Fibroblast Growth Factors (FGFs) in neural induction

Abstract

Neural induction represents the first stage in the formation of the vertebrate nervous system from embryonic ectoderm. Fibroblast Growth Factors (FGFs), initially identified for their mitogenic and angiogenic roles in bovine brain extracts, are now known to have many developmental roles in particular that of neural induction, comprising of a family of 22 FGFs.

Spemann and Mangold (1924) pioneered the study of neural induction through the identification of the organizer. Early work in amphibians suggested that neural fate was instructed by signals from Spemann's organiser or dorsal mesoderm. Over a decade ago, the default model proposed that neural induction was the direct consequence from inhibition of bone morphogenetic proteins (BMPs) found in Xenopus laevis, not taking into consideration neural induction in avian embryos. Consequently many experimental studies, in the chick, subsequent to this finding conflicted the idea that BMP inhibition was the only necessary step required suggesting that FGFs were required at an earlier stage prior to BMP inhibition.

Much controversy has surrounded the role of FGFs in neural induction but now it is widely accepted to have a role in both amphibians and amniotes.

Fibroblast Growth Factors in neural induction

Structure and Function: FGFs broken down

Fibroblast Growth Factors (FGFs) regulate a vast array of developmental processes, including, limb development, neural induction and neural development (Böttcher and Niehrs, 2005). FGFs play an important role in development of an organism by regulating cellular differentiation, proliferation and migration and are involved in tissue-injury repair (Itoh and Ornitz, 2004). The early FGFs, FGF1 and FGF2 (also known as acidic and basic FGF, respectively) were first discovered from bovine brain and pituitary extracts and identified for their mitogenic and angiogenic activities (Gospodarowicz et al., 1974). Additionally, a number of family members were found revealing a total of 22 FGFs in humans ranging from 17 to 34 kDa in molecular mass in vertebrates. The nomenclature extends to FGF23 but in humans FGF19 is the equivalent to mouse Fgf15 (Ornitz and Itoh, 2001). Also the FGFs have been organised into seven subfamilies based on sequence comparisons.

FGFs show conservation through species, especially across the vertebrate species in gene structure and amino-acid sequence. FGF sequences are yet to be found in unicellular organisms such as yeast (Saccharomyces cerevisiae) and bacteria (Escherichia Coli) (Itoh and Ornitz, 2004). Interestingly, an Fgf-like gene has been encoded in the nuclear polyhedrosis virus genome (Ayres et al., 1994). In protostomes, there are far fewer FGFs in contrast to vertebrates, as two (let-756 and egl-17) have been found in Caenorhabditis elegans and three (branchless, pyramus and thisbe) in Drosophila (Mason, 2007).

Most FGFs have amino-terminal signal peptides (Fig. 1 (a)) and are secreted from cells. FGFs 9, 16 and 20 lack this signal peptide but nevertheless are still secreted (Ornitz and Itoh, 2001). FGF1 and FGF2 lack these signal sequences and are secreted by non-canonical pathways, however they can be found on the cell surface and within the extracellular matrix. Golfarb (2005) suggests that FGFs 11-14 do not interact with FGF receptors (FGFRs) and are not secreted but instead localise to the cell nucleus.

Fig. 1 (above) illustrates the structural features of the FGF polypeptide (a). A signal sequence (shaded grey) can be seen here within the amino terminus and is present in most FGFs.

All FGFs contain a core region (Fig. 1 (a)) containing around 120 amino acids of which 6 are identical amino acids residues and 28 are highly conserved (Goldfarb, 1996). The black boxes (numbered 1 to 12) represent the location of β strands within the core. The three dimensional structure of FGF2 (b) can also be seen where the heparin binding region (yellow) includes residues between β1 and β2 strands and in β10 and β11 strands.

FGFs have a high affinity for heparan sulfate proteoglycans (HSPG) and require heparan sulphate to activate one of four transmembrane receptor tyrosine kinases (FGFR1-4) in all vertebrates. FGFR5 has been identified recently, however most action is mediated via FGFR1-4 (Powers et al., 2000). FGFRs are membrane associated class IV receptor tyrosine kinases (RTKs). The FGFR tyrosine kinase receptors (Fig. 2 B) include 3 immunoglobulin (Ig) domains and a heparin binding sequence which requires heparan sulphate to be activated (McKeehan et al., 1998). HSPG are low affinity receptors that are unable to transmit a biological signal but act as co-factors for activation and regulation of an interaction between FGFs and FGFRs.

Fig. 2 Illustrates the structure of a FGF molecule (A) indicating that the core region is where FGFR and HSPG binding occurs. The FGFR (B) has three Ig-domains which lie extracellularly. Ig-domain I affects binding affinity whereas Ig-domain II is where FGF binding occurs and Ig-domain III is involved in ligand selectivity. An acidic box (AB) lies between Ig-domain I and Ig-domain II which optimises interaction between HSPG and FGFR. Adjacent to the AB is the heparin-binding domain and CHD. The tyrosine kinase domain is split for catalytic activity and binding of adaptor proteins. Ig, Immunoglobulin; ECM, Extracellular matrix; CAM, Cell adhesion molecules; CHD, CAM homology domain; PKC, Protein kinase C; FRS-2, FGF receptor substrate-2. Image taken from: Böttcher and Niehrs, (2005)

Fig. 2 (above) illustrates a two dimensional generic FGF (A) and a FGFR (B) protein. The structure of a FGF (A) coincides with that of Fig. 1, containing a signal sequence in the amino-terminus and the conserved core region containing HSPG and receptor-binding sites. The main features of FGFRs (B) include 3-Immunoglobulin domains, an acidic box (AB) which lies between IgI and IgII, heparin-binding domain, Cell Adhesion Molecule (CAM)-homology domain, transmembrane domain and a split tyrosine kinase enzyme domain for catalytic activity and binding of adaptor proteins. The Ig domains in the extracellular region of a FGFR are required for FGF binding and regulate binding affinity and ligand specificity.

