Download Early posterior neural tissue is induced by FGF in the

473
Development 125, 473-484 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV1253
Early posterior neural tissue is induced by FGF in the chick embryo
Kate G. Storey1,†, Anne Goriely1, Catherine M. Sargent1, Jennifer M. Brown1, Helen D. Burns2,
Helen M. Abud2,* and John K. Heath2
1Department
2Department
of Human Anatomy, University of Oxford, South Parks Rd, Oxford, OX1 3QX, UK
of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
*Present address: Peter MacCallum Cancer Institute, Smorgon Family Building, Andrews Place, East Melbourne, Australia
†Author for correspondence (e-mail: [email protected])
Accepted 21 November 1997: published on WWW 13 January 1998
SUMMARY
Signals that induce neural cell fate in amniote embryos
emanate from a unique cell population found at the anterior
end of the primitive streak. Cells in this region express a
number of fibroblast growth factors (FGFs), a group of
secreted proteins implicated in the induction and patterning
of neural tissue in the amphibian embryo. Here we exploit
the large size and accessibility of the early chick embryo to
analyse the function of FGF signalling specifically during
neural induction. Our results demonstrate that
extraembryonic epiblast cells previously shown to be
responsive to endogenous neural-inducing signals express
early posterior neural genes in response to local,
physiological levels of FGF signal. This neural tissue does
not express anterior neural markers or undergo neuronal
differentiation and forms in the absence of axial mesoderm.
Prospective mesodermal tissue is, however, induced and we
present evidence for both the direct and indirect action of
FGFs on prospective posterior neural tissue. These findings
suggest that FGF signalling underlies a specific aspect of
neural induction, the initiation of the programme that leads
to the generation of the posterior central nervous system.
INTRODUCTION
hindbrain reticular neurons and spinal interneurons, the
precursors of which arise adjacent to the anterior primitive streak
during the early phase of neural induction (Sechrist and BronnerFraser, 1991; also see Lawson et al., 1991). The precursors of
the posterior CNS (posterior hindbrain and spinal cord),
therefore, appear to be among the first to respond to neuralinducing signals from Hensen’s node. We have recently shown
that signals from the node induce expression of the basic helixloop-helix (bHLH) transcription factor cash4 and the
homeodomain-containing gene Sax-1 (Henrique et al., 1997).
These genes are expressed in the precursors of the posterior CNS
at HH5-6 (Hamburger and Hamilton, 1951 stages) and therefore
after L5, Sox 2 and FLIK. cash4 is a homologue of the
Drosophila achaete-scute genes which are required for the
formation of neural precursors in the fly (for review, see
Campuzano and Modolell, 1992; Campos-Ortega, 1993).
Overexpression of cash4 in the Xenopus embryo results in the
expansion of the neural plate and an increase in the number of
neurons produced. Further, expression of this chick gene in
Drosophila reveals that it is a functional as well as structural
homologue of the fly achaete-scute genes; it is able to rescue
sensory bristle formation in flies lacking both achaete and scute
(Henrique et al., 1997). Our functional studies therefore
implicate cash4 in a similar early neural specification process in
the posterior regions of the chick embryo (Henrique et al., 1997).
We have also demonstrated that cash4 expression can be
induced in anterior neural tissue following grafting of fibroblast
growth factor (FGF)-soaked beads at primitive streak stages
In higher vertebrates, the formation of the central nervous system
(CNS) is initiated by signals that emanate from the anterior end
of the primitive streak (Waddington, 1932; Gallera, 1971; Storey
et al., 1992, 1995; Beddington, 1994). Grafts of this region
(known as Hensen’s node in the chick embryo) induce
extraembryonic epiblast cells to form an organised neural axis
that expresses a range of anteroposterior marker genes and which
contains differentiating neurons (Dias and Schoenwolf, 1990;
Storey et al., 1992). However, the neural-inducing signals
produced by Hensen’s node have yet to be identified.
In the chick, cells competent to respond to such inducing
signals express L5220 (a cell-surface glycoprotein, Roberts et
al., 1991; Streit et al., 1997) whose domain of expression at
early primitive streak stages includes the prospective neural
plate and the region of extraembryonic epiblast cells responsive
to Hensen’s node (Roberts et al., 1991). Expression of the L5
antigen can be maintained by Hepatocyte growth factor/scatter
factor, a secreted factor that is present in Hensen’s node, but
which does not induce neural tissue (Streit et al., 1995, 1997).
A number of genes expressed in the early neural plate have also
been identified and shown to be induced by Hensen’s node,
these include the transcription factor Sox2 (Streit et al., 1997)
and a follistatin-like (FLIK) molecule (Patel et al., 1996).
However, the functions of these genes and the identity of the
signals that regulate them have yet to be established.
Among the first nerve cells to differentiate in the chick are
Key words: Fibroblast growth factor, Neural induction, Hensen’s
node, Chick
474
K. G. Storey and others
(Henrique et al., 1997). This raises the possibility that FGF
signalling plays a role in the anteroposterior patterning of the
chick CNS. This result also implies that FGF signalling acts
upstream of cash4 and therefore suggests that an early step in
the neural programme could be the induction of cash4 by FGF
signals. A number of FGFs are expressed in the anterior
primitive streak when neural induction is taking place (FGF2,
FGF3, FGF4, FGF8; Riese et al., 1995; Mahmood et al.,
1995a,b; own observations) and might therefore play a role in
the initiation of the neural programme. FGFs are characterised
by shared features of sequence conservation and gene structure
as well as the ability to bind to a conserved family of
transmembrane signalling receptors of the tyrosine kinase class
and heparan sulphate proteoglycans present in the extracellular
matrix (reviewed Ornitz et al., 1996; Wilkie et al., 1995). FGF
signals are therefore unlikely to diffuse far from their source and
probably act locally on cells in their immediate vicinity. It is thus
striking that FGFs are expressed during neural induction in and
adjacent to the cells that will give rise to the posterior CNS.
In the early amphibian embryo, FGFs have been shown to
have general posteriorising effects (Slack and Tannahill, 1992)
which act on both mesodermal and neural tissues (e.g. Isaacs et
al., 1994; Cox and Hemmati-Brivanlou, 1995; Pownall et al.,
1996). However, experiments assessing the function of FGFs in
the induction of the amphibian nervous system are at present
inconclusive. While FGFR1-mediated signalling appears not to
be required for the induction and anteroposterior patterning of
the nervous system in the intact embryo (Kroll and Amaya,
1996) and some explants of prospective epidermis (the animal
cap) do not form neural tissue in response to FGF (Cox and
Hemmati-Brivanlou, 1995), other reports suggest that this tissue
does express neural genes in response to FGF and that FGF
signalling in the ectoderm is required for its later response to
neural-inducing signals (Kengaku and Okamoto 1993, 1995;
Lamb and Harland, 1995; reviewed by Doniach, 1996; Launay
et al., 1996; Sasai et al., 1996). Further, mice mutant for FGFR1
do not survive long enough for the role of FGF signalling in
anteroposterior patterning to be assessed (Deng et al., 1994;
Yamaguchi et al., 1994). Indeed, while some neural tissue
appears to be induced in these embryos, they fail to undergo
regression movements and may therefore fail to form more
posterior regions of the CNS; a phenotype similar to that
observed in chick embryos treated globally with molecules that
bind FGF, heparin or suramin (Riese et al., 1995). Finally, while
mouse embryos chimeric for FGF4 exhibit apparent overgrowth
of the CNS (Abud, 1995), mice homozygous for the null allele
of Fgf4 fail to develop far enough to assess the role of FGF
signalling in early neural development (Feldman et al., 1995).
In all these studies, cells are subjected to activation or
inhibition of FGF signalling over prolonged periods of time in
a spatially unrestricted manner. It is therefore difficult, using
these experimental approaches, to define the action of FGF
signalling at specific times during neural development. While
the role of Fgf8 has recently been specifically assessed during
midbrain development (Crossley et al., 1996a; Lee et al.,
1997), there has been no study of FGF function during neural
induction in higher vertebrates. We have accordingly addressed
the role of FGF in the induction of neural tissue in the chick
embryo by making use of local application of FGFs bound to
beads, which permits tightly defined temporal and spatial
activation of FGF-signalling pathways.
Using this approach, we have assessed the neural-inducing
activity of FGFs presented to extraembryonic epiblast cells that
we have shown to be responsive to neural-inducing signals from
Hensen’s node, but which normally give rise to extraembryonic
membranes (Storey et al., 1992; 1995). We show that FGFsoaked heparin beads can maintain and/or induce the L5 epitope
in extraembryonic epiblast cells and induce neural tissue that
specifically expresses markers of the early posterior nervous
system, including Sax-1 and cash4. Strikingly, this FGFinduced tissue does not express anterior neural markers and
does not undergo neuronal differentiation. Induction of early
posterior neural genes takes place in the absence of axial
mesoderm. However, FGF signalling does induce prospective
mesoderm (indicated by the presence of cells co-expressing
Brachyury (Bra) and Delta-1). This raises the possibility that
posterior neural genes are induced indirectly, by signals
provided by prospective mesoderm. Significantly, this ‘indirect’
effect may also be FGF mediated as Fgf8 is induced shortly
after Bra in response to FGF beads and is expressed in
prospective mesoderm cells and the adjacent posterior
neuroepithelium itself. The co-localisation of Bra and cash4 in
epiblast cells around FGF beads also supports the possibility
that FGF acts directly within the prospective neuroepithelium,
downstream of bra expression in this tissue.
