close

Вход

Забыли?

вход по аккаунту

?

959

код для вставкиСкачать
DEVELOPMENTAL DYNAMICS 206:41-26
(1996)
FGF Receptor-1 (fZg)Expression Is Correlated With Fibre
Differentiation During Rat Lens Morphogenesis
and Growth
R.U. DE IONGH, F.J. LOVICU, A. HANNEKEN, A. BAIRD, AND J.W. MCAVOY
Department of Anatomy and Histology and The Sydney Institute for Biomedical Research, The University of Sydney, NSW
2006, Australia (R.U.d.I., F.J.L., J.W.M.); and Department of Cell Biology, The Scripps Institute, La Jolla, California 92037
(A.H., A B . )
Our previous studies indicate an
ABSTRACT
important role for fibroblast growth factor (FGF)
in lens development. Here we study the expression
of the flg variant of FGF receptor 1(FGFR1) during lens development by immunohistochemistry
and in situ hybridisation. FGFRl was expressed
throughout lens development. Prominent FGFRl
immunoreactivity was associated with cell nuclei,
particularly in differentiating lens fibres, suggesting internalisation and nuclear translocation of
the receptor. FGFRl immunoreactivity was also
associated with basolateral membranes of cells in
the equatorial region and at lens sutures. FGFRl
mRNA was only weakly expressed during early
lens morphogenesis but expression increased with
the onset of lens fibre differentiation. Once the lens
acquired its distinct polarity, an anteroposterior
gradient in both protein reactivity and mRNA s i g
nal was evident. Anteriorly, central epithelial cells
showed weak expression for FGFR1, whereas
more posteriorly, in the germinative and transitional zones of the lens where cells maximally proliferate and undergo early stages of fibre differentiation, respectively, expression was significantly
stronger. The anteroposterior gradient of increased expression of FGFRl in the lens coincides
with the previously documented anteroposterior
gradient of FGF stimulation. In lens epithelial explants, FGF stimulation was found to upregulate
FGFRl expression. Such upregulation may be an
important mechanism for generating a high level
of FGF stimulation and ensuring a fibre differentiation response. In postnatal rat lenses, there was
a significant age-relateddecline in FGFRl expression; this correlates with the reduced rate of lens
fibre differentiation with age. Overall, these studies support the hypothesis that FGF and FGFRl
are important for regulation of lens fibre differentiation throughout lens development.
ferentiates into a spheroidal, transparent lens which
has a highly ordered and polarised cytoarchitecture.
The differentiated lens is composed of two distinct cell
types, contained within a thick basement membrane,
the lens capsule; elongated fibres, arranged in an anteroposterior axis, make up the bulk of the lens while a
monolayer of cuboidal epithelial cells covers the anterior surface of the fibres (Fig. 1).Within the lens there
are distinct spatial patterns of proliferation and differentiation which are established during morphogenesis
and are maintained throughout life (Hanna and
O’Brien, 1961; McAvoy, 1978a,b). Epithelial cells in
the germinative region of the lens, just anterior to the
lens equator, undergo proliferation and the progeny of
these divisions migrate, or are displaced, posteriorly
into the transitional zone of the lens where they elongate into fibres (see Fig. 1).
In vitro studies have established that two members
of the fibroblast growth factor (FGF) family (FGF-1
and FGF-2) stimulate rat lens epithelial cells to proliferate, migrate and undergo the structural (Lovicu and
McAvoy, 1989,1992) and molecular (Chamberlain and
McAvoy, 1989; Peek et al., 1992) changes characteristic of fibre differentiation. Moreover, these responses
occurred in a progressive dose-dependent manner; cell
proliferation, migration and fibre differentiation were
induced at low, medium and high concentrations of
FGF, respectively (McAvoy and Chamberlain, 1989).
As the progression of these FGF-induced responses was
similar to the anteroposterior pattern of cell behaviour
in the intact lens, it was hypothesised that an anteroposterior gradient of FGF stimulation may influence
lens polarity and growth patterns (McAvoy and Chamberlain, 1989; Schulz et al., 1993).
Recent studies of FGF distribution in the eye have
provided evidence for anteroposterior variations in the
distribution and activity of FGF in the lens and ocular
media. Cytoplasmic immunoreactivity for FGF-1 was
found predominantly in equatorial regions of the de-
0 1996 Wiley-Liss, Inc.
Key words: Lens development, FGF, FGF receptor, Fibre differentiation
INTRODUCTION
During vertebrate lens development, a sheet of competent ectodermal cells thickens, invaginates and dif0
1996 WILEY-LISS. INC.
Received October 30,1995; accepted February 9, 1996.
Address reprint reauestslcorresDondence to Dr. J. W. McAvov. Department of Anatomy and Histology, University of Sydney, Sydney
NSW Australia 2006.
F.J. Lovicu’s present address is Department of Cell Biology, Baylor
College of Medicine, Houston, TX 77030-3498.
FGFRl EXPRESSION DURING LENS DEVELOPMENT
413
FGFRl cDNA
M6
R803
Fig. 2. Schematic diagram of the structure of FGFRl (adapted from
Johnson and Williams, 1993). The structural features of the extracellular
portion of FGFRl include an amino-terminal signal sequence (ss), three
immunoglobulin-like domains (I, II, Ill), and an acidic box domain (a).
Following a single transmembrane domain (tm), the cytoplasmic portion
of the receptor contains a split tyrosine kinase domain (tk) and a carboxyterminal tail. The regions of the receptor recognised by the anti-FGFRI
antibodies (designated R803, M15 and M6), used for immunohistochemistry, and the region encoded by the cDNA, used to prepare riboprobes,
are indicated.
Fig. 1. Midsagittal section of a 3 day-postnatal (P3)lens. Guboidal
lens epithelial cells cover the anterior surface of the highly elongated fibre
cells (fib). The six regions (I-VI) used for image analysis in this study are
indicated: region I,incorporating the anterior epithelium; II, incorporating
the germinative zone; 111, incorporating the transitional zone; IV, incorporating inner cortical fibres; V, incorporating mature fibres in the deeper
cortex, and VI, incorporating the most mature fibres. aq, aqueous; c,
cornea; cb, ciliary body; i, iris; vit, vitreous. Scale bar: 100 pm.
veloping lens where cells actively proliferate and differentiate (de Iongh and McAvoy, 1992, 1993; Lovicu
and McAvoy, 1993). Differential immunoreactivity for
FGF-2 was found in the lens capsule with the posterior
region staining more intensely than the anterior (de
Iongh and McAvoy, 1993; Lovicu and McAvoy, 1993).
Consistent with this, posterior but not anterior capsule
was found to have FGF-associated biological activity
(Schulz et al., 1993). Furthermore, analysis of the ocular media showed that more FGF was recovered from
vitreous than aqueous (the former bathes the posterior
part of the lens and the latter the anterior part of the
lens; see Fig. 1)and only vitreous had fibre-differentiating activity. Fractionation of vitreous showed that
virtually all of this activity was associated with FGF.
These results are consistent with the hypothesis that
anteroposterior differences in levels of FGF stimulation influence lens polarity and growth patterns. Recent support for this hypothesis comes from studies of
transgenic mice which overexpressed a secreted form of
FGF-1 in the lens (Robinson et al., 1995).In these mice,
anterior epithelial cells differentiated into fibres resulting in a loss of lens polarity.
A requirement for cellular responsivness t o FGF is
the expression of appropriate receptors. FGFs mediate
cellular responses by binding and activating specific
cell surface receptor tyrosine kinases (Johnson and
Williams, 1993). Four distinct but highly homologous
FGF receptor genes (FGFR1-4) have been identified
and within these four genes alternative splicing gives
rise to numerous subtypes. Common features of these
receptors include an extracellular region composed of
two or three immunoglobulin-like domains, a single
transmembrane domain and a cytoplasmic region containing a split tyrosine kinase domain (Fig. 2). High
affinity binding sites for FGF have been described in
lens epithelial cells (Blanquet et al., 1989) and several
studies have shown FGFR to be expressed in the lens of
the mouse (Orr-Urtreger et al., 1991,1993; Marcelle et
al., 1994; Peters et al., 1992, 1993), chicken (Heuer et
al., 1990; Ohuchi et al., 1994) and rat (Wanaka et al.,
1991) a t various stages of development. However, to
date, no detailed study has been carried out on the
temporal and spatial distribution of these receptors in
the developing mammalian lens.
