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The Journal of Neuroscience, September 1, 2000, 20(17):6517-6528
Laminin Expression in Adult and Developing Retinae: Evidence of
Two Novel CNS Laminins
Richard T.
Libby1,
Marie-France
Champliaud2,
Thomas
Claudepierre1, 2,
Yin
Xu3,
Erin P.
Gibbons1,
Manuel
Koch2,
Robert E.
Burgeson2,
Dale D.
Hunter3, and
William J.
Brunken1, 2
1 Department of Biology, Boston College, Chestnut Hill,
Massachusetts 02467, 2 Cutaneous Biology Research Center,
Massachusetts General Hospital, Harvard Medical School, Charlestown,
Massachusetts 02129, and 3 Departments of Neuroscience,
Anatomy and Cell Biology, and Ophthalmology, Tufts University School of
Medicine, Boston, Massachusetts 02111
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ABSTRACT |
Components of the extracellular matrix exert myriad effects on
tissues throughout the body. In particular, the laminins, a family of
heterotrimeric extracellular glycoproteins, have been shown to affect
tissue development and integrity in such diverse organs as the kidney,
lung, skin, and nervous system. Of these, we have focused on the roles
that laminins play in the differentiation and maintenance of the
nervous system. Here, we examine the expression of all known laminin
chains within one component of the CNS, the retina. We find seven
laminin chains  3, 4, 5,  2, 3,  2, and 3outside
the retinal basement membranes. Anatomically, these chains are
coexpressed in one or both of two locations: the matrix surrounding
photoreceptors and the first synaptic layer where photoreceptors
synapse with retinal interneurons. Biochemically, four of these chains
are coisolated from retinal extracts in two independent complexes,
confirming that two novel heterotrimers423 and
523are present in the retinal matrix. During development, all four of these chains, along with components of laminin 5 (the 3,
3, and 2 chains) are also expressed at sites at which they could
exert important effects on photoreceptor development. Together, these
data suggest the existence of two novel laminin heterotrimers in the
CNS, which we term here laminin 14 (composed of the 4, 2, and
3 chains) and laminin 15 (composed of the 5, 2, and 3
chains), and lead us to hypothesize that these laminins, along with
laminin 5, may play roles in photoreceptor production, stability, and
synaptic organization.
Key words:
retina; synapse; matrix; photoreceptor; interphotoreceptor matrix; laminin
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INTRODUCTION |
The laminins are heterotrimeric
glycoproteins of the extracellular matrix, each composed of an , a
, and a chain (Burgeson et al., 1994 ). Currently, 11 laminin chains have been identified: five , three , and three chains (for review, see Timpl, 1996; Koch et al., 1999).
Although the exact combinations of chains that can combine to form
heterotrimers are unknown, it has been suggested that there are at
least 12 distinct laminin heterotrimers (Timpl, 1996; Miner et al.,
1997; Koch et al., 1999), termed laminins 1-12.
Defects in several of the component chains have been shown to cause
disease in humans. Mutations in the 2 chain have been linked to a
muscular dystrophy that is also marked by CNS demyelination (for
review, see Arahata et al., 1995); defects in all three chains of
laminin 5 (3, 3, and 2) have been linked to a degenerative skin disorder, junctional epidermolysis bullosa (for review, see Timpl,
1996); a muscular dystrophy (Walker-Warburg syndrome) that also has
ocular involvement shows reduced expression of the 2 chain (Wewer et
al., 1995); and finally, the gene encoding 3 maps to the site of a
wide variety of neurodevelopmental disorders that include
eye-brain-muscle disease and retinitis pigmentosa 21 (Koch et al.,
1999). Targeted mutations of laminin genes also support critical roles
for laminins during development (for review, see Ryan et al.,
1996).
Expression of laminins has been studied in several areas of the nervous
system; of these, we have focused on the retina. In the inner retina,
several groups have associated laminin chains with retinal ganglion
cells (Cohen et al., 1987; Sarthy and Fu, 1990; Dong and Chung, 1991;
Morissette and Carbonetto, 1995). In the outer retina, we have shown
that the laminin 2 chain is associated with photoreceptors (Hunter
et al., 1992b; Libby et al., 1996). The 2 chain promotes the
expression of the rod photoreceptor phenotype in vitro
(Hunter et al., 1992b; Hunter and Brunken, 1997) and is vital for
proper photoreceptor development in vivo (Libby et al.,
1999). In the peripheral nervous system, laminins appear to mediate
synaptic formation or stability (Hunter et al., 1989; Noakes et al.,
1995); within the retina, they may subserve similar functions in the
outer plexiform layer (Libby et al., 1999).
The identity of the laminin(s) present in the retina is currently
unknown. To define the functional retinal heterotrimers and their roles
in retinal development and maintenance, it is important to ascertain
with which and chain(s) the laminin 2 chain is associated.
Here, we identify several laminin chains in the interphotoreceptor
matrix and plexiform layers, allowing us to hypothesize that two novel
laminins containing the laminin 2 chainlaminins 14 and 15exist;
we also demonstrate biochemically that these heterotrimers are
components of the retinal matrix. These laminins, together with laminin
5, are likely to be important during neuronal maturation and
maintenance in the retina and, by extension, the rest of the CNS.
