Vol.

174,

January

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

31, 1991

Expression

of mRNA for activin-binding early embryonic development

Kosuke TashiroI, Ryutaro YamadaI, Masami Muramatsu2,

ILaboratory

protein (follistatin) of Xenopus Zaeuis

Misaki Asanol. Makoto and Koichiro Shiokawal

during

Hashimoto2.

of Molecular Embryology, Zoological Institute, Faculty of Science, The University of Tokyo, Hongo, Bunkyo-ku. Tokyo 113, Japan

2Department

Received

1022-1027

Pages

of Biochemistry, Faculty of Medicine, The University Bunkyo-ku, Tokyo 113. Japan

December

25,

of Tokyo, Hongo,

1990

SUMMARY: Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xcnopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xcnopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84 % homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable. and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.

Morphogenesis different

embryonic

development

significant

regions.

is mesoderm

cells of marginal

factors

in development

factors

for natural

axial mesoderm Activin

modulation

Copyright All rights

The results is provided

regulators

structure

of the differentiation

rudiment

At this

activin

in amphibian is induced

of localized

moment,

embryos

and have various

of follicle-stimulating of follicular

1022

that the

peptide-growth

as the most

plausible

to induce

dorsal

(3). biological

hormone granulosa

in

Recently,

suggest

B has been reported

in Xenopus

among

of the mesoderm-inducing

from several laboratories

$1.50

0 1991 by Academic Press, hc. of reproduction in any form reserved.

a mesodermal

by a combination

of the TGF-l3 family

of the secretion

interactions

identification

(1. 2).

MIF, endogenous

and anterior

is a member

as enhancement

0006-291X/91

signal

inductive

interactions

of the signals from vegetal cells.

has been made towards

(PGFs) and their

candidate

in which

the influence

(MIFs) in amphibians.

mesoderm-inducing

involves cell-cell

One of the earliest induction,

zone under

progress

of all organisms

activities,

by pituitary cells

gland

(5). regulation

such (4). of

Vol.

174,

No.

2, 1991

erythropoiesis modulation protein

(6). stimulation

for activin

Because

processes

which

If the

activin

embryogenesis. inactivation

acts

the follistatin

function

by preventing

cells (8).

from porcine,

bovine,

the activity

of activin

mesoderm

also control

in diverse

binding.

the mesoderm

For example,

as

direct

regulatory

excessive spreading Generally,

of activin activin

of follistatin

Materials

the

of cells not

manner

with the activin

in the

in amphibian

inducing

follistatin

cDNA and

of a Xenopus

mRNA during

regulate

in a reaction-diffusion

to start study on follistatin the isolation

through

acts

induction,

we report

might

to the regions

mesodenn

it is important

in amphibian

induction

follistatin

acts in a concerted

of the expression

through

inducer

systems, and if follistatin

analysis

(7), and

and rat, and named

participates

as a natural

mesoderm.

Here,

islets

On the other hand, a binding

patterning

system as well.

COMMUNICATIONS

by rat pancreatic

that follistatin

could

by direct

to become

RESEARCH

by activin.

B really

of activin

secretion

inhibits

thought

are induced

BIOPHYSICAL

pituitary

follistatin

(12). it is generally

destined

of insulin

has also been purified

(9- 12).

binding

AND

of several types of anterior

follistatin

activin

BIOCHEMICAL

early Xenopus

development.

and Methods

Isolation and sequence determination of Xenopus folllstatin cDN& Xenopus ovary cDNA library was constructed into hgtl0 with a random primer by Amersham cDNA cloning system and subjected for screening. The probe used was a fragment of human follistatin cDNA. which was generated by PCR method. In situ plaque hybridization was performed according to standard methods under low stringency condition (13). From the screening of 8 X 105 phages. one positive plaques was isolated. Its inserted DNA was subcloned into plasmid pBluescriptIISK(-) and its nucleotide sequence was determined by the dideoxy nucleotide method (14). Extraction of RNA and Northern blot hybridization: Xenopus laeuis embryos were obtained as described previously (15). Total RNAs were extracted from embryos at various stages by AGPC methods (16) and Poly(A)+ RNAs were isolated with oligotexdt30. Poly(A)+ RNAs corresponding to the thirty embryos were denatured with formaldehyde and electrophoresed in a 1 % agarose gel under denaturing conditions (13). The blotting onto filters and hybridization were carried out according to the standard methods under high stringency conditions (13). Results Isolation

of Xenopus

follistatin

cDNA

We first prepared

a fragment

of human

a probe for isolation

of Xenopus

follistatin

coding

region

of human

(16) . Since follistatin ovary cDNA library

follistatin

follistatin

cDNA. The probe

corresponding

cDNA of other animals

(18) we screened

cDNA by a PCR method

the kgtl0 1023

obtained

to the region had been isolated cDNA library

to use as

contained

the

from a.a. 1 to a.a. 200 successfully

of Xenopus

from their

ovary to clone

Vol.

174, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

cALLKAKcm3vPEmv

XFOll m

~~KPRc~CAPD~?SNITWX~~~OI-E ---------.-----------p---L-----RN-------R--EQ---E-

XFOll

QY@3KCKKTCRDVLCwSSScwDQTNuAY ----R--------F-----T--------

XFOll

NDQITYQsAcxLRxa ---v--s----------------------------

CVTCNRICPEPTSPDQYLCQ -------------A-SE-----

TCLLGRSIaL?iYE5KcIKAXS~IQCSAQXXcLw ---m-m..

XFOll

DSRVGRGRCQLSDDLCXSD -FIT------s-C-E--PD----EP------A--A------

XFoll w

EVlUTSQSC!BTSI~S~ ---------.-s--------D-DQ----P---ILEW

RESEARCH COMMUNICATIONS

TQ-----/& I ‘,"d

DTVCASDNTTYPSECAMXQAACSTQILL J&---S-V--

Fig. 1. Deduced amino acid sequence of Xenopus folllstatin (a) and Northern blot analysis of follistatin mRNA in ovary (b). a) A comparison of deduced amino acid sequence of Xenopus follistatin with that of human folllstatin is shown. Dashes are placed at positions of ammo acid identity. Only the amlno acid sequences encoded by isolated Xerwpus cDNA (XFoll) Is presented. b) Two microgram poly(A)+ RNA extracted from

ovary

was analyzed

by Northern

blot

hybridlzatlon

using

DNA

fragment

of XFoll

as a probe. Arrows indicate the position of 28s and 18s ribosomal RNA.

the cDNA for Xenopus

follistatin.

797 bp insert and contains dideoxy method acid sequence of human

(17) and porcine

follistatin

lb).

A considerable

been reported

Expression

conditions

sequence

of follistatin

of high stringency

has a by the

we found that the amino

has a homology very strongly

of 84 % with those that XFoll

isolated

protein.

of mRNA population

(17) and porcine

mRNA during

from embryos

which

When sequenced

ovary poly(A)+ RNA in a Northern

heterogeneity

also in human

RNAs

frame.

a major band (about 2 kb) and a minor

To study the expression poly(A)+

clone, XFoll,

as a template,

(18) (Fig. la), suggesting

When tested on Xenopus bands were obtained:

strand plasmid

from its nucleotide

here encodes the Xenopus

one positive

a part of the open reading

using a double deduced

We obtained

of follistatin at various

blot analysis, band (about

due to alternative

two RNA 1.2 kb] (Fig.

splicing

has

systems (18).

Xenopus embryogenesis mRNA during

Xenopus

stages were hybridized

(0.1 X SSC-0.1

laeuts embryogenesis. with XFoll

% SDS at 65 oC). In embryos,

under

the

two major

bands (3.5 and 2 kb) and several other minor bands ( 5, 2.7, and 1.8 kb ) were obtained and the amounts 10).

of all of these FINAs increased

The 3.5 kb mRNA was not detected

greatly

in later stages (see Fig. 2, lane

tn RNA from ovary (cf. Fig. lb). 1024

Vol.

174,

No.

2, 1991

BIOCHEMICAL

12

m.

Expression

3

of follistatin

AND

4

5

6

7

BIOPHYSICAL

6

RESEARCH

COMMUNICATIONS

910

mRNA durtng early embryogenesis of Xenopus

laevls

FUWs extracted from thirty embryos at various stages were analyzed by blot hybridization using DNA fragment of XFoll as a probe. Lane 1, egg: lane 2, stage 3 (16 cell-stage): lane 3. stage 6 (late cleavage) ; lane 4,

Poly(A)+ Northern unfertilized

stage 8 (mid-blastula)

; lane 5, stage 10 (mid gastrula) ; lane 6. stage 11 (late gastrula) ;

lane 7, stage 15 (early neurula) : lane (tailbud) ; lane 10. stage 25 (tadpole).

8. stage 20 (late neurula) : lane 9. stage 23 Arrows indicate the position of 28s and 18s

ribosomal RNA.