In light of the distinct patterns of cell behaviour and
FGF distribution in the lens, it was important to identify the expression patterns of FGF receptors. To
elucidate further the role of FGFs during lens development, we examined the spatial and temporal expression of FGFRl (fig) during morphogenesis, differentiation and growth of the rat lens. At all ages studied,
correlation of the patterns of lens cell behaviour and
FGFRl expression suggest that FGFRl is linked with
fibre differentiation, further implicating FGF as an important regulator of lens polarity and growth patterns
throughout life.
414
de IONGH ET AL.
CORNEAL EXPRESSION OF TYPES XI1 AND XIV COLLAGEN
RESULTS
synthesis
of
corneal-specific
components of the secondImmunolocalisation of FGFRl
ary
stroma
and stromal compaction. These data are
During
Development
presented in Figure 2A.
In
thetypes
epithelial
cells, we
found that the were
level obof
Two
of FGFRl
immunoreactivity
is highest
dayspepof
mRNA
for type
XIV collagen
tained with
our panel
of antibodies.
Usingat
the9 two
development.
It then
a lower
tide antibodies,
M15decreases
and R803and
(seeremains
Fig. 2), aat predomlevel
at all
the subsequent
inantly
punctate
reactivitydevelopmental
was found in stages
all lenstested
cells.
(Fig.
2A).
Counterstaining with Hoechst dye (data not shown)
In contrast,
stromal
cells
show a different
al(X1V)
of this
reactivity
was intranuclear.
showed
that most
9 to 13, thedomain
stromal
collagen
mRNA
profile. From
In contrast,
an antibody
to thedays
extracellular
of
concentration
normalized
to
G3PDH
remains
relaFGFRl, M6, revealed predominantly membrane-assotively
At day 9, this is approximately equal to
ciatedconstant.
reactivity.
that
of the epithelium.
However,
since
the epithelial
Nuclear
immunoreactivity
for
FGFR1.
Antibod11 days, the
stromal
values
subsequently
decrease
ies R803
and M15 had
similarat
distribution
patterns
of
value
a
t
this
timepoint
is
three
times
higher.
After
day
reactivity in the developing lens at all stages studied.
13,
stromal
cells exhibit
a sharp
in their
content
of
Initial
experiments,
carried
outrise
using
antibody
R803
type
mRNA.and
By paraformaldehyde-fixed
day 17, a stromal cell
and XIV
M15 collagen
on methanolcontains
approximately
more type
XIV collagen
frozen sections,
showed 10-fold
strong specific
immunoreactivmRNA
than
an
epithelial
cell.
ity associated with cell nuclei at all stages of development. Weak cytoplasmic or membrane-associated reactivity XI1
wasCollagen
also observed at some stages. In general,
Type
paraformaldehyde fixation favoured the cytoplasmic
Immunohistochemistry.
antibody
reactivity,
but also resultedA inmonoclonal
greater nonspecific
against
a region
of theFor
amino-terminal
domain
background
staining.
this reason NC3
all the
data
common
both study
the short
andobtained
long form
splicemethanolvariants
were
using
shown into this
5 dayshave
of developof
typefrozen
XI1 collagen
was
used. Atstudies
fixed
sections.
Previous
shown
ment,
the
antibody
indicates
that
type
XI1
that, in the case of FGF localisation, differentcollagen,
antibodlike
collagen
XIV,regimes
appears can
to bemarkedly
distributed
throughout
ies and
fixation
influence
imthe
primary stromapatterns
(Fig. 3A).(Hanneken
By 7 days, after
primunolocalisation
and the
Baird,
mary
1992).stroma has begun to swell, we observe immunoreactivity
for type
XI1
collagen largely
concentrated
in
During early
lens
morphogenesis
(embryonic
day 11;
the
subepithelial
and
subendothelial
regions
of
the
corFig. 3A), the optic vesicle which is the precursor of the
nea
(Fig.
3B). The
immunoreactivity
for
retina
comes
into subendothelial
contact with the
overlying presumpis At
present
throughout
all stages
obtype
tive XI1
lenscollagen
ectoderm.
this stage,
punctate
reactivity
served
(Fig.was
3A-E).
immunoreactivity
is present
for FGFRl
foundThis
ubiquitously
in ectoderm
(includseveral
days
before
the
onset
of
deposition
of
Desceming lens ectoderm), optic vesicle and the surrounding
et’s
membrane a t mesenchyme
about 9 days of
development,
and is
3B). Additional
undifferentiated
(Fig.
probably
to the
interface
Descemet’s
treatmentconfined
of sections
with
Hoechstbetween
dye confirmed
that
membrane
and
the stroma
Discussion).
the
the punctate
reactivity
was(see
associated
with Under
cell nuclei
corneal
epithelium,
the
matrix
undergoes
considerable
(data not shown). The lens placode and optic vesicle
modification
its contentonofembryonic
type XI1 collagen
during
day 12 (El21
to
subsequentlyofinvaginate
development.
detect(Fig.
form the opticInitially,
cup andimmunohistochemically
the lens pit, respectively
able
XI1 collagen
is progressively
from
this
3C). type
F'unctate
nuclear FGFRl
reactivitylost
was
seen
in
that
by 10
days
it is virtually
being
region,
the lenssopit,
optic
cup,
ectoderm
and theabsent,
surrounding
comparable
that 3D).
givenBybyEthe
negative
control
antimesenchymeto(Fig.
l 4 the
lens pit
has closed
body
(Fig.
3C,H).
Procedures
to expose
a vesicle
(Fig.
3E). Elongation
and potentially
differentiato form
masked
epitopes
(seelens
Experimental
Procedures)
didleads
not
tion of the
posterior
cells into primary
fibres
reveal
any masked
immunoreactivity
in this orstrucany
to the lens
vesicle assuming
the characteristic
other
ocular
region
(dataisnot
shown). By
12 days, howture of
the lens
which
maintained
throughout
life;
ever,
subepithelial
for type
XI1with
colof the lensimmunoreactivity
is composed of primary
fibres
the bulk
lagen
returns
to
this
zone
(Fig.
3D).
This
correlates
a monolayer of cuboidal epithelium covering their antemporally
withAtthe
onset
of deposition
of the mature
terior surface.
this
stage,
strong punctate
nuclear
Bowman’swas
membrane
(Fitch
et al.,
1994).cells, particureactivity
found in
all lens
vesicle
Finally,
the region
theprimary
corneoscleral
junction
larly
a t thein
equator
and inofthe
lens fibres
(Fig.
(ocular limbus), the NC3 specific type XI1 antibody re3F).
acts
withfurther
anterior
and posterior
the “scleral
of the
With
development
and(termed
maturation
spur”)
in a temporospatial
that reactivity
is identiof FGFRl
lens ansites
anteroposterior
patternpattern
cal to that
described
(Sugrue,
1991)
(see
Disbecame
evident
withpreviously
E20, postnatal
and
adult
lenses
cussion).
51
showing similar patterns of reactivity (Figs. 4A,C,D
mRNA
and 5). In anterior epithelial cells the nuclei showed
4Efor
andthe
5).
diffuse
and fine
punctate
In isolated
epithelia,
thereactivities
normalized(Figs.
values
At the oflens
the germinative
and
mRNA
bothequator,
forms ofincluding
al(XI1) collagen
is extremely
transitional
there
was diffuse cytoplasmic
reac11 days
of development
(Fig. 2B and
C,
low
a t 9 and zones,
tivity
as
well
as
fine
punctate
nuclear
reactivity.