Multiple alternative splicing that generates a range of FGFR1-4 receptor isoforms with transformed ligand binding properties provides diversity (Olsen et al., 2006). For example, FGF2 interacts with all four receptors FGFR1-4 whereas FGF7 only interacts with the FGFR2 IIIb isoform (a splice variant of FGF2; expressed in epithelial cells). Ligand-receptor binding specificity is affected by alternative splicing particularly in the C-terminal region of the third immunoglobulin loop in FGFR1-3 which produces IIIb or IIIc isoforms (Mason, 2007). Table 1 (below) illustrates the specificity of the FGF ligands for particular FGFR isoforms. This table is useful yet evidence from in vitro may appear misleading as in vivo involves influence from co-factors such as HSPG (Mohammadi et al., 2005).

FGF subfamily

FGFR1b

FGFR1c

FGFR2b

FGFR2c

FGFR3b

FGFR3c

FGFR4

FGF1

FGF1

+++

+++

+++

+++

+++

+++

+++

FGF2

FGF1

++

+++

-

++

-

+++

+++

FGF3

FGF7

++

-

++

-

-

-

-

FGF4

FGF4

-

+++

-

+++

-

++

+++

FGF5

FGF4

-

++

-

+

-

-

-

FGF6

FGF4

-

++

-

++

-

-

+++

FGF7

FGF7

-

-

+++++

-

-

-

+

FGF8

FGF8

-

++

-

+++

+

++++++

++++

FGF9

FGF9

-

-

-

++

++

+++

-

FGF10

FGF7

++

-

++++++

-

-

-

-

FGF11

FGF11

-

-

-

-

-

-

-

FGF12

FGF11

-

-

-

-

-

-

-

FGF13

FGF11

-

-

-

-

-

-

-

FGF14

FGF11

-

-

-

-

-

-

-

FGF15/19

FGF19

-

+++

++

++++++

+

++++

++++++++

FGF16

FGF9

-

-

-

+

-

+

-

FGF17

FGF8

-

+

-

+

-

+++

+++

FGF18

FGF8

-

-

-

+

-

++

++

FGF20

FGF9

-

+

-

++

++

+++

+

FGF21

FGF19

+

+

+++

+++

+

+

++++

FGF22

FGF7

++

-

++++++

-

-

-

-

FGF23

FGF19

-

+

++

+++

+

++

++++++

Table 1 shows the FGF/FGFR (ligand/receptor) interactions as determined by the Baf3 cell mitogenicity assay (which express FGFRs at higher levels than in most cell types in vivo). FGF1 is used as a reference as it activates all seven FGFR isoforms efficiently. FGFS 11-14 are nuclear and therefore have no reported activity on FGFRs. The level of activity relative to FGF1 (100%) is displayed by the number of '+' signs. The '-' illustrates a 10% less mitogenic activity approximately when compared to FGF1. This table provides useful information of FGF-FGFR associations even though in vivo heparan sulphate proteoglycans (HSPGs) can alter receptor specificity and that recombinant ligands may differ from post-translationally modified forms (occur in vivo). Taken from: Mason (2007)

Table 1 (above) shows there are seven FGFR isoforms (FGFR1b; FGFR1c; FGFR2b; FGFR2c; FGFR3b; FGFR3c and FGFR4) that FGF1 through to FGF23 variously bind. Alternative mRNA splicing of FGFR1-3, particularly in the carboxy-terminal half of the third extracellular immunoglobulin loop (Ig-domain III), derives the b and c isoforms. HSPGs are necessary co-factors in activation of FGFRs by FGFs and evidence has found the ternary complex to comprise of FGF-FGFR-HSPG in a 2:2:1 ratio (Mohammadi et al., 2005). The co-binding of HSPG prevents proteolysis and thermal denaturation (Itoh and Ornitz, 2004). HSPG binding of FGF induces dimerization of FGFR, followed by transphosphorylation of receptor subunits, initiating an intracellular signalling cascade.

