Vol. 168, No. 1, 1990 April 16, 1990

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 343-349

EXPRESSION PATTERN OF ACIDIC AND BASIC FIBROBLAST GENES IN ADULT RAT EYES

GROWTH FACTOR

Sumihare Noji', Toshihiko Matsuo2, Eiki Koyamal, Tomoichiro Tsutomu Nohno3, Nobuhiko Matsuo2, Yamaail, and Shigehiko Taniguchi

2

Department

1"

'Okayama University Dental School and of Ophthalmology, Okayama University Medical School, 2-5-l Shikata-cho Okayama City, Okayama 700, Japan

3Kawasaki

Medical

Received

, Kurashiki

School

February

28,

City,

Okayama

701-01,

Japan

1990

Although the retinal angiogenic and mitogenic factors have been identified to be acidic and basic fibroblast growth factors (aFGF and bFGF), little information has so far been available about the cells producing them and their function in retinal tissues. We found, by --in situ hybridization, that the expression pattern of the aFGF gene differed remarkably from that of the bFGF gene in adult rat eyes.Our results demonstrated thattheaFGF gene was produced by photoreceptor visual cells, neuronal cells in the inner nuclear layer and ganglion cells of the retina, in addition to pigment epithelial cells of the choroid, iris and ciliary body, and epithelial cells of the cornea, conjunctiva and lens, while bFGF was synthesized solely by the photoreceptor visual cells. 0 1990 Academic Press, Inc. The

existence

proposed

by Michaelson

retina-derived activity

vitro

endothelial

acidic

(4).

cells, and their

identify

the

factors

was

and more recently

by Patz

(2).

been

the

the little

these

were

fibroblast

basis of

been

brain,

the cells

Thus,

it

of primary

producing

*Correspondence to: Shigehiko Taniguchi, Biochemistry, Okayama University Dental cho, Okayama City, Okayama 700, Japan.

343

of their capillary

the Ph.D., School,

to be (aFGF

in

various vascular

cartilage,

about

specifically

These

factors

found

known

is

first

demonstrated

growth

FGFs have

retina, is

on the

the proliferation

and basic

functions. cells

isolated

Recently,

Although

including

in vivo

have

(3,4).

with

bFGF)

tissues,

(1)

vasculogenic

to stimulate

cells

identical

retinal

factors in

endothelial

and

of

producing importance FGFs

in

them to these

Department of 2-5-l Shikata-

0006-291x190 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

168, No. 1, 1990 tissues. the situ

BIOCHEMICAL

In the

expression

present

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

study , we demonstrated

pattern

of both

FGF genes

for

in

adult

the

first

rat

time

eyes by

b

hybridization.

Materials

and Methods

Eyes of 6-week rats were enucleated and cut into halves after perfusion from the left ventricle with phosphate-buffered 4% paraformaldehyde (pH7.4) at 4"C, post-fixed for 3 hours in the same fixative, dehydrated with alcohol series at 4 'C, and embedded in paraffin (Paraplast). The sections with 5 pm thickness were pretreated, hybridized and washed, according to Ingham et a1.(5) and Noji et a1.(6). We performed h m hybridization 99 paraffin sections of the rat eyes with single-stranded S-labeled riboprobes synthesized from a -HI fragment of the rat bFGF cDNA (7,8) and a chemically synthesized DNA fragment (454 bp) coding the human aFGF (The sequence will be published elsewhere.), which were generous gifts from Takeda Chemical Industries, LTD. To prepare antisense and sense riboprobes, the chemically synthesized aFGF cDNA and the bFGF cDNA were inserted in a plasmid pGEM and a Specific activity of the riboprobeg ~~ba~~~~ "tP:i r'?~~~i~$~'Ci/mol Amersham) was about 5x10 dpm/pg. Probes for the hybridizaiion were subjected to limited alkaline hydrolysis to shorten the transcripts to about 50-150 base pairs. For autoradiography, the slides were immersed in Kodak NTBZ emulsion (diluted 1:l with water), air-dried and exposed for 7 days at 4'C. The slides were developed and finally stained with hematoxylin and eosin.

Results Hybridization level

with

distribution

sections With

lc,

the of

photoreceptor

visual

inner

nuclear

layer

cells

at the the

top

synthesis. secretion

2d,

3c and 3d).

riboprobe

gave

grains

for

silver

grains

cells

at the

bottom

of Fig.

of Fig.

lb,

in the middle of Fig.

because result

lb.