Comsolid line). This is consistent with the reduced level of
mencing in immunof
the transitional
zone
continuing
subepithelial
luorescence
seenand
a t day
10. The
through
theshort
maturing
cortical
fibre
cells, the
nuclear
rises
sharply,
to peak
at
value
for to
the
form then
reactivity
became
progressively
more
intense
day
15 at a value
about
20-fold higher
than
that at(Figs.
day
4A,ItC,then
D, G
andto 5).
Control
sections
with
about
half this
levelincubated
by day 17.
In
11.
falls
either adsorbed
immune
serumof(Fig.
4H),form
nonimmune
contrast,
the epithelial
content
the long
mRNA
IgG or a4%gradual
bovineincrease
serum albumin
(BSA,todata
not
exhibits
after 11 days,
a level
or no
reactivity.
shown)
littleshort
form
a t 17 days.
equal
to showed
that of the
Membrane
associated
immunoreactivity
for
The
isolated stromal
tissue includes
the adherent enFGFRl. Reactivity
with M6from
appeared
to be predomidothelial
cell layer, which,
our immunofluoresnantlyresults,
associated
with source
membrane
and/or
adjacent
cence
is the likely
of mRNA
for collagen
cytoplasm
lens cells.
Atrecently
embryonic
stages,
weak
type
XII. Inofsupport
of this,
in situ
hybridizareactivity
was
observed
in
epithelial
and
early
differtion has revealed that rabbit corneal endothelial cells,
entiating
fibre cells
(data not
shown). In
postnatal
rats,
but
not stromal
fibroblasts,
synthesize
al(XI1)
collagen
reactivity
wasetstronger
and At
more
defined.
For of
example,
11days
develmRNA
(Zhan
al., 1995).
9 and
lens were
reactive
epithelialthecells
of the P3
opment,
normalized
values
for theweakly
short form
of
along
their
basal
surfaces,
closely
apposed
to
the
lens
type XI1 mRNA (Fig. 2B, dashed line) are vanishingly
6A). "his
reactivity
was is
strongest
a t the
capsulewhile
(Fig. that
substantially
small,
for the
long form
equatorial
Reactivity
was
also apparent
t the
greater
at 9region.
days, but
becomes
negligible
at 11adays.
apical11
surface
of cells
(epithelial-fibre
howdays, both
forms
of the moleculejunction);
are expressed
After
this waslevels
shown
to be
nonspecific
normal
aever,
t significant
with
mRNA
for the when
long form
of
mouse
IgG was
substituted
for the
primary
antibody
the
molecule
becoming
relatively
more
abundant
over
6B). 2B
Strong
specific
reactivity
wasthat
ob(Fig.(Fig.
time
and C,
dashedfibrillar
lines). This
suggests
and posterior
served
a t the anterior
the
subendothelial
type(Fig.
XI16C)
collagen
after 11(Fig.
days6D)
is
lens sutures
where the
endslong
of opposing
fibres
meetwith
and
probably
a mixture
of both
and short
forms,
overlap.
mouse IgG
wastime
substituted
for
the
ratio When
of longnormal
form increasing
with
of developM6, no specific labelling at lens sutures was observed
ment.
(Fig. 6E).
DISCUSSION
Expression
of FGFRl
mRNAis initiated in the emAvian corneal
development
During
Development
bryo
a t about
3.5 days with the deposition of an epithe10
lially
derived
acellular
primary stroma
that is about
During
early
lens development
(E10-E12),
FGFRl
Fm
thick
(for
reviews ubiquitously
see Hay and in
Revel,
1969;
Hay,
mRNA
was
expressed
the optic
primor1980).
By 7).
5 days
development,
periocular
that
dia (Fig.
Priorof to
commencement
of lenscells
morphoof
migrated
centripedally
along
the
posterior
surface
genesis (ElO), uniformly weak signals for FGFRl tranthe
stroma
a continuous
layer.
scripts
were form
detected
in ectodermendothelial
(arrowheads,
Fig.
Shortly
thereafter,
cells invade
the
7A), optic
vesicle andmesenchymal
in the undifferentiated
mesenstroma;
this
coincides
with
stromal
swelling
and
furchyme between the optic vesicle and the ectoderm. In
ther
expansion
continues
to about day 12.
earlymatrix
E l l embryos,
thethat
signal
in the neuroepithelium
The
invading
fibroblasts
secrete a set of
fibrillar
of the
optic vesicle
and diencephalon,
relative
to collaother
V (Linsenmayer
gens,
composed
of to
collagen
types I and
tissues,
appeared
have increased,
particularly
in the
et
al., 1985)
that differs
thoseFig.
deposited
earlier by
7D). Transcripts
basal
ependymal
layer from
(arrows,
the
I and
I1 (Linsenmayer
et Fig.
al.,
wereepithelium,
still foundcollagens
in the lens
ectoderm
(arrowhead,
1990).
Theundifferentiated
complement of mesenchyme.
fibril-associated
7D) and
At collagens
E12, the
also
changes
withfordevelopment,
IX, for
strongest
signal
FGFRl was type
detected
in example,
the early
being
associated
specifically
with
the
primary
lens vesicle. Elongating cells in the posterior stroma
vesicle
(Fitch
al., 1988b),
while
and XIV,
as
andcollagens
anteriorXI1
epithelial
cells
(open et
arrow,
Fig. 7G)
shown
appear
in distinct
in the
secondshowedhere,
strong
signals.
Signalslocations
for FGFRl
expression
ary
stroma.
The fibrils
that
are deposited
into
were
also evident
in the
anterior
margins
of the
the swoloptic
len
immature
secondary
stroma,
which
has
not yet
cup (arrowheads, Fig. 7G) and the optic stalk (arrows,
compacted,
form orthogonally
orientedundifferentiated
bundles, sepaFig. 7G), whereas
the surrounding
rated
by largeshowed
spaces. weak signal for FGFRl tranmesenchyme
By day
the secondary
stroma
compacts
to about
No17,
hybridisation
signal
above
background
was
scripts.
50% of its thickness a t day 12, and has become com-
Fig. 3. lmmunolocalisation of FGFRl during early lens morphogenesis. A, C, E Haematoxylinand phloxine stained sections of El 1, E l 2 and
E l 4 embryos, respectively. 6: At E l l , strong punctate reactivity was
detected in presumptive lens ectoderm, the optic vesicle and the surrounding mesenchyme.This reactivity appeared to be associated primarily with cell nuclei. D: At E12, similar punctate nuclear reactivity was
detected in the lens pit, optic cup, ectoderm and to a lesser extent in the
extraocular mesenchyme. F: At E14, punctate reactivity was detected in
nuclei of all lens vesicle cells, and was particularly strong in cells at the
equator and in the primary lens fibres. Cells of the optic cup, ectoderm
and surrounding extraocular mesenchyme were also labelled. e, ectoderm; lv, lens vesicle; Ip, lens pit; m, mesenchyme; oc, optic cup; ov, optic
vesicle; plf, primary lens fibres. Scale bars: (A,B), 50 pm; (C, D), 60 pm;
(E, F), 100 pm.
416
de IONGH ET AL.
detected when analogous sections at each stage were
hybridised with the control sense probe (Fig. 7C, F, I).
At later stages of ocular development, FGFRl expression appeared to be more intense and tissue-specific
(Fig. 8). Consistent with the immunolocalisation of
FGFRl in the lens, the hybridisation signal also increased anteroposteriorly. For example a t E16, weak
hybridisation signals were detected in anterior lens epithelial cells (arrow, Fig. 8A) and stronger signals for
FGFRl were detected in the equatorial regions of the
developing lens where cells elongate into fibres (arrowheads, Fig. 8A). In more differentiated fibres, in the
centre of the lens, the signal diminished markedly (asterisk, Fig. 8A). Relatively strong hybridisation signals
were also detected in extraocular muscles (open arrow,
Fig. 8A) and in mesenchyme surrounding the optic cup.
The anteroposterior pattern of FGFRl expression in
the lens was more evident a t E20 (Fig. 8C) and persisted in postnatal and adult lenses (data not shown).
Transcripts for FGFRl were found in lens epithelial
cells and early differentiating cortical fibres. A relatively weak signal was found in central epithelial cells,
a moderate signal in the germinative zone, with the
strongest signal in the transitional zone where epithelial cells differentiate into fibres (arrowheads, Fig. 8C).
