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Development 109, 387-393 (1990)
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Fibroblast growth factor during mesoderm induction in the early chick
embryo
E. MITRANI 1 , Y. GRUENBAUM 2 , H. SHOHAT 1 and T. Z I V 1 2
Departments of Zoology1 and Genetics2, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Summary
A chick genomic clone that reveals a high degree of
homology to the mammalian and Xenopus bFGF gene
has been isolated. The pattern of expression of bFGF has
been examined during early chick embryogenesis. RNA
blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis
indicated that bFGF protein is present at these early
developmental stages and is distributed evenly in the
epiblast, hypoblast and marginal zone of the chick
blastula. Substances that can inhibit FGF action were
applied to early chick blastoderms grown in vitro under
defined culture conditions (DCM). Both heparin and
suramin were capable of blocking the formation of
mesodermal structures in a dose-dependent manner.
Our results indicate that FGF-like substances may need
to be present for axial structures to develop although
they may be acting earlier during the induction of nonaxial mesoderm.
Introduction
the entire lower layer. Interaction between the epiblast
and the hypoblast results in the formation of axial
mesoderm through the process of primitive streak
formation (Azar and Eyal-Giladi, 1981; Mitrani and
Eyal-Giladi, 1981; Mitrani et al. 1983).
In the present work, we have isolated a chick
genomic clone that reveals a high degree of homology
to the mammalian and Xenopus bFGF gene. We have
studied the transcription pattern of the chick bFGF
homologue and the localization of the bFGF protein
during early stages of chick development. We have also
performed experiments in which agents that can block
FGF-like substances, were applied to early chick blastoderms grown in vitro under defined culture conditions
(DCM). Application of both heparin and suramin
blocked the formation of mesodermal structures in a
dose-dependent manner.
Basic fibroblast growth factor (bFGF) can induce the
formation of non-axial mesoderm in ectodermal animal
caps of Xenopus blastula (Slack et al. 1987). The limited
capacity of bFGF to induce muscle actin, which is the
earliest marker for muscle differentiation (Gurdon,
1987), can be increased by transforming growth factor-/?
(TGF-/3, Kimelman and Kirschner, 1987). Rosa et al.
(1988), however, have claimed that TGF-/32, but not
TGF-/31 is active in o^actin induction while addition of
bFGF has a small synergistic effect. The Xenopus cell
line XTC secretes mesodermal-inducing capacity
(Smith, 1987). Partial characterization of the secreted
protein showed that its properties closely resemble
those of TGF-/3 (Smith et al. 1988). FGF is probably
involved in the induction of more ventral non-axial
mesodermal structures (Godsave et al. 1988 and see
Slack and Isaacs, 1989). 1NT-2 and kFGF, two oncogene-related factors related to FGF induce mesoderm
formation in Xenopus animal caps as assayed by cardiac
actin mRNA levels (Paterno et al. 1989). Recently, it
has been shown that aFGF can induce mesoderm
formation in colonies from single cells derived from
animal caps of stage 8 Xenopus blastoderms (Godsave
and Slack, 1989).
The chick blastula [stage XIII of Eyal-Giladi and
Kochav (E&K), 1976] is formed of two physically
distinct layers. The upper layer comprises the primary
ectoderm or epiblast, the marginal zone and the area
opaca. The primary endoderm or hypoblast constitutes
Key words: FGF, mesoderm induction, heparin, suramin,
chick embryo.
Materials and methods
Library screening
A chick genomic library (kindly provided by D. Engel) was
screened with a 600 bp human bFGF probe containing the
entire protein coding region (Abraham et al. 1986a, 1986b).
The hybridization solution contained 37% formamide,
5xSSC, lxDenhardt's, O.lmgmP 1 salmon sperm DNA and
^
n i m i "i1 of labeled probe. Hybridization was
performed at 37°C. Washes were 3 times, 20min each with
2xSSC, 0.1 % SDS at room temperature and twice with the
1
388
E. Mitrani and others
same solution at 50°C. Radiolabeled probe was prepared
according to the method of Feinberg and Vogelstein (1983).