This study is the first to assess the role of FGF signalling in
the induction of neural tissue in an amniote embryo. It shows
that FGF signals can mimic a very specific aspect of the neuralinducing capacity of the organiser region; the initiation of the
programme that leads to the generation the posterior nervous
system.
MATERIALS AND METHODS
Preparation of FGF proteins
Human FGF-2 protein was expressed under the control of the trp
promoter of plasmid pFC80 in E. coli strain FICE127 (both a gift of
Dr Antonella Isacchi, Pharmacia Biopharmaceuticals) and purified by
heparin affinity chromatography. Murine FGF-8A, FGF-8B and FGF9 were expressed in E. coli as glutathione-s transferase fusion
proteins, purified by glutathione affinity chromatography, proteolytic
cleavage and heparin affinity chromatography. Recombinant human
FGF-4 protein was a gift of Dr David Rogers (Genetics Institute).
Recombinant FGF-7 was purchased from R and D Systems (Europe)
Ltd. The identity of all recombinant proteins was verified by amino
acid sequencing.
Preparation and detection of DIG-FGF-2
Digoxigenin-labelled FGF-2 (DIG-FGF-2) was prepared as described
by Gleizes et al. (1994). Digoxigenin malemide was purchased from
Boehringer Mannheim. DIG-FGF-2 exhibited comparable affinity and
biological activity to wild-type FGF-2 as determined by binding to
immobilised FGF receptors and the mitogenic response of fibroblasts.
DIG-labelled FGF2 was detected in whole-mount embryos with an
anti-DIG antibody conjugated with alkaline phosphatase (Boehringer
Mannheim) using standard immunocytochemical techniques.
Implanting FGF-soaked beads
Heparin-coated acrylic beads (selected to be approx. 75-100 µm in
diameter; Sigma H5263) were soaked in a FGF of choice for 30 minutes
at room temperature and washed three times in phosphate-buffered saline
(PBS) prior to implantation. Heparin-coated beads simply rinsed in PBS
served as controls. Embryos at stages HH3-4+ were set up in New
culture (New, 1955) ventral side up and individual beads were positioned
Early posterior neural genes induced by FGF
in contact with extraembryonic epiblast cells. Embryos with implanted
beads were incubated at 37oC for desired periods of time up to 36 hours.
DiI-labelling
Epiblast cells were labelled with the lipophilic dye DiI
(1,1′-dioctadecyl-3,3,3′,3′ tetramethyl indocarbocyanineperchlorate,
Molecular Probes) using standard procedures (e.g. Stern, 1990;
Izpisúa-Belmonte et al., 1993). The spatial relationship between the
bead and labelled cells was confirmed using a cooled CCD camera to
detect low level fluorescence through a fluorescein filter set (to reduce
photobleaching and the production of toxic free-radicals) prior to
incubation of embryos for the desired period. Embryos were then
fixed in formal saline/EGTA and the pattern of fluorescently labelled
cells observed using a rhodamine filter set to reveal cell movement.
Fluorescent and bright-field images were superimposed using Adobe
Photoshop.
In situ hybridisation and immunocytochemistry
Following New culture, embryos were fixed in 4% formaldehyde/PBS
for 1 hour to overnight. Whole-mount in situ hybridisation protocol
was adapted after Izpisúa-Belmonte et al. (1993). In some cases,
embryos were further processed for immunocytochemistry with the
Bra antibody (TN-1, kindly provided by Prof. Bernhard Herrman).
Briefly, embryos were fixed for 30 minutes in formol saline at 4°C,
washed in PBS and placed in a blocking solution (PBS containing 3%
bovine serum albumin (BSA), 1% Triton X-100, 0.01% thimerosal and
5% heat-inactivated normal goat serum for 4 hours at room
temperature). Polyclonal antibody TN-1 was added 1:500 and embryos
incubated for 6 hours at room temperature. After extensive washing in
PBS, embryos were incubated in blocking solution with
peroxidase-conjugated goat anti-rabbit IgG antibody (Jackson
ImmunoResearch) (1:50) overnight at 4°C. Embryos were then
washed, underwent the usual diaminobenzidine tetrahydrochloride
reaction and were postfixed in 4% formol saline. Whole-mount
immunocytochemistry with Not-1, Inv4D9 and 3A10 antibodies were
as described in Storey et al. (1992, 1995) and the L5 antibody was used
as described by Streit at al. (1995).
Marker gene expression patterns
Mesoderm markers
All the following genes or proteins are expressed in mesodermal
tissues, however, all but the first are also expressed in the other tissues
including the nervous system:
•The Not-1 antibody recognises a notochord-specific antigen
(Yamada et al., 1991) and is expressed shortly after this axial
mesoderm emerges from Hensen’s node at HH7.
•goosecoid is expressed in the node and later in the emerging
prechordal plate (mesoderm and endoderm) and in the anterior neural
folds from HH8 (Izpisúa-Belmonte et al., 1993).
•Brachyury (Bra) is expressed in posterior regions of the chick
embryo from primitive streak stages; it is found in prospective
mesodermal cells in the primitive streak and in surrounding epiblast cells
(Kispert et al., 1995). These domains of expression retain their spatial
relationship with the regressing node as the posterior CNS is laid down.
Bra is later also expressed in the notochord from HH5, in prospective
posterior endoderm, and in the neural tube anterior to the first formed
somite (Kispert et al., 1995; Knezevic et al., 1996). Bra is therefore a
marker of all three germ layers in posterior regions of the embryo.
•Fgf8 is also expressed in multiple germ layers; it is found in the
anterior primitive streak (excluding the node) and flanking epiblast
and in a number of discrete sites in the developing CNS, including
the forebrain, the isthmus of the midbrain and the newly formed
posterior neural plate (Heikinheimo et al., 1994; Crossley and Martin
1995; Mahmood et al., 1995; own observations).
•cDelta-1 is expressed in presomitic mesoderm and nascent
neurons (Henrique et al., 1995).
475
Neural markers
•tailless is a steroid-hormone receptor expressed in the
diencephalon from HH10 (Yu et al., 1994).
•Engrailed-2 (En-2) is expressed in the posterior midbrain and
rhombomere 1 of the hindbrain (Patel et al., 1989).
•Krox-20 is expressed in the hindbrain in rhombomeres 3 and 5
from HH9 (Wilkinson et al., 1989).
•cash4 is expressed at HH5 in the epiblast flanking Hensen’s node
which will give rise to the posterior CNS and maintains its spatial
relationship with the regressing node during the laying down of this
region of the nervous system. (Henrique et al., 1997). cash4
expression extends laterally into prospective epidermal tissue and also
has an extraembryonic domain in the forming blood islands. Thus
while it is the first gene to distinguish prospective posterior neural
tissue from the anterior neural plate, it is not a neural-specific gene.
•Sax1 is expressed after cash4 in the prospective posterior CNS
either side of the regressing Hensen’s node at HH6-7 (Spann et al.,
1994; own observations). In contrast with cash4, Sax-1 expression is
confined to the nervous system and is therefore the earliest neuralspecific marker expressed in the posterior regions of the embryo.
•Hox b9 is expressed (at HH8-9) in prospective spinal cord and
posterior mesoderm, its anterior boundary of expression is later
established below the level of the somite 9 in the central nervous
system, while its mesodermal domain is located more posteriorly at
the 18/19 somite level.
•The L5 antibody recognises an antigen expressed in the epiblast
cells throughout the anterior two thirds of primitive streak stage
embryos but which becomes restricted to the neural plate by HH8
(Roberts et al., 1991; Streit et al., 1995, 1996).
•The 3A10 antibody (a gift of Dr Jane Dodd) recognises a
neurofilament-associated antigen expressed in the chick by in early
differentiating neurons in the posterior hindbrain, anterior spinal cord
and diencephalon at HH11-12 (Yamada et al., 1991; Storey et al.,
1992).
RESULTS
Grafts of Hensen’s node can induce extraembryonic epiblast
cells to form an ectopic neural axis consisting of both anterior
and posterior CNS that contains differentiating neurons (Storey
et al., 1992). We have used this in vivo approach to assess the
extent to which FGF signalling can mimic these organiser
properties by presenting FGF4 on heparin-coated beads to the
same extraembryonic epiblast cells. FGF4 was chosen because
it contains a signal sequence and because it binds to a number
of FGF receptors and is therefore likely to emulate the activity
of other FGFs. In all cases, beads were soaked in 50 µg/ml FGF4
(FGF beads) (see below) grafted into HH3-3+ embryos (Fig. 1)
and then incubated for 20-22 hours in New culture (New, 1955)
until they reached HH8-9 (unless stated otherwise).
FGF induces early posterior neural tissue in
extraembryonic epiblast
FGF beads, but not control PBS washed beads (PBS beads),
induce ectopic structures expressing the early posterior neural
markers Sax1 and cash4 (Table 1; Fig. 2A-D). Hoxb9, which is
also expressed posteriorly in neural tissue and mesodermal tissue
at HH8-9, is also induced in response to FGF beads (Table 1;
Fig. 2E). FGF beads implanted in embryos incubated until they
reached HH5 (n=4/4) as well as to HH8-9 (Table 1) maintain
and/or induce expression in extraembryonic tissue of the L5
epitope (Fig.2H), an early pan-neural marker and possible
indicator of neural competence (Roberts et al., 1991; Streit et al.,
476
K. G. Storey and others
accumulation of epiblast cells. In contrast with grafts of the
young Hensen’s node, therefore, FGF signalling does not
induce anterior neural tissue, but specifically elicits expression
of genes characteristic of the inchoate posterior CNS.
