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Keywords:

  • central nervous system;
  • fibroblast growth factor receptors;
  • fibroblast growth factors;
  • neural precursor cell proliferation;
  • neurogenesis;
  • neurotrophism;
  • patterning

SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

1. It is now clear that members of the fibroblast growth factor (FGF) family have multiple roles during the formation of the central nervous system (CNS).

2. There are at least 23 members of the FGF family and, of these, 10 are expressed in the developing CNS, along with four FGF receptors (FGFR-1–4).

3. The present review discusses the roles of these FGFs, with emphasis on FGF-2, FGF-8, FGF-15 and FGF-17. Fibroblast growth factors-2 and -15 are generally expressed throughout the developing CNS, whereas FGF-8 and FGF-17 are tightly localized to specific regions of the developing brain and are only expressed in the embryo during the early phases of proliferation and neurogenesis.

4. Expression studies on FGFRs in the chick and mouse indicate that FGFR-1 is most generally expressed, whereas FGFR-2 and FGFR-3 show highly localized but changing patterns of expression throughout CNS development. The FGFR-4 has been localized to the developing CNS in fish but not at a detailed level, as yet, in chick or mouse.

5. A picture is emerging from these studies that particular FGFs signal through specific receptors in a highly localized manner to regulate the development of different regions of the brain.

6. This picture has been demonstrated so far for the developing cortex (FGF-2–/– mice), the forebrain and midbrain (FGF-8 hypomorphs) and the cerebellum (FGF-17/FGF-8 mutant mice). In addition, generation of mutant animals deleted for FGFR-1 and FGFR-2b IIIb demonstrate their importance in FGF signalling.

7. However, there are significant gaps in our knowledge of the localization of members of the FGF family and their receptors. More detailed information on the spatio-temporal mapping of FGFs and FGFR isoforms is required in order to understand the molecular mechanisms through which FGFs signal.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

The entire central nervous system (CNS) in vertebrates is derived from a group of epithelial cells contained within the neural tube of the developing embryo. The neural plate is induced in an area of surface ectoderm overlying newly formed mesoderm during gastrulation. The neural tube forms when the neural plate along the dorsal surface of the embryo invaginates, rolls up and closes off from a thin strip of overlying ectoderm. At this early stage in the mouse, there are approximately 250 000 cells contained within its length. These cells form the precursor or stem cell pool for the entire brain, spinal cord and peripheral nervous system. Most of the cells at the early stages of development are undergoing active proliferation. This proliferative phase continues throughout embryogenesis, but is restricted to the cells at the ventricular and subventricular zones of the developing brain and spinal cord. As neural development proceeds, many of the cells exit the proliferative region, migrate out and differentiate into neurons and glia.1 It is now clear that members of the fibroblast growth factor (FGF) family have multiple critical roles during the formation of the CNS from the stage of neural induction through to the stage of terminal differentiation2.

We now know that there are at least 23 different members of the FGF family. These FGFs are classified as a family on the basis of a conserved 120 amino acid core region and share a 30–60% amino acid identity across the family. Fibroblast growth factor family members have diverse functions, being potent modulators of cell proliferation, migration, differentiation and survival (for reviews see Goldfarb3 and Ornitz4). There are four FGF receptor genes, FGFR-1–4, and, within these, alternative splicing creates receptor isoforms with distinct specificities for particular FGFs. There have been multiple studies on the expression patterns of FGF ligands and receptors during CNS development that indicate the sites where they may be active. There have also been many functional in vitro and in vivo assays that, together, provide a framework for deciphering the actions of FGF during different phases of CNS development.

Expression studies demonstrate that several members of the FGF family are highly expressed early in the developing CNS (Fig. 1; Table 1). Of these, FGF-1, FGF-2 and FGF-15 are more generally expressed throughout the developing neural tube. These FGFs are present in both the embryonic and adult CNS, while FGF-8 and FGF-17 are tightly localized to specific regions of the developing brain and are only expressed in the embryo during the early phases of proliferation and neurogenesis. In the present review, we focus on the expression patterns and possible functions of these five FGFs in the development of the nervous system. Significant findings pertaining to functions in the developing CNS for several other FGFs are also discussed more briefly. These data are then discussed in the context of the current knowledge of receptor expression in the developing brain.

image

Figure 1. Fibroblast growth factor (FGF) expression in the developing brain: a diagrammatic representation. The expression domains of members of the FGF family at early times in the developing (embryonic day (E) 9.5–E10) mouse neural tube are shown in relation to the major divisions of the embryonic brain at the five-vesicle stage. The expression domains represent the anterior–posterior extent of mRNA distribution in the neural tube. The adult structures that derive from these divisions are also given. MHB, midbrain–hindbrain junction.

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Table 1.  Expression of fibroblast growth factors in the developing central nervous system
 VZ: ProliferatingMZ: Differentiating
FGFcellscellsTimingLocationMethod
  1. Fibroblast growth factors (FGF)-11–14 have not been included in this review. They are members of a distinct branch of the FGF family originally known as the fibroblast growth factor homologous factors (FHF)-1–4.104 All are prominently expressed in the developing nervous system.104,105 However, studies suggest that the FHF do not bind FGF receptors105 and may, in fact, be intracellular signalling molecules (M Goldfarb, pers. comm., 1998; see also Ornitz and Itoh41). This table was compiled from references cited in the text.