DNA sequencing
The desired fragments were subcloned into the vector
pUC118. The nucleotide sequence of the cloned DNA was
determined for both strands using the dideoxy chain termination method of Sanger et al. (1977).
Northern blot analysis
Northern blot analysis of total RNA was performed as
described previously (Mitrani et al. 1987). Briefly, embryonic
tissue was digested in proteinase K (200[igml"1) at 42°C for
45min. The digest was extracted twice with phenol-chloroform and precipitated in 0.3 M sodium acetate and 2 volumes
of ethanol. Total nucleic acids were resuspended in 10 mM
Tris-HCl/lOmM MgCl2, and digested with DNAse I
(10//gmr') for 30min at 37°C. The RNA was denatured and
electrophoresed on a 1 % a"garose/formaldehyde gel, transferred to a Genescreen (Dnnlop) nylon membrane crosslinked with UV and hybridized to the chicken bFGF probe
(pGF0.9). The hybridization solution contained 50% formamide, 5xSSC, lxDenhardt's, O.lmgmP 1 salmon sperm
DNA and 5xlCr'disintsminimi" 1 of labeled probe. Hybridization was performed at 42°C. Washes were 3 times, 20min
each with 0.3xSSC, 0.1% SDS at 50°C and twice with the
same solution at 65 °C.
Dissection of embryos
Staging of the early blastoderms was done according to EyalGiladi and Kochav (E&K) (1976). Gastrulating blastoderms
were staged according to Hamburger and Hamilton (H&H)
(1951). When necessary, the marginal zone was dissected
from stage X1I1 blastoderms (Kahner et al. 1985).
Growing whole blastoderms in vitro
We have recently developed a reliable bioassay that allows for
the growth of early chick blastoderms, or explants derived
from them, in the absence of the vitelline membrane, and
under defined culture conditions (DCM). This system allows
for the controlled application of chosen factors directly to the
blastoderm. The blastoderm is layered onto 1.8% agarose in
Roswell Park Memorial Institute (RPMI) medium (Mitrani
and Shimoni, 1990). The advantage of this culture system is
that factors can be directly applied to the embryo via the
culture medium in a dose-controlled fashion. Heparin
sodium-salt grade I was obtained from Sigma. Suramin was a
gift from Dr I. Vlodavsky.
Growing cells in agarose
The culture medium used was RPMI 1640 containing 10%
fetal calf serum and 1 % penicillin and streptomycin. Cells
were grown in agarose as described previously (Mitrani,
1984). Briefly, 0.8 ml of 0.33 % agarose in culture medium was
prepared as an underlayer in 35 mm Petri dishes. Stage X-XJI
blastoderms (E&K) were incubated in trypsin-EDTA for
lOmin and then transferred to culture medium and dissociated by gently forcing them through a fine Pasteur pipette.
Cells were plated on the same medium at a final agarose
concentration of 0.3% and layered onto the previously
agarose-coated Petri dish at 5X104 cells ml"1 (Mitrani, 1984).
The cells were incubated at 37°C in 5% CO2 and 100%
humidity. Duplicate dishes were plated with varying concentrations of heparin or suramin ranging from 10 to 200 /jgmP 1 .
Indirect immunofluorescence
Chicken blastoderms were dissected into Ringer's solution
and processed for indirect immunofluorescence as described
previously (Mitrani, 1982). Briefly, the blastoderms were
transferred to a fixative solution containing 9 parts of formalin, 3 parts ethanol and one part acetic acid. Following 30min
fixation, the blastoderms were subjected to three rinses,
lOmin each, with 70% ethanol and transferred to Ringer's
solution. The fixed blastoderms were then carefully placed
into a drop of Tissue-Tek II mounting medium (Lab-Tek
products), kept at -40°C until frozen and sectioned in a
standard cryostat. 8/tm sections were obtained and stained
with the anti-bFGF antibodies (kindly provided by Dr I.