FGF signalling: It's a cellular game

Following formation of the FGF-HSPG-FGFR complex several downstream signalling pathways are activated (Fig. 3 below). This includes three pathways, the Ras/Mitogen-activated protein kinase (MAPK) pathway, Phosphoinositide 3-kinase (PI3K)/ Akt pathway and phospholipase C- (PLC )/ Ca2+/ protein kinase C (PKC) pathway. These pathways are mediated via docking proteins (such as FGF receptor substrate (FRS) and Grb2 in the Ras/MAPK pathway) that recruit downstream enzymes. The Ras/MAPK pathway (Fig. 3) is initiated via Grb2 (a docking protein) where its SH2 domain binds to the tyrosine phosphorylated FRS2 in response to activation of the FGFR receptor (Kouhara et al., 1997). Grb2 binds to SOS (son of sevenless; a guanine nucleotide exchange factor) via a SH3 domain on the Grb2 molecule. This Grb2-SOS complex activates SOS which promotes the dissociation of GDP from Ras so it is able to bind GTP for its activation. Activated Ras activates RAF (MAPKKK) which is normally held in a closed conformation by the 14-3-3 protein. Once activated, RAF phosphorylates and activates mitogen-activated and extracellular signal-regulated kinase (MEK (MAPKK)) which in turn phosphorylates ERK1/2 (MAPK). MAPK then translocates into the nucleus to phosphorylate specific transcription factors of the Ets family which in turn activate expression of FGF target genes. In addition, it is also evident from Fig. 3 that active ERK itself can antagonise FRS activity.

Activation of the PI3K/Akt pathway (Fig. 3) is by binding of Gab1 (Grb2-associated-binding protein 1) to FRS2 indirectly via Grb2. In the presence of Gab1, activation of PI3K stimulates the Akt pathway which suggests FGFs have anti-apoptotic effects in the developing nervous system (Mason, 2007). In addition, PI3K can bind to a phosphorylated tyrosine residue of FGFR directly. The third way in which the PI3K/Akt pathway is activated is by activated Ras inducing membrane localisation of the PI3K catalytic subunit.

Fig. 3 The three main signalling pathways activated by FGFs are illustrated above. The negative feedback signals imposed on or mediated by FRS2 are shown by the dotted lines. Image taken from: Cotton et al. (2008)

PLC- /Ca2+/PKC pathway is also activated when a tyrosine residue is autophosphorylated in the carboxy terminal of the FGFR. PLC- hydrolyses phosphatidylinositol to produce inositol trisphosphate (IP3) and diacylglycerol (DAG) which stimulates calcium release and activates PKC, respectively. PKC has also been found to activate the Ras/MAPK pathway independent of Ras but dependent on c-Raf (Ueda et al., 1996). Fig. 3 also indicated that the final activated components, of the three signalling pathways mentioned, translocate into the nucleus to activate specific transcription factors of the Ets family (particularly Ets1, Pea3, and Erm) which activate expression of FGF target genes and in turn these feedback (Fig, 4) to regulate intracellular signalling (Dailey et al., 2005).

Most of the proteins produced function as feedback inhibitors (as seen in Fig. 4), including Sprouty (Spry), Sef and MAP Kinase phosphatase 3 (MKP3) which modulate particularly the Ras/Erk pathway at different levels (Mason, 2007). In contrast, stimulation of the fibronectin leucine-rich transmembrane type III (XFLRT3) protein causes FGF signalling to be positively regulated (Böttcher et al., 2003).

Fig. 4 Shows the feedback regulators of the Ras/MAPK pathway. The red arrows illustrate feedback loops which regulate the FGF signalling pathway. The black arrows indicate the direction of the Ras/MAPK signalling pathway. Three of the four target genes shown here (Spry, SEF and MKP3) function as feedback inhibitors which regulate the Ras/MAPK pathway at different levels. The red blind-ended arrows illustrate this. Spry antagonises FGF signalling at the Grb2-SOS-Ras and Raf levels. MKP3 blocks at level of MAPK. SEF blocks both phosphorylation of MAPK and its translocation to the nucleus aswell as at the membrane. XFLRT3 positively regulates FGF signalling at the level of the membrane. Spry, Sprouty; MKP3, MAP kinase phosphatase 3; XFLRT3, fibronectin leucine-rich transmembrane type III; SOS, son of sevenless. Image taken from: Cotton et al. (2008)

Sprouty (Spry) was one of the first identified feedback regulators of the FGF pathway. Thisse and Thisse (2005) found Spry to antagonise FGF Signalling by gain and/or loss of function experiments in mouse. Spry acts at the level of Raf and/or Grb2 (Fig. 4). Gain and/or loss of function experiments in zebrafish demonstrated that Sef antagonises FGF signalling (Fig. 4) acting at level of MEK and ERK (Tsang et al., 2002). Mouse studies have suggested that FGFR signalling is required for Dusp6 transcription which codes for MKP3 (Ekerot et al., 2008). From this study it was also found that MKP3 acts as a negative regulator of ERK activity (as seen in Fig. 4). Sef and XFLRT3 are located at the membrane (Fig. 4) and carry out antagonising actions with FGFR directly.

FGF signalling can be regulated at different levels, from the membrane all the way down to the level of phosphorylation of MAPK and it is important also to know that FGFs have been detected in the nucleus (Mason, 2007). Most of the downstream target genes as described earlier are feedback inhibitors (Spry, Sef and MKP3) but FGF signals are also known to interact with many other important pathways such as transforming growth factor-β (TGF-β), Hedgehog (HH), Notch and Wnt (Gerhart, 1999). Therefore, in conjunction with these, FGFs are responsible for development of most organs of the vertebrate body. In the nervous system, FGFs have been implicated to play a role in early developmental processes, such as neural induction, patterning and proliferation (Umemori, 2009).

Neural induction: The Default Model

Fig. 5 Illustrates the famous two-headed tadpole identified by Spemann and Mangold (1924) showing a developed second nervous system by implantation of organizer tissue onto a host embryo. Image taken from: De Robertis (2006).