Fig. 1. FGF or control PBS beads were positioned against the
extraembryonic epiblast in HH3-3+ host embryos and maintained in
New culture for the desired period.
1997). These findings show that FGF signalling can induce
neural tissue, which expresses a combination of transcription
factors characteristic of early posterior neural tissue.
FGF does not induce anterior neural markers
To ascertain that FGF4 specifically initiates the generation of
the posterior neural tissue, we assessed its ability to induce
ectopic expression of a panel of neural genes characteristic of
anterior regions: a forebrain marker, tailless, a
midbrain/hindbrain boundary marker, Engrailed 2 and a marker
of rhombomeres 3 and 5 of the hindbrain, Krox20. FGF beads
do not induce ectopic expression of any of these genes when
placed in the extraembryonic epiblast of HH3-3+ embryos
(Table 2; Fig. 2F,G and 2I-L).
Hensen’s nodes grafted into later stage hosts (HH4) induce
truncated axes, which appear to constitute largely anterior CNS
(Storey et al., 1992). It has also been observed in the amphibian
embryo that competence to respond to FGF changes during
development (Lamb and Harland, 1995) such that ageing
ectoderm loses competence to respond to FGF by expressing
posterior neural markers, but gains the ability to express
progressively more anterior neural genes. We therefore assessed
the ability of the extraembryonic epiblast to respond to FGF
beads in later stage embryos. While the ability to induce ectopic
Sax1 and cash4 is lost by HH4, this activity is not replaced by
the induction of the more anteriorly expressed markers (tailless,
Engrailed and Krox20) (Table 2). An alternative hypothesis is
that, while high FGF concentrations induce expression of
posterior neural genes, low levels induce genes characteristic of
the anterior CNS (Kengaku and Okamoto, 1995; Lamb and
Harland, 1995). We therefore also assessed the ability of FGF
to induce expression of tailless and Krox-20 at lower
concentrations (Fig. 4). Neither Krox-20, nor tailless are
induced by FGF concentrations 10-fold lower (beads soaked in
5 µg/ml FGF4) than those able to induce cash4 and Sax-1 (Fig.
4). Indeed, FGF beads placed in HH4 hosts and beads soaked
in this lower FGF concentration elicit only a small ectopic
Table 1. Induction of neural genes by FGF in HH3-3+
extraembryonic epiblast
FGF beads
PBS beads
cash4
sax1
L5
Hoxb9
12/18 (66%)
0/18 (0%)
9/16 (56 %)
0/8 (0%)
10/10 (100%)
0/6 (0%)
5/7 (71%)
0/4 (0%)
FGF signalling does not elicit neuronal
differentiation
To determine whether FGF expression in the extraembryonic
epiblast is sufficient to elicit a neural programme that proceeds
to the onset of neuronal differentiation, embryos were grafted
with FGF beads or PBS beads and allowed to develop to a later
stage, HH12-13. The resulting ectopic structures were then
assessed for the expression of a neurofilament-associated protein
recognised by the antibody 3A10. Hensen’s nodes derived from
HH3-4 embryos and implanted in extraembryonic epiblast of
HH3-3+ embryos induce ectopic neural axes expressing this
neurofilament-associated antigen within this period of time
(Storey et al., 1992). FGF beads however, do not induce 3A10positive cells within the same period (n=12; Fig. 3A) nor is this
antigen detected around FGF beads implanted within the
extraembryonic epiblast in ovo and cultured for a much longer
period to HH20-24 (n=6; Fig. 3B,C). These data suggest that,
while FGF can initiate the neural programme in extraembryonic
epiblast cells, it is not sufficient to elicit neuronal differentiation,
which therefore requires the action of additional signals.
FGF DOES NOT INDUCE AXIAL MESODERM IN
EXTRAEMBRYONIC EPIBLAST
It is important to establish in this assay whether FGF beads
induce posterior neural tissue directly or as a consequence of
the prior induction of axial mesoderm. To test this, we therefore
assayed for the induction of the Not-1 antigen, which
specifically identifies the notochord from HH7 and for the
expression of goosecoid, which at the time assessed is
expressed in prechordal mesendoderm anterior to the emerging
notochord as well as in the ventral diencephalon. Neither the
Not-1 antigen (0/6 FGF beads, 0/4 PBS beads) nor goosecoid
mRNA (0/7 FGF beads, 0/6 PBS) were induced in response to
FGF or PBS beads placed in contact with the extraembryonic
epiblast at HH3+ (Fig. 5A-C). Together these findings suggest
that axial mesoderm is not formed in response to FGF.
However, brachyury (bra) (Herrman et al., 1990; see
Beddington et al., 1992 for review) is induced in response to FGF
beads at HH3-3+ and in older HH4-4+ hosts (Table 2; Figs 4,
5C). Expression of this T-box-containing transcription factor is
an immediate early response to FGF signalling (Smith et al.,
Table 2. FGF induces posterior, but not anterior neural
genes in HH3-3+ hosts and does not elicit neural genes in
older HH4-4+ host embryos
tlx
En2
Krox20
sax1
cash4
Bra
HH3-3+FGF
beads
0/2
(0%)
0/11
(0%)
0/8
(0%)
9/16
(56%)
7/10
(70%)
12/12
(100%)
PBS beads
0/4
(0%)
0/4
(0%)
0/4
(0%)
0/8
(0%)
0/18
(0%)
0/6
(0%)
HH4-4+FGF
beads
0/5
(0%)
0/6
(0%)
0/6
(0%)
0/4
(0%)
0/4
(0%)
4/4
(100%)
PBS beads
0/4
(0%)
0/4
(0%)
0/4
(0%)
0/4
(0%)
0/4
(0%)
0/4
(0%)
Early posterior neural genes induced by FGF
477
Fig. 3. The neurofilament-associated antigen recognised by the
antibody 3A10 is not present in ectopic structures induced by FGF
beads implanted in extraembryonic epiblast at HH3-3+ and
maintained in New culture to HH12-13 (A) or cultured in ovo to
HH20-24 (B,C). (A) Whole-mount showing 3A10-positive neurons
in the diencephalon and hindbrain of the host embryo and the
absence of this antigen in tissue around the FGF bead (arrowhead);
(B) 3A10-negative ectopic structure associated with a FGF bead
(arrowhead) after 4 days culture in ovo, only brown background
staining is visible; (C) Section of ectopic growth in B confirming the
absence of the 3A10 antigen.
Fig. 2. FGF beads, but not control PBS beads, induce expression
of a combination of transcription factors characteristic of posterior
neural tissue and maintain and/or induce expression of the L5
epitope in the extraembryonic epiblast (A-E,H). However, neither
FGF beads nor PBS beads induce anterior neural markers (F,G,JL). In all cases, beads were implanted at HH3-3+ and gene
expression assessed at HH8-9, except for (F,G,J-L) which were
allowed to develop to HH10-12, when the host embryos express
the marker genes assessed). Whole-mount in situ hybridisation
techniques using DIG-labelled RNA probes were used to detect
relevant genes, unless stated otherwise. (A) FGF beads induce
local ectopic expression of Sax-1, which is expressed in
prospective posterior neural tissue in the host embryo; (B) closeup view showing ectopic Sax-1 expression; (C) PBS beads do not
induce expression of cash4 (shown here) or any other gene
assessed; (D) cash4 is induced in epiblast cells close FGF beads;
(E) Hoxb9 is induced in response to FGF beads; (F) The steroid
hormone receptor tailless is expressed in the diencephalon and is
not induced by FGF beads; (G) Close-up view of FGF bead in F
showing absence of tailless expression; (H) The L5 antigen is
maintained and/or induced by FGF signalling in the
extraembryonic epiblast, as revealed by immunocytochemistry
using a peroxidase conjugated secondary antibody; (I) Krox-20, a
marker of rhombomeres 3 and 5 of the hindbrain is not induced by
FGF beads; (J) Close up view of FGF bead in I showing absence
of Krox20 expression; (K) Close up view of FGF bead in L
showing absence of En2 expression, detection by
immunocytochemistry with a peroxidase conjugated secondary
antibody; (L) The posterior midbrain/anterior hindbrain marker
Engrailed 2 is not induced by FGF beads (arrowhead).
1991; Isaacs et al., 1994) and in the chick Bra is expressed in
presumptive mesodermal tissues of the primitive streak and
subsequently in notochord and endodermal tissues as well as in
the posterior CNS (Kispert et al., 1995; Knezevic et al., 1996).
As Bra is only co-expressed with Delta-1 in the primitive streak,
we combined in situ hybridization for Delta-1 with
immunocytochemistry for the Bra antibody in order to identify
cells that have characteristics of the prospective mesoderm. In
most cases (16/17), Bra and Delta-1 are co-expressed in response
to FGF. In many cases (8/16), they are expressed together in a
block of cells at a distance from the bead which resembles the
regressed primitive streak (Fig. 5D-F). However, not all Braexpressing cells co-express Delta-1 (Fig. 5F) and some Brapositive cells are also found in the immediate vicinity of the bead
(Fig. 5E). Together, these findings indicate that, while ectopic
FGF does not induce axial mesoderm, it can induce expression
of genes characteristic of prospective mesoderm.