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MATERIALS AND METHODS |
Animal and tissue preparation. All procedures
involving animals were approved by the Boston College, Massachusetts
General Hospital, and Tufts University animal care committees and were in accordance with the National Institutes of Health Guide for the Care and Use of Animals and the policies of the Society for Neuroscience. Rats were killed by exposure to
CO2. Human retinal sections, unfixed and
fixed, were a gift of Dr. Ann Milam (Scheie Eye Institute, University
of Pennsylvania, Philadelphia, PA).
Immunohistochemistry. Immunohistochemistry was performed as
described previously (Libby et al., 1996, 1997). Adult rat eyecups were
embedded in OCT compound (Miles, Elkhart, IN) and frozen by immersion
in liquid nitrogen-cooled isopentane; transverse, 10-µm-thick
sections were cut with a Leica cryostat and placed onto Superfrost Plus
slides (Fisher Scientific, Pittsburgh, PA). Human retina specimens were
obtained as unfixed transverse sections. Slides were stored at  20°C
until use. For use, slides were returned to room temperature, immersed
briefly in acetone (or, interchangeably, for all but the 5, 3,
and 2 chains, MeOH) at 20°C, washed in PBS (137 mM NaCl, 2.68 mM KCl, 10 mM
Na2HPO4, and 1.76 mM KH2PO4, pH
7.4), and then incubated in primary antibodies for 2 hr at room
temperature or overnight at 4°C. Primary antibodies (see below) were
diluted in PBS containing 2% goat serum, 2% bovine serum albumin, or
both. Sections were washed in PBS and incubated in species-appropriate,
affinity-purified, fluorescently labeled secondary antibodies diluted
in 2% goat serum in PBS for 1 hr at room temperature. After washes in
PBS, slides were mounted in 90% glycerol and 10% water, containing
para-phenylenediamine (1 mg/ml; Sigma, St. Louis, MO) to
reduce photobleaching, or in Prolong (Molecular Probes, Eugene, OR).
The antibodies used were as follows: laminin 1, 111 [Life
Technologies, Gaithersburg, MD; rabbit polyclonal to mouse
Engelbreth-Holm-Swarm (EHS) tumor protein]; laminin 2 chain (Life
Technologies; mouse monoclonal to human protein); laminin 3 chain
[BM-2; made in one of our laboratories (R.E.B.); mouse
monoclonal to human protein]; laminin 4 chain [Miner et al.
(1997); rabbit polyclonal to mouse fusion protein or R17; made in one
of our laboratories (R.E.B.); rabbit polyclonal to human fusion
protein]; laminin 5 chain (Miner et al. [1995]; rabbit polyclonal
to mouse fusion protein or 4C7; Engvall et al. [1986] [see Tiger et
al. (1997) for 5 reactivity]; mouse monoclonal to human protein);
laminin 1 chain [C21; Sanes and Chiu (1983); mouse monoclonal to
rat protein]; laminin 2 chain [GP1; Sanes et al. (1990a); guinea
pig polyclonal to rat fusion protein or C4; Sanes and Chiu (1983);
mouse monoclonal to bovine protein or D5; Hunter et al. (1989); mouse
monoclonal to bovine protein]; laminin 3 chain [6F12; Rouselle et
al. (1991); mouse monoclonal to human protein]; laminin 1 chain
[D18; Sanes et al. (1990a); mouse monoclonal to rat fusion protein];
laminin 2 chain [Sugiyama et al. (1995); rabbit polyclonal];
laminin 3 chain [R16 and R21; Koch et al. (1999); rabbit
polyclonals to human protein and human fusion protein, respectively];
and laminin 5, 332 [4101; Rouselle et al. (1991); Marinkovich
et al. (1992) or 8Ln5 and 9Ln5; made in one of our laboratories
(R.E.B.); rabbit polyclonals to human protein]. 8Ln5 and 9Ln5 were
made to the same antigen as the published antiserum 4101 and have the same reactivity.
In situ hybridizations. Adult rat eyecups were
dissected and fixed overnight at 4°C in 4% paraformaldehyde in PBS,
pH 7.4, dehydrated, and embedded in paraffin. Fifteen-micrometer-thick sections were cut and placed onto Probe-on Plus slides (Fisher Scientific). Human retina specimens were obtained as fixed transverse sections. Rehydrated rat sections or frozen human sections were then
processed for in situ hybridizations as described previously (Libby et al., 1997).
cRNA probes for the laminin chains were generated as described
previously (Libby et al., 1997). Probes for the laminin 1 and 2
chains and for cellular retinaldehyde-binding protein were those used
previously (Libby et al., 1997). A cRNA probe for the laminin 5
chain (Miner et al., 1995) was generated from a plasmid obtained from
J. Sanes (Washington University, Saint Louis, MO). All other laminin
chain probes were generated from plasmids (containing fragments of
human laminin cDNAs) obtained in one of our laboratories (R.E.B.).
cRNAs were labeled during transcription by the incorporation of
digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN); ~1 µg/ml
cRNA was used for hybridization.