In the cortical fibres the signal was associated with the
cell nuclei constituting the characteristic bow zone of
the lens. As the cortical fibre cells become more differentiated and are displaced deeper into the lens cortex,
the signal for FGFRl transcripts decreased until no
appreciable signal was detected in the terminally differentiated fibres (Fig. 8C, asterisk).
Quantitation of hybridisation signals using image
analysis confirmed the anteroposterior changes in
FGFRl expression within the E20 lens (see Fig. 9).
Measurements of signal density in six defined lens regions (see Fig. 1) showed a progressive increase in
FGFRl expression from region I to region I11 and a
progressive decline in deeper regions of the lens (regions IV to VI) where fibres are more mature and undergoing terminal differentiation. There was an approximate doubling of signal between regions I and I11
(P < 0.001) and a progressive significant decrease to
region VI (P < 0.03). This pattern was similar at all
ages analysed (data not shown for P3, P21, P100).
In addition to spatial changes in FGFRl expression,
temporal changes were also observed during lens development. Whilst the signal appeared to increase during early development (E10-E20), there was a marked
age-related decrease in signal from P3 to PlOO (Fig.
10). Quantitation of hybridisation signals in region I11
of P3, P21 and P l O O rat lenses (arrowheads, Fig. 10)
confirmed that there was a significant decrease
(P<0.05) in FGFRl expression a t each age.
Effect of FGF-2 on FGFRl Expression in
Lens Explants
Lens epithelial cell explants can be induced to differentiate into fibres by addition of FGF. To determine if
fibre differentiation in vitro was associated with
changes in FGFR1 expression, we quantified the hybridisation signal for FGFRl mRNA by grain counting
in sections of 3-day-old lens explants cultured for four
days with or without FGF-2 (Table 1). In control explants, which remained as a monolayer of epithelial
cells, there were relatively low levels of FGFRl expression, with the peripheral region showing significantly
greater FGFRl expression (P < 0.001) than the central
region of the explant. Similar levels of expression were
found in central (3.9 * 0.2 grains/100 pm2) and germinative (5.8 f 0.4 grainst100 p,m2) regions of 3-day-old
rat lenses in situ, which had been sectioned and processed in parallel with the explants; these regions are
equivalent to central and peripheral regions of lens
explants, respectively. This indicates that the culture
conditions did not affect receptor expression. Lens explants cultured with FGF-2 increased in thickness due
to multilayering and cell elongation, which are morphological changes characteristic of early fibre differentiation (Lovicu and McAvoy, 1989). After 4 days
exposure to FGF there was a three- to fourfold
increase (P < 0.005) in density of hybridisation signal
in the peripheral and central regions compared to control explants. The slight difference in levels of expression between the peripheral and central regions of neonatal rat lens explants cultured with FGF-2 was not
significant.
DISCUSSION
In the present study we have investigated the spatiotemporal expression patterns of FGFRl during lens
morphogenesis and differentiation using immunohistochemical and in situ hybridisation techniques. There
was a strong concordance between the FGFRl protein
and mRNA localisation patterns.
The antibodies, R803 and M15, were raised against
distinct peptide regions of the intracellular and extracellular domains of the receptor, respectively. The
FGFRl reactivity of both antibodies was predominantly in the cell nuclei, although weak cytoplasmic
reactivity was also detected. Nuclear reactivity for
Fig. 4. lmmunolocalisationof FGFRl in eyes from E20 (A) P3 (E-G),
P21 (C) and PlOO (D) rats. A: Adistinct anteroposterior pattern of FGFRl
reactivity was evident at E20. Cells in the germinative zone (gz) showed
fine punctate reactivity whereas cells in the transitional zone (tz) and
cortical fibres showed a more intense and coarse punctate reactivity. In
general, the punctate reactivity appeared to be primarily associated with
cell nuclei and this was most obvious in the maturing fibres. Diffuse
cytoplasmic and fine punctate reactivity was also found in the ciliary body
and iris. Weak reactivity was also detected in the ganglion cell and neuroblast layers of the retina. B: Similar section to that in A, stained with
haematoxylin and phloxine. A similar anteroposterior pattern of reactivity
to that described for E20 was found in the P21 (C) and PlOO lens (D).
E-G: Higher magnification of cells in the anterior epithelium (E), equatorial region (F) and early fibres (G) of the P3 lens showing FGFRl
reactivity. H: No specific reactivity was observed with adsorbed control
serum for R803 (Pi00 lens). cb, the ciliary body: g, ganglion cell layer; i,
iris. Scale bar: (A-D,H), 100 km; (E-G) 40 km.
418
de IONGH ET AL.
Fig. 5. lmmunolocalisation of FGFRI in P3 rat eye using antibody
M15. The distribution pattern of FGFRl reactivity was similar to that
obtained with R803. Reactivity was predominantly associated with lens
cell nuclei; weakly in the lens epithelium and increasing anteroposteriorly
with the strongest signal detected in differentiating fibres. The inset
shows the fine punctate nuclear reactivity obtained with MI 5 in early lens
fibres. Scale bar: 100 pm; inset, 18 ym.
FGFRl has also been demonstrated recently in foetal
cardiac myocytes using a peptide antibody similar to
R803, raised against the carboxy terminus of f l g (Engelmann et al., 1993). Such nuclear localisation suggests that the whole receptor may be internalised and
translocated to the nucleus. It has been demonstrated
that, after FGF-1 stimulation, FGFR-1 trafficks to or
near the nucleus as a cell cycle-regulated event (Prudovsky et al., 1994) similar to that observed for FGF-1
(Zhan et al., 1993). There is also some evidence that
covalent complexes of FGF and receptor components
are internalised and translocated to the nucleus (Shi et
al., 1991a, b). In agreement with this we have shown
that one of the FGFRl ligands, FGF-1, is also localised
in lens cell nuclei in a similar distribution to that of
FGFRl (Lovicu and McAvoy, 1993). It has been suggested that internalisation and nuclear translocation
of peptide ligands andlor their receptors may partici-
pate directly in regulation of gene expression (Jans,
1994).
During early lens morphogenesis (ElO-E14), FGFRl
was expressed in both lens and retinal precursors, suggesting that FGF may play a role in the inductive interactions and subsequent differentiation of both these
tissues. Similarly, other studies have documented
FGFRl expression in ectodermal and mesodermal lineages of early mouse (Yamaguchi et al., 1992) and rat
(Wanaka et al., 1991) embryos and in presumptive lens
and neural retina of chick (Heuer et al., 1990; Ohuchi
et al., 19941, mouse (Orr-Urtreger et al., 1991) and rat
(Wanaka et al., 1991) eyes. Together with previous
studies which localised FGF-1 protein (de Iongh and
McAvoy, 1993) and mRNA (unpublished observations)
in the developing lens placode and retinal disc, the
present findings are consistent with the suggestion
that FGF-1 plays a role in the formation of these structures and this may involve an autocrine mechanism. In
agreement with this, both FGF-1 and FGF-2 have been
shown to induce proliferation and differentiation responses in cultured neuroepithelial cells (Murphy et
al., 1990) and embryonic retinal cells (Guillemot and
Cepko, 1992; Lillien and Cepko, 1992). However, to
date, no studies on the influence of FGF on embryonic
lens cells have been carried out.
We have previously proposed that an anteroposterior
gradient of increased FGF stimulation is involved in
the maintenance of lens polarity and growth patterns
(McAvoy and Chamberlain, 1989; Schulz et al., 1993).