  2. VZ, ventricular zone; MZ, marginal zone; Tel., telencephalon (cortex); di., diencephalon (thalamus etc.); mes., mesencephalon (midbrain); met., metencephalon/myelencephalon (hindbrain); MHB, midbrain–hindbrain boundary or isthmus; SC, spinal cord.

FGF-1+E11 onwardsTel., di., mes., met, SCImmuno and in situ
FGF-2++< E9 onwardsTel., di., mes., met, SCImmuno and in situ
FGF-3+E8–E11Otocyst, hindbrainIn situ
FGF-4In situ
FGF-5In situ
FGF-6In situ
FGF-7+E14.5–E15.5Lateral ventricleIn situ
FGF-8+E8.5–E12.5Tel., di., midline, MHBIn situ
FGF-9+E10–E14SC motor neuronsIn situ
FGF-10 OtocystIn situ
FGF-15+E8–E14Tel., di., mes., met., SCIn situ
FGF-16In situ
FGF-17++E9–E16Tel., midline, MHBIn situ
FGF-18++E10–E15.5Tel., MHBIn situ

FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

There is accumulating evidence that FGFs have a critical role in the initial generation of neural tissue at the stage of neural induction. Neural tissue is induced in adjacent ectoderm by signals emanating from the anterior region of the primitive streak. The current model of neural induction involves the inhibition of bone morphogenetic protein (BMP) signalling by the secretion of BMP antagonists from the anterior streak, which then allows ectoderm cells to become neural tissue. The role of the FGFs in this process has been very controversial. There have been a number of conflicting reports on the role of the FGFs as direct neural inducers in Xenopus5–7 and some studies in the chick, which show that FGFs can induce posterior neural tissue.8,9 One of the latest studies has suggested that the process of neural induction is a multistep process with FGF signalling being required at a very early stage and inhibition of BMP signalling being required later.10 This is supported by the observation that the expression of a dominant negative FGFR in Xenopus precludes the induction of neural tissue by BMP antagonists.11 A number of FGFs are present in the streak, namely FGF-8, FGF-2, FGF-4 and FGF-3,8,12,13 during the phase of neural induction and may be involved in this process.

FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

Fibroblast growth factor-2

Fibroblast growth factor-2 and FGF-1 are the prototypical FGFs and have been most extensively studied. They were the first discovered in 198414 and were found to be mitogenic for fibroblasts. Subsequently, FGF-1 and FGF-2 have been found to be potent modulators of proliferation in the developing nervous system. Fibroblast growth factor-2 is expressed early in brain development and, analogous to FGF-1, persists throughout development into adulthood. Expression studies on FGF-2 in the mouse and the rat show that, in addition to its abundance in the nervous system, it is expressed in a wider range of tissues than FGF-1 (for a review see Vaccarino et al.2). The expression studies also reveal distinct differences between FGF-1 and FGF-2 in the developing CNS, with FGF-2 localized to both neuronal and non-neuronal cell types and FGF-1 being mostly neuronal. Fibroblast growth factor-2 immunoreactivity is abundant at early stages in the cortex. Fibroblast growth factor-2 is sequestered in the basement membrane around the neural tube15 and, in association with heparan sulphate proteoglycans (HSPG), within the neuroepithelium.16 Our in situ hybridization analysis shows specific expression of FGF-2 within the mouse neural tube at embryonic day (E) 10.17 Recent studies have shown that FGF-2 is expressed in the ventricular region of the developing cortex in a dorsoventral gradient.18 It is seen in the cortical plate at E18, with expression then diminishing in neuronal cell types. In the postnatal and adult brain, highest levels are present in astrocytes.19,20

Fibroblast growth factor-2 and the regulation of stem cell survival and proliferation

We initially found that both FGF-1 and FGF-2 stimulated the proliferation and survival of neuroepithelial cells isolated from the telencephalon and mesencephelon of E10 mice.21 At least 50% of neuroepithelial cells divided in the presence of FGF-2, whereas in the absence of FGF all cells died within 6 days of culture. Fibroblast growth factor-2 not only influenced the proliferation and survival of the cells, but also their morphology; at high concentrations of FGF-2, cells begin to adhere to the substrate and acquire both neuronal and glial morphologies. This morphological change is accompanied by the expression of both neurofilament and glial fibrillary acidic protein (GFAP), which are definitive markers for neurons and glia, respectively. These results suggest that FGF-2 is stimulating the differentiation of the neuroepithelial cells into mature neurons and glia,21 a proposal that is supported by results from studies of Qian et al.22 However, this latter response may have resulted from the secondary production of other factors within the cultures or from direct effects on adhesive interactions with extracellular matrix proteins.23 Other studies indicate that the role of FGF-2 is associated with proliferation and additional signals are required for differentiation.24 Clonal studies provide a more precise means of analysing these potential influences. Using clonal analysis, we found that FGF-2 alone was unable to induce the differentiation of these stem cells25 and, thus, its primary activity at this stage of development may be proliferation and survival.

Neural precursor cells from the embryonic spinal cord also proliferate in response to FGF-2 (M Murphy, unpubl. data, 1992) and Ray and Gage26 have shown that FGF-2 stimulates proliferation of neuronal precursor cells isolated from embryonic spinal cord. Thus, it is possible that FGF-2 can stimulate proliferation of neural precursor cells for the entire CNS.