Biochemical isolation of laminin heterotrimers. Bovine eyes
were obtained from Pel-Freeze Biologicals (Rogers, AR) and dissected to
isolate the retinae. Approximately 50 retinae were pooled, washed in
PBS containing the protease inhibitors phenylmethylsulfonyl fluoride
(150 mg/l) and N-ethylmaleimide (650 mg/l), frozen in liquid
nitrogen, ground in a Waring blender, resuspended in 100 ml of 2 M urea, 0.5 M NaCl, 10 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 5 mM
N-ethylmaleimide, and 50 mM Tris-HCl, pH 7.8, and then stirred for 24 hr at 4°C. The retinal extract was cleared by
centrifugation at 30,000 × g for 60 min, dialyzed in
0.5 M NaCl and 50 mM
Tris-HCl, pH 7.8, and then cleared by centrifugation at 100,000 × g for 60 min. Glycoproteins were isolated by applying the
extract to a concanavalin A-Sepharose column (Pharmacia, Piscataway, NJ); unbound material was removed by washing with 0.5 M NaCl, 5 mM
CaCl2, 5 mM
MgCl2, and 50 mM Tris-HCl,
pH 7.4. The column was washed with 10 mM
-D-methylmannopyranoside in 0.5 M NaCl and 50 mM Tris-HCl,
pH 7.4, and then eluted with 1 M
-D-methylglucopyranoside in 0.5 M NaCl and 50 mM Tris-HCl,
pH 7.4.
To isolate laminin 14 and laminin 15, the concanavalin A eluate was
separated without sulfhydryl reduction on a 3-5% polyacrylamide-SDS gel (Laemmli, 1970). After staining with Coomassie brilliant blue R-250
(Sigma), bands containing the high-molecular weight proteins were
excised, washed in 0.5 M Tris-HCl, pH 6.8, and incubated in
SDS sample buffer containing 10% -mercaptoethanol for 30 min at
ambient temperature, and the different laminin chains were separated on
a 5% polyacrylamide-SDS gel. Proteins were analyzed by protein
transfer (Western) blot analysis (Towbin et al., 1979) using an
anti-laminin 4 chain antiserum (R17), an anti-laminin 2 chain
antibody (D5), and an anti-laminin 3 chain antiserum (R21).
The 380 kDa protein isolated by this method was not reactive with any
of our anti-laminin antibodies. Therefore, after digestion by the
protease Lys-C, peptide fragments of this protein were sequenced by the
use of matrix-assisted laser desorption time-of-flight mass
spectrometry (Chait and Kent, 1992) performed on a Finnegan Lasermat
2000 in the Harvard Microchemistry laboratories.
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RESULTS |
Protein expression
Antibodies that recognize the 11 known laminin chains were used to
catalog the laminin chains in adult rat and human retinae. We describe
here the reactivity for antibodies directed against each of these chains.
Laminin chains
A polyclonal antiserum that recognizes the three chains of laminin
1 (111) reacts only with the vasculature in the rat (Fig.
1A) and human (Fig.
1B) and not with the matrix of the neural retina
itself. Laminin 1 immunoreactivity was seen on the basal side of the
retinal pigmented epithelium, that is, Bruch's membrane (Fig.
1A) [Hunter et al. (1992b), compare their Fig.
2E]; in those sections in which the inner limiting
membrane is present, laminin 1 is expressed there as well (data not
shown). These observations are consistent with numerous other reports
(see Kohno et al., 1987; Morissette and Carbonetto, 1995) and
suggest that the laminin 1 chain, a component of laminin 1, is not
associated with the matrix of either the neural retina or the
interphotoreceptor matrix (IPM) but is a component of the basement
membranes of the retina: Bruch's membrane and the internal limiting
membrane.
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Figure 1.
Expression patterns of laminin chains in
mature rat and human retinae. Unfixed frozen sections of rat
(top row; A, C, E, G, I) and human
(bottom row; B, D, F, H, J)
retinae were probed for the presence of the laminins 111
(A, B), 2 (C, D), 3 (E,
F), 4 (G, H), and 5
(I, J) chains by the use of chain-specific (or
trimer-specific for 111) antibodies. Although several chains
are present in the vasculature, only three (3, 4, and 5) are
expressed within the interphotoreceptor matrix
(arrowheads) and outer plexiform layer
(arrows). In addition, 4 is present in fibers
extending through the outer nuclear layer (ONL) and
inner nuclear layer (INL). GCL, Ganglion
cell layer. Scale bar, 25 µm.
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The laminin 2 chain, in agreement with a previous report (Morissette
and Carbonetto, 1995), is also present in the retinal vasculature (Fig.
1C,D). However, in contrast to this previous report, we did
not detect the laminin 2 chain associated with ganglion cell bodies.
We have not systematically studied the internal limiting membrane;
however, despite the fact that this membrane can be easily removed
during isolation of the retina, we regularly had the internal limiting
membrane in our sections, and we have not detected the laminin 2
chain associated with this basement membrane. The 2 chain does not
appear to be a component of Bruch's membrane.
In contrast, the laminin 3 chain is present in the
interphotoreceptor matrix (Fig. 1E,F); the
laminin 3 chain is prominent at the external limiting membrane and
at the tips of the photoreceptor inner segments. Laminin 3 chain
immunoreactivity is also present in the outer plexiform layer; however,
in contrast to the chains of laminin 1 and the laminin 2 chain,
which are associated with elements of the vasculature in the outer
plexiform layer, the laminin 3 chain does not appear to be
associated with the larger vessels in this region. Nevertheless, the
laminin 3 chain does appear to be present in the outer plexiform
layer; we cannot discern whether the laminin 3 chain is associated
with small vessels or associated with the synaptic connections in this
layer. In the human, weak immunoreactivity for the laminin 3 chain
is also present around cell bodies of the outer and inner nuclear
layers (Fig. 1F). Finally, in the human, the laminin
3 chain is diffusely associated with the inner plexiform layer.
In contrast to the laminin 1-3 chains, the laminin 4 chain
appears to have a broad distribution in rat and human retinae. Immunoreactivity for the laminin 4 chain is present in the IPM, as
well as diffusely in both the inner and outer plexiform layers (Fig.