Clearly, this could be determined by the anteroposterior gradient of increased FGF distribution and activity that we have identified in the lens and the ocular
media (see Introduction). However, the extent to which
FGF stimulates a cell also depends on the level of
FGFR expression. The present study has demonstrated
an anteroposterior gradient of FGFRl expression in
the intact lens with weak expression of both protein
and mRNA in the central epithelium anteriorly, but
increasingly strong expression in the germinative and
transitional zones where cells maximally proliferate
and undergo early stages of differentiation, respectively. The increased nuclear reactivity for FGFRl,
shown by antibodies R803 and M15, in the germinative
and transitional zones may reflect increased internalisation and nuclear translocation of the receptor during
cell proliferation and early fibre differentiation. Increased expression of FGFRl in these regions, indicated by both the increased membrane-associated reactivity, shown by M6, and increased signal for mRNA,
may indicate upregulation of FGFRl expression during
cell proliferation and early fibre differentiation. Increased membrane-associated reactivity, shown by M6,
was also detected in outer cortical fibres at the lens
sutures. The significance of this reactivity at the lens
sutures is not clear at present.
Interestingly, although FGFRl protein persisted in
nuclei of terminally differentiating fibres, the mRNA
gradually decreased in mature fibres (regions V and
FGFRl EXPRESSION DURING LENS DEVELOPMENT
Fig. 6. lmmunolocalisationof FGFR in neonatal rat lens using monoclonal antibody M6. A: Lens epithelial cells showed specific reactivity
along their basal surface which closely appose the lens capsule (arrowheads). 8 : If primary antibody was substituted with normal mouse IgG,
there was no labelling of basal cell surfaces but there was nonspecific
419
labelling (curved arrow) at the apical surface of cells (epithelial-fibrejunction). C and D: At both anterior (C) and posterior (D) poles of the lens,
strong fibrillar reactivity was observed at lens sutures. When normal
mouse IgG was substituted for M6,no labelling was observed (E; posterior suture). Scale bars: (A, B), 100 pm; (C-E), 65 pm.
420
de IONGH ET AL.
Fig. 7. FGFRI expression during early lens development (E10-E12).
The darkfield micrographs show hybridisation signals obtained with antisense (A, D, G) and corresponding sense (C, F, I)FGFRI probes. B, E
and H are brightfield micrographs of haematoxylin-stainedsections in A,
D and G respectively. At E l 0 (A-C) uniformly weak signal was detected
in presumptive lens ectoderm (arrowheads), optic vesicle and diencephalon, and in the surrounding mesenchyme. At E l 1 (D-F) there was no
change in the ectodermal signal (arrowhead) but a slightly increased
signal was detected in the basal ependymal layer of the neuroepithelium
of the diencephalon (arrows) and optic vesicle which includes me reiinai
disc. At E l 2 (G-I), strong signal was found in the early lens vesicle (open
arrow), margins of the optic cup (arrowheads) and in the ependymal layer
of the optic stalk (arrows). No specific hybridisation signal was evident
with the sense probe at any age (C, F, I). d, diencephalon; e, ectoderm;
Iv, lens vesicle; m, mesenchyme; oc, optic cup; os, optic stalk; ov, optic
vesicle; rd, retinal disc. Scale bars: (A-C), 50 pm; (D-F), 100 pm; (G-I),
100 wm.
FGFRl EXPRESSION DURING LENS DEVELOPMENT
Fig. 8. FGFRl expression at El6 and E20. A At E16, strong signal
for FGFRl was found in the equatorial lens where fibres differentiate
(arrowheads) but only weak signals were detected in the anterior epithelium (arrow) and in more differentiated fibres in the centre of the lens
(asterisk). Strong signals were also found in extraocular muscles (open
arrow) and extraocular mesenchyme which may include the retinal pigmented epithelium. B: Brightfield micrograph of section shown in A. C: In
the E20 lens there was a distinct anteroposterior pattern of expression
with weak signals in the anterior epithelium which increased towards the
42 1
lens equator in the germinative zone. The strongest signals were in the
transitional zone of the lens (arrowheads). Strong signals were also detected in ciliary body (arrow), dermis and hair follicles (curved arrow) of
the eyelid and in mesenchyme of choroid and sclera. Weaker signals
were detected in the cornea and stroma of the eyelids. D: Brightfield
micrograph of same section shown in C, stained with haematoxylin. c,
cornea; cs, choroid-sclera; e, epidermis; g, ganglion cell layer; I,lens; le,
lens epithelium; nl, neuroblast layer. Scale bars (A, B), 250 pm; (C, D),
300 pm.
422
de IONGH ET AL.
TABLE 1. Effect of FGF-2 on FGFR-1 Expression in
Lens Epithelial Explants*
Treatment
Control
FGFS
Grain density (graindl00 pm2)
Peripheral region
Central region
3.8 ? 0.2 (36)
5.0 0.2 (50)**
17.0 2 0.6 (19)*
15.4 1.0 (22)*
*
*
*
S.E.M. The number of fields
aData presented as mean
countedkreatment is indicated in parentheses.
*Significantly greater than corresponding control value
(P<0.005).
**Significantly greater than corresponding value for the central region (P<O.OOl).
E
9a,
.->
-Qa,
.I-
U
Lens Regions
Fig. 9. Quantitativeanalysis of FGFRl expression in different regions
of the E20 lens. Using Tracor Northern Image Analysis System on darkfield images, the density of FGFRl hybridisation signals in six defined
regions of the lens ( 1 4 1 , see Fig. 1) were measured. Values were normalised with respect to the mean value for lens region I. Each bar represents the mean ? S.E.M. of 4-7 determinations from separate sections. A similar pattern of FGFRl expression was found in lenses from
P3, P21 and PI00 rats.
VI) suggesting downregulation of FGFRl mRNA (possibly by decreased synthesis andfor increased degradation) during terminal differentiation. Another interpretation of the decreased signal density in the mature
fibres is dispersement of the mRNA in these highly
elongated cells. However, this would seem a less likely
explanation, as the cells in region 111, which have undergone substantial cell elongation, have the highest
signal density.
Support for such a gradient of FGFRl expression in
the lens contributing to the maintenance of the anteroposterior gradient of FGF stimulation is provided by in
vitro studies (Lovicu and McAvoy, 1992; Richardson et
al., 1992) of lens epithelial explant responses to FGF.
These studies showed that cells from the periphery of
lens epithelial explants, which incorporates the germinative zone (region I1 in Fig. l), were more responsive
to FGF than cells from the centre of the explant (region
I in Fig. 1).In the present study, in situ hybridisation
with FGFRl probe on sections of lens epithelial explants showed that cells in the peripheral region of
control explants had significantly greater FGFR
mRNA expression than in the central region. Thus, a
higher level of FGFRl expression in the cells of the
peripheral region (germinative zone) may underlie
their greater responsiveness to FGF.
The fact that the anteroposterior increase in FGFRl
expression coincides with the previously documented
anteroposterior increase in FGF activity in the lens
and ocular media led us to propose that FGF stimula-
tion itself may upregulate expression of FGFRl. To investigate this we treated lens explants with a fibredifferentiating dose of FGF-2 and showed that, after 4
days treatment, FGFRl expression was indeed enhanced in both central and peripheral regions of the
explants. These data suggest that when lens cells are
exposed to high concentrations of FGF, such as would
occur when they are exposed t o vitreous, they upregulate expression of FGFR1, thus potentially increasing
their sensitivity to FGF stimulation. Such upregulation of FGFRl by FGF in the transitional zone of the
lens would amplify the already existing gradient of
FGF stimulation present in the eye and may be an
important mechanism for generating a high level of
FGF stimulation and ensuring a fibre differentiation
response.
As well as the regional differences in FGFRl expression, this study revealed a significant age-related decline in FGFRl expression in postnatal rat lenses. This
may be related to the previously reported age-related
decline in the ability of epithelial explants to respond
to FGF (Richardson et al., 1990, 1992; Lovicu and McAvoy, 1992). Other studies have reported substantially
greater levels of FGF receptors in embryonic compared
with adult rodent tissues (Olwin and Hauschka, 19891,
as well as a decline in FGF receptor levels during chick
ocular development (Olwin and Hauschka, 1990). The
age-related decline in FGFR expression in postnatal
rat lenses coincides with the reduced rate of fibre differentiation that occurs with age (Hanna and O'Brien,
1961; Cenedella, 1989). Overall, these results suggest
that the responsiveness of lens cells to FGF, both
within the lens and with aging, may be influenced, at
least in part, by the level of FGFRl expression.