An in vivo demonstration that FGF-2 plays a role in the development of the brain came through studies on FGF-2-deficient mice.27,28 The FGF-2 null mutant mice are viable, but display defects in the cerebral cortex. There is a reduction in parvalbumin-positive neurons in cortical layers, most pronounced in the frontal motor sensory area. In addition, there are neuronal deficiencies in the cervical spinal cord. However, cell density in many other areas of the CNS appears normal. Further studies show that both cortical neurons and glia are reduced in FGF-2-deficient mice.29 Bromodeoxyuridine (BrdU) studies in these animals demonstrated that FGF-2 increased the proportion of dividing cells without affecting the cell cycle length. The degree of apoptosis within the ventricular zone (VZ) was not changed in FGF-2 knockouts, which suggests that FGF-2 does not act as a survival molecule in vivo. A more recent study from that laboratory has shown the major effects of FGF-2 loss are found in dorsal regions of the brain (the cortex).18 Fibroblast growth factor-2 and one of its receptors, FGFR-1, are expressed predominantly in early stages of neurogenesis (E10–E12 in the mouse), when the precursor cells for the deepest layers of the cortex are dividing and differentiating. This correlates with the finding that the neurons lost in FGF-2-deficient mice are the large neurons in deep cortical layers.18

Combined with our findings in vitro, these data indicate that FGF-2 is involved in brain development. It has a spatially and temporally defined role, which is to initiate or maintain cell division of neural precursor cells in the dorsal regions of the telencephelon during the early stages of neurogenesis. Other roles for FGF-2, such as in migration and differentiation, are also possible. Where these studies clearly point to a direct involvement of FGF-2 in the development of the brain, it is also clear that this factor cannot be the only factor responsible for proliferation and differentiation in the brain because, in many respects, the brains of these animals are relatively normal.

Our early in vitro studies suggested that FGF-2 was involved in cell survival, but it is more likely that insulin-like growth factor-1 (IGF-1) is a necessary requirement for the survival of the E10 precursor cells.30 In cultures of the precursor cells, FGF-induced proliferation is dependent on the presence of IGF and the neuroepithelial cells also endogenously produce IGF-1. Blocking of endogenous IGF-1 activity with anti-IGF-1 antibodies results in complete inhibition of FGF-mediated proliferation and in cell death. Insulin-like growth factor-1 alone acts as a survival agent. These observations correlate with the detection of transcripts for IGF-1 and FGF-2 in freshly isolated neuroepithelium and are consistent with an autocrine action of these factors in early brain development in vivo.

Fibroblast growth factor-2 and the neural crest

The neural crest (NC) is a transient embryonic structure that arises from the most dorsal aspect of the neural tube. The NC cells migrate away from the neural tube at around the time of closure and migrate into a series of discrete regions of the embryo. They give rise to a multitude of cell types, including most of the peripheral nervous system, adrenal chromaffin cells, melanocytes of the skin and many mesectodermal structures, including muscle, cartilage and bone in the head and upper body. We have shown that FGF-1 or FGF-2 act directly on neural crest cells in vitro to stimulate proliferation17 in the presence of serum. These findings correlate with in situ hybridization analysis, which shows FGF-2 mRNA in cells in the neural tube and within newly formed sensory ganglia at E10 in the mouse, where NC precursors are proliferating. These data infer an autocrine/paracrine loop for FGF-2 regulation of proliferation, similar to that described for CNS precursor cells in the brain.

We have also found that FGF-2 treatment of NC cultures stimulates the expression of Pax3, a paired box protein involved in NC and dorsal neural tube development.31 The Pax-3 mRNA in NC cultures treated with FGF-2 is initially expressed in all cells, but is retained only in neurons, suggesting a role for Pax3 in neuronal generation. Further experiments involving the blocking of Pax3 expression in neural precursor cells demonstrated a requirement for Pax3 in neuronal development. Thus, FGF-2 may not only be involved in NC cell proliferation, but also in the specification of NC cells to a neuronal fate.

Whether the endogenous FGF mediating these activities in vivo is FGF-2 or whether it emulates the activity of another FGF family member is unknown. There has not been a comprehensive analysis of the effects of FGF-2 gene deletion on NC derivatives, but neuronal deficiencies have been observed in the cervical spinal cord of FGF-2-knockout mice.27 Fibroblast growth factor-15 shows a striking pattern of expression in the developing neural tube. Expression in the spinal cord is predominantly in the most dorsal aspect and includes premigratory NC cells.32

Fibroblast growth factor-8 and FGF-17

Fibroblast growth factor-8 was first identified as a secreted androgen-induced growth factor33 and was subsequently identified as a Wnt1-cooperating proto-oncogene in mammary tumorigenesis.34 Fibroblast growth factor-8 has been studied in some detail and, like many FGFs, has a wide spectrum of activities in the developing embryo. Fibroblast growth factor-17 has only recently been discovered.35 It is closely related to FGF-8, with 60% amino acid identity. Both FGFs have similar patterns of splicing in the 5′ coding region.36,37 Fibroblast growth factor-8 and FGF-17 have very similar expression patterns in the developing CNS, which suggests they may have a functional relationship in patterning some areas of the brain.37–39 There are also some distinct expression domains. Both factors are predominantly expressed in the midline region of the developing forebrain and the midbrain–hindbrain junction (MHB). Fibroblast growth factor-8 is expressed earlier than FGF-17, whose expression persists a little longer.40