Vlodavsky and by Dr G. Neufeld), diluted 1:200 to 1:1000.
Fluorescein-labeled anti-mouse, for monoclonal antibodies,
or anti-rabbit, for polyclonal antibodies, were used as secondary antibodies. In control experiments, normal pool serum
was used instead of anti-bFGF antibodies. Also, in a separate
control, anti-bFGF antibodies, diluted 1:200, were preincubated with 3j/g of cloned human bFGF protein (kindly
provided by Dr G. Neufeld) for 60min at 37°C prior to
incubation with the tissue.
Results
Cloning of a chicken bFGF homologue
A chicken genomic library (kindly provided by D.
Engel) was screened under low-stringency conditions,
using a human bFGF cDNA clone as probe (Abraham
et al. 1986a,b). Three phages, which turned out to
contain the same insert, 4.2kb in length, were picked.
Fig. 1A shows a partial restriction map of this clone
A
EcoRI
Bglll
Pst I
Bgl II Eco Rl
B
cgtatatagga
12
gaataacttaccttacccttgccaatttcgataataactttgtcctgtttttCicagTC
71
AAA CTG CAG CTT CAA GCA GAA GAA AGA GGA GTA GTA TCA ATC AAA
lys leu gin leu gin ala glu glu arg gly val val ser ile Iy3
61
'
70
116
GGC GTA AGT GCA AAC CGC TTT CTG GCT ATG AAG GAG GAT GGC AGA
gly val «*r ala asn arg ph« leu ala met lys glu asp gly arg
80
90
161
TTG CTG GCA CTG otaascagcattJxttaatgtatctctctgtatcttactgcta
leu leu ala l«u
94
216
ggtcctcttttcggtttgtgcgttgcataggggagaaattaagctcggaaaaaacttgt
275
300
gtcaattaagagagagatttctgttg
Fig. 1. Restriction map and partial sequence of pGF4.2
clone. (A) The exon part is shown as an open box. (B) The
putative protein translation of bFGF sequence is shown
under the sequence. The three amino acids substituted in
the chicken compared to the mammalian bFGF are shown
in bold letters. Also shown underlined, the nucleotides that
are conserved in the chicken and the human second intron.
bFGF during chick mesoderm induction
389
-pGF4.2-. The pstl-Bglll fragment -pGF0.9-, derived
from one of the clones, was used as probe in this study.
The entire coding region of the putative chicken bFGF
second exon contained in pGF4.2 and flanking regions
at both ends are shown in Fig. IB. The homology
between the chicken exon sequence and the corresponding bFGF exon sequence in other species is 85 %
at the nucleotide level and 86-91 % at the amino acid
level (Fig. IB). Residue Cys78 in human is not essential
for bFGF biological activity (Seno et al. 1988) and is not
conserved either in chicken or Xenopus [the chicken
Ser78 is substituted in Xenopus by Thr (Kimmelman
and Kirschner, 1987)]. The change of Ser94 to Leu is
conserved both in chicken and Xenopus. Tyr82, which
is conserved in all bFGF genes analyzed to date is
substituted in the chicken by Phe. All bFGF genes
analyzed so far contain a second exon of identical size.
Also, the first seven nucleotides of the putative chicken
second intron are identical to those of the human
second intron (Fig. IB). After the first 14 nucleotides,
the two intron sequences completely diverge.
Transcription of bFGF during embryonic development
FGF transcription was analyzed in early chicken embryos by the Northern blot technique, using pGF0.9 as
probe. About one hundred stage XIII blastoderms
(E&K) were dissected in order to obtain enough RNA
from the marginal zone region. RNA samples from
stages X-XII (E&K) and early primitive streak stages
were also tested. The lane in Fig. 2 contains 5 ^ig of total
RNA from the marginal zone region. Three weak
transcripts 7.8, 1.9 and 1.5 kb in size, could be detected
only after exposing the autoradiogram for two weeks.