FGF has been shown to induce different responses
(proliferation, migration, differentiation) in lens cells
depending upon its concentration (McAvoy and Chamberlain, 19891.However, it is still not known how these
different responses are elicited by FGF. As different
FGFRs display different ligand binding affinities
(Johnson and Williams, 1993) as well as the potential
for activating different signalling pathways (Vainikka
et al., 1992; Wang et al., 1994), it is possible that the
different responses of lens cells to FGF may be related
to the differential expression of FGF receptors. This
FGFRl EXPRESSION DURING LENS DEVELOPMENT
423
study clearly shows that expression of FGFRl changes
in relation to the lens fibre differentiation response.
Therefore, it will be important in future work to determine how expression of the other members of the FGF
receptor family relate to patterns of lens cell behaviour.
EXPERIMENTAL PROCEDURES
Animals
At various stages of gestation (E10, E l l , E12, E14,
E l 6 and E20), pregnant Wistar rats were euthanased
by C02 asphyxiation or an overdose of barbiturate, and
the uterine horns were removed. Embryos were removed from the uterine horns and dissected free of
extra-embryonic membranes in cold (4°C) phosphatebuffered saline (PBS), pH 7.4. Neonatal (3-day postnatal; P3), weanling (P21) and adult (P100) rats were
euthanased by C02 asphyxiation and their eyes removed. Dissected embryos and eyes were immersed in
Tissue Tek OCT compound (Miles Inc., Elkhart, IN)
and frozen in isopentane cooled by liquid nitrogen.
Specimens were stored in liquid nitrogen until sectioned.
All procedures involving animals were in accordance
with the National Health and Medical Research Council (Australia) guidelines and The Association for Research in Vision and Ophthalmology Handbook for the
Use of Animals in Biomedical Research (USA).All protocols were approved by the Animal Ethical Review
Committee of the University of Sydney, Australia.
Lens Explant Culture
Untrimmed lens explants from neonatal rats (P3)
were prepared as described previously (McAvoy and
Fernon, 1984) and cultured in Medium 199 with Earle’s
salts (Cytosystems, Sydney, Australia), supplemented
with 0.1%bovine serum albumin (Sigma, Sydney, Australia), 0.1 Fgiml L-glutamine, 50 IUlml penicillin, 50
p.g/ml Streptomycin and 2.5 Fg/ml Fungizone (all from
Cytosystems, Sydney, Australia), with or without
FGF-2 (40 ng/ml) for four days. Previous in vitro studies (Lovicu and McAvoy, 1989) have shown that after 4
days culture with FGF-2, explanted neonatal lens epithelial cells elongate and undergo early stages of fibre
differentiation. At the end of the culture period, explants were quickly rinsed in ice-cold sterile PBS, im-
Fig. 10.
Fig. 10. Age-related decrease in FGFRl expression in postnatal rat
eyes. Sections of whole eyes from P3 (A), P21 (6)and PlOO (C) rats,
hybridised with anti-sense FGFRI probes under identical conditions,
were exposed to the same autoradiographic film. Signal for FGFR1
mRNA was demonstrated in the lens (I) and surrounding ocular tissues,
particularly in the ciliar body and iris, and the intensity clearly diminished
with age. At all ages, strongest expression for FGFRl mRNA in the lens
was in the cells of the transitional zone at the equatorial region (arrowheads). A strong signal was detected in the P3 lens (A); however, only a
faint trace of signal was detected in the PlOO lens (C). In the older
animals, signal present in the centre of the lens (the lens nucleus) was
nonspecific. Scale bar: 1.5 mm.
424
de IONGH ET AL.
mersed in OCT compound and frozen as described
above.
FGFRl Probes
FGFRl antibodies. A panel of three anti-FGFRl
antibodies (R803, M15, M6) were used to localise
FGFRl immunoreactivity (see Fig. 2). R803 was an
affinity-purified antiserum raised in rabbits against a
synthetic amino acid fragment corresponding to 17
amino acids (806-822; CLPRHPAQLANGGLKRR)a t
the carboxy terminal of human FGFR1. This antibody
is specific for FGFRl (Gonzalez et al., 1996). M15 was
a monoclonal antibody raised against a synthetic
amino acid fragment corresponding to 20 amino acids
(240-259; NHTYQLDVVERSPHRPILQA) between
immunoglobulin-like (Ig) domains I1 and I11 of chicken
FGFRl. By immunoblotting, this antibody has been
shown to recognise the chicken form of FGFRl (cek-1)
and the recombinant protein corresponding to the extracellular domain of human FGFRl (Hanneken et al.,
1994, 1995). M6 was a monoclonal antibody raised
against a baculovirus-expressed recombinant protein
corresponding to the extracellular domain of human
FGFRl. It recognises high molecular weight FGFRl
from 3T3 and BHK cells and low molecular weight
truncated FGFRl receptors (FGF binding proteins)
from bovine calf serum but also has low levels of crossreactivity with FGFR2 and FGFR3 (Allen and Maher,
1993; Hanneken et al., 1994, 1995).
FGFRl cDNA. A 300-bp rat FGFRl (flg)cDNA, isolated from a rat embryonic cDNA library (Wanaka et
al., 1990), was used for in situ hybridisation experiments (see Fig. 2). This cDNA has been characterised
(Wanaka et al., 1990) and used previously to identify
FGFRl transcripts in rat tissues (Wanaka et al., 1990,
1991).The cDNA was linearised using Eco RI and Kpn
I to obtain DNA templates. Using a RNA transcription
kit (Stratagene, La Jolla, CA) and 35S-UTP (Amersham, Sydney, Australia), radiolabelled antisense and
sense RNA probes were transcribed from these templates with T3 and T7 RNA polymerases, respectively.
nus, Hawthorn, Vic. Australia): sheep anti-mouse IgG
(for monoclonal antibody) or sheep anti-rabbit IgG (for
polyclonal antibody). Occasional sections were also
treated with 1 kg/ml bisbenzimide (Hoechst dye, Calbiochem, La Jolla, CA) to label nuclei. Sections were
then rinsed with PBS-BSA and mounted. Representative sections were subsequently stained with haematoxylin and phloxine.
In Situ Hybridisation
Frozen sections of ocular primordia, eyes and explants were mounted on poly-L-lysine-coated gelatinised slides and fixed in 2.5% paraformaldehyde in 0.1
M phosphate buffer, pH 7.4 for 20 minutes. Sections
were rinsed in distilled water and then processed for in
situ hybridisation based on a protocol described by
Simmons et al. (1989). Sections were hybridised with
the radiolabelled probe (specific activity lo7 cpdml) at
approximately 55°C for 16 hours. After hybridisation,
sections were digested with RNase A (20 kg/ml; Sigma,
St. Louis, MO) a t 37°C for 30 minutes and rinsed in
decreasing salt solutions for increasing stringency
(from 2 x SSPE to 0.1 x SSPE, where 1 x SSPE = 15
mM NaCl, 1 mM NaH,PO,, 1 mM EDTA) supplemented with 1 mM dithiothreitol. Following a final
high stringency wash (0.1 x SSPE, 45 minutes a t
65"C), slides were dehydrated and dried for autoradiography.
To initially assess the strength of hybridisation signal, sections were exposed for 5 days to autoradiographic film @-Max Hyperfilm, Amersham, Sydney,
Australia) which was developed according to manufacturer's instructions. Slides were then coated with a fine
film of NTB-2 emulsion (Kodak, Sydney, Australia)
and stored in light-tight boxes with dessicant at 4°C.
Based on the signal obtained on the initial autoradiograph, slides were exposed for 2-3 weeks, after which
they were developed (D-19, Kodak), rinsed and stained
with haematoxylin.
Photography
Sections were photographed using a Leitz Dialux 20
microscope equipped with normal, darkfield and epiillumination. Immunofluorescence micrographs were
photographed with 400 ASA film (T-Max, Kodak)
pushed to 1,600 ASA during processing and brightfield
micrographs were photographed on 50 ASA film
(Pan-F, Kodak), processed according to manufacturer's
instructions. In situ hybridisation slides were viewed
by darkfield illumination and photographed using 400
ASA film (T-Max, Kodak) processed according to manufacturer's instructions.