In the forebrain, the first domain of expression is the anterior neural ridge (ANR). The ANR contributes to the olfactory placode, nasal epithelium and the dorsal structures of the telencephalon. Fibroblast growth factor-8 is expressed in the ANR at E8.5, whereas expression of FGF-17 begins at E9.5. By E11.5, both FGF-8 and FGF-17 are expressed in the septal neural epithelium and the telencephalon–diencephalon junction. During this time, FGF-8 is also broadly expressed along the dorsal midline of the diencephalon and in ventral hypothalamic regions, while FGF-17 is restricted to a small domain in the telencephalon–diencephalon transition region.

The MHB is a major signalling centre for the patterning and growth of future midbrain and hindbrain structures. At E8.5, both FGF-8 and FGF-17 are expressed at similar patterns and intensities in the MHB. Analogous to the developing forebrain, there are also significant spatio–temporal differences between FGF-8 and FGF-17 expression in the MHB. At E8.5, FGF-8 expression is broader and stronger in the MHB than that of FGF-17. From E11.5, the reverse applies and FGF-17 expression is broader and stronger than that of FGF-8. This expression ceases by E15.5.40,41

Fibroblast growth factor-8 and patterning of the forebrain, midbrain and cerebellum

There have been a number of very elegant studies that indicate that FGF-8 is a key signalling molecule in the patterning and development of the midbrain, isthmus and cerebellum. The implantation of an FGF-8 bead rostral to the isthmus, close to the midbrain– diencephalon boundary, has the dramatic effect of inducing an ectopic midbrain.42 Recently, Martinez et al.43 have shown that FGF-8 patterns the brain both rostrally and caudally by suppressing the expression of the Otx2 gene. An appropriate gradient of Otx2 in this region of the developing brain is required both for midbrain development and cerebellar development. Thus, implantation of FGF-8 in slightly more caudal regions induces both ectopic midbrain and cerebellum.43 Fibroblast growth factor-8 hypomorphs (leaky knockouts) show defects in midbrain and cerebellar tissue.44 In zebrafish acerebellar mutants (which is an FGF-8-loss of function), the cerebellum does not form;45Hox genes are also repressed by FGF-8. A recent study in the chick has shown that FGF-8 signalling from the isthmus establishes the anterior limit of Hox genes and thereby patterns the anterior hindbrain, the territory of the brain that gives rise to the cerebellum.46

Several reports also indicate that FGF-8 has a role in patterning of the forebrain. Removal of the ANR in explant culture eliminates the expression of brain factor-1 (BF-1), a transcription factor that is essential for the development of the telencephalic vesicles. It has been shown that BF-1–/– mutants have severe cerebral cortex defects47 as a result of massive premature differentiation of neural precursor cells. Implantation of FGF-8-coated beads can restore BF-1 expression.48 Fibroblast growth factor-8 hypomorphs also show midline deletions in the development of the telencephalon and a lack of midline structures, such as olfactory bulbs.44 The zebrafish aussicht mutants exhibit widespread overexpression of FGF-8 and show defects in the differentiation of the forebrain.49

The aforementioned experiments suggest that FGF-8 influences cell specification by repressing or activating the expression of a variety of transcription factors. Another study showing the effects of FGF-8 on cell specification is that of Ye et al.,50 who showed that FGF-8 was necessary for the development of midbrain dopaminergic and hindbrain serotonergic neurons. Fibroblast growth factor-8 may also have an effect on cell proliferation, because studies on transgenic mice in which FGF-8 is ectopically expressed in the mesencephalon show an expanded mesencephelon with an abnormally thick VZ of mitotically active cells.51

Fibroblast growth factor-17 and patterning of the cerebellum

Disruption of the FGF-17 gene in mice leads to defects in the late phase of cerebellar development. These mice show decreased precursor cell proliferation from E11.5 in the cerebellar vermis, the midline of the primordial cerebellum. Mice that have an additional FGF-8 hypomorphic mutation show an enhanced phenotype with accelerated onset. As a consequence, there is a progressive dose-dependent loss of the most anterior lobe of the cerebellar vermis. This demonstrates that both FGF-8 and FGF-17 cooperate to regulate the size of the precursor pool of cells that develop into the cerebellar vermis.40 These mice also show accelerated rostro–medial progression of Purkinje cell differentiation in the vermis primordium. Therefore, these studies suggest that FGF-8 and FGF-17 normally function to inhibit differentiation in the vermis primordium. The vermis cerebellum functions in motor coordination and maintenance of body equilibrium and some of the FGF-17/FGF-8 mice also show gait defects.40 Further studies of these mice may reveal additional sites of FGF-17/FGF-8 cooperativity at other sites (the forebrain, for example) where they are coexpressed.