More

localized

segments

presents

of aFGF in the

This

344

all

have

cells

tissue

extensive

observed lb,

in

cells

and the the

inner

ganglion

signals

that are

of is

the ability

of production

closely

the

in the

segments

localization

a possibility

photoreceptor

background-

mRNA,

precisely,

on the

cells. these

were

a at

aFGF

exposed

visual

This

silver

2c,

aFGF mRNA were

photoreceptor reasonable,

Id,

probes

exposed

antisense

accumulation

the

sense-strand

of

(Fig.

and Discussion

for the quite

protein and related

Vol.

BIOCHEMICAL

168, No. 1, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fiz. 1. Expression pattern of the aFGF gene in retina. a and b: Bright and dark field photographs with the antisense probe; c and d: bright and dark field photographs with the sense probe.

to

photo-stimulation.

cells

of

bipolar,

the

The aFGF mRNAs were

inner

nuclear

and horizontal

cells

Mueller.

Since

the

of

inner

nuclear

this

possibility express

that

not

cells

the

grains than

but

specific

all

in

Fig.

observed the ganglion These

with

cells

in

is large,

clear

whereas (smaller

to

of

more

intensive

its

middle, in

this

did

the

selectively nuclei

with

cells

of

in

the edge

there

is

layer

would

other

a

cells. ganglion

in ganglion

dark

the

amacrine,

in

no significant cells

in

supporting

than

expressed

detected

consist

cells

intensively

la and lb),

in glial

are

layer

aFGF gene

cells

(larger

which

in addition

signal

the aFGF gene more

Furthermore,

layer

layer,

also

expression

cell was around

nuclei)

cells. findings

immunohistochemical

are

consistently

localization

of the 345

correlated aFGF protein:

with the

the aFGF

Vol.

168, No. 1, 1990 proteins

BIOCHEMICAL

were

localized

photoreceptors, bovine

the

retina

(9).

mRNA with

that

range

intercellular

aFGF

in the

expressed not

suppose

that

among the

Fig.

well

neuronal

to a far

network

retina.

in the

2.

and d: probe.

of the

among the of

the

cells

as in

of the

neurons

those

neuronal facts of the

and also

a

short-

rat

brain and

signal

in maintaining

is

is (data

that

retinal it

through

aFGF gene

retina

in

of the

cells

that

the

of aFGF

indicates

bFGF (lo), somehow

cells

localization

of the postnatal than

of

and

reasonable transduction

normal

neuronal

pattern of the aFGF gene in lens, iris, and conjunctiva of a rat eye. a and b: photographs with the antisense probe; c dark

field

346

photographs

with

aFGF

ganglion

retina.

Expression body, cornea and dark field bright

and ganglion

in neuronal

degree

segments

proteins

aFGF participates

retinal

ciliary Bright

layer

In view

outgrowth

greater

inner

correlation

interaction

as

the

corresponding

intensively

promotes

in nuclear

This

of the

rat

shown)

cells

inner

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

sense

to

Vol. 168, No. 1, 1990 We cells

BIOCHEMICAL

also

detected

of the

cells

choroid,

of the

The

aFGF

maintain

growth

autocrine

factor,

differentiation

and

differentiation

in

the

and ciliary

by the and

of (11).

aFGF mRNA in the

conjunctiva lens

lens

epithelial

of themselves of aFGF in

epithelial

several

subtypes

splicing

of

the

created

the

stimulate

and/or

their

stromal

by such a

2.

likely

to

is

in vitro

may

aFGF proteins

in Fig.

as

aFGF and bFGF

aFGF produced

cells

epithelial

themselves

cells

the neuronal

cells,

cells

same manner,

epithelial

of that

epithelial

and the

cells

differentiation fact

pigment

as shown

epithelial

on the

RESEARCH COMMUNICATIONS

body,

and lens,

based

In the

conjunctival

function

iris

cornea,

produced

previously

the

AND BIOPHYSICAL

as

promote reported

by the

cornea1

growth cells.

seems to differ

and As the

from

may be classified mechanism

an

as

that into

alternative

common aFGF gene.

pattern of the bFGF gene in retina. a and b: Fig. 3. Expression Bright and dark field photographs with the antisense probe; c and photographs with the sense probe. d: bright and dark field 347

Vol.