Immunofluorescence
Frozen sections through the ocular primordia (E10E20) and eyes (P3-PlOO) were mounted on gelatinised
slides, fixed briefly in cold (- 20°C) methanol and allowed to dry at -20°C. Occasional sections were also
fixed using 2.5% paraformaldehyde in 0.1 M phosphate
buffer (pH 7.4) for 20 minutes a t room temperature
followed by several PBS rinses. Sections were incubated for 25 minutes in PBS containing 0.1% BSA
(PBS-BSA),supplemented with 3% normal goat serum,
to block nonspecific binding. All antisera were diluted
with this goat serum-supplemented PBS-BSA. Sections Image Analysis of FGFRl Hybridisation Signal
Image analysis was used to compare the expression
were incubated with the primary antibodies overnight
at 4°C in a humidified chamber. After incubation, sec- of FGFRl in different regions of the intact lens from
tions were rinsed in PBS-BSA and incubated for one rats of different ages and in lens explants cultured with
hour a t room temperature with f luoroscein isothiocy- or without FGF.
Intact lens. To quantify FGFRl expression in differanate (FITCI-conjugated secondary antibodies (Sile-
FGFRl EXPRESSION DURING LENS DEVELOPMENT
ent regions of the lens, the intensity of hybridisation
signal was measured in six distinct regions (I-VI, see
Fig. 1)of lens sections. Darkfield images were captured
directly from slides by a sensitive videocamera (slowscan, Dage/MTI Inc., Michigan City, IN) and the hybridisation signals were measured as an optical density
using a Tracor Northern Image Analysis system (Trator Inc., Middleton, WI). For each region, the area that
contained specific signal was delineated using the cursor and the optical density of that area was measured.
This technique offered a high level of resolution. As
hybridisation and autoradiography conditions may
have varied between experiments, values were first
corrected for nonspecific background measurements
and the data from each experiment were then normalised with respect to the mean count for region I from
that experiment. Data from a t least three sections from
each of three different experiments were then pooled
and analysed. Values obtained for each of the different
regions were compared using one-way analysis of variance and Student’s t-test.
To compare the intensity of FGFRl hybridisation
signal between lenses from rats of different ages, it was
important that all samples be processed under identical conditions. To do this, sections of whole eyes from
P3, P21 and PlOO rats, all hybridised with anti-sense
FGFRl probes under identical conditions, were exposed to the same autoradiographic film for a period of
5 days. The density of hybridisation signal in region I11
(see Fig. 1)of the P3, P21 and PlOO lenses was determined by scanning the autoradiographic films with a
personal HeNe laser densitometer (Molecular Dynamics, Sunnyvale, CA; wavelength 632.8 nm, resolution
50 pm) and quantifying the signal by image analysis
software (ImageQuant, Molecular Dynamics). Data
were compared using one-way analysis of variance and
Student’s t-test as above.
Lens explants. To examine the effects of FGF on
FGFRl expression in different regions of lens explants,
the intensity of hybridisation signal was measured by
manual counting of exposed silver grains directly from
darkfield images on the microscope. Representative
transverse sections through the centre of lens explants
from each treatment group were processed in parallel
to ensure that hybridisation and autoradiography conditions were similar for each section. For each section,
the lens explant was divided into central and peripheral regions (see Richardson et al., 1993): (i)the central
region comprised the central third of the section and
included lens epithelial cells from the anterior pole of
the lens (equivalent to region I; see Fig. 1)and, (ii)the
peripheral region comprised the remaining two-thirds
of the section and included cells from the germinative
zone (equivalent to region 11). An eyepiece graticule
was used to divide each region into equal-sized fields
which corresponded to a 21 x 21 Fm field when used in
conjunction with a 40 x objective and the numbers of
silver grains per field were counted. The numbers of
silver grains were counted in every field which in-
425
cluded cells, with correction for partial filling of the
field where necessary. For each treatment group, grain
counts were made from a t least four different sections
from two different explants. Counts were corrected for
background which was quantified from regions of the
slide adjacent to the sections; analysis of variance
showed that there was no significant difference between different slides. One-way analysis of variance
and Student’s t-test were used to compare values obtained from different regions of explants cultured with
or without FGF.
ACKNOWLEDGMENTS
This work was supported by grant R01 EY03177 to
J. McA from the National Eye Institute, Department of
Health, Education and Welfare, USA, and by a grant
from the National Health and Medical Research Council (NH & MRC), Australia. R.de I. acknowledges the
support of a NH & MRC Biomedical Research Scholarship and a travelling scholarship from the Faculty of
Medicine, University of Sydney, Australia. F.J.L. acknowledges the support of a Postdoctoral Research Fellowship from the Medical Foundation, University of
Sydney, Australia. We gratefully acknowledge Dr. P.A.
Maher and Dr. D. Lappi for providing FGFRl antibodies and Dr. J. Milbrandt for the rat FGFRl cDNA. Roland Smith is thanked for technical assistance with
photography. We also thank The University of Sydney
Electron Microscope Unit for the use of their image
analysis facilities and Dr. C. Chamberlain for critical
reading of the manuscript.
REFERENCES
Allen, L.E., and Maher, P.A. (1993) Expression of basic fibroblast
growth factor and its receptor in an invasive bladder cell carcinoma
cell line. J. Cell Physiol. 155:368-375.
Blanquet, P.R., Patte, C., Fayein, N., and Courtois, Y. (1989) Identification and isolation from bovine epithelial lens cells of two basic
fibroblast growth factor receptors that possess bFGF-enhanced
phosphorylation activities. Biochem. Biophys. Res. Commun. 160:
1124-1 131.
Cenedella, R.J. (1989) Aging and rates of lens cell differentiation in
vivo, measured by a chemical approach. Invest. Ophthalmol. Vis.
Sci. 30575-579.
Chamberlain, C.G., and McAvoy, J.W. (1989) Induction of lens fibre
differentiation by acidic and basic fibroblast growth factors. Growth
Factors 1:125-134.
de Iongh, R., and McAvoy, J.W. (1992) Distribution of acidic and basic
fibroblast growth factors (FGF) in the foetal rat eye: Implications
for lens development. Growth Factors 6:159-177.
de Iongh, R., and McAvoy, J.W. (1993) Spatio-temporal distribution of
acidic and basic FGF indicates a role for FGF in rat lens morphogenesis. Dev. Dynam. 198:190-202.
Engelmann, G.L., Dionne, C.A., and Jaye, M.C. (1993) Acidic fibroblast growth factor and heart development: Role in myocyte proliferation and capillary angiogenesis. Circ. Res. 72:7-19.
Gonzalez, A.M., Berry, M., Maher, P.A., Logan, A,, and Baird, A.
(1996) A comprehensive analysis of the distribution of FGF-2 and
FGFR-1 in the rat brain. Brain Res. (In press).
Guillemot, F., and Cepko, C.L. (1992) Retinal fate and ganglion cell
differentiation are potentiated by acidic FGF in an in uitro assay of
early retinal development. Development 114:743-54.
Hanna, C., and O’Brien, J.E. (1961) Cell production and migration in
the epithelial layer of the lens. Arch. Ophthalmol. 66:103-107.
426
de IONGH ET AL.
Hanneken, A,, and Baird, A. (1992) Immunolocalization of basic fibroblast growth factor: Dependence on antibody type and tissue
fixation [letter]. Exp. Eye Res. 54:lOll-1014.
Hanneken, A,, Ying, W., Ling, N., and Baird, A. (1994) Identification
of soluble forms of the fibroblast growth factor in blood. Proc. Natl.
Acad. Sci. USA 91:9170-9174.
Hanneken, A., Maher, P., and Baird, A. (1995) High affinity immunoreactive FGF receptors in the extracellular matrix of vascular
endothelial cells-implications for the modulation of FGF2. J . Cell
Biol. 128:1221-1228.
Heuer, J.G., von Bartheld, C.S., Kinoshita, Y., Evers, P.C., and Bothwell, M. (1990) Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system. Neuron 5:283-296.