Fibroblast growth factor-15

Fibroblast growth factor-15 was first discovered as a downstream target of the homeodomain oncoprotein E2A-Pbx1.32 In the mouse, the expression of FGF-15 is largely confined to the nervous system throughout development, where it is both highly and dynamically expressed. In the forebrain, FGF-15 expression begins around E9, where it is expressed in the septal region from which the olfactory bulbs develop. From E12 to E14, FGF-15 continues to be strongly expressed in the septal region of the telencephalon and is seen in differentiating neurons within the olfactory neuroepithelium. It is also highly expressed in the neuroblast layer of the optic cup. This layer gives rise to the retina.

In the diencephalon, FGF-15 is predominantly expressed in the dorsal thalamus from E9.5 and, by E12, expression also extends to the ventral thalamus. In the mesencephalon, FGF-15 is expressed in a rostro–caudal gradient, initially (E9.5–E12) being high caudally and later (E12–E14) being higher rostrally. Significantly, there is a sharp boundary of expression at the borders between the midbrain and the diencephalon and between the midbrain and hindbrain. In the developing hindbrain, FGF-15 is strongly expressed dorsally from E8.5 to E10; from E12 to E14 it is strongly expressed in the developing cerebellum.

Fibroblast growth factor-15 is also strongly expressed in distinct regions of the developing mouse spinal cord from E8 to E14. Most expression is in the most dorsal parts of the spinal cord, probably including premigratory NC cells as well as roof plate cells.32

In the absence of a study that directly compares the spatio– temporal expression domains of FGF-15 with those of FGF-8 and FGF-17 expression, it is difficult to determine the exact degree of overlap and difference in expression boundaries, but some are clearly apparent. All three factors are coexpressed in the midline regions of the developing telencephalon, in the regions of the future olfactory bulb and nasal placode. Notably, FGF-15 is absent from the diencephalon–midbrain junction and MHB, where both FGF-8 and FGF-17 are expressed. In contrast, FGF-15 is expressed throughout the midbrain neuroepithelium where both FGF-8 and FGF-17 are absent. Fibroblast growth factor-15 is expressed in the neuroepithelium of the developing eye, where neither FGF-8 nor FGF-17 have been detected. Neither FGF-8 nor FGF-17 are expressed in the developing spinal cord.

There are no functional studies published to date on the activity of FGF-15 to provide us with information on its activity on neural precursor cells. However, by comparison with results obtained for other FGFs, the expression data suggest that FGF-15 may play an important role in regulating cell division and patterning within the embryonic brain and spinal cord. It is possible that FGF-15 cooperates in a sequential and/or reciprocal manner (similar to the situation in limb development) with other FGFs in regions of the developing neural tube, as has been shown for the FGF-17/ FGF-8 double mutant mice in the developing cerebellum. It has been suggested that FGF-15, which is also heavily expressed in the hindbrain, may play a role in other aspects of cerebellar development.40

Fibroblast growth factor-1

Fibroblast growth factor-1 is found almost exclusively in nervous tissue. The cellular localization of FGF-1 message and its protein is largely neuronal, with little or no glial component. In the early developing mouse brain, the expression of FGF-1 is very low and only begins to become detectable at E12.52 Studies in the rat53 and mouse54 show that expression remains low until E16, when levels increase significantly to highest concentrations in the adult brain. A study of the developing chick brain55 showed that expression increased during embryonic development, reaching its highest level in the adult brain. This study also demonstrated that expression was localized to neurons such as adult telencephalic neurons, Purkinje cells, deep cerebellar and brainstem neurons and neurons of the peripheral nervous system (e.g. dorsal root ganglia). In the rat spinal cord, FGF-1 is restricted mainly to the cytoplasm and nucleus of mature sensory and motor neurons.53

Fibroblast growth factor-1 and a possible role in neuronal maturation

Studies on the timing and localization of FGF-1 expression suggest that FGF-1 may be involved in the maturation and maintenance of neurons. Fibroblast growth factor-1 has previously been reported to stimulate neuronal process regrowth in retinal ganglion cell cultures,56 spiral ganglion explants57 and adult dorsal root ganglion cells.58 Downregulation of FGF-1 mRNA with antisense oligonucleotides in cultured neural retinal cells resulted in an inhibition of neuronal differentiation and survival with no effects on proliferation.59 High levels of FGF-1 in mature cochlea neurons are believed to be important to the maintainance of the neuronal circuitry in the organ of Corti.60 Experiments in our laboratory have shown that FGF-1 stimulates the extension and maturation of neurites from neurons differentiating in FGF-1 treated neural precursor cell cultures. These studies revealed that the effects of FGF-1 were specific to neurite extension and maturation, the percentage of neurons differentiating in culture were similar to controls. (AE Apedaile et al., unpubl. obs., 1999).

Fibroblast growth factor-1 null mutant mice are apparently normal in appearance and behaviour and show no obvious defects in the brain when compared with wild-type mice.61 Furthermore, FGF-1–FGF-2 double knock-out animals show only defects previously described for FGF-2 null mutant mice. However, the nervous systems of these animals have not been thoroughly investigated and it is possible that there are more subtle neural defects in the FGF-1 mutant animals. In addition, given that FGF-1 is expressed at highest levels in the adult brain, it may be involved in neuronal maintenance, plasticity or following trauma.