1G,H). This extensive immunoreactivity in both
plexiform layers and the lack of any association with the retinal
vasculature suggest that the laminin 4 chain is contained within the
extracellular matrix of the plexiform layers. However, the most
prominent reactivity for the laminin 4 chain is in what appear to be
Müller cell fibers coursing through the retina. These fibers have
been confirmed as Müller cell processes, on the basis of
colocalization of the laminin 4 chain with a Müller cell
marker [vimentin (see Libby et al., 1997)]. Reactivity for the
laminin 4 chain is also present in the ganglion cell layer; this may
reflect laminin 4 chain associated with the end feet of Müller
cells. The presence of the laminin 4 chain within the Müller
cell suggests that the Müller cell is a source of the laminin
4 chain in the neural retina, consistent with the data that
confirmed the Müller cell as a source of another laminin chain,
2 (Libby et al., 1997).
Our initial localization studies using a polyclonal antiserum raised
against the laminin 5 chain (Miner et al., 1995) suggested that the
laminin 5 chain was only a component of the true basement membranes
of the retina, that is, the internal limiting membrane, Bruch's
membrane, and vascular basement membranes (data not shown). However, a
monoclonal antibody [4C7 (Engvall et al., 1986)] that specifically
recognizes the laminin 5 chain (Tiger et al., 1997) demonstrates
that the laminin 5 chain is more broadly distributed within the
neural retina: the laminin 5 chain has a distribution similar to
that of the laminin 3 chain (Fig. 1I,J).
Specifically, the laminin 5 chain is present in both rat and human
interphotoreceptor matrices, as well as in the outer plexiform layer in
the rat. In addition, the laminin 5 chain, like the laminin 1 and
2 chains, is associated with the retinal vasculature; this is
particularly notable in the human (Fig. 1J). Laminin
5 chain immunoreactivity is present in the choroid, the hyaloid
vessels, the outer plexiform layer vessels, and the vasculature that
extends through the retina from the hyaloid vessels to the outer
plexiform layer. This expression in the vasculature is similar to the
expression pattern of the laminin 5 chain in the brain (our
unpublished observations).
Together, these data suggest that all five laminin chains are
expressed in the retina, but twothe laminin 1 and 2 chainsmay be associated exclusively with the retinal vasculature. Of these, the
laminin 1 chain has not been directly associated with the basement
membrane of vessels; indeed, protein transfer blots of retinal extracts
fail to detect the 1 chain (Hunter et al., 1992b). In the case of
the laminin 2 chain, these data conflict somewhat with previous
reports of expression in some vertebrates (Morissette and Carbonetto,
1995), although they are consistent with others (Toti et al., 1997)
that show that the laminin 2 chain is restricted to the vasculature
in the human retina. In contrast to these two laminin chains,
threethe laminin 3, 4, and 5 chainsare associated with the
IPM and, potentially, are associated with the neural retina at synapses
in the plexiform layers. Laminins at each of these locations could be
provided from the cell that spans the entire thickness of the retina,
the Müller cell; the Müller cell is the likely source for
at least one other laminin chain, 2 (Libby et al., 1997).
Laminin chains
As noted above, a polyclonal serum that recognizes all three
chains of laminin 1, including the laminin 1 chain, reacts only with
the vasculature in rat (Fig. 1A) and human (Fig.
1B) retinae; for the human, this pattern is
consistent with the previously reported expression of laminin 1 (Kohno
et al., 1987; Toti et al., 1997). Thus, the laminin 1 chain cannot
be an element of the matrix of either the IPM or the neural retina. A
rat-reactive antibody against the 1 chain confirms this observation
(Fig. 2A). However,
because there is little authentic laminin 1 chain in the retina and
little authentic laminin 1 chain in the retinal vasculature of the
rat, it is likely that the polyclonal serum against laminin 1 (e.g.,
Fig. 2B) is detecting primarily the laminin 1
chain in the vasculature of both rat and human.
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Figure 2.
Expression patterns of laminin chains in
mature rat and human retinae. Unfixed frozen sections of rat
(top row; A, C, E) and human
(bottom row; B, D, F) retinae were
probed for the presence of the laminin 1 (A),
111 (B), 2 (C, D), and
3 (E, F) chains by the use of chain-specific
(or trimer-specific for 111) antibodies. Two chains, 2
and 3, are expressed within the neural retina, specifically,
in the interphotoreceptor matrix (arrowheads); in
addition, the 2 chain is present in the outer plexiform layer
(arrows). GCL, Ganglion cell layer;
INL, inner nuclear layer; ONL, outer
nuclear layer. Scale bar, 25 µm.
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As reported previously in the rat (Hunter et al., 1992b; Libby et al.,
1997), the laminin 2 chain is present in the interphotoreceptor matrix (Fig. 2C) and appears to be associated with the
external limiting membrane. Here, we also demonstrate a similar
distribution in the human retina (Fig. 2D). The
laminin 2 chain, a known component of brain vasculature (Hunter et
al., 1992a), was also associated with the vessels of the retina. In the
human, immunoreactivity is also present surrounding cell bodies in the
inner nuclear layer, as well as in the inner limiting membrane. In both
species, the laminin 2 chain is also diffusely associated with the
outer plexiform layer. A comparison of this diffuse immunoreactivity
with that for laminin 1 or the laminin 2 chain suggests that the
laminin 2 chain is not only associated with the vasculature within
the outer plexiform layer. As was true for the laminin 3 chain, we cannot say conclusively that the laminin 2 chain is associated with
the extracellular matrix of the outer plexiform layer. However, it is
intriguing to speculate that the laminin 2 chain may be localized to
synapses in the CNS, as it is in the peripheral nervous system (Hunter
et al., 1989).