Induction of early posterior neural genes is
preceded by expression of brachyury and Delta-1
The presence of Bra/Delta-expressing cells in this assay
indicates that prospective mesodermal cells might be induced
by FGF and it is possible that, in turn, these cells produce
signals that elicit expression of early posterior neural genes. We
investigated this possibility further by assessing the temporal
sequence in which bra, Delta-1 and cash4 are expressed. FGF
induces bra and Delta-1 in extraembryonic epiblast cells within
6 hours, while cash4 is only expressed 10-12 hours after FGF
478
K. G. Storey and others
is presented to extraembryonic epiblast cells (Table 3; Fig. 6AD). The expression of bra and Delta-1 is therefore a prelude to
cash4 induction. It is thus possible that FGF signalling acts
‘indirectly’ via non-FGF signals provided by prospective
mesodermal tissue, to induce early posterior neural genes.
FGF4 induces Fgf8
In view of the different kinetics of bra/Delta-1 and cash4
induction by FGF signalling, we investigated the possibility
that FGF4 might also induce activation of other FGF genes (as
reported by Crossley et al., 1996b). We find that Fgf8 is
induced by FGF4 beads in the extraembryonic epiblast within
8 hours and therefore prior to cash4 (Fig. 6E; Table 4). Fgf8
is normally expressed in both prospective mesoderm (the
primitive streak) and also in the posterior neural plate where
its domain of expression overlaps with Bra and cash4 as well
as Sax1 (see below Fig. 7). This finding therefore identifies
Fgf8 as a signal downstream of FGF4 signalling which may
act ‘directly’ on or within prospective neural tissue to induce
early posterior neural genes (see below).
Bra and cash4 are co-expressed in a subset of
epiblast cells
After 6 hours incubation, Bra is expressed in cells surrounding
FGF beads, however, by 12 hours cells closest to the bead now
express cash4. In embryos in which cash4 in situ hybridisation
is combined with the Bra antibody, we find that some cells
express both cash4 and Bra (Fig. 6F). This suggests that
exposure to FGF elicits a succession of gene expression from
Bra to cash4, in the same cell population. Strikingly, this
sequence of events mirrors the temporal pattern in which these
two genes are normally expressed in prospective posterior neural
tissue. Bra is expressed prior to the onset of cash4 at HH5-6 in
Table 3. Response times of Bra, Fgf8 and cash4 to FGF
signalling
bra
Delta-1
Fgf8
cash4
4 hours
6 hours
8 hours
10 hours
0/4 (0%)
ND
0/4 (0%)
0/4 (0%)
8/8 (100%)
6/12 (50%)
0/4 (0%)
ND
10/10 (100%)
10/10 (100%)
6/10 (60%)
0/8 (0%)
ND
ND
ND
5/12 (71%)
the epiblast adjacent to the primitive streak, (Fig. 3 in Kispert et
al., 1995; Henrique et al., 1997) and, by HH8-9, the overlap
between these two genes (and Sax-1 and Fgf8) is confined to the
most recently formed posterior neural plate (Fig. 7A-J; and see
Fig. 4 in Kispert et al., 1995). These findings further support the
direct action of FGF signals on the prospective neuroepithelium,
which could act via brachyury expression in this tissue.
A specific subset of FGFs induce cash4 and Bra
We assessed the ability of a variety of FGF family members with
differing receptor specificities to induce expression of cash4 and
Bra in order to establish the ligand-specificity of this effect and
to deduce the possible receptor-mediated signalling pathways
involved (Table 5). While FGF7 does not induce Bra or cash4,
FGF2, FGF4 FGF9 and FGF8b all induce expression of both
these genes. Given the binding specificities of these FGFs for
specific receptors (reviewed by Orntiz et al., 1996), we can rule
out transduction through FGFR2 (IIIb) which specifically
mediates the activity of FGF7. Chick FGFR1 and FGFR2 are
both expressed in the extraembryonic epiblast at HH3-3+, while
FGFR3 is expressed most highly in the primitive streak and only
weakly in surrounding epiblast cells (data not shown). The most
likely receptors transducing FGF presented to extraembryonic
epiblast are therefore the IIIC splice variants of FGFR1 and/or
FGFR2. It is significant that FGF8b is able to induce cash4 as
well as Bra, as this supports an endogenous role for this gene in
the induction of early posterior neural genes.
Concentration effects and cell movements in
response to FGF
Previous studies of the activity of FGF bound to heparin-coated
beads (e.g., Cohn et al., 1995; Crossley et al., 1996) have not
defined the concentration of FGF required for signalling. It is,
however, important to establish that FGF-induced neural
induction occurs at physiologically relevant concentrations of
ligand (Slack, 1994). We soaked heparin-coated beads in a
range of FGF4 concentrations (0.5-50 µg/ml) and assessed
their ability to induce expression of Bra (Fig. 4). We find that
beads soaked in a concentration between 0.5 and 5 µg/ml elicit
Bra expression in half the cases examined, while a maximal
response is achieved when beads are soaked in 50 µg/ml FGF4.
By comparison, the data of Ornitz et al. (1996) indicate that
Fig. 4. Heparin-coated beads were soaked in a range of FGF4
concentrations (µg/ml) and juxtaposed with extraembryonic epiblast
cells in HH3-3+embryos, which were then cultured until they
reached HH8-9. The activity of different concentrations of FGF
presented by the beads was assessed in terms of the ability to induce
expression of Bra, cash4, Sax-1, Krox20 and Tlx. While Bra is
induced by FGF presented by beads soaked in 0.5 µg/ml. cash4 and
Sax-1 are only expressed in response to higher FGF concentrations.
Krox20 and tailless expression are not elicited by FGF signalling.
(‘n’ values on columns tops).
Table 4. A subset of FGFs induce expression of Bra and
cash4
FGF2
FGF4
FGF7
FGF8a
FGF8b
FGF9
conc.
Bra
cash4
50 µg/ml
50
300
60
6
50
4/4
6/6
0/12
0/10
2/2
4/4
4/4
4/6
0/12
0/10
2/7
4/4
Early posterior neural genes induced by FGF
the Ed50 for FGF4 activating FGF-R2c and R1c in transfected
cell lines is about 0.5 nM. Combined with a clear doseresponse curve (Fig. 4), this suggests that FGF beads present
physiological levels of ligand, although we cannot calculate the
exact amount of FGF available to surrounding cells.
FGFs bind locally to cell surface receptors and heparan
sulphate proteoglycans present in the extracellular matrix
(Wilkie et al., 1995). FGFs should therefore only be presented
to cells in the immediate vicinity of a bead. In order to define
the cell population directly exposed to FGFs in this assay, the
distance of diffusion from heparin beads was assessed by
immunocytochemical detection of digoxigenin (DIG)-labelled
FGF2. While this growth factor is detected only on the surface
of beads soaked in 0.5 µg/ml DIG-FGF (Fig. 8A), DIG-FGF
is restricted to cells immediately adjacent to beads soaked in
concentrations of 5 µg/ml (Fig. 8B). At 50 µg/ml DIG-labelled
FGF2 is detected 4-6 cell diameters from the beads (Fig. 8C;
also assessed in sections, not shown). The lower level of our
immunocytochemical detection of DIG-FGF therefore
correlates with the lowest concentrations at which we can
detect Bra expression around a bead. Together, these data
indicate that beads soaked in 50 µg/ml FGF present local
physiological levels of growth factor.
FGFs are known to stimulate both cell movement and
proliferation (Wilkie et al., 1995). In order to define further the
cell population exposed to FGF, we compared the movement of
DiI-labelled extraembryonic epiblast cells in response to beads
washed in PBS or soaked FGF. We observe no dramatic
difference between the movement of cells in contact with PBS
(n=14) or FGF beads (n=23). In both cases, DiI-labelling is found
in the vicinity of the bead and in trails of cells that have moved
away from the bead. Thus, although cells move away from FGF
beads placed in the extraembryonic epiblast, this behaviour
appears to reflect normal morphogenetic movements in this
region, rather than a response evoked by FGF signalling (Fig.
8D,E). We also assessed the possibility that FGF-stimulated
extraembryonic epiblast cells recruit cells from the embryonic
epiblast. Epiblast cells at the lateral edge of the prospective neural
plate were labelled with DiI at HH3-3+ and an FGF bead placed
in the adjacent extraembryonic epiblast (Fig. 8F). In all cases
(14/14), no DiI-labelled cells were found in the vicinity of the
bead, following overnight culture (to HH8-9) (Fig. 8G). Thus,
while cell proliferation may expand the population of cells
responding to this local source of growth factor, we show that
embryonic epiblast cells are not recruited in response to local
application of FGF to the extraembryonic epiblast.
DISCUSSION
This is the first study to examine the role of FGF signalling in
the induction of neural tissue in an amniote embryo. FGFs are
expressed in the organiser region Hensen’s node/anterior
primitive streak; a source of signals that induces both anterior
and posterior neural tissue which subsequently undergoes
neuronal differentiation. We demonstrate that, in comparison
with Hensen’s node, FGF signalling at primitive streak stages
leads to the induction of a very specific subset of genes
associated with the laying down of the posterior nervous system.