Spemann and Mangold (1924) pioneered the study of neural induction, which is defined as the process by which naive ectodermal cells aquire a neural fate. Their work involved demonstrating that tissue from the dorsal lip of the frog Xenopus laevis blastopore could induce a second ectopic nervous system (Fig. 5 above left) when implanted onto the ventral side of a host gastrula embryo. The second ectopic nervous system was host derived indicating that the graft was important in determining cell fate. This region, located on the dorsal side of an amphibian embryo, was named the Spemann organizer as it could direct the neighbouring ectodermal cells to form nervous system instead of epidermis.

Although the organizer (group of dorsal mesodermal cells) was found to be present in many species (Hamburger, 1988) it was the Xenopus laevis which gave an insight into the molecular events involved in neural induction in vertebrates (Hemmati-Brivanlou et al., 1994). This was particularly because amphibians were found to be ideal experimental models for the study of neural induction as neurulation initiated within twelve hours after fertilisation (Weinstein and Hemmati-Brivanlou, 1997).

It was implied that signals from the organizer provide instructions to the ectoderm to form neural tissue therefore for many decades the view was that the 'default' state of the ectoderm was to produce epidermis. The first challenges to this model came from studies making use of dissociated cell cultures (Sato and Sargent, 1989). It was found that when animal caps were cultured intact that epidermis formed but neural tissue arose from animal caps that had been dissociated for prolonged periods (as seen in Fig. 6 below). This led to the idea that intact tissue may block the formation of neural tissue by presence of neural inhibitors which are diluted out when the tissue is dissociated. Recent research has found that the default nature of the ectoderm is to produce neural tissue that requires inhibition of a neural inhibitor from the ectoderm.

Before considering the process of neural induction I would like to take a step back and describe the three germ layers of the embryo. Following fertilisation, the zygote undergoes stages of cleavage to eventually form a gastrula with three germ layers (in triploblastic animals) usually only visible in vertebrate animals. The Germ layers will eventually give rise to all of the animal's organs through a process known as organogenesis. The three layers include, the ectoderm (outermost), endoderm (innermost) and mesoderm (which is between the ectoderm and endoderm) layers. The Endoderm gives rise to the lung, thyroid and pancreas. The mesoderm forms the skeleton, skeletal muscle, the urogenital system, heart and blood. The outermost layer, the ectoderm which is of concern here, gives rise to the epidermis and nervous system. It is at gastrulation that the vertebrate ectoderm is competent to differentiate into neural tissue or epidermis. Unless told otherwise, the default nature of the ectoderm is to produce neural tissue and this was outlined as the default model.

The Default model of vertebrate neural induction, discovered over a decade ago in Xenopus, proposed that in the presence of bone morphogenetic protein (BMP), a signalling molecule of the TGF-β superfamily, causes the ectoderm to give rise to an epidermal cell fate (Stern, 2006; Muñoz-Sanjuan and Brivanlou, 2002). In support of this model, consistent with the idea that BMP activity inhibits neural fates, animal caps which had been injected with RNA encoding effectors of BMP4 (Smad 1/5 or Msx1) neuralization did not occur. Conversely, it was found that inhibition of BMP activity in the ectoderm is essential for a neural fate which forms the basis of the default model of neural induction. Inhibition of BMP is achieved through direct binding of BMP antagonists emitted from the organizer (Wilson and Hemmati-Brivanlou, 1997). These BMP antagonists include chordin (Sasai et al., 1995), noggin (Lamb et al., 1993) and follistatin (Hemmati-Brivanlou et al., 1994) which bind to BMPs extracellularly to prevent its interaction with its own receptor (Hemmati-Brivanlou and Melton, 1997). These molecules have direct neural activity which means they induce formation of neural tissue in the ectoderm without forming mesoderm.

It was initially believed that these molecules acted as ligands to bring about neural tissue formation. Experiments found that there was conservation through species, identifying that chordin was homologous to the short gastrulation (sog) gene found in Drosophila which has been shown to antagonize the BMP homologue decapentaplegic (dpp) (Wharton et al., 1993), suggesting that these molecules might act as inhibitors rather than inducers and that these inhibitory mechanisms have been conserved from arthropods through to vertebrates. It was experiments (Fig. 6) showing that dissociated ectodermal explants would become neural tissue in absence of 'inducing' signals from the organizer (Sato and Sargent, 1989). Evidence found that neural induction resulted from inhibition of the TGF-β pathway as expression of dominant-negative activin receptor gave rise to neural fates in amphibian ectoderms (Hemmati-Brivanlou and Melton, 1994). It was found that chordin, noggin, follistatin and molecules such as Cerberus and Xnr3 (Xenopus nodal related 3) bound to BMP in the extracellular space inhibiting its action (Hemmati-Brivanlou and Melton, 1997) leading to the much debated default model of neural induction.