168, No. 1, 1990 In

BIOCHEMICAL

contrast

expressed

to

the results

exclusively

visual

cells

were

reported

inner

nuclear

in the

as shown

of the

interpretation in the

photoreceptor

cells

in

the

analyses the

are

the

cornea1

find

the

required

our

amount

induced

in

capillaries

is

the

secretion abnormal

is with

Since

formation

ocular

FGF is

by

the

proliferative fluid

(14). 348

did

implies in a

may depleted. retinal

as

from

our

results,

considered

to

to

abnormal

an angiogenic

factor,

fact

be

so as to result

occur.

those

the

condition

leads

retinopathy

than

in

normal

is

damage

in not

or not

when

Judged

been

found

result

occurs

then

substantiated

have

of bFGF is

that

may

hand,

transcription

capillaries

and this

other

constitutively

formation

retinal

precise

RNA

a pathological

(2).

FGF proteins.

advanced

FGF in the

under

cells

capillary

consideration

known

bFGF

we could

This

storage

target

case,

were

However,

its

possible

More

cells

that

complications

neuronal of the

well

the

the

epithelial

when the

new vessel

of

is

On the

not

but

obstructed

clinical

obstruction

damage

more

only

retinopathy,

in serious

patient

cornea,

it are

diabetic

the

the

transcribed

of

to the

point.

conditions.

the

synthesized

of the bFGF messenger

experimental is

A

retina.

(12,13).

amount

in

localization

the bFGF proteins

membranes

intermittently

Clinically,

this

bFGF, because

detectable

that

in the

and/or

the

and diffuse If

is

the bFGF proteins

bFGF proteins

secreted

factor

Since

gene product.

the

layer.

endothelial

the bFGF gene

large

are

gene

of the photoreceptor

f9),

of the that

bFGF

microvasculature

retina

to clarify

basement

under

that

is

nuclear

to produce

cornea

fact

angiogenic

cornea1

supposed

that

cells

inner

the

bovine

the

segments

around

from

of this

aFGF,

3a and 3b.

to be localized layer

be

for

inner

in Fig.

the bFGF mRNA differs

should

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that had without

The a nine

above diabetic times

retinopathy

Vol.

168,

No.

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Acknowledgments We thank Drs. Makoto Iwane, Koichi Igarashi and Atsushi Kakinuma in Biotechnology Laboratories of Takeda Chemical Industries, LTD. for aFGF and bFGF cDNAs. Supported in part by grants from The Ministry of Education, Science and Culture of Japan. References 1. Michaelson, I.C. (1948) Trans. Ophthalmol. Sot. UK 68,137-180 2. Patz, A. (1982) Am. J. Ophthalmol. 94,715-743 3. Baird, A., Esch, F., Mormede, P., Ueno, N., Ling, N., Boehlen, P., Ying, S.A., Wehrenberg, W.B., and Guillemin, R. (1986) Rec. Prog. Hormone Res. 42,143-205 4. Baird, A., Esch, F., Gospodarowicz, D., and Guillemin, R. (1985) Biochemistry 24,7855-7860 5. Ingham, P.W., Howard, K.R., and Ish-Horowitz, D.(l985) Nature 318,439-445 6. Noji, S., Yamaai, T., Koyama, E., and Taniguchi, S. (1989) Radioisotopes 38,366-372 7. Kurokawa, T., Sasada, R., Iwane, M., and Igarashi, K. (1986) FEBS Lett. 213,189-194 8. Kurokawa, T., Seno, M., and Igarashi, K. (1988) Nucl. Acids Res. 16,5201 9. Hanneken, A.M., Mcleod, D.S., Lutty, G.A., and Hjelmeland, L.M. (1987) Invest. Ophthalmol. Vis. Sci. 28(supp1.),259 10. Lipton, S.A., Wagner, J.A., Madison, R.D., and D'Amore, P.A. (1988) Proc. Natl. Acad. Sci. USA 85,2388-2392 11. Chamberlain, C.G., and McAvoy, J.W. (1987) Curr. Eye Res. 6,1165-1168 12. Folkman, J., Klagsbrun, M., Sasse, J., Wadzinski, M., Ingber, D., and Vlodavsky, I. (1988) Am. J. Pathol. 130,393400 13. Morton, K., Hutchinson, C., Jeanny, J.C., Karpouzas, I., Pouliquen, Y., and Courtois, Y. (1989) Curr. Eye Res. 8,975987 14. Baird, A., Culler, F., Jones, K.L., and Guillemin, R. (1985) Lancet 2:563

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