Laminin 3 chain immunoreactivity was also present in the mature rat
retina (Fig. 2E), as well as in the mature human
retina (Fig. 2F). The 3 chain seems primarily
limited to the interphotoreceptor matrix, suggesting that laminins
containing the laminin 3 chain are components of this matrix.
Because laminin 3 has a "tightly restricted tissue distribution"
in the rodent (Utani et al., 1995) and has, to date, only been
demonstrated as a component of laminin 5 (332), it is likely
that this reflects the presence of laminin 5 in the interphotoreceptor matrix.
Together, these data suggest that, although the laminin 1 chain is
associated with the basement membrane of the retinal vasculature in
both rat and human retinae, only two chainsthe laminin 2 [as
reported previously in rat, rabbit, and skate (Hunter et al., 1992b)]
and 3 chainsare expressed in the matrix of the IPM. Moreover, the
laminin 2 chain is also expressed in the matrix of the outer
plexiform layer.
Laminin chains
As noted above, a polyclonal serum that recognizes all three
chains of laminin 1, including the laminin 1 chain, reacts primarily with the vasculature. Consistent with this observation, an antibody directed against the laminin 1 chain reacts only with the
vasculature in both rat (Fig.
3A) and human (Fig.
3B), suggesting that the anti-laminin 1 serum is reacting
with at least the 1 chain. In addition, in the human, the laminin
1 chain is present at the internal limiting membrane (Fig.
3B); this may reflect production by astrocytes, the hyaloid
blood vessels, and retinal ganglion cells (Sarthy and Fu, 1990; cf.
Sarthy, 1993). There is also some punctate immunoreactivity for the
laminin 1 chain within the ganglion cell layer. Importantly, there
is no laminin 1 chain reactivity in the IPM or plexiform layers;
thus, the laminin 1 chain is confined to the vitread side of the
retina.
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Figure 3.
Expression patterns of laminin chains in
mature rat and human retinae. Unfixed frozen sections of rat
(top row; A, C, E) and human
(bottom row; B, D, F) retinae were
probed for the presence of the laminin 1 (A, B), 2
(C, D), and 3 (E, F) chains by
the use of chain-specific antibodies. Two chains, 2 and
3, are expressed within the neural retina, specifically in
the interphotoreceptor matrix (arrowheads); in addition,
the 3 chain is present in the outer plexiform layer
(arrows). GCL, Ganglion cell layer;
INL, inner nuclear layer; ONL, outer
nuclear layer. Scale bar, 25 µm.
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In contrast to the laminin 1 chain, the laminin 2 chain is
present in the interphotoreceptor matrix of rat (Fig. 3C)
and human (Fig. 3D) retina. It is also present in the
hyaloid vessels and, to a limited extent, the intraretinal capillaries
of the human. Some laminin 2 chain is also present in the outer
plexiform layer of the rat; this immunoreactivity may reflect
capillary-associated laminins. As for the laminin 3 chain, previous
reports have suggested a restricted distribution of the laminin 2
chain (Kallunki et al., 1992).
The laminin 3 chain is the most recently isolated of the growing
family of laminins (Koch et al., 1999). The tissue distribution of this
chain is quite limited; however, it seems most extensively expressed in
the nervous system (our unpublished observations), including the
mouse retina (Libby et al., 1999). Here, we document the presence of
the laminin 3 chain in a portion of the human and rat CNS. Prominent
laminin 3 chain immunoreactivity is present in the
interphotoreceptor matrix, notably, throughout the region of
photoreceptor inner segments (Fig. 3E,F). In
addition, there is marked laminin 3 chain immunoreactivity
associated with the external limiting membrane in the rat (Fig.
3E) and surrounding cell bodies within the outer and inner
nuclear layers in the human (Fig. 3F). Finally, the
laminin 3 chain is diffusely present in the outer plexiform layer,
at least in the rat. As with the laminin 3, 4, and 2 chains,
we cannot say conclusively that the laminin 3 chain immunoreactivity
in the outer plexiform layer is concentrated at points of synaptic
contacts in the outer plexiform layer. However, the laminin 3 chain
is not associated with the vasculature present at the vitread side of
the retina, and its pattern of expression is distinct from that for
laminin chains in the vasculature, such as the 1 chain. Therefore,
it is probable that the laminin 3 chain in the outer plexiform layer
is contained within the matrix of the plexiform layer.
Together, these data suggest that the laminin 2 and 3 chains are
the only known laminin chains in the IPM. Furthermore, the laminin
3 chain appears to be the only laminin chain found potentially
associated with the synaptic regions of the outer plexiform layer in
both rat and human.
Summary of protein expression
In the IPM, we have shown the presence of seven laminin chains:
3, 4, 5, 2, 3, 2, and 3. This is consistent with
the presence of one previously isolated laminin, laminin 5 (332), as well as several novel laminin heterotrimers. If the
other chains were to combine, then, potentially, there would be two
such novel laminin trimers in the IPM: 423 and 523.
In the matrix of the outer plexiform layer, these two trimers also
appear to be present, because their component chains are present. In
contrast, only one laminin chain, 4, is prominent in the matrix of
the inner plexiform layer, suggesting that other, uncharacterized, and chains may be expressed in the retina.