Fig. 6 The Default Model. Ectodermal cells acquiring a neural identity in absence of signals forms the basis of the neural default model. It is the inhibition of an inhibitor (BMP) which leads to neural tissue from the ectoderm. The experiment above shows that culture of an intact animal cap of a blastula-stage (stage 9) Xenopus ectoderm gives rise to epidermal tissue. In contrast, it can be seen in a dissociated ectodermal animal cap cultured for >5 hours with no other factors or serum, absent in cell-cell signalling, becomes neuralised. Addition of BMPs to dissociated ectoderm can restore epidermal fate (Wilson PA & Hemmati-Brivanlou A, 1995). Addition of a dominant negative activin receptor (BMP signalling inhibitor) to an intact explant results in neural fate. A cement gland fate is adopted by explants that have been briefly dissociated and can be transformed by exposure to FGFs to a neural fate. Image taken from: Muñoz-Sanjuan and Brivanlou, (2002)

Neural Induction: FGFs get it started

Support for the default model still remains, mainly in Xenopus, but other work (especially in chick and mouse) suggests a more complex mechanism (Streit et al., 1998). It has been established that the BMP pathway is involved in determining ectodermal cell fate (Wilson and Hemmati-Brivanlou, 1997) but it still remains to be proved conclusive if BMP inhibition is required for neural induction alone or if other pathways act separately or with BMP inhibition.

In the chick embryo it has been found that naive epiblast cells do not respond to BMP antagonists until previous exposure to organizer signals for five hours (Streit et al., 1998). Striet et al. (2000) grafted an organizer to observe the genes induced in the epiblast within this time period. A gene ERNI (early response to neural induction) was identified as a coiled coil domain with a tyrosine phosphorylation site and found to be expressed throughout the region that later contributes to the nervous system at pre-primitive streak stages (Hatada and Stern, 1994). Striet et al. (2000) findings made ERNI the earliest known marker after a response to organizer signals, prior to even Sox3 (induced by the node in 3 hours (Streit and Stern, 1999)).

FGFs are becoming more evident that they have a major role in neural induction as it has been shown to begin before gastrulation, before BMP antagonists even appear (Wilson et al., 2000). In the chick, it has been found that FGFs have the role of blocking BMP signalling and promoting neural differentiation (Wilson et al., 2000). In ascidians, FGF signalling is the main mechanism of neural induction with BMP antagonism playing a role in later development (Lemaire et al., 2002). In frogs and fish, in contrast, FGFs do not have a certain role in neural induction and is believed their primary role is BMP inhibition (Pera et al., 2003).

Fig. 7 (redooo) FGF Signalling a part of neural induction. Hensen's node (brown) induces cERNI (a, arrow), Sox3 (d, arrow) and Sox2 (left in g, i). The FGF receptor inhibitor SU5402 (arrowheads in b, e) inhibits induction of all three genes (b, e; right in g, h) by the node, which still elongates and expresses the organizer marker chordin (g-i; Sox2 in purple, chordin in red). Cells secreting a soluble form of the FGFreceptor (outlined) also greatly reduce induction of cERNI (c) and Sox3 (f) by the node. The endogenous expression of Sox3 is reduced in embryos treated with SU5402 (k) as compared with untreated embryos (j) (embryos processed simultaneously in the same vial). Image taken from: Streit et al. (2000)

Exposure of the chick epiblast to an implanted organiser for around 5 hours induces Sox3 (an early neural plate marker) (Stern, 2005). After removal of the implanted organiser, chordin can be used to stabilise it (Striet et al., 1998) which implies that before the ectoderm can respond to BMP antagonists it must be exposed to 5 hours of signals from the organizer. During these 5 hours, several genes become activated such as, ERNI (early response to neural induction) which becomes active after 1 hour (Streit et al., 2000) and Churchill (Chch) after about 4 hours (Sheng et al., 2003). These are both induced by FGF and not BMP inhibition, indicating the importance of FGFs in early neural induction. Churchill which is expressed in the neural plate inhibits brachyury, a transcription factor, which as a result suppresses mesoderm formation by preventing cell ingression.

In the chick, FGF8 is expressed in the hypoblast, prior to gastrulation before Hensen's node appears (the chick equivalent to the organizer) indicating that neural induction is in fact able to begin before gastrulation. This is important because ERNI and Sox3 mark neural induction and require FGF signalling (Stern, 2005). Streit et al. (2000) found that FGF8 coated beads induce ERNI as efficiently as the node within 1-2 h without inducing brachury and also the expression of Sox3. These results indicate FGFs to be possible early signals in neural induction. It is FGF8 which has been identified as the best candidate because it is expressed in the anterior part of the streak as well as the node in primitive streak stages and as the node loses neural inducting ability it is also downregulated (Streit and Stern, 1999). In xenopus, more recently similar conclusions have been reached (Delaune et al., 2005).

To find if FGF expression in Hensen's node is actually required to induce ERNI and Sox3 experiments involving loss of function were undertaken. By using a FGF receptor inhibitor in the chick, such as SU5402, FGF signalling can be blocked preventing the early phase of neural induction (Delaune et al., 2005; Mohammadi et al., 1997). SU5402 is able to greatly reduce induction by a grafted node of Sox3 (Fig. 7e) and of ERNI (Fig. 7b). Streit et al. (2000) also found a large reduction in expression of Sox3 and ERNI particularly in the normal neural field of host embryo in the existence of FGF inhibitors (seen in Fig. 7k and j when compared). Pera et al. (2003) has shown that FGF signalling has a role in neural induction as it activates the MAPK cascade which phosphorylates the linker region of Smad1 (a BMP effector), inhibiting Smad1 and hence the BMP pathway. This is compared to phosphorylation of the C-terminus of Smad1 by BMP normally, which activates it. Also Kuroda et al. (2005) also suggested that FGF induces neural induction through inhibiting BMP signalling by phosphorylation of Smad1. A recent study by Linker and Stern (2004) indicated that, independent of downregulating BMP targets, FGF is required for neural induction. Therefore, though MAPK signalling is able to downregulate BMP signalling this is not able to explain fully why FGF is required for inducing neural fate.