The hybridization signal with RNA prepared from
whole embryos at the above stages of development was
below our level of detection. When the filter was
reprobed with a-actin probe, the hybridization signal
was stronger in the lane containing RNA derived from
whole embryos compared to that of the marginal zone
(data not shown).
kb
7.8-
t
1.91.5-
Fig. 2. bFGF RNA is present in the
marginal zone of stage XIII blastoderms.
Northern blot analysis of marginal zone
of stage XIII blastoderms. 5j*g of total
RNA were loaded on the lane and
pGF0.9 fragment was used as a probe.
The length of the hybridized transcripts in
kb is marked on the left. Three faint
bands (7.8, 1.9 and 1.5 kb) could be
discerned after exposure of the
autoradiogram for 14 days.
Fig. 3. bFGF protein is expressed in the chick blastula.
Indirect immunofluorescence analysis of bFGF in stage XII
blastoderms. An antibody that recognizes the M-terminus of
bFGF was used as the primary antibody (35). Transverse
section through the central region (A) and the more
peripheral region (B) of a stage XII blastoderm. bFGF
immunostaining pattern seems to be evenly distributed
between the epiblast (e), the emerging hypoblast (h) and
the marginal zone (mz). Control section is shown in C
Magnification x800.
390
E. Mitrani and others
Expression of the protein bFGF during early
embryonic stages
Monoclonal antibodies or polyclonal antibodies directed against the bFGF protein were used for indirect
immunofiorescence experiments. Various antibodies
were tested. In particular, a polyclonal antibody raised
against a synthetic peptide corresponding to the bovine
bFGF amino terminal positions 1 to 12 (R4/R6 kindly
provided by Dr I. Vlodavsky, Vlodavsky et al. 1987)
and a polyclonal antibody also directed against the Nterminus of bovine bFGF (kindly provided by Dr G.
Neufeld, (Schweigerer et al. 1987)), gave the more
consistent results. We found positive labeling already
by stage XI (E&K). The staining seemed to be distributed evenly throughout most of the blastodermic area
with some scattered cells giving a higher signal but
distributed evenly throughout the epiblast, the emerging hypoblast and the marginal zone (Fig. 3A,B).
During primitive streak formation, with the onset of
migration of mesenchymal cells through the primitive
streak, no significant change in the labeling pattern
could be observed (data not shown).
Two sets of controls were also performed. The first
control entailed replacing the anti-bFGF antibody with
pooled rabbit serum. The second control entailed
preincubating the anti-bFGF antibody with excess human bFGF, before applying it to the tissue section. No
positive immunofluorescence signal could be detected
in any control experiment. Instead a very weak uniform
background was observed (Fig. 3C).
Effect of FGF- inhibitors on early blastoderm
development
Blastoderms from preblastula to gastrula stages (stages
X-XIII (E&K) and stages 1-3 (H&H)) were grown in
DCM in the presence of heparin or suramin. Both
substances are known to block bFGF interaction with
its receptor (Neufeld and Gospodarowicz, 1985, 1987).
Blastoderms treated with heparin or with suramin at
doses ranging from 1 to 200 ^g ml" 1 responded in a
dose-dependent manner (Table 1 and Fig. 4). Pregastrula blastoderms when grown in the presence of
heparin at a dose of 50/.igm\~l were incapable of
forming a primitive streak in 42 % of cases and when
treated with heparin at 100/UgmF1 in 72% of cases
(Table 1). Gastrulating blastoderms, when treated at
SO^gml"1, continued through gastrulation, formed a
full-length primitive streak and in 60 % of cases developed further. 10 % were arrested after forming a fulllength notochord but no somites, whilst the remainder
50% continued to develop into normal embryos. At
100 ^g ml" 1 , gastrulating blastoderms developed a fulllength primitive streak in 75 % of the cases but in the
remaining 25 % formed a full-length notochord but
were incapable of forming somites. Suramin-treated
blastoderms behaved similarly except that the effects
were observed at slightly lower doses (Table 1A and see
Fig. 5). In contrast whole stage XI-XIII blastoderms
when grown in DCM for 48 h developed into normal
embryos in 77 % of the cases. The rate of development
corresponded to those of normal blastoderms grown in
ovo (Figs 4A,C).