RNA expression
cRNA probes that recognize the RNAs encoding the 11 known laminin
chains were used to catalog these RNAs in the retina and to localize
them to particular cell types. Because laminin trimers are assembled
before secretion, the RNAs encoding all three chains of any given
trimer should be present in the same cell.
Laminin chains
RNAs encoding the laminin 1 (Fig.
4A,B) and 2 (Fig.
4C,D) chains were not readily detected in the rat or human
retina, suggesting that both of these RNAs are not abundant in the
retina. However, for both chains, some RNA was detected in the inner
nuclear layer. This may reflect production of these two chains by
components of the vasculature.
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Figure 4.
Expression patterns of the RNA encoding laminin
chains in mature rat and human retinae. Fixed sections of rat
(top row; A, C, E, G, I)
and human (bottom row; B, D, F, H,
J) retinae were probed for the presence of RNA encoding
the laminin 1 (A, B), 2 (C, D),
3 (E, F), 4 (G, H),
and 5 (I, J) chains by the use of
chain-specific cRNA probes. Two RNAs (those encoding 3 and 4) are
expressed within the neural retina of both species; in addition, the
RNA encoding 5 is readily detected in human retina.
GCL, Ganglion cell layer; INL, inner
nuclear layer; ONL, outer nuclear layer. Scale bar, 25 µm.
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In contrast, the RNA encoding the laminin 3 chain is readily
detectable in the rat and human retina (Fig.
4E,F). This expression agrees with the high
expression levels of the laminin 3 chain in the retina from the
human expressed sequence tag database. Interestingly, laminin 3
chain RNA is not localized to perinuclear sites; rather, the RNA is in
fibers coursing through the inner and outer nuclear layers and the
outer plexiform layer. This location is consistent with production of
laminin 3 chain RNA by Müller cells.
The RNA encoding the laminin 4 chain is present in a pattern similar
to that encoding the laminin 3 chain: the RNA appears to be located
in fibers coursing through the inner and outer nuclear layers (Fig.
4G,H), which are likely to be Müller cell
processes. Unlike laminin 3 chain RNA, there does seem to be
perinuclear laminin 4 chain RNA in the inner nuclear layer,
particularly of the human retina (Fig. 4H),
suggesting that the source of the RNA encoding the laminin 4 chain
is a cell whose nucleus resides in the inner nuclear layer;
Müller cell nuclei are in this layer. Finally, in human, laminin
4 chain RNA is present in the ganglion cell layer, in what we
presume to be Müller cell end feet, as we have shown for laminin
2 chain RNA (Libby et al., 1997).
Similar to the laminin 1 and 2 chain, RNA encoding the laminin
5 chain is not detectable within the rat retina (Fig.
4I); this suggests that the RNA encoding the laminin
5 chain is not abundant in the rat retina. In an example of species
variation, we detected RNA encoding the laminin 5 chain within the
neural retina of the human (Fig. 4J). The pattern of
expression for laminin 5 chain RNA in the human retina is similar
to, albeit considerably less intense than, that detected with a probe
for laminin 4 chain RNA (Fig. 4, compare H,J).
Together, the patterns of expression of the RNAs encoding the laminin
chains suggest that the laminin 3, 4, and 5 chain RNAs are
expressed in the neural retina, consistent with the presence of laminin
3, 4, and 5 chain protein noted above. Specifically, they
suggest that laminin 3, 4, and 5 chains are produced in the
neural retina and deposited in the matrices of the IPM and outer
plexiform layer and, in the case of the laminin 4 chain, the inner
plexiform layer.
Laminin chains
RNA encoding the laminin 1 chain is not highly expressed in the
neural retina (Fig. 5A,B), as
reported previously (Libby et al., 1997). These data are consistent
with the lack of laminin 1 chain protein in neural structures within
the retina.
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Figure 5.
Expression patterns of the RNA encoding laminin
chains in mature rat and human retinae. Fixed sections of rat
(top row; A, C, E, G) and human
(bottom row; B, D, F, H) retinae
were probed for the presence of the RNA encoding laminin 1
(A, B), 2 (C, D), and 3 (G,
H) chains by the use of chain-specific cRNA probes. RNA
encoding the 2 and 3 chains is expressed within the neural
retina. Expression of an RNA encoding a Müller cell antigen,
cellular retinaldehyde-binding protein (CRALBP), is
shown for comparison (E, F). GCL,
Ganglion cell layer; INL, inner nuclear layer;
ONL, outer nuclear layer. Scale bar, 25 µm.
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We have shown previously that the laminin 2 chain is expressed in
the adult rat retina (Libby et al., 1997). As also shown in Figure
5C, RNA encoding the laminin 2 chain is present in fibers
in the outer and inner nuclear layers of the rat. In the human, RNA
encoding the laminin 2 chain is present in what appear to be fibers
in the inner and outer nuclear layers; it is striking at the external
limiting membrane and is also present in the ganglion cell layer (Fig.
5D). We have ascribed previously the laminin 2 chain RNA
in the ganglion cell layer to Müller cell end feet (Libby et al.,
1997). There is also perinuclear RNA present in and around some cell
bodies in the inner nuclear layer, suggesting that a cell in the inner
nuclear layer, possibly the Müller cell, is a source of the
laminin 2 chain in the neural retina. Finally, as shown here (Fig.
5E) and previously (Libby et al., 1997) for the rat and here
for the human (Fig. 5F), this pattern of RNA expression is similar to that of cellular retinaldehyde-binding protein, an authentic marker of the Müller cell (Bunt-Milam and Saari, 1983).