Further experiments in the chick were carried out to strongly suggest the role of FGF signalling in the neural induction pathway initiated by the organizer. These included investigation of whether FGFs had a role in later stages of neural induction. The FGF inhibitor SU5402 was grafted with quail nodes into the area opaca (which part of the blastoderm that surrounds the area pellucida) of chick hosts and left overnight to give results where Sox2 induction was reduced to 20% (Fig. 7g, h) by the node (Streit et al., 2000). However, the node elongated as normal and chordin (organizer marker) was continued to be expressed eliciting that the graft remains unaffected by the FGF inhibitor.

FGF8 was found to induce msx1 expression as well as repress expression of GATA2/3 which are both targets of the BMP pathway (Fig. 8 below) (Streit et al., 2000). This shows that FGF and BMP pathways relate and antagonise each other. The finding that FGF8 upregulates msx1 leads to idea that BMP signalling is maintained which is required if BMP is to be inhibited later. It was also found that the activity of FGF8 can not be explained by simply antagonism with the BMP pathway as FGF8 beads were used in host embryos with BMP4 and it was found that FGF8 continued to induce expression of Sox3 and ERNI (Streit et al., 2000).

Delaune et al. (2005) demonstrated that FGF signalling is required prior to gastrulation within the ectoderm and that the FGF pathway contributes to BMP inhibition, which is not enough to cause neural induction alone in vivo. It is established that FGFs are required as an early step for neural induction in amniotes, therefore it is interesting to consider the roles of FGF and BMP pathways during neural induction in Xenopus. Work by Delaune et al. (2005) found FGF to function as a conserved initiator of neural specification amongst chordates. The in vivo results were consistent with FGF signalling prior to gastrulation with BMP inhibition to give rise to the nervous system. Also observations in amphibian embryos found that in the presence of a dominant-negative FGFR1 construct neither Noggin (Launay et al., 1996) nor Chordin (Sasai et al., 1996) could induce neural tissue. This suggests that FGF signalling is required for BMP inhibitors to induce neural markers. Further studies showed that injecting Smad6, an intracellular BMP antagonist, into the A4 blastomere (which does to contribute to neural plate or its border) of Xenopus embryos was inadequate to neuralise unless FGF4 was co-injected.

There has been much prior controversy regarding whether FGFs were required in amphibians for neural induction as injection of dominant-negative FGFR1 inhibits mesoderm formation but not formation of anterior neural features (Ribisi et al., 2000). Reports have also shown that use of antimorphic FGFR1 or FGFR4 have suggested that FGF plays a role during neural development (Launay et al., 1996). It has also been proposed that FGFR4 rather than FGFR1 is involved in neural induction and as previously stated that an inhibitor of FGFRs has led to the clear need for FGF signalling in neural induction in Xenopus (Delaune et al., 2005).

Recent findings by Wilson et al. (2001) in chick epiblasts have indicated, that during neural induction, FGF signalling functions in a BMP-independent way. Experiments by Bertrand et al. (2003) in the ascidian Ciona intestinalis further supports the role of FGFs, particularly FGF9/16/20 in this case, as neural inducers. Ascidians are not vertebrates, but prior to development into an adult there is an intermediate tadpole phase which resembles a simple vertebrate-like larva with a dorsal neural tube and notochord. It does not appear that BMP inhibitors such as noggin and chordin are involved in induction of neural tissue (Darras and Nishida, 2001) and instead in the embryo, it is FGF that instructs the animal cells to become neural tissue and the vegetal cells to form mesoderm. The earliest known marker of ascidian neural tissue was identified to be the Otx gene. Bertrand et al. (2003) found that Fgf9/16/20 all activate an enhancer of Otx expression through action of Ets1/2 and GATAa transcription factors.

Work by Kudoh et al. (2004) in zebrafish shows that rather than Bmp antagonism, Fgf activity initiates development of potential vegetal neural tissue that aids in trunk and tail CNS and has brought about the debate about a possibility of more than one organizer. It has been proposed that both FGF signalling and BMP inhibition act as direct neural inducers in Zebrafish (Kudoh et al., 2004) BMP inhibition has been found to induce anterior neural CNS by the shield (the zebrafish equivalent of the Spemann organizer) where FGFs induce posterior neural plate (Mason, 2007). The marginal zone, which is a second more ventral organizer, induces posterior neural tissue via FGF signalling without influence of BMP.

In vertebrates, the role of FGFs in neural induction has been controversial. It has been suggested that FGFs direct ectodermal cells to a neural fate in chick (Streit et al., 2000), amphibians (Delaune et al., 2005) and zebrafish (Kudoh et al., 2004). An ancient role for FGF signalling in neural induction has also been identified in the starlet sea anemone (Matus et al., 2007).

Add Fig. 8 here from Dev. Neuro book of BMP and FGF summarising the cascade.page 28

In mammals it was suggested that in mouse ESCs (mESCs) neuralization was independent of FGF signalling (Smukler et al., 2006). However, more recently Pollard et al. (2008) identified autocrine FGF signalling to be involved in neural induction of mESCs. Cohen et al. (2010) came to the conclusion that FGF signalling has an instructive role in human neural specification (Fig. 9). In this study when MEK1/2 were inhibited, FGF induced neuralization was blocked suggesting that active Erk1/2 is required for FGF to maintain its neuralization effect. This coincides with results obtained from mESCs (Stavridis et al., 2007).