Jans, D.A. (1994) Nuclear signaling pathways for polypeptide ligands
and their membrane receptors. FASEB J . 8:841-847.
Johnson, D.E., and Williams, L.T. (1993) Structural and functional
diversity in the FGF receptor multigene family. Adv. Cancer Res.
6O:l-41.
Lillien, L., and Cepko, C. (1992) Control of proliferation in the retina:
Temporal changes in response to FGF and TGF-a. Development
115:253-266.
Lovicu, F.J., and McAvoy, J.W. (1989) Structural analysis of lens
epithelial explants induced to differentiate into fibres by fibroblast
growth factor (FGF). Exp. Eye Res. 49:479-494.
Lovicu, F.J., and McAvoy, J.W. (1992) The age of rats affects the
response of lens epithelial explants to fibroblast growth factor: An
ultrastructural analysis. Invest. Ophthalmol. Vis. Sci. 33:26292278.
Lovicu, F.J., and McAvoy, J.W. (1993) Immunolocalisation of acidic
FGF, basic FGF and heparan sulphate proteoglycan in the rat lens:
Implications for lens polarity and growth patterns. Invest. Opbthalmol. Vis. Sci. 34:3355-3365.
Lovicu, F.J., de Iongh, R., and McAvoy, J.W. (1996) Expression of
FGF-1 and FGF-2 mRNA during lens morphogenesis, differentiation and growth. (unpublished).
Marcelle, C., Eichmann, A., Halevy, O., BrBaut, C., and Le Douarin,
N.M. (1994) Distinct developmental expression of a new avian fibroblast growth factor receptor. Development 120:683-694.
McAvoy, J.W. (1978a) Cell division, cell elongation and distribution of
a-,p- and y-crystallins in the rat lens. J . Embryol. Exp. Morphol.
44:149-165.
McAvoy, J.W. (197813) Cell division, cell elongation and the co-ordination of crystallin gene expression during lens morphogenesis in
the rat. J . Embryol. Exp. Morphol. 45:271-281.
McAvoy, J.W., and Chamberlain, C.G. (1989)Fibroblast growth factor
(FGF) induces different responses in lens epithelial cells depending
on its concentration. Development 107:221-228.
McAvoy, J.W., and Fernon, V.T.P. (1984) Neural retinas promote cell
division and fibre differentiation in lens epithelial explants. Curr.
Eye Res. 3:827-834.
Murphy, M., Drago, J., and Bartlett, P.F. (1990) Fibroblast growth
factor stimulates the proliferation and differentiation of neural precursor cells in uitro. J . Neurosci. Res. 25:463-475.
Ohuchi, H., Koyama, E., Myokai, F., Nohno, T., Shiraga, F., Matsuo,
T., Matsuo, N., Taniguchi, S., and Noji, S. (1994) Expression patterns of two fibroblast growth factor receptor genes during early
chick eye development. Exp. Eye Res. 58:649-658.
Olwin, B.B., and Hauschka, S.D. (1989) Cell type and tissue distribution of the fibroblast growth factor receptor. J . Cell Biochem. 39:
443-454.
Olwin, B.B., and Hauschka, S.D. (1990) Fibroblast growth factor levels decrease during chick embryogenesis. J . Cell Biol. 110:503-509.
Orr-Urtreger, A,, Givol, D., Yayon, A,, Yarden, Y., and Lonai, P.
(1991) Developmental expression of two murine fibroblast growth
factor receptors, flg and bek. Development 113:1419-1434.
Orr-Urtreger, A,, Bedford, M.T., Burakova, T., Arman, E., Zimmer,
Y., Yayon A,, Givol, D, and Lonai, P. (1993) Developmental localization of the splicing alternatives of the fibroblast growth factor
receptor-2. Dev. Biol. 158:475-486.
Peek, R., McAvoy, J.W., Lubsen, N.H., and Schoenmakers, J.G.G.
(1992) Rise and fall of crystallin gene messenger levels during fibroblast growth factor induced terminal differentiation of lens cells.
Dev. Biol. 152:152-160.
Peters, K., Ornitz, D., Werner, S., and Williams, L.T. (1993) Unique
expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev. Biol. 155:423-430.
Peters, K.G., Werner, S., Chen, G., and Williams, L.T. (1992) TWO
FGF receptors are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the
mouse. Development 114:233-243.
Prudovsky, I., Savion, N., Zhan, X., Friesel, R., Xu, J., Hou, J., McKeehan, W.L., and Maciag, T. (1994) Intact and functional fibroblast growth factor (FGF) receptor-1 trafficks near the nucleus in
response to FGF-1. J . Biol. Chem. 269:31720-31724.
Richardson, N.A., and McAvoy, J.W. (1990) Age-related changes in
fibre differentiation of rat lens epithelial cells exposed to fibroblast
growth factor. Exp. Eye Res. 50:203-211.
Richardson, N.A., McAvoy, J.W., and Chamberlain, C.G. (1992) Age
of rats affects response of lens epithelial explants to fibroblast
growth factor. Exp. Eye Res. 55:649-656.
Richardson, N.A., Chamberlain, C.G., and McAvoy, J.W. (1993) IGF-1
enhancement of FGF-induced lens fiber differentiation in rats of
different ages. Invest. Ophthalmol. Vis. Sci. 343303-3312.
Robinson, M.L., Overbeek, P.A., Verran, D.J., Grizzle, W.E., Stockard,
C.R., Friesel, R., Maciag, T., and Thompson, J.A. (1995) Extracellular FGF-1 acts as a lens differentiation factor in transgenic mice.
Development 121:505-514.
Schulz, M., chamberlain, C.G., de Iongh, R.U., and McAvoy, J.W.
(1993) Acidic and basic FGF in ocular media: Implications for lens
polarity and growth patterns. Development 118:117-126.
Shi, E., Kan, M., Xu, J., and McKeehan, W.L. (1991a) 16-kilodalton
heparin binding (fibroblast) growth factor type one appears in a
stable 40-kilodalton complex after receptor-dependent internalization. J . Biol. Chem. 2665774-5779.
Shi, E., Kan, M., Xu. J., Morrison, R., and McKeehan, W.L. (1991b)
Direct linkage of heparin binding (fibroblast) growth factor type
one to a fragment of its extracellular domain after receptor-dependent internalization. J. Cell Biol. (Suppl.) 115:416a.
Simmons, D.M., Arriza, J.L., and Swanson, L.W. (1989) A complete
protocol of in situ hybridization of messenger RNA in brain and
tissues with radiolabelled single-stranded RNA probes. J. Histotechnol. 12:169-181.
Vainikka, S., Partanen, J., Bellosta, P., Coulier, F., Birnbaum, D.,
Basilico, C., Jaye, M., and Alitalo, K. (1992) Fibroblast growth fact o r 4 shows novel features in genomic structure, ligand binding and
signal transduction. EMBO J . 11:4273-4280.
Wanaka, A., Johnson, E.M., and Milbrandt, J . (1990) Localization of
FGF receptor mRNA in adult rat central nervous system by in situ
hybridization. Neuron 5:267-281.
Wanaka, A., Milbrandt, J., and Johnson, E.M. (1991) Expression of
FGF receptor gene in rat development. Development 111:455-468.
Wang, J.K., Gao, G., and Goldfarb, M. (1994) Fibroblast growth factor
receptors have different signalling and mitogenic potentials. Mol.
Cell. Biol. 14181-188.
Yamaguchi, T.P., Conlon, R.A., and Rossant, J . (1992) Expression of
the fibroblast growth factor receptor FGFR-l/fZg during gastrulation and segmentation in the mouse embryo. Dev. Biol. 152:75-88.
Zhan, X., Hu, X.,, Friesel, R., and Maciag, T. (1993) Long term growth
factor exposure and differential tyrosine phosphorylation are required for DNA synthesis in BALB/c 3T3 cells. J . Biol. Chem. 268:
9611-9620.
Документ
Категория
Без категории
Просмотров
4
Размер файла
2 789 Кб
Теги
959
1/--страниц
Пожаловаться на содержимое документа