assessed by intensity of rhodopsin staining in the ONL [bracketed in (J)], is delayed in central and peripheral regions of the retina in
PtchlacZ þ/ mice compared wildtype littermates [compare (I), (K) and (J), (L)]. cen, central retina; per, peripheral retina.
Human Molecular Genetics, 2003, Vol. 12, No. 24
MATERIALS AND METHODS
Clinical details
Patients with Gorlin syndrome known to the North-West
Regional Genetics Service were ascertained according to
guidelines approved by the North-West Region Ethics
Committee. A thorough medical history was obtained and
examinations were performed seeking evidence of developmental, dermatological, and dental problems. Thirty individuals
underwent a complete eye examination, including slit-lamp
biomicroscopy, applanation tonometry and dilated fundus
examination.
Transgenic mice and immunohistochemistry
PtchlacZ þ/ mice (12) were purchased from Jackson
Laboratories (Bar Harbor, Maine) and maintained on a
C57Bl/6 background. To harvest tissues, an anesthetic overdose (euthanyl) was administered intraperitoneally and the
animals were perfused with 4% paraformaldehyde in 0.1 M
phosphate buffer pH 7.4. Eyes were enucleated and the lens
removed. The posterior segment tissues were then cryoprotected in 30% sucrose/PBS and embedded in a 1 : 1 mixture of
30% sucrose : OCT (Tissue Tek compound). Serial sections of
the eye were cut at 9–14 mm and collected in series of four
slides. To assess the retinal architecture, every 4th slide was
processed for immunostaining with anti-GFAP antibodies using
established protocols, as described previously (21), followed by
counterstaining with hematoxylin. Other slides in each
series were stained with anti-glutamine synthetase antibodies
(BD Pharmingen) to identify Müller cells. To detect cells in
S-phase, postnatal day 5 PtchlacZ þ/ mice were given two
intraperitoneal injections 2 h apart with 30 ml of a 16 mg/ml
solution of BrdU (Sigma Aldrich) in MEM (ICN Biomedicals
cat#12-104-54). Two hours after the last injection the tissues
were harvested, as described above, and processed for
immunohistochemistry with anti-BrdU antibodies (Becton
Dickinson), as previously described (22), anti-rhodopsin
[B630, (42)] to detect rod photoreceptors and anti-syntaxin
antibodies (HPC-1, Sigma Biosciences) to identify horizontal
and amacrine cells. Primary antibodies were detected with the
appropriate horseradish peroxidase conjugated secondary
antibodies and developed using DAB. Sections were analyzed
on a Zeiss Axioplan microscope and digital images were
captured using an AxioVision 2.05 (Zeiss) camera and
processed with Adobe1 Photoshop.
ACKNOWLEDGEMENTS
We thank M. Raff for antibodies, and R. Bremner, D. Picketts
and C. C. Hui for criticism of the manuscript. V.A.W’s
laboratory was supported by Canadian Institutes of Health
Research and the National Cancer Institute of Canada. V.A.W.
has a Canadian Institutes of Health Research Scholarship.
G.C.M.B. is a Wellcome Trust Senior Research Fellow in
Clinical Science.
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A role for Hh signaling in the adult retina is indicated by
sustained Shh expression in both RGCs and a subset of inner
nuclear layer amacrine cells, and of Ptch expression in Müller
cells (21,23). Moreover, we have detected Shh and Ptch
expression by RT–PCR analysis of adult human retinas (data
not shown). ERM formation from glial activation in BCNS
patients and intraretinal gliosis in PtchlacZ þ/ mice represent
the retinal response to dysregulation of this pathway and raise
the possibility that inappropriate activation of this pathway
could underlie non-genetic disorders with these features. In the
general population idiopathic ERMs are seen in 25% of postmortem eyes after the age of 75 years and are a wellrecognized, predominantly unilateral, cause of visual loss in
later life (39,40). The ERMs have an initial glial component
derived from extension of Müller processes through and over
the inner limiting lamina (41), but their pathogenesis remains
undefined. In the light of our findings, we speculate that altered
Müller cell responsiveness to Shh signaling, for example a
localized reduction in Müller cell Ptch expression, could
represent one such mechanism. Such epiretinal gliotic lesions
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