We show that ectopic FGF maintains and/or induces expression
of the early neural marker L5 in extraembryonic epiblast cells
and elicits the expression of the transcription factors cash4 and
479
Sax-1, genes expressed transiently during the formation of the
posterior nervous system. Further, this early posterior neural
tissue forms without the induction of anterior neural markers and
does not undergo the complete neural programme culminating
in neuronal differentiation. We present evidence for both direct
and indirect actions of FGF signalling on the prospective
neuroepithelium. While axial mesoderm does not form in
response to FGF signalling, the co-expression of Bra and Delta1 in some cells suggests that presumptive mesodermal tissue is
formed. Neural-inducing signals could therefore be provided
indirectly by presumptive mesoderm. However, these ‘indirect’
signals may also be FGF-mediated as FGF4 beads induce Fgf8,
which is expressed in both prospective mesoderm and the
forming posterior neural plate. The co-localisation of Bra and
cash4 in cells around FGF beads further suggests that the
induction of posterior neural genes could be mediated directly
via Bra expression in the prospective neuroepithelium. These
findings therefore show that FGF signalling in an early amniote
embryo can mimic a specific subset of node activities, which
include the maintenance of neural competence as well as the
induction of early posterior neural genes.
The assay
We have used the large and accessible chick embryo to analyse
the effects of FGF activity in a spatiotemporally defined
manner. By placing FGF-soaked heparin beads in contact with
extraembryonic epiblast cells, previously shown to be
competent to respond to endogenous neural-inducing signals,
we have tested the neural-inducing activity of FGF. While the
extraembryonic epiblast may not be a completely naïve tissue,
it contrasts favourably with Xenopus animal cap ectoderm
which contributes to embryonic structures and appears to be
prepatterned (Sokol and Melton, 1991). In comparison, chick
extraembryonic epiblast is fated to give rise to extraembryonic
membranes and is physically remote from the embryonic axis.
Our DiI-labelling studies also demonstrate that FGF beads
placed in the extraembryonic epiblast do not recruit embryonic
epiblast cells that might have been exposed to neural-inducing
signals from Hensen’s node. Further, although only the medial
half of the extraembryonic epiblast is competent to respond to
neural-inducing signals (Storey et al., 1992), it is striking that
ectopic gene expression is often found on the side of FGF beads
furthest from the host embryo (see for example Figs 2A, 6B).
This pattern is inconsistent with the influence of other neuralinducing signals emanating from the embryonic axis and may
reflect the radial expansion of the embryo at these stages.
By assessing the effects of FGF signalling with respect to the
FGF concentration in which beads were soaked, we have also
identified a ‘soaking’ concentration that delivers physiologically
relevant levels of FGF. Further, using DIG-labelled FGF2, we
show that at these concentrations FGF diffuses from the heparin
bead and becomes associated with cells in its immediate vicinity.
The induction of Fgf8 in response to FGF4, however, may relay
FGF signals beyond this region. Indeed, Fgf8 expression, along
with the morphogenetic movements of extraembryonic epiblast
cells may account for patterns of gene expression that extend
outside the domain defined by the spread of DIG-labelled FGFs.
Comparison of the effects of six different FGF members has also
allowed us to establish the ligand-specificity of observed effects
and to deduce the possible receptor-mediated signalling
pathways, which include IIIC splice variants of FGFR1 and/or
480
K. G. Storey and others
Fig. 5. Axial mesoderm is not induced in response to FGF beads
(A-C), but expression of Bra and Delta-1 is detected (D-F).
(A) goosecoid expression marking the cells of the prechordal region
(as well as overlying diencephalic tissue) is not induced by FGF
beads; (B) the notochord-specific antigen Not-1 is shown in the host
embryo; (C) Not-1 is not induced by FGF beads; (D) Delta-1 is
expressed, frequently in a block of cells at a distance from the FGF
bead; (E) Delta-1-expressing cells (purple) also express Bra (brown)
as revealed by immunocytochemistry with the TN antibody following
in situ hybridisation for Delta-1 (shown in D). Not all Bra-expressing
cells co-express Delta-1, some Bra only positive cells are also found
in the vicinity of the bead (arrowhead); (F) Section (level indicated by
bar in E) through region of Delta-1-expressing cells in E, showing
that a central group of Delta-1 cells co-express bra (arrowhead).
FGFR2. Our study therefore further defines the extraembryonic
epiblast assay and characterises this in vivo method of delivering
secreted factors.
FGF can initiate the neural programme in the chick
embryo
In common with grafts of Hensen’s node (Streit et al., 1997), FGF
signalling is able to maintain and/or induce expression of the L5
antigen in the extraembryonic epiblast. The L5 antibody
recognises the LeX oligosaccharide epitope present in two
different glycoproteins one of which (L5220) is present early in
extraembryonic tissue and is associated with neural competence,
while the second (L5450) is a later pan-neural marker (Streit et
al., 1996, 1997). As FGF beads implanted for only a short period
(6-8 hours) are surrounded by an ectopic region of L5 expression,
it is possible that FGF signalling maintains L5220. However, FGF
may also induce neural tissue that subsequently expresses L5450.
Thus, while these findings and the induction of neural-specific
Fig. 6. Temporal sequence of gene expression (detected by in situ
hybridisation) in response to FGF beads; bra and Delta-1 are induced
prior to Fgf8 and all three are induced prior to cash4 (A-E, see Table
3). (A) bra is induced within 6 hours in response to FGF signalling
(arrowhead); (B) Delta-1 is also present after 6 hours (although the
signal shown here is stronger than that shown for bra in A, this is not
typical and Delta-1 is detected in only half the cases assessed at 6h
while is bra is always present, see Table 3); (C) cash4 is first
detected around FGF beads (arrowhead) after 10-12 hours; (D) High
magnification of ectopic cash4 expression around the FGF bead in C;
(E) Fgf8 is first detected after 8 hours (arrowhead); (F) Following
overnight culture (16-18 hours) cash4 and Bra are co-expressed in
cells close to FGF beads (arrowhead); shown here in section
following in situ hybridisation for cash4 (blue labelling; same bead is
shown in Fig. 2C) in combination with immunocytochemistry with
an anti-brachyury antibody (brown labelling).
genes (see below) implicate FGF in neural competence, the
specific maintenance of L5220 by FGF signalling has yet to be
determined. Like Hensen’s node, FGF also induces ectopic
expression of the transcription factors cash4 and Sax1, which are
markers of early posterior neural tissue (Henrique et al., 1997;
Spann et al., 1994). However, unlike organiser activity (Storey et
al., 1992), FGF signalling in extraembryonic epiblast does not
elicit the expression of the early neuronal marker recognised by
the 3A10 antibody even in embryos cultured to HH24, suggesting
that FGF does not trigger the complete neural programme leading
to neuronal differentiation.
The failure to induce neurons is consistent with the induction
of only early posterior neural genes (cash4 and Sax-1). cash4 is
expressed transiently in precursors of the posterior CNS in a
rostrocaudal wave of expression that moves in concert with the
regressing node/primitive streak and is induced by signals
emanating from the node (Henrique et al., 1997). It can substitute
for the neural specification activity of homologous genes
(achaete and scute) in Drosophila and expands the neural plate
Early posterior neural genes induced by FGF
481
Fig. 7. During normal development Bra, cash4, Sax-1
and Fgf8 are expressed in overlapping domains in the
posterior neural plate at HH9-10 (A-J). (A) The
posterior neural plate (white arrowhead) expresses bra
as well as the cells in the regressing primitive streak
(PS) and the notochord (black arrowhead); (B) section
through A (white bar) showing expression of bra in
both open neural plate and underlying mesoderm.
(C) cash4 is expressed in the neural plate at this time,
the caudal-most region of this domain overlaps with
bra expression; (D) Section through B (white bar)
showing cash4 expression in the open neural plate but
not in underlying mesodermal tissues; (E) Sax-1 is
expressed in a similar domain to cash4 in the posterior
neural plate; (F) Section through E (white bar) showing
Sax-1 expression confined to the neural plate; (G) Fgf8
is expressed in the regressing primitive streak and in
the caudal end of the forming posterior neural plate in a
domain that overlaps with cash4, Sax-1 and bra at HH9; (H) Section through (white bar) G showing Fgf8 expression in the mesodermal and
prospective neural tissue; (I) By HH10 Fgf8 is clearly expressed in the elevating posterior neural folds; (J) Section through I (white bar)
showing localisation of Fgf8 throughout neural folds as well as underlying mesodermal tissues.
when overexpressed in the frog embryo (Henrique et al., 1997).
The expression of cash4 in neural precursors in the chick,
together with these functional studies therefore suggests that
cash4 acts early to assign neural cell fate in posterior regions of
the chick embryo. Our current findings suggest that expression
of cash4 is however, insufficient to trigger the cascade of gene
expression that leads to neuronal differentiation.