Fig. 9 The role of FGF in early neural differentiation events in human embryonic stem cells (hESCs). Floating hESC clusters differentiate towards the primitive ectoderm lineage independent of FGF-signaling. Further neuralization is instructed by FGF-signaling. FGF-signaling induces neuralization, at least in part, through a mechanism which is independent of inhibition of BMP-signaling. In line with the default model, inhibition of BMP-signaling promotes neuralization of hESCs. FGF-signaling encourages the neuralization tendency in the presence of noggin, though it is not essential, since neuralization still occurs when both BMP and FGF-signaling are blocked. Image taken from Cohen et al. (2010)

FGFs are clearly not enough to induce a complete nervous system but are able to sensitize the epiblast for BMP antagonists and allow expression of later neural markers. It was concluded that FGF or 5 hours of signals from the node were not sufficient even with BMP inhibition to induce Sox2 (Streit and Stern, 1999).

Downstream of FGFs in neural induction

Sox2, a definitive neural plate marker, is one of the earliest markers for the neural plate (Kishi et al., 2000) which will give rise to the complete central nervous system (CNS). It was found that the chick epiblast requires exposure for 5 hours from a grafted organizer (requiring 11-13 hours for neural induction) which induces SOX3 expression. The events that take place during these first 5 hours include induction of ERNI after 1 hour and churchill (ChCh; a zinc finger gene) after about 4-5hours (Sheng et al., 2003). These two genes are induced by FGF and not BMP inhibitors.

FGF signalling is required when the neural plate is established from within the chick epiblast and when gastrulation causes mesoderm and endoderm to develop from epiblast. Churchill, induced by FGF, activates Smad-interacting-protein-1 (Sip1) which blocks brachury (a mesoderm marker) and acts to switch FGF between different roles (Sheng et al., 2003). The main role of Churchill is to repress mesoderm markers such as brachury and Tbx6 or upregulate genes that will do, such as Sip1.

Competitive interactions between ERNI, BERT and Geminin control the repressors and control Sox2 expression. Brahma (Brm; a chromatin remodelling enzyme) is able to activate Sox2 through direct binding with the N2 enhancer and is normally expressed ubiquitously in the embryo. Premature Sox2 expression is normally inhibited by a transcriptional repressor HP1α (heterochromatin protein; also expressed ubiquitously in embryo) which binds to Brm in the basal state (Fig. 10A below). FGF8 which is known to induce ERNI (Streit et al., 2000), also induces Geminin.which competes to displace HP1α bound to Brm at the N2 enhancer (Fig. 10B).

Geminin is known to be expressed at the start of gastrulation before Sox2 appears therefore it is ERNI which prevents premature Sox2 expression by binding through its coiled coil domain with Geminin (Fig. 10C). During gastrulation ERNI recruits the repressor HP1γ (which interacts with the C-terminus of ERNI) to prevent premature activation of Sox2 by Geminin which is bound to Brm at the time (Fig. 10D). At the end of gastrulation, another protein, BERT (a coiled coil domain which an endogenous ERNI antagonist) competitively binds to both ERNI and Geminin, displacing the repression by HP1γ and activating Sox2 (illustrated in Fig. 10E). The induction of BERT is not via FGF or BMP inhibition and currently research remains in this field to determine the factor(s) that regulate its expression. Overall, the mechanism described above regulates the timing of Sox2 preventing its early release, defining the domain that will become the nervous system.

In xenopus, it has been proposed that neural induction requires early FGF signalling in addition to BMP inhibition (Delaune, 2005). Overall, it is now accepted that both amphibians and the chick require FGF signalling for neural induction.

Fig. 10 HP1α is bound to Brm (A) in a basal state and acts as a transcriptional repressor of Sox2 (a definitive neural plate marker). (B) Geminin is able to displace HP1α from Brm releasing its inhibition on Sox2. (C) It is proposed that ERNI via Geminin blocks induction of Sox2. (D) HP1γ is recruited by ERNI to the N2 enhancer (earliest enhancer of Sox2) to inhibit Sox2 expression. (E) Proposed that BERT, a coiled coil domain, interrupts interaction between Geminin and ERNI to free the HP1γ repressor from the N2 enhancer allowing Sox2 induction via Geminin/Brm. Image taken from: Papanayotou et al. (2008)

In the chick it has been proposed that FGF initiates two separate pathways including one that involves FGF inducing neural tissue independent of BMP inhibition and another where FGF represses BMP transcription requiring inhibition of the Wnt pathway additionally (Wilson et al., 2001). It has been found that Wnt signalling plays a role in the vertebrate embryo. Wnt functions by binding to its receptor frizzled, an integral membrane protein, which in turn activates the cytoplasmic protein Dishevelled. β-catenin, an intracellular protein, is bound to several proteins such as Axin, GSK3 and APC (canonical pathway). Glycogen synthase kinase 3 (GSK3) phosphorylates β-catenin, targeting it for proteolysis so this complex only exists momentarily.