OTHER SIGNIFICANT FGFs

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

Fibroblast growth factor-3 and FGF-10

Studies in mouse and Xenopus have shown that FGF-3 is expressed in the presumptive hindbrain adjacent to the otic placode and early otic vesicle.62–64 A more recent study in the mouse has shown that both FGF-3 and FGF-10 are present in the presumptive sensory and neuronal regions of the otocyst from E8 to E11.65 Fibroblast growth factor-3 is downregulated in the hindbrain by E11, but expression continues in the vestibular sensory epithelia and organ of Corti at later ages. This evidence implicates FGF-3 and FGF-10 in inner ear development and FGF-3 knockouts show inner ear defects.66,67 More recent studies show that ectopic expression of FGF-3 induces the formation of otic placodes and otic marker genes.68 Both FGF-3 and FGF-10 signal through the FGFR-2 IIIb isoform.69,70 Studies on mice where this isoform has been specifically deleted show severe defects in the inner ear caused by a failure in morphogenesis at the otocyst stage.65,71 These findings indicate that signalling between either FGF-3 and/or FGF-10 and its cognate receptor is crucial for inner ear development.

Fibroblast growth factor-7

Fibroblast growth factor-7 is expressed in a tightly regulated spatio–temporal manner in the developing brain. The FGF-7 message is detected transiently in the VZ of the lateral ventricle from E14.5 to E15.5.72 Expression is restricted to three sites, the ganglionic eminence, anterior horn and the presumptive parietal cortex, regions from which the frontal cortex and parietal cortex are derived. Fibroblast growth factor-7-deficient mice, other than for ‘rough’ hair, have no apparent nervous system defects.73 Fibroblast growth factor-7 binds and signals exclusively through FGFR-2 IIIb70 and, therefore, a study of the developing CNS in FGFR-2 IIIb mice may be enlightening.

Fibroblast growth factor-9

Fibroblast growth factor-9, initially referred to as a glia-activating factor (GAF), was isolated from a glioblastoma cell line.74,75 Studies on human and rat tissues show that it is strongly expressed in the spinal cord. Expression was initially detected in the adult in motor neurons and in the neurons of the dorsal root ganglia adjacent to the spinal cord.76 Later studies demonstrated expression of FGF-9 in astrocytes, in the spinal cord and brainstem and in oligodendrocytes in the cerebellum and corpus callosum.77 During development of the CNS, expression is more restricted to developing spinal motor neurons from E10.5 to E14, as well as in the olfactory bulb. In addition, FGF-9 is coexpressed with other FGF genes in some skeletal myoblasts, the future targets of the spinal motor neurons.78

Fibroblast growth factor-18

Fibroblast growth factor-18 is most related to FGF-8 and FGF-1779 and they have similar biochemical properties.40 Studies in the mouse have shown that FGF-18 is also expressed in the MHB region from E8.5 to E10.5, albiet at lower levels when compared with FGF-8 and FGF-17. Like FGF-8, FGF-18 expression diminishes in intensity by E12.5 and is no longer detectable by E14.40 Another study has shown that FGF-18 is expressed in the cerebral cortex of the mouse at E15.5.80 Unlike FGF-8 and FGF-17, FGF-18 is expressed in the trigeminal ganglion and dorsal root ganglia in the trunk.79

A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

During development of the nervous system, many classes of neurons undergo a period of naturally occurring cell death, wherein up to 50% of neurons of a particular class are lost. The cell death occurs at a time when neuronal axons and dendrites are innervating their targets. It is generally considered that the cell death represents pruning of excess neurons and a selection for neurons that have most appropriately innervated their targets. Spinal motor neurons directly innervate muscle and the survival of these neurons is dependent on their successful innervation of muscle. It is still unclear which growth factors are responsible for the neurotrophic support supplied by muscle. In cultures of embryonic spinal motor neurons, some of the most potent survival factors are members of the FGF family. Fibroblast growth factor-2 can support the survival of 50% of embryonic motor neurons isolated from E6 chick, a time of naturally occurring cell death.81 Other members of the FGF family can also support the survival of developing motor neurons. Fibroblast growth factor-5 is an interesting candidate as a target derived neurotrophic factor for motor neurons because it is expressed in skeletal muscle during the period of motor neuron death and in the adult.82 It also supports the survival of embryonic chick motor neurons.82 In addition, a significant proportion of motor neuron survival activity contained within muscle extracts could be immunoprecipitated with an anti-FGF-5 antibody.82 Fibroblast growth factor-5 has also been hypothesized as a regulatory molecule in the formation, maturation and/or maintenance of the neuromuscular junction. However, analysis of FGF-5-knockout mice does not show any loss of motor neurons or defects in the neuromuscular junction.83

Another FGF, FGF-9, is strongly expressed by spinal motor neurons and, thus, it is possible that FGF-9 acts as an autocrine survival factor for these neurons, as opposed to a target-derived survival factor. There is some evidence to support this idea; in spinal cord cultures from E13 rat, FGF-9 stimulates both survival of putative motor neurons and the levels of choline acetyl transferase,84 the synthetic enzyme for the motor neuron transmitter acetylcholine. It has been hypothesized that FGF-9 may be neurotrophic for motor neurons after they cease to be dependent on their target muscle.84

FIBROBLAST GROWTH FACTOR RECEPTORS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