During neural development cash4 is expressed prior to Delta1, which in the CNS identifies the first post-mitotic neurons
(Henrique et al., 1995). In this assay, Delta-1 is always coexpressed with Bra (a combination indicative of prospective
mesodermal tissue in the primitive streak; Fig. 5F) suggesting that
post-mitotic neurons (which do not express Bra) do not form in
response to FGF. Consistent with this interpretation,
overexpression of Fgf8 in the developing midbrain leads to a
dramatic expansion of the neural precursor population in the
ventricular zone (Lee et al., 1997). It is therefore tempting to
speculate that, in line with in vitro data (Gensburger et al., 1987;
Murphy et al., 1994; Temple and Qian, 1995) a common function
of FGF signalling in the developing nervous system is the
induction and/or maintenance of precursor cells. Indeed, the
generation of post-mitotic cells and their subsequent
differentiation as neurons or glia may require the down regulation
of cash4 and Sax-1 and the activity of other signals, such as
retinoic acid, bone morphogenetic proteins, sonic hedgehog and
neurotrophins (Papalopulu and Kintner, 1996; Sharpe and Cross,
1997; Liem et al., 1995; Shah et al., 1996; Averbuch-Heller et al.,
1994; Roelink et al.,1995; Jungbluth et al., 1997). FGF signalling
in this assay, therefore, appears to elicit only the initial steps in
the neural programme, which includes maintenance of neural
competence and the induction of a combination of transcription
factors characteristic of posterior neural precursors.
FGF does not induce anterior neural tissue
FGF does not induce any of the anterior neural markers tested
in this assay; these include genes expressed in the diencephalon
(tailless), at the midbrain/hindbrain boundary (Engrailed 2)
and in the hindbrain (Krox20). This suggests that neural tissue
induced in response to FGF has a more posterior regional
character, which may include posterior hindbrain (at least
Fig. 8. Digoxigenin-labelled FGF2 was used to detect the spread of
FGF from beads implanted in the extraembryonic epiblast (A-C), and
DiI was used to follow the movement of cells exposed to FGF or
PBS beads (D,E) as well as to test the possibility that FGF beads
recruit embryonic epiblast cells from the host embryo (F,G). The
spread of DIG-FGF2 from beads soaked in concentrations of (A) 0.5
µg/ml, (B) 5 µg/ml and (C) 50 µg/ml, was detected with an antiDIG-alkaline-phosphatase conjugated antibody following
implantation in HH3-3+ embryos and overnight incubation in New
culture. The dorsal surface of the extraembryonic epiblast cells was
labelled with DiI and (D) PBS beads or (E) FGF beads were placed
over the labelled cells and in HH3-3+ embryos, which were cultured
overnight. Cells exposed to FGF did not appear to move more than
those exposed to PBS beads; (F) DiI labelling of embryonic epiblast
cells at the lateral edge of the prospective neural plate at HH3-3+
adjacent to a FGF bead (arrowhead); (G) Following overnight culture
no DiI-labelled cells are recruited by FGF beads (arrowhead).
482
K. G. Storey and others
below the level of Krox20 expression in rhombomere 5) and
spinal cord, as judged by the induction of Hoxb9. The absence
of more anterior neural markers might also reflect the
undifferentiated state of this FGF-induced neural tissue, which
is characterised by the expression of only early markers of the
posterior neural precursor population.
In this assay, anterior neural markers are also not induced by
low FGF concentrations. This contrasts with reports in
amphibian embryos in which high FGF concentrations elicit
expression of posterior neural genes, while low levels of FGF
induce anterior neural markers (Kengaku and Okamoto, 1995).
However, these experiments were carried out with ectoderm
cells which had been previously dissociated, a treatment that can
in itself lead to the formation of anterior neural tissue (Grünz
and Täcke, 1989; Godsave and Slack 1991; Wilson and
Hemmati-Brivanlou, 1995). Indeed, Lamb and Harland (1995)
report only weak concentration effects, but they do find anterior
neural markers, such as Engrailed, induced when old (late
gastrula) animal caps are exposed to FGF. Ectoderm in these
experiments, however, also underwent treatment that may have
led to the partial dissociation of cells. A further problem with
these Xenopus experiments is that it is not possible to distinguish
between multiple actions of FGF signalling during development.
Using more precise methods afforded by the use of FGFcoated beads, our results show that in the chick there is a
defined window of competence during which epiblast cells
respond to FGF by expressing early posterior neural genes and
that anterior neural genes are not induced in their stead in older
epiblast cells. Further, using the same technique Crossley et al.
(1996a) have demonstrated that much later during the
regionalisation of the nervous system and formation of the
midbrain, FGF signals do now elicit Engrailed expression. This
emphasises, along with other studies (e.g. Shimamura and
Rubenstein, 1997) that FGF signalling acts at specific times
and in distinct regions of the developing nervous system. By
showing that FGF signalling underlies the initial steps in the
generation of posterior neural tissue, our study identifies a new
role for such signals at primitive streak stages.
Do FGF signals act directly or indirectly to induce
early posterior neural genes?
It is possible that non-FGF signals provided by mesodermal
tissues induce the expression of early neural genes in this assay.
Axial mesodermal tissue has been shown to provide neuralinducing signals in many vertebrates, including the chick
embryo (Storey et al., 1995; A. Rowan, C. D. Stern and K. G.
S., unpublished data). We demonstrate that FGF signalling in
this assay does not induce markers of axial mesoderm, the
prechordal plate marker goosecoid and the notochord-specific
antigen Not-1, confirming observations made in the Xenopus
embryo (Cho et al., 1991; Pownall et al., 1996). However, we
do find that FGF beads induce the co-expression of genes
characteristic of prospective para-axial mesoderm in the
primitive streak (Delta-1 and Bra).
Evidence for the indirect action of FGF
Several lines of evidence suggest that early posterior neural
genes could be induced indirectly by signals emanating from
these prospective mesoderm cells: (i) bra and Delta-1
expression precede cash4 induction by at least 4 hours; and (ii)
the competence to respond to FGF by expressing bra persists
beyond that for cash4 and Sax-1 (Table 2). As neural
competence is lost shortly after HH4 (Gallera, 1971; Storey et
al., 1992; Streit et al., 1995), it may be that signals produced
by bra/Delta-1-expressing tissue are not produced soon enough
to elicit cash4 and Sax-1 expression.
However, the proposition that these prospective mesoderm
cells are a source of neural-inducing signals is not consistent
with the results of grafting experiments that compare the neuralinducing abilities of prospective axial and non-axial mesoderm
cells in the anterior primitive streak (Storey et al., 1995). These
experiments show on the one hand that, while prospective axial
mesoderm (medial mesendoderm) is a potent neural inducer,
prospective non-axial mesoderm (posterolateral mesendoderm)
does not induce neural tissue in extraembryonic epiblast cells
(Storey et al., 1995). On the other hand, this failure to induce
neural tissue was assessed only on morphological grounds and
it may be, therefore, that prospective non-axial mesoderm can
induce expression of early posterior neural genes.
Evidence for direct effects
In this context, it is significant that an early response to the FGF
beads is the induction of Fgf8. During development Fgf8 is
expressed in both this prospective non-axial mesoderm in the
primitive streak, as well as later in adjacent developing posterior
neural tissue in a domain that overlaps with those of cash4 and
Sax-1 (Fig. 7G-I). It is therefore possible that FGF signalling
mediates the action of non-axial mesoderm and also that it may
act directly within the posterior neural plate. We show that Fgf8
is induced prior to cash4 in response to FGF beads and also that
FGF8 protein can induce cash4 (as well as bra) identifying it
as a potential endogenous regulator of this early neural gene.
The presence of cells that co-express Bra and cash4 close to
FGF beads (Fig. 6F) further suggests that direct action of FGFs
on prospective neural tissue could be mediated via Bra expression
in this tissue. This Bra-expressing cell population most likely
corresponds to those Bra-positive cells close to FGF beads that
do not co-express Delta-1 (Fig. 5E). In fact, the spatiotemporal
pattern in which Bra and cash4 are expressed in the embryo
during the laying down of the posterior nervous system suggests
that bra is a prelude to the expression of cash4 in the same cell
population (Fig. 7A-D) (Kispert et al., 1995; Henrique et al.,
1997). Strikingly, a requirement for bra for the maintenance of
Sax-1 expression has been demonstrated in the T-mutant mouse
(Schubert et al., 1995), although this may reflect the failure to
form mesodermal tissue as well the absence of bra in the posterior
neural plate. A role for bra in the induction of early posterior
neural genes might also account for the formation of an
apparently normal posterior nervous system in transgenic frogs
in which FGFR1-mediated-signalling is blocked at early gastrula
stages, but after the induction of bra (Kroll and Amaya, 1996).
The concentration effects that we observe (Fig. 4) may also
support the direct action of FGFs on prospective
neuroepithelium. These data show that bra is induced by both
low and high FGF doses while posterior neural genes are only
induced at high FGF concentrations. This is consistent with the
early expression of bra in the vicinity of the bead and its later
expression at a distance, while cash4 is expressed close to FGF
beads. One interpretation of these results is that cells close to
the FGF bead initially express bra, but later acquire a neural
fate. Alternatively, cells may initially turn on mesodermal
genes, move away and induce neural tissue in their wake.
Early posterior neural genes induced by FGF
However, as DiI-labelling of the extraembryonic epiblast
exposed to FGF (or PBS) beads shows that some cells do
remain in the vicinity of these beads while others move away
we cannot distinguish between these two possibilities.
In short, we find evidence for both direct and indirect effects
of FGF signalling on prospective neural tissue. These two
routes are not mutually exclusive. Indeed, given the induction
of Fgf8 in this assay and its expression in both prospective
mesodermal and neural tissue, direct and indirect effects could
be mediated by the same signal in this region of the embryo.