When Wnt binds and Dishevelled is activated it functions by blocking GSK3 which in turn increases the β-catenin within the cell forming a complex with the protein TCF. The β-catenin /TCF complex binds to DNA at specific points to activate target genes. The canonical (β-catenin dependent) Wnt signalling is required early on to specify the dorsal side of the embryo where the organiser will form. The Wnt targets Xnr3 and siamois have been shown previously to have neuralizing activity when overexpressed.

It has been reported by Baker et al. (1999) that the canonical Wnt pathway requires activation for neural induction in Xenopus as it was shown that Wnt8 can inhibit BMP4 expression at early gastrula stages. This study also showed that inhibition of β-catenin activity in the neural ectoderm results in a decrease in neural development proposing that Wnt activation is required for neural induction in xenopus. The work done on Wnts has been very controversial as in the chick epiblast, it was suggested by Wilson et al. (2001) that Wnt inhibition collaborates with FGF for neural fate acquisition. A more recent study by Heeg-Truesdell and Labonne, (2006) constructed in xenopus which is consistent with that of the chick shows that blocking canonical Wnt signalling results in a larger neural plate. Also Aubert et al. (2002) concluded that Wnt antagonism, in stem cells, can stimulate neural differentiation.

The inconsistency with the findings previously can be corrected if taking into account differences in timing. As stated above at early stages of development Wnt signalling is required to specify the dorsal side of the embryo. It may be necessary by the blastula stage to inhibit Wnt signalling for FGF to downregulate BMP expression (Wilson et al., 2001). The signals involved in inhibition of Wnts at this late stage are still not completely understood. Experiments in other vertebrates such as the mouse have shown that mouse Wnt3 (mWnt3) and mouse Wnt8 (mWnt8) are neuralising molecules (Baker et al., 1999).

Experiments by Linker and Stern (2004) have found that a combination of FGFs, BMP antagonists (Smad6, Chordin and Noggin) and Wnt antagonists were still unable to induce the neural marker Sox2 in chick epiblast (Fig. 11 below). They found that FGF8 is unable to induce Sox2, the definitive neural plate marker, when combined with Smad6 (a BMP inhibitor; Fig. 11F, H, I). This is consistent with the finding by Streit et al. (2000) that FGF8 and Chordin fail to induce Sox2. When FGF8 in combination with three Wnt antagonists (Dkk1, Crescent and NFz8; Fig. 11C-E) is misexpressed, similar results are seen. These experiments suggest that, in vivo, at least in the chick, signals other than FGFs, BMP antagonists and Wnt antagonists are required.

Fig. 11 Use of FGF8, BMP antagonist (Smad6) and Wnt antagonists have appeared unsuccessful to induce neural fate. (D-G) Shows histological sections that the different factors, in the epiblast, are unable to express Sox2. (A) FGF8 as well as (B, D) Wnt signalling by Dkk, Crescent and NFz8 (αWnt) are unable to induce Sox2 expression in the area opaca epiblast. Even in a combination of FGF8+NFz8+Dkk+Crescent the same conclusions are seen (C, E). In (F, H, I) FGF8+Smad6 (BMP inhibitor) is unable to induce Sox2 also. Finally combinations of all these factors (FGF8+NFz8+Dkk+Crescent+Cerberus+Smad6) are also unable to induce the definitive neural plate marker, Sox2, suggesting the role of other factors (G, J, K). Image taken from: Linker and Stern (2004).

In a very recent study by Cohen et al. (2010) with use of human embryonic stem cells (hESCs) it was found that FGF signalling is able to induce neuralization via a mechanism independent of BMP signalling. The data suggests that human neural induction involves FGF signalling and is instructed via this pathway but in hESCs, neuralization can occur in its absence.

Conclusion

Since isolation of FGFs in pituitary extracts for their mitogenic and angiogenic activites, our understanding of their involvement in neural development has come a long way over the last 35 years. FGFs have an important role in many developmental processes in particular limb development, neural induction and development. Recently it has become more evident that FGFs are involved in axon growth and guidance.

Neural induction is a very complex multi-step process, though well understood, still remains to be fully uncovered. Work in Xenopus ectodermal explants have suggested that BMP inhibition gives rise to a neural fate, whereas epidermis is induced by BMP signalling. For many years this was considered to be the case, however, in amniotes and ascidians, BMP inhibition did not prove to be sufficient for acquisition of neural fate and FGF signalling was found to initiate it. Loss of function experiments with use of a pharmacological FGF inhibitor, SU5402, made it possible to identify that FGFs are required prior to gastrulation in the ectoderm to induce a neural fate.

In the chick it was found that a grafted organizer would need to be exposed to the chick epiblast for atleast 5 hours to induce the early neural plater marker SOX3 and ERNI which can be stabilised by Chordin after the grafted organizer has been removed. These findings imply that before the ectoderm can respond to BMP inhibitors it must be exposed to 5 hours of signals from the organizer.

There has been much controversy regarding whether FGFs are required for neural induction but is now generally accepted that FGFs are required in chick and amphibians as well as ascidians and zebrafish. FGFs have been suggested to induce neural induction through inhibiting BMP signalling by phosphorylation of Smad1 (Kuroda et al., 2005) or by limiting expression of BMP signalling genes (Wilson et al., 2000). Also Delaune et al. (2005) suggested that FGF signalling, independent of BMP signalling, directs neural diffentiation.

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Randeep Dhariwal