Expression

of the 3.5 kb follistatin

and its level increased detected

gradually

also in ovary

unfertilized

during

RNA (Fig.

significantly

results

By contrast,

lb),

existed

embryo-spectfic

at the early neurula

indicate

embryogenesis

that

results

a significant

of early Xenopus,

type of follistatin

or the time of the expression

regulation

mRNA.

together

of inductive

suggest

at early gastrula

before gastrula

the 2 kb mRNA.

at a very low level constant

which

expression

in the RNA of

through

gastrulation

of follistatin

mRNA

increase

potent MIF so far reported

events through

induction,

(activin

may play some important

its direct binding

occurs

in the possibly

mFWA (3.5 kb) at the time of mesodermal

that follistatin

was

stage.

and there is a sudden

of the most

stage

Since this mRNA was not

1) and embryos

eggs, and the level of this RNA remained

until it increased These

proceeded.

eggs (Fig. 2, lane

2-4). it is not a maternal

detected

These

as development

in the RNA of unfertilized

(lanes

mRNA was itrst detected

PB) (3).

roles in the

to activins.

Discussion Activins

and the other members

and neural

tissues

interactions

are involved.

inductive

events,

(l-3).

of TGF-P family are potent inducers

In the course If’ different

they should

of

embryogenesis.

types of PGFs. including

be regulated

precisely

gene level but also at the level of the functional 1025

protein.

multiple activins,

and concertedly Here,

for mesodennal inductive

operate

in the

not only at the

we showed

the specific

Vol.

174,

No.

2, 1991

expression embryos

BIOCHEMICAL

of follistatin

mRNAs

of different

BIOPHYSICAL

RESEARCH

sizes during

development

by using newly cloned cDNA of Xenopus

Follistatin

directly

level probably follistatin

binds to activin

by some masking

mRNA occurred

mesoderm-inducing

factor,

acts as a regulator

embryonic

development.

There

are at least

in an inactive embryonic mechanism. developmental

function

Second,

follistatin

might

fate is not mesodermal

region of follistatin

Therefore,

expression

together

of

B. the most potent the follistatin

it is quite likely its direct

for follistatin

itself that the

binding

during

to regulate

with activin

activin

to keep the activin

from the activin at the appropriate

developmental

time

be synthesized

and secreted

and might

prevent

it is very interesting

in Xenopus

Though

through

mechanisms

appropriate

(3).

at the protein

that the expression

activin

interactions,

might be secreted

at an

by the act&in.

of mesoden-n

stage when

or latent form and might be eliminated region

of Xenopus

functions

We observed

cellular

of activin

two possible

First, follistatin

the activin

first detectable

in various

follistatin

functions.

and inhibits

mechanism.

becomes

COMMUNICATIONS

follistatin.

at the early gastrula

may have its own function

induced

AND

embryos,

by some

those

to identify

in relation

unmasking

by cells whose cells from

the timing

being and the

to the region and timing

induction.

Acknowledgments This work was supported Ministry Foundation

of Education.

in part by Grant-in-Aid

Science

and Culture

( 1990). and The Fujisawa

for Scientific

of Japan,

Foundation

grants

Research

from Takeda

from the Science

( 1990).

References Smith, J. C., Cooke, J.. Green. J. B. A., Howes, G., and Symes. K. (1989) Development Supplement, 149-159 Smith, J. C. (1989) Development 105, 665-577 Thomsen, G.. Woolf, T., Whitman, M., Sokol. S.. Vaughan, J.. Vale, W., and Melton, D. A. (1990) Cell 63, 485-493 Miyamoto, K., Hasegawa. Y., Fukuda. M., Nomura. M., Igarashi. M., Kanagawa, 129. 396-403 K., and Mastuo. H. (1985) Biochem. Biophys. Res. Commun. Sugino, H.. Nakamura, T.. Hasegawa. Y.. Miyamoto. K., Abe, Y., Igarashi. M., Eto, Y., Shibai, H., and Titani. K. (1988) Biochem. Biophys. Res. Commun. 153. 281288 6. 7.

Yu, J., Shao, L.. Lemas, V.. Yu, A. L., Vaughan. J.. Rivier, J., and Vale, W. (1987) Nature 330, 765-767 Totsuka. Y.. Tabuchi, M.. Kojima. I.. Shibai, H.. and Ogata. E. (1988) Biochem. Biophys. Res. Commun. 156.335-339 1026

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AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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