Table 1. (A) Maximum degree of axial mesoderm development of stage X-XII blastoderms expressed as a
percentage
Factor
Oigml"1)
no axis
primitive streak
chorda
chorda+somites
Stage
n
(%)
(%)
(%)
(%)
X-XII
-
35
13
2
8
77
25
7
21
18
3
10
4
20
29
42
72
_
_
-
8
42
28
17
0
40
75
10
20
23
16
10
5
20
15
13
69
100
0
0
5
16
0
0
0
heparin
X-XII
Primitive streak
10
25
50
100
25
50
100
X-XII
Primitive streak
suramin
1
10
25
50
100
25-50
4
68
0
29
9
0
0
10
14
11
100
50
0
25
0
10
38
25
0
40
(B) Cloning efficiency of stage XII celb as percentage of untreated celb
Factor added
(,/gmr1)
Goning efficiency
(%)
heparin
200
100
suramin
200
100
80
70
33
6
0
60
bFGF during chick mesoderm induction
391
Fig. 4. Suramin and heparin
block the formation of axial
structures. Control and treated
stage XII blastoderms grown in
DCM. (A) Untreated
blastoderm grown for 24 h has
reached the characteristic head
process stage. (B) Blastoderm
grown in the presence of
lO^gml"1 suramin for 24 h: no
axis develops. (C) Untreated
blastoderm grown for 48 h: a
normal stage 13 (H&H)
embryo develops.
(D) Blastoderm grown in the
presence of 10/igml"1 suramin
for 48 h: a very rudimentary
primitive streak develops.
(E) Blastoderm grown in the
presence of 25 jjg ml"1 heparin
for 48 h: no axis develops.
(F) Blastoderm grown in the
presence of 25;/gml"1 suramin
for 48 h: no axial structures
develop. Magnification x!5.
Single cells derived from either pregastrulation or
gastrulation blastoderms were grown as colonies in soft
agar in the presence of either heparin or suramin.
Concentrations of each substance as high as
200 jig ml"1, did not inhibit agar growth of the early
cells (Table IB).
Discussion
We have isolated a chick genomic clone that reveals a
high degree of homology to the mammalian and Xeno-
pus bFGF gene. A fragment of this clone, containing
the full second exon, was used as probe in RNA
hybridization experiments. We found that bFGF is
already expressed at the blastula stage. We could only
detect RNA messages in the marginal zone of stage
XIII blastoderms. In contrast, we detected bFGF protein throughout the developing blastoderm. One possible explanation for that difference would be that bFGF
transcripts are present in other embryonic cells, but in
amounts that are below our level of detection. It is
nevertheless interesting that bFGF transcripts were
392
E. Mitrani and others
Fig. 5. Different levels of abnormalities caused by suramin. Whole stage XII blastoderms grown in DCM for 48 h in the
presence of 25 fig ml"' suramin for48h. (A) No axial structures develop; (B) an embryonic axis develops but there are no
somites; (C) both a notochord and somites developed from this treated blastoderm. Magnification x!5,
detected in the marginal zone since this region has the
capacity to regenerate a hypoblastic layer, which, when
in contact with a stage XIII epiblast, can induce the
formation of axial structures (Azar and Eyal-Giladi,
1979).
Slack and Isaacs (1989) have recently reported that
bFGF-like molecules are present in Xenopus blastulae.
There have also been previous attempts at examining
FGF levels in the early chick embryo (Seed et al. 1988).
These workers, however, reported that FGF is present
in the chick egg yolk and egg white, but did not address
the question of whether the early embryo itself is
synthesizing FGF.