The activity of the FGFs in the developing neural tube is ultimately controlled by their receptors, the cell surface tyrosine kinases or FGFRs. The binding of the FGF to these receptors is both facilitated and stabilized by a family of macromolecules called heparan sulphate proteoglycans (HSPGs). The HSPGs are localized to cell surfaces or within the extracellular matrix and, in the developing embryo, they bind the FGFs soon after they are secreted from cells. Fibroblast growth factors are, therefore, localized close to the site of production and there is evidence to suggest that they do not stray more than a few cell diameters8 (digoxigenin-labelled FGF only diffuses three to four cell diameters). The heparan sulphate sugar chains on the HSPG are believed to confer the specificity of binding of particular FGFs to their receptors.4,85 Recent studies have demonstrated that there is a remarkable degree of specificity in heparan sulphate for both the FGF ligand and FGFR that dictates ligand–receptor specificity.86

There are four FGF receptor genes, FGFR-1–4. The receptors are transmembrane glycoproteins containing three Ig-like loops (loops I, II and III) in the extracellular domain and a split tyrosine kinase intracellular domain. Loop III determines the FGF binding specificity of the receptor.87 Alternative splicing of two exons that encode the C-terminal half of loop III creates different isoforms (IIIb or IIIc) for FGFR-1–3 but not FGFR-4. In vitro assays show that both the individual receptors and their isoforms have quite distinct FGF-binding specificities. Table 2 shows the relative activities of the FGFs expressed in the developing brain for the different FGFR variants, as determined by an in vitro binding assay with BaF3 cells.40,70 The FGF/FGFR binding profiles show the remarkable characteristic of a combination of a high degree of cross-reactivity between receptors and ligands with distinct binding profiles for individual FGFRs. In vivo, the particular endogenous HSPG to which the FGFs are initially bound will also affect FGFR binding. Therefore, the relative affinities shown in Table 2 may not reflect the binding of particular FGF to their receptors in vivo.

Table 2.  Receptor specificity (%) for members of the fibroblast growth factor family
 Fibroblast growth factor receptor
 1IIIb1IIIc2IIIb2IIIc3IIIb3IIIc4
  1. Potential ligand–receptor pairs have been determined using cell lines (BaF3) that were engineered to express the major splice variants of all known fibroblast growth factor (FGF) receptors. FGF-1 is the only FGF that can activate all FGF receptor splice variants. The relative mitogenic activity of FGF on individual FGFR splice variants is normalized to data obtained with FGF-1 (100%). Potential pairs (high values) are indicated in bold type. Data were compiled from from Ornitz et al.70 and Xu et al.40

FGF-1100100100100100100100
FGF-2601049641107113
FGF-3340.34541.50.65.8
FGF-415.61021594169108
FGF-76.30.3812.51.211.9
FGF-84.41.45.920.62.243.964.6
FGF-93.5217.389429675
FGF-10111001111
FGF-175.72.75.153.8289.295.1
FGF-182.72.25.37.42.522.378.3

Many of the FGFR localization studies do not distinguish the expression of the splice variants. The few studies that have, show they are differentially expressed. In situ hybridization studies with probes that would not distinquish the isoforms have shown that FGFR-1 is expressed in the VZ of the mouse and chick neural tube during early to mid-phases of neurogenesis.88 Fibroblast growth factor receptor-1 appears to be most strongly expressed in the developing forebrain VZ.18,89,90 This expression pattern corresponds with that of FGF-2, which binds to FGFR-1 with high affinity in vitro and, when deleted, results in significant loss of forebrain neurons, as described earlier. Both FGFR-1 and FGF-2 are downregulated in this region by mid- to late stages of neurogenesis.18 At later stages of development (E16.5), FGFR-1 is expressed in maturing neuronal populations of the brain.90 It is also expressed by motor neurons in the developing spinal cord.91

A summary of the results obtained on the localization of FGFR-1–3 in the developing chick neural tube is given in Fig. 2. These studies provide the most detailed spatio–temporal mapping of these FGFR in the developing neural tube to date and illustrate that FGFR-2 and FGFR-3 expression is highly dynamic. In situ hybridization studies with probes that do not distinguish the isoforms show that FGFR-2 is expressed throughout the brain and strongly in the developing midbrain and hindbrain from E9.5 to E16 in the mouse,90 which is supported by more recent studies in the chick.88,92 The splice variants for FGFR-2 have been distinguished in a study of the developing embryo,93 but not with a detailed focus on the developing neural tube. It appears that FGFR-2IIIb is more diffusely expressed throughout the neuroepithelium within the dorsal and ventral regions of the telencephalon of the developing brain, whereas FGFR-2IIIc expression appears to be more restricted to the VZ of the basal forebrain and other more ventral structures.

image

Figure 2. Fibroblast growth factor receptor (FGFR) expression in the developing chick neural tube: a diagrammatic representation. The expression domains of FGFR at early times are shown in relation to the major divisions of the embryonic brain at the five-vesicle stage. The expression domains represent the anterior–posterior extent of mRNA distribution in the neural tube. HH, Hamburgher and Hamilton stages of chick development (the developmental age is given in brackets). This diagram has been compiled from data in Wilke et al.88 and Walshe and Mason.92 The dashed line indicates a lower level of expression. MHB, midbrain–hindbrain junction.