Laminin 3 chain RNA appears to be expressed in the adult rat retina:
RNA encoding the laminin 3 chain is located in fibers coursing
through the inner and outer nuclear layers, in the outer plexiform
layer, and at the external limiting membrane (Fig. 5G). In
another example of species variation, laminin 3 chain RNA could not
be detected within the human neural retina (Fig.
5H).
Together, these data suggest that, in both rat and human, the laminin
2 chain is the prominent chain expressed in the neural retina.
In addition, the laminin 3 chain appears to be a component of the
neural retina. Finally, the laminin 1 chain is not likely to be
expressed in the mature neural retina.
Laminin chains
The RNA encoding the laminin 1 chain could not be detected in
the neural retina (Fig.
6A,B). This suggests
that the laminin 1 chain protein in the internal limiting membrane
is not derived from the neural retina. The laminin 1 chain in the
internal limiting membrane must, therefore, be derived from one of the
non-neural retinal cells that contact it. Both astrocytes and the
hyaloid vessels contact the internal limiting membrane and have been
suggested as sources of the protein components of the internal limiting membrane (Sarthy and Fu, 1990; Sarthy, 1993).
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Figure 6.
Expression patterns of the RNA encoding laminin
chains in mature rat and human retinae. Fixed sections of rat
(top row; A, C, E) and human
(bottom row; B, D, F) retinae were
probed for the presence of the RNA encoding laminin 1 (A,
B), 2 (C, D), and 3 (E,
F) chains by the use of chain-specific cRNA probes. Only
the RNA encoding the 3 chain is readily detected within the neural
retina, although 2 can be detected as well. GCL,
Ganglion cell layer; INL, inner nuclear layer;
ONL, outer nuclear layer. Scale bar, 25 µm.
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RNA encoding the laminin 2 chain was consistently difficult to
detect in the retina. However, the RNA is detectable in the inner
nuclear layer of the human and, to a lesser extent, the rat retina
(Fig. 6C,D).
In contrast, RNA encoding the laminin 3 chain is readily detected in
both the rat (Fig. 6E) and human (Fig.
6F) retina. Laminin 3 chain RNA is expressed in a
pattern that is similar to that of several other laminin chain RNAs: in
fibers coursing through the outer nuclear layer, at the external
limiting membrane, and in presumed Müller cell end feet in the
ganglion cell layer. The 3 chain is, therefore, a likely component of mature retinal laminins.
Summary of RNA expression
The expression patterns of the laminin chain RNAs detected in the
neural retina demonstrate that RNAs encoding the laminin 3, 4,
2, 2, and 3 chains are expressed in the rat and human retina;
in addition, RNA encoding the laminin 5 chain was detected in human
retina and that encoding the laminin 3 chain was detected in rat
retina. Although slightly different, the basic distribution of all of
these RNAs was the same: primarily within fibers coursing through the
inner and outer nuclear layers. RNAs for the laminin 4 and 2
chains also appear to be present at perinuclear sites in the inner
nuclear layer as well as within the ganglion cell layer. Together,
these data suggest that the Müller cell is the source of these
laminin chain-encoding RNAs; in addition, they support our assertion
that the retina produces two novel laminin trimers: laminin 14 (423) and laminin 15 (523).
Biochemical identification of laminin 14 and 15 in the retina
Our protein and RNA localization data suggest that laminins 5, 14, and 15 are expressed in the neural retina. We have extended these
findings by isolating laminins, and their component chains, from the retina.
Although we can demonstrate the presence of the laminin 3 chain
protein on protein transfer blots of retinal extracts (data not shown),
we have been unable to isolate any heterotrimeric laminins containing
the laminin 3 chain from retinal extracts and have, therefore, been
unable to confirm biochemically the presence of laminins 5 (332) or 13 (323). This may reflect a relative dearth
of these trimers in the retina or a difficulty in extracting them in a
native form. However, we have shown previously that the laminin 2
chain is present in retinal extracts (Hunter et al., 1992b). In
addition, laminins eluted from an anti-laminin 2 chain resin contain
the 4 chain, demonstrating that the 2 chain is associated with at
least this chain in the retina (data not shown). These studies lead us
to ask whether the laminin 14 and 15 trimers are components of the
retinal matrix.
Retinal laminins were isolated from retinal matrix by chromatography on
concanavalin A-Sepharose followed by size fractionation on
polyacrylamide gels. Two high-molecular weight components were selected
from this purification scheme (Fig. 7).
Each was reduced and separated on polyacrylamide gels. The first (band
"A") resolved into components of ~190, 220, and 380 kDa (Fig. 7).
Two of these proteins were identified immunologically as the laminin
2 (190 kDa) and 3 (220 kDa) chains; the third did not react with
any of our anti-laminin antibodies (e.g., anti-4, Fig. 7). The
second high-molecular weight component (band "B") resolved into
components of ~190 and 220 kDa. The 190 kDa component consisted of
both the 4 and 2 chains, and the 220 kDa component was identified
as the 3 chain (Fig. 7). No other chains were detected as components of this complex; therefore, band B consists of the novel laminin composed of 4, 2, and 3 chains, which we term laminin 14.
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Figure 7.
Purification of laminins 14 and 15 from retinal
extracts. Glycoproteins were isolated from retinal matrix by
concanavalin A chromatography, separated on a nonreducing
polyacrylamide gel, and stained with Coomassie blue
(CB). Two components (A, B) were further
separated on reducing polyacrylamide gels and then probed for the
presence of the 4, 2, and 3 chains by protein transfer blots.