Direct experiments on induction of non-axial mesoderm cannot be performed in the chick since, by the
time the epiblast and hypoblast tissues sort themselves
out into two separate entities, non-axial mesoderm has
already been predetermined (Azar and Eyal-Giladi,
1979; Mitrani and Eyal-Giladi, 1982). Since bFGF is
already expressed in the chick gastrula, we had to resort
to applying agents that block bFGF action in order to
assay for the possible function of bFGF. Neither
heparin nor suramin are specific for bFGF but both are
known to block bFGF action (Neufeld and Gospodarowicz, 1985, 1987; Slack et al. 1987; Moscatelli and
Quarto, 1989). The two substances, although rather
different, had very similar effects on early blastoderms.
Application of either heparin or suramin also resulted
in mesoderm malformations when applied after the
primitive streak was already induced, suggesting that
FGF-like substances may be required throughout the
process of axial mesoderm formation.
It is unlikely that both substances acted in a nonspecific toxic manner since, when applied at higher
concentrations to cells derived from the same blastoderms and grown in agarose, anchorage-independent
growth (AIG) was not inhibited. This behavior should
be contrasted with the behavior of the same cells when
treated with retinoic acid (RA). RA can block AIG at
doses as low as 10~ 9 M whilst effects on whole blastoderms can only be felt at concentrations which are two
orders of magnitude higher (Mitrani and Shimoni,
1989).
The doses of heparin that were shown to inhibit the
formation of axial structures although relatively high
were similar to those that were shown previously to
inhibit FGF induction of mesoderm in Xenopus animal
caps in transfilter experiments (Slack et al. 1987).
Heparin at lmgml" 1 had no toxic effects in Xenopus
isolated animal caps, and did not block the formation of
mesoderm in animal-vegetal combinations (Slack et al.
1987).
If an animal cap of a Xenopus blastula is grown in
isolation, differentiation of non-axial mesoderm does
not take place. In the chick, however, isolated epiblasts
can give rise to non-axial mesoderm (blood islands)
without the addition of exogenous factors (Azar and
Eyal-Giladi, 1979; Mitrani and Eyal-Giladi, 1982; Stern
and Canning, 1990). Presumably by stage XI-XII the
signals responsible for the induction of non-axial mesoderm are already present in the chick blastoderm. The
fact that bFGF transcripts and protein are already
present in pregastrula blastoderms suggests that in the
chick bFGF may be involved, as in Xenopus, in the
induction of non-axial mesoderm.
We have recently shown that bFGF cannot induce
axial mesoderm (notochord and somites) in isolated
chick epiblasts (Mitrani and Shimoni, 1990). In contrast, we showed that conditioned medium derived
from the Xenopus XTC cell line (XTC CM) (Smith,
1987), induced the formation of mesoderm which
became organized into structures such as a full-length
notochord and rows of bilaterally symmetric somites
(Mitrani and Shimoni, 1990). These experiments
suggested that bFGF is not sufficient to induce axial
structures in the chick. The blocking experiments
bFGF during chick mesoderm induction 393
reported here, however, indicate that FGF-like substances may still be necessary for that process to take
place.
FGF-like substances may thus be acting throughout
the whole process of mesoderm formation: earlier in
development in the induction of non-axial mesoderm
and later, in combination with inductive factors provided by the hypoblast (Mitrani and Eyal-Giladi, 1981;
Mitrani and Shimoni, 1990), during the generation of
axial mesodermal structures.
We wish to thank Dr I. Vlodavski for bFGF antibodies,
bovine bFGF protein and suramin, Dr G. Neufeld for bFGF
antibodies and for providing the human bFGF cDNA clone,
Y. Grinfeld and I. Raibstein for help during the cloning
process and Dr A. Fainsod for his comments. This work was
partly supported by the Israel Academy of Sciences (E.M and
Y. G) and by a grant from the American-Israel Binational
Science foundation No. 85-00296/3 (E.M).
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(Accepted 2 March 1990)