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While FGFR-1 and FGFR-2 are both expressed throughout tissues of the developing embryo, FGFR-3 is largely confined to the CNS and developing bone.94 In addition, FGFR-3 is strongly expressed in the VZ of the neural tube from E9 through to E16 in the developing mouse embryo. At later stages (post-E16), FGFR-3 expression appears to become largely confined to glia.94 At this time, it begins to be expressed in the differentiating hair cells of the cochlea where it has been colocalized with FGF-1.60 Within the developing chick spinal cord, there is intense expression of FGFR-3 in a subset of motoneurons in the medial subdivision of the median motor column. The onset of expression within these neurons correlates with axonal growth95 and with the expression of FGF-9, an FGF with high affinity for FGFR-3.76

These FGFR-3 expression studies may only represent the IIIc isoform, because no expression of the IIIb isoform has been observed in the CNS by exon-specific probes96 and reverse transcription–polymerase chain reaction.92 The study of Wuechner et al.96 showed that the IIIc isoform was abundant throughout the developing CNS and was most intense in the mouse hindbrain. Its expression decreased by E20 in the brain, but was still strong in the ventral spinal cord. Essentially similar results have been found in the chick.92

Fibroblast growth factor receptor-4 has not been studied in detail in the mouse or the chick nervous system. However, an early study in the chick does indicate that FGFR-4 is expressed in the VZ of the developing spinal cord and the dorsal root ganglia.97 A more recent study in the mouse shows that the expression of FGFR-4 in the developing CNS is initially low and rises to a peak at E16, where there is strong localization in the VZ of the cortex.98 The most detailed studies on FGFR-4 expression in the neural tube have been conducted in fish.99,100 In both studies, the expression of FGFR-4 was highly dynamic and intense in the telencephalon, the diencephalon, the hindbrain and the dorsal-most portion of the rostral spinal cord. However, the extent of localization in the midbrain and MHB region differs in the two studies. In one, FGFR-4 was not expressed in a narrow band in the midbrain region, but appeared to be expressed in the MHB region,99 while in the other study there was no apparent expression of FGFR-4 in the MHB region where FGF-8 (and FGF-17) is expressed.100 The latter result is consistent with data on FGFR-2 and FGFR-3 in the chick (see Fig. 2). Overall, it would seem that further studies are necessary to comprehensively evaluate FGFR-4 expression in the chick and the mouse and to resolve conflicts in the data pertaining to FGFR-4 expression in the developing CNS.

Thus, there are still some significant gaps in our knowledge of the localization of the FGFs and their cognate receptors. Both FGF-8 and FGF-17 are strongly expressed and function in some key areas, such as the MHB in the patterning of the brain; however, the receptors that bind most strongly to these FGFs in vitro, FGFR-3IIIc and FGFR-4, have not been colocalized in the appropriate regions.88,92 While FGFR-1 is expressed in regions such as the MHB, in vitro studies of the binding affinities of these FGFs (see Table 2) would suggest that neither FGF-8 nor FGF-17 bind this receptor. This suggests that FGFR-1 may not mediate the signalling of FGF-8 and FGF-17 and that another FGFR (possibly FGFR-4), which has yet to be localized by in situ hybridization studies, may be present. An alternative explanation is that the endogenous HSPG may stabilize binding of FGF-8 and/or FGF-17 to FGFR-1 sufficiently to cause signalling in vivo.

Fibroblast growth factor receptor knock-out studies in the mouse have not been particularly illuminating with respect to the function of FGFRs in the developing CNS. It has been shown that FGFR-1 and FGFR-2 null mutants are lethal prior to the formation of the mature CNS. However, a study of chimeric mice in which the effects of the FGFR-1 deletion were attenuated has revealed that FGFR-1 is essential for neural tube development.101 The FGFR-3 null mice exhibit bone defects.102 The FGFR-4 null mutant is superficially normal, but the only published report of the phenotype is a study on a double knock-out of both FGFR-3 and FGFR-4, where mice have lung defects.103 The understanding of the function of FGFRs in the CNS would be greatly enhanced by the generation of both tissue-specific and receptor isoform-specific knock-outs.

CONCLUSIONS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES

In summary, there is now a great deal of evidence that FGF signalling plays multiple roles in neural development. Currently up to 10 different FGFs may play a role in brain development from the very earliest stages of neural induction through to establishment of appropriate connectivity and beyond. In addition, there is highly specific expression of at least three of the four different FGFRs throughout brain development. These studies are beginning to support the idea that specific FGF signal through specific receptors in a highly localized manner to regulate brain development. The coexpression of FGF-2 and FGFR-1 during the proliferative phase of cortical development is one example. However, there are still some significant gaps in our knowledge of the localization of FGFs and their cognate receptors. Furthermore, the specific interactions between unique combinations of FGF and FGFR isoforms signal a whole array of different developmental functions. These include primary neural induction, neural precursor proliferation, patterning, neuronal specification and neurotrophism.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. FIBROBLAST GROWTH FACTORS AND NEURAL INDUCTION
  5. FIBROBLAST GROWTH FACTORS DURING THE PROLIFERATIVE AND NEUROGENIC PHASES OF NEURAL DEVELOPMENT
  6. OTHER SIGNIFICANT FGFs
  7. A NEUROTROPHIC ROLE FOR MEMBERS OF THE FGF FAMILY
  8. FIBROBLAST GROWTH FACTOR RECEPTORS
  9. CONCLUSIONS
  10. REFERENCES
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