The role of FGF in neural induction
Our results implicate FGF signalling in neural competence and
show that FGF signalling can elicit expression of early posterior
neural genes in the absence of anterior neural tissue. This
suggests that the initial steps in the generation of posterior neural
tissue take place downstream of FGF signalling independently
of other node-derived signals. This contrasts with many studies
in the Xenopus embryo which show that initial neural-inducing
signals (such as Noggin and Chordin) elicit formation of anterior
neural tissue and that this step is a prerequisite for the later
induction of posterior neural genes (Nieuwkoop et al., 1952;
reviewed by Hemmati-Brivanlou and Melton, 1997; see Taira et
al., 1997). It is also striking that in our assay FGF signalling
elicits only early and transient markers of prospective posterior
neural tissue and it is possible that expression of these genes may
not be indicative of caudal neural character, but rather reflects
the induction of a particular cell state associated with the
formation of neural precursors in this region.
In conclusion, while our data strongly suggest that FGF
signalling functions during neural induction and that it
specifically plays a role in the generation of precursors of the
posterior nervous system, spatiotemporally defined loss-offunction experiments are required to confirm these activities.
The early posterior neural genes induced in this assay are
present only transiently; their expression ceases when the cells
of the posterior neural plate are left behind by the regressing
Hensen’s node/FGF source and no longer express Fgf8. It will
be intriguing to assess the regulation of this transition in the
context of signals that instigate later phases of the neural
programme leading to neuronal differentiation.
We thank Helen Skaer, Jonathan Slack, Claudio Stern and Andrea
Streit for critical reading of the manuscript. We are grateful to Yosef
Gruenbaum for the gift of Sax-1 probe; Bernard Herrman for the gift
of the Bra antibody; Domingos Henrique for gifts of cash4 and Delta1 plasmids; Robb Krumlauf for the gift of Hoxb9 plasmid; Gail Martin
and Clive Dickson for the gift of mouse Fgf8 plasmids; Jim Smith for
the gift of the bra plasmid and to Andrea Streit for the L5 antibody.
This work was supported by Joint MRC small project grant for the
MRC to K. G. S. and J. K. H. and by grants from the Wellcome Trust
to J. K. H. and K. G. S.
REFERENCES
Abud, H. E. (1995). In Examination of methods for the study of FGFs during
mouse development. D. Phil. Thesis, University of Oxford.
Averbuch-Heller, L., Pruginin, M., Kahane, N., Tsoulfas, P., Parada, L.,
Rosenthal, A. and Kalcheim, C. (1994). Neurotrophin-3 stimulates the
differentiation of motoneurons from avian neural tube progenitor cells. Proc.
Natl. Acad. Sci. USA 91, 3247-3251.
Beddington, R. S. P. (1994). Induction of a second neural axis by the mouse
node. Development 120, 613-620.
483
Beddington, R. S. P., Rashbass, P. and Wilson, V. (1992). Brachyury – a
gene affecting mouse gastrulation and early organogenesis. Development
1992 supplement, 157-165.
Campos-Ortego, J. (1993). Early neurogenesis in Drosophila melanogaster.
In The Development of Drosophila (ed. M. Bat and A. Martinez Arias) Vol.
2. pp 1132-1172. New York: Cold Spring Harbour Laboratory Press.
Campuzano, S. and Modolell, J. (1992). Patterning of the Drosophila nervous
system: the achaete-scute gene complex. Trends. Genet. 8, 202-208.
Cho, K. W. Y., Blumberg, B., Steinbreisser, H. and De Robertis, E. M.
(1991). Molecular nature of Spemann’s organiser: the role of the Xenopus
homeobox gene goosecoid. Cell 7, 1111-1120.
Cohn, M. J., Izpisua-Belmonte, J-C, Abud, H., Heath, J. K. and Tickle, C.
(1995). Fibroblast growth factors induce additional limb development from
the flank of chick embryos. Cell 80, 739-746.
Cox, W. G. and Hemmati-Brivanlou, A. (1995). Caudalisation of neural fate
by tissue recombination and bFGF. Development. 121, 4349-4358.
Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a
family of polypeptides and is expressed in regions that direct outgrowth and
patterning in the developing embryo. Development. 121, 439-451.
Crossley, P. H., Martinez, S. and Martin, G. R. (1996a). Midbrain
development induced by FGF8 in the chick embryo. Nature. 380, 66-68.
Crossley, P. H., Minowada, G., MacArthur, C. A. and Martin, G. R.
(1996b). Roles for FGF8 in the induction, initiation, and maintenance of
chick limb development. Cell 84, 127-136.
Dias, M. and Schoenwolf, G. C. (1990). Formation of ectopic
neuroepithelium in chick blastoderms : age related capacities of induction
and self-differentiation following transplantation of quail Hensens’s node.
Anat. Rec. 229, 437-448.
Deng, C-X., Wynshaw-Boris, A., Shen, M., Daugherty, C., Orntiz, D. and
Leder, P. (1994). Murine FGFR-1 is required for early postimplantation
growth and axial organisation. Genes Dev. 8, 3045-3057.
Doniach, T. (1995). Basic FGF as an Inducer of Anteroposterior Neural
pattern. Cell 83, 1067-1070.
Feldman, B., Poueymirou, W., Papaioannou, V. E., DeChiara,T. M. and
Goldfarb, M. (1995). Requirement of FGF4 for post-implantation mouse
development. Science 267, 246-249.
Gallera, J. (1971). Primary induction in birds. Adv. Morph. 9, 149-180.
Gensburger, C., Labourdette, G. and Sensenbrenner, M. (1987). Brain
basic fibroblast growth factor stimulates the proliferation of rat neuronal
precursor cells in vitro. FEBS Lett. 217, 1-5.
Gleizes, P. E., Noaillac-Depeyre, J. and Gas, N. (1994). Labelling of basic
fibroblast growth factor with digoxigenin: a non-radioactive probe for
biochemical and cytological applications. Anal. Biochem. 219, 360-7.
Godsave, S. and Slack, J. M. W. (1991). Single cell analysis of mesoderm
formation in the Xenopus embryo. Development 111, 523-530.
Grunz, H. and Tacke, L. (1989). Neural differentiation of Xenopus laevis
ectoderm takes place after disaggregation and delayed reaggregation without
inducer. Cell Diff. Dev. 28, 211-218.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the
development of the chick embryo. J. Morph. 88, 49-92.
Heikinheimo, M., Lawshe, A., Shakleford, G. M., Wilson, D. B. and
MacArthur, C. A. (1994). FGF8 expression in the post-gastrulation mouse
suggests roles in the development of the face limbs and central nervous
system. Mech. Dev. 48, 129-138.
Hemmati-Brivanlou, A. and Melton, D. (1997). Vertebrate Embryonic Cells
Will Become Nerve cells Unless Told Otherwise. Cell 88, 13-17.
Henrique, D. Tyler, D. Kintner, C. Heath, J. K., Lewis, J., Ish-Horowicz,
D. and Storey, K. G. (1997). cash4 a novel achaete-scute homologue
induced by Hensen’s node during the generation of the posterior nervous
system. Genes Dev. 11, 603-611.
Henrique, D., Adam, J., Myatt, A., Chitnis, A., Lewis, J. and IshHorowicz, D. H. (1995). Expression of a Delta homologue in prospective
neurons in the chick. Nature 375, 787-790.
Herrmann, B. G., Labeit, S. Poustka, A., King, T. R. and Lehrach, H.
(1990). Cloning of the T gene required in mesoderm formation in the mouse
embryo. Development 113, 891-911.
Isaacs, H. V., Pownall, M. E. and Slack, J. M. (1994). eFGF regulates Xbra
expression during Xenopus gastrulation. EMBO J. 13, 4469-4481.
Izpisua-Belmonte, J. C., De-Robertis, E. M., Storey, K. G. and Stern, C.
D. (1993). The homeobox gene goosecoid and the origin of organiser cells
in the early chick blastoderm. Cell 74, 645-659.
Jungbluth, S. Koentges, G. and Lumsden, A. (1997). Coordination of early
neural tube development by BDNF/trkB. Development 124, 1877-1885.
Kengaku, M. and Okamoto, H. (1993). Basic fibroblast growth factor induces
484
K. G. Storey and others
differentiation of neural tube and neural crest lineages of cultured ectoderm
cells from Xenopus gastrula. Development 119, 1067-1078.
Kengaku, M. and Okamoto, H. (1995). bFGF as a possible morphogen for
the anteroposterior axis of the central nervous system in Xenopus.
Development 121, 3121-3130.
Kispert, A., Ortner, H., Cooke, J. and Herrmann, B. G. (1995). The chick
Bra gene: Developmental expression pattern and response to axial induction
by localised activin. Dev. Biol. 171, 458-470.
Knezevic, V., De santo, R. and Mackem, S. (1996). T-box genes in chick
gastrulation. Development 123, 411-419.
Kroll, K. L. and Amaya, E. (1996). Transgenic Xenopus embryos from sperm
nuclear transplantations reveal FGF signalling requirements during
gastrulation. Development 122, 3173-3183.
Lamb, T. M. and Harland, R. M. (1995). Fibroblast growth factor is a direct
neural inducer, which combined with noggin generates anterior-posterior
neural pattern. Development 121, 3627-3636.