Both A and B contain the 2 and 3
chains; B additionally contains the 4 chain. The 3
chain antibody reacts with two bands: the full-length 3 chain and a
faster-migrating protein, a degradation product of the 3 chain (or,
potentially, cross-reactivity with the 2 chain) induced by the
multiple purification steps used to prepare these proteins. Position of
200 kDa marker is shown.
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What is the identity of the third component of band A? The high
molecular weight of this protein suggested that it was a laminin chain, perhaps the laminin 5 chain. However, because our antibodies did not react with this protein, it was excised from a polyacrylamide gel, digested, and microsequenced. The resultant fragments were compared with known laminin sequences, and all were identical to
sequences within the laminin 5 chain (Table
1), demonstrating that this third
component is the laminin 5 chain. Therefore, band B consists of the
novel laminin composed of 5, 2, and 3 chains, which we term
laminin 15.
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Table 1.
Comparison of sequences of peptide fragments of the 380 kDa
component of bovine band A with the deduced amino acid sequence of the
mouse laminin 5 chain
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Expression of laminins 5, 14, and 15 during
retinal development
We have shown previously that the laminin 2 chain is expressed
throughout retinal development (Libby et al., 1996, 1997), at first in
the subretinal space and subsequently in the interphotoreceptor matrix
and the outer plexiform layer. In addition, we have shown recently that
the laminin 2 chain is critical for the proper formation and
function of synapses in the outer plexiform layer (Libby et al., 1999).
We have extended these observations by examining the expression of
potential partners for the laminin 2 chain, that is, components of
laminins 14 and 15, during development of the interphotoreceptor matrix
and outer plexiform layer. In the course of these experiments, we also
found that the components of laminin 5 (332) are expressed in
the developing retina.
We have examined the expression of the components of laminins 14 and 15 as well as those of laminin 5 (the laminin 3, 4, 5, 2,
3, 2, and 3 chains) from postnatal day 0 (P0) through P15. We compared the appearance of these chains with that for two
components of the photoreceptor synapse, dystrophins and
-dystroglycan.
At P0, few rod photoreceptors have differentiated in the rat retina,
and the outer plexiform layer has not yet formed (and, therefore, no
dystrophins are present; Fig. 8). At this
age, the laminin 2 chain is prominently expressed in the subretinal
space, as we have reported previously at embryonic day 21 (Libby et
al., 1996), and in fibers spanning the width of the retina (Fig.
8C), as are the other components of laminins 14 and 15, the
laminin 4 (Fig. 8B), 5 (data not shown), and
3 (Fig. 8D) chains. In addition, the laminin 3,
3, and 2 chains are also expressed in these locations, and an
antiserum against laminin 5 displays similar immunoreactivity (Fig.
8H). These immunohistochemical data are consistent
with the expression of laminins 14 (423), 15 (523),
and 5 (332) at P0. However, none of the laminin chains is
concentrated in the region that will eventually become the outer
plexiform layer.
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Figure 8.
Expression patterns of laminin chains in P0 rat
retina. At PO, the outer plexiform layer has not yet formed, as
demonstrated by the lack of dystrophin (Dys) expression
in the retina (A). However, the laminin 4
(B), 2 (C), and 3
(D) chains are prominently expressed in the
subretinal space (SRS) and in fibers spanning the
neuroepithelium (NE) and penetrating through the inner
plexiform layer (IPL) and ganglion cell layer
(GCL). In addition, the components of laminin 5the 3 (E), 3 (F),
and 2 (G) chainsare expressed at P0, as is
immunoreactivity for laminin 5 (Lam-5;
H). Scale bar, 25 µm.
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At P5, the central portion of the retina has begun to elaborate an
outer plexiform layer in which dystrophins are expressed (Fig.
9A), whereas the peripheral
portion has not (Fig. 9B). At this age, the components of
laminins 14 and 15 are still present in the subretinal space and in
fibers spanning the thickness of the retina (Fig.
9C-H); in addition, in the central portion of the
retina, these laminin chains are beginning to be concentrated in the
developing outer plexiform layer (Fig. 9C,E,G). Components of laminin 5 remain associated with the subretinal space and in fibers
spanning the thickness of the retina (Fig. 9I,J).
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Figure 9.
Expression patterns of laminin chains in central
(top row; A, C, E, G, I) and
peripheral (bottom row; B, D, F, H,
J) P5 rat retina. At P5, the outer plexiform layer has
begun to form in the central retina, as judged by dystrophin
(Dys) expression (A), whereas it
has not yet formed in the peripheral retina, as demonstrated by the
lack of dystrophin expression (B). The laminin
4 (C, D), 2 (E, F), and 3
(G, H) chains are expressed in the subretinal
space (SRS) and in fibers spanning, centrally, the outer
nuclear layer (ONL) and inner nuclear layer
(INL) and, peripherally, the neuroepithelium
(NE). These fibers penetrate through the inner plexiform
layer (IPL) and ganglion cell layer
(GCL). All three chains are also present in the
developing outer plexiform layer (arrows). In
addition, immunoreactivity for laminin 5 (Lam-5) is
present at P5 (I, J). Scale bar, 25 µm.
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At P10, the entire retina has developed an outer plexiform layer
in which dystrophins are prominently expressed (Fig.
10A). Interesting |
TOP
ABSTRACT
INTRODUCTION