Launay, C., Fromentoux, V., Shi, D-L. and Boucaut, J-C. (1996). A
truncated FGF-receptor blocks neural induction by endogenous Xenopus
inducers. Development 122, 869-880.
Lawson, K. A., Meneses, J. J. and Perdersen, R. A. (1991). Clonal analysis
of the epiblast during germ layer formation in the mouse embryo.
Development 113, 891-911.
Lee, S. M. K., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997).
Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates
growth and polarity in the developing midbrain. Development 124, 959-969.
Liem, K. F., Tremml G., Roelink, H. and Jessell, T. M. (1995). Dorsal
differentiation of neural plate cells induced by BMP-mediated signals from
epidermal ectoderm. Cell 82, 969-979.
Mahmood, R. Bresnick, J., Hornbruch, A., Mahony, C., Morton, N.,
Colquhoun, K., Martin, P., Lumsden, A., Dickson, C. and Mason, I.
(1995a). A role for FGF-8 in the initiation and maintenance of vertebrate
limb bud outgrowth. Curr. Biol. 5, 797-806.
Mahmood, R., Kiefer, P., Guthrie, S., Dickson, C. and Mason, I. (1995b).
Multiple roles for FGF-3 during cranial neural development in the chicken.
Development 121, 1399-1410.
Murphy, M., Reid, K., Ford, M., Furness, J. B. and Bartlett, P. F. (1994).
FGF2 regulates proliferation of neural crest cells, with subsequent neuronal
differentiation regulated by LIF or related factors. Development 120, 35193528.
New, D. A. T. (1955). A technique for the cultivation of the chick embryo in
vitro. J. Embryol. Exp. Morph. 3, 326-331.
Nieuwkoop, P. D., Boterenbrood, E. C., Kremer, A., Bloesma, F. F. S. N.,
Hoessels, E. L. M. J., Meyer, G. and Verheyen, F. J. (1952). Activation
and organisation of the central nervous system in amphibians. J. Exp. Zool.
120, 1-108.
Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C.,
Coulier, F., Gao, G. and Goldfarb, M., (1966). Receptor specificity of the
fibroblast growth factor family. J. Biol. Chem. 271, 1592-15297.
Patel, K., Connolly, D. J., Amthor, H., Nose, K. and Cooke, J. (1996).
Cloning and early dorsal axial expression of Flik, a chick follistatin-related
gene: evidence for involvement in dorsalization/neural induction. Dev. Biol.
178, 327-342.
Patel, N. H., Martin-Blanco, E., Coleman, K. G., Poole, S. J., Ellis, M. C.,
Kornberg, T. B. and Goodman, C. S. (1989). Expression of engrailed
proteins in arthropods, annelids and chordates. Cell 58, 955-968.
Papalopulu, N. and Kintner, C. (1996). A posteriorising factor, retinoic acid,
reveals that anteroposterior patterning controls the timing of neuronal
differentiation in Xenopus neuroectoderm. Development 122, 3409-3418
Pownall, M. E., Tucker, A. S., Slack, J. M. W. and Isaacs, H. V. (1996).
eFGF, Xcad3, and Hox genes form a molecular pathway that establishes the
anteroposterior axis in Xenopus. Development 122, 3881-3892.
Riese, J., Zeller, R. and Dono, R. (1995). Nucleo-cytoplasmic translocation
and secretion of fibroblast growth factor-2 during avian gastrulation. Mech.
Dev. 49, 13-22.
Roberts, C., Platt, N., Streit, A., Schachner, M. and Stern, C. D. (1991).
The L5 epitope: an early marker for neural induction in the chick embryo
and its involvement in inductive interactions. Development 112, 959-970.
Roelink,H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P.
A. and Jessell, T. M. (1995). Floor plate and motor neuron induction by
different concentrations of the amino-terminal cleavage product of sonic
hedgehog autoproteolysis. Cell 81, 445-455.
Sasai, Y. Lu, B. Piccolo, S. and De Robertis, E. M. (1996). Endoderm
induction by the organiser secreted factors chordin and noggin in the
Xenopus animal caps. EMBO J. 15, 4547-4555.
Schubert, F. R., Fainsod, A., Gruenbaum, Y. and Gruss, P. (1995).
Expression of a novel murine homeobox gene Sax-1 in the developing
nervous system. Mech. Dev. 51, 99-114.
Sechrist, J. and Bronner-Fraser, M. (1991). Birth and differentiation of
reticular neurons in the chick hindbrain: Ontogeny of the first neuronal
population. Neuron 7, 947-963.
Shah, N., Groves, A. and Anderson, D. J. (1996). Alternative neural crest fates
are instructively promoted by TGFb superfamily members. Cell 85, 331-343.
Sharpe, C. R. and Goldstone, K. (1997). Retinoid receptors promote primary
neurogenesis in Xenopus. Development 124, 515-523.
Shimamura, K. and Rubenstein, J. L. (1997). Inductive interactions direct
early regionalisation of the mouse forebrain. Development 124, 2709-2718.
Slack, J. M. W. (1994). Inducing factors in Xenopus early embryos. Current
Biology 4, 116-125.
Slack, J. M. W. and Tannahill, D. (1992). Mechanisms of anteroposterior
axis specification in vertebrates: lessons from amphibians. Development
114, 285-302.
Smith, J. C., Price, B. M. J., Green, J. B. A., Weigel, D. and Herrmann,
B. G. (1991). Expression of the Xenopus homologue of Bra (T) is an
immediate early response to mesoderm induction. Cell 67, 79-87.
Sokol, S. and Melton, D. A. (1991). Pre-existent pattern in Xenopus animal
cap pole cells revealed by induction with activin. Nature 351, 409-11.
Spann, P., Ginsburg, M., Rangini, Z., Fainsod, A., Eyal-Giladi, H. and
Gruenbaum, Y. (1994). The spatial and temporal dynamics of
Sax1(CHox3) homeobox gene expression in the chick’s spinal cord.
Development 120, 1817-1828.
Stern, C. D. (1990). The marginal zone and its contribution to the hypoblast
and primitive streak of the chick embryo. Development 109, 667-682.
Storey, K. G., Crossley, J. M., De Robertis, E. M., Norris, W. E. and Stern,
C. D. (1992). Neural induction and regionalisation in the chick embryo.
Development 114, 729-741.
Storey, K. G., Selleck, M. A. and Stern, C. D. (1995). Neural induction and
regionalisation by different subpopulations of cells in Hensen’s node.
Development 121, 417-428.
Streit, A., Stern, C. D., Thery, C., Ireland, G. W., Aparicio, S., Sharpe, M.
J. and Gherardi, E. (1995). A role for HGF/SF in neural induction and its
expression in Hensen’s node during gastrulation. Development 121, 813824.
Streit, A.,Yuen, C. T., Loveless, R. W., Lawson, A. M., Finne, J., Schmitz,
B., Feizi, T. and Stern, C. D. (1996). The Lex carbohydrate sequence is
recognised by the antibody L5, a functional antigen in early neural
development. J. Neurochem. 66, 834-844
Streit, A., Socknathan, S., Pérez, L. Rex, M., Scotting, P. J., Sharpe, P.
T., Lovell-Badge, R. and Stern, C. D. (1997). Preventing loss of
competence for neural induction: HGF/SF, L5 and Sox2. Development
124, 1191-1202.
Taira, M., Saint-Jeannet, J.-P. and Dawid, I. (1997). Role of the Xlim-1 and
XBra genes in anteroposterior patterning of neural tissue by the head and
trunk organsier. Proc. Natl. Acad. Sci. USA 94, 895-900.
Temple, S. and Qian, X. (1995). bFGF, neurotrophins and the control of
cortical neurogenesis. Neuron 15, 249-252.
Thomas, P. and Beddington, R. (1996). Anterior primitive endoderm may be
responsible for patterning the anterior neural plate in the mouse embryo.
Current Biol. 6, 1487-1496.
Waddington, C. H. (1932). Experiments on the development of chick and
duck embryos cultivated in vitro. Phil. Trans. Roy. Soc. Lon. B 221, 179230.
Wilkie, A. O. M., Morriss-Kay, G. M., Jones, E. Y. and Heath, J. K. (1995).
Functions of fibroblast growth factors and their receptors. Curr. Biol. 5, 500507.
Wilkinson, D. G., Bhatt, S., Chavrier, P., Bravo, R. and Charnay, P. (1989).
Segment-specific expression of a zinc-finger gene in the developing nervous
system of the mouse. Nature 112, 959-970.
Wilson, P. A. and Hemmati-Brivanlou, A. (1995). Induction of epidermis
and inhibition of neural cell fate by BMP-4. Nature 376, 331-333.
Yamada, T., Placzek, M., Tanaka, H., Dodd, J. and Jessell, T. M. (1991).
Control of cell pattern in the developing nervous system: polarising activity
of the floorplate and notochord. Cell 64, 635-647.
Yamaguchi, T., Harpal, K., Henkemeyer, M. and Rossant, J. (1994). Fgfr1is required for embryonic growth and mesoedrm patterning during mouse
gastrulation. Genes Dev. 8, 3032-3044.
Yu, R. T., McKeown, M., Evans, R. M. and Umesono, K. (1994).
Relationship between Drosophila gap gene tailless and a vertebrate nuclear
receptor Tlx. Nature 370, 375-379..