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EVOLUTIONARY HOMOLOGS

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Response of neural stem cells to FGF

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. Neural stem cells that have the capacity to self-renew and differentiate into neurons and glia can be cultured from the adult SVZ. These cells grow as spherical floating clusters (neurospheres) in the presence of epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF). The SVZ germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, it has been shown that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. It is concluded that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain (Doetsch, 1999).

Many astrocytes express EGF and bFGF receptors. Interestingly, using these growth factors, neural stem cells can be isolated in vitro not only from the adult SVZ but also from other brain regions, including the spinal cord, diencephalon, and hippocampus. The results described here raise the intriguing possibility that neural stem cells that have been cultured from other brain regions may actually be derived from astrocyte-like cells in vivo. Astrocytes encompass a heterogeneous population of cells that are widely distributed in the adult brain and continue to divide in situ. After injury, astrocytes proliferate to form glial scars but not neurons. It is possible that a neurogenic potential is latent in many astrocytes throughout the CNS but that inhibitory signals may suppress these cells from producing neurons. Neurogenic factors may only be present close to the brain ventricles or within the adult SVZ. Alternatively, SVZ astrocytes that act as stem cells in vitro and generate neurons in vivo may correspond to a fundamentally different cell type that resembles astrocytes and expresses GFAP and other glial markers. If this is the case, then this work suggests that use of markers such as GFAP, a hallmark of glial cells, may not be a reliable indication of fully differentiated glia. Further work is required to characterize those SVZ astrocytes that can act as primary neuronal precursors and to determine how they differ from other astrocytes within the SVZ and the rest of the brain (Doetsch, 1999 and references).

During neural development, astrocytes are derived from radial glia. Adult cortical astrocytes assume the characteristics of radial glia upon exposure to embryonic brain extracts. Interestingly, radial glia divide and have been hypothesized to function as neuronal precursors. The identification of SVZ astrocytes as neuronal precursors in the adult brain further suggests that what has classically been considered an astrocytic lineage, including radial glia, may in fact correspond to embryonic and adult neuroepithelial cells that retain some of the properties of neural stem cells. The present results demonstrate that SVZ astrocytes are the in vivo primary precursors for new neurons during regeneration and under normal conditions. Furthermore, neurospheres in vitro can be derived from SVZ astrocytes labeled in vivo, indicating that these cells can act as neural stem cells. Exploitation of the regenerative capacity and neurogenic potential of SVZ astrocytes may have powerful implications for brain repair (Doetsch, 1999).

Temporal changes in progenitor cell responses to extrinsic signals play an important role in development, but little is known about the mechanisms that determine how these changes occur. In the rodent CNS, expression of epidermal growth factor receptors (EGFRs) increases during embryonic development, conferring mitotic responsiveness to EGF among multipotent stem cells. Cell-cell signaling controls this change. Whereas EGF-responsive stem cells develop on schedule in explant and aggregate cultures of embryonic cortex, co-culture with younger cortical cells delays their development. Exogenous BMP4 mimics the effect of younger cells, reversibly inhibiting changes in EGFR expression and responsiveness. Moreover, blocking endogenous BMP receptors in progenitors with a virus transducing dnBMPR1B accelerates changes in EGFR signaling. This involves a non-cell-autonomous mechanism, suggesting that BMP negatively regulates signal(s) that promotes the development of EGF-responsive stem cells. FGF2 is a good candidate for such a signal: it antagonizes the inhibitory effects of younger cortical cells and exogenous BMP4. These findings suggest that a balance between antagonistic extrinsic signals regulates temporal changes in an intrinsic property of neural progenitor cells (Lillien, 2000).

What then triggers the onset of the appearance of EGF-responsive stem cells? It is appealing to consider a feedback mechanism, whereby cells that produce BMPs or FGF2 achieve appropriate numbers or states of maturation at mid-embryonic development, resulting in the generation of a net positive signal. BMPs are produced by radial glial cells and by the choroid plexus. FGF2 is made by progenitor cells and choroid plexus. The numbers of these cells do not change in an appropriate manner at mid-embryonic development to provide a trigger, suggesting that the cellular event(s) that initiates the change in EGFR expression may be more complex and involve a change in the level of expression of FGF, BMP and/or their receptors. It has been reported that the level of FGF2 increases during mid-late stages of embryonic development. Thus, an increase in FGF2 expression could be the trigger. The level of expression of BMPRs in the brain appears to decline during embryonic development, suggesting that BMP signaling might decline. Given the observation that BMPs in the limb negatively regulate the expression of FGFs, together with the finding that dnBMPRs have a non-cell-autonomous positive effect, it is possible that a reduction in BMP signaling triggers the increase in FGF2 expression in the CNS. In explant cultures, it has been observed that a lower concentration of FGF2 (1 ng/ml) has a greater effect on E15 explants than on E12 explants. This observation is consistent with an increase in the level of an endogenous positive signal, such as FGF2 with age, or with a decline in the level or effectiveness of endogenous BMPs (Lillien, 2000).

In addition to the change in EGFR expression and responsiveness, cortical progenitor cells and stem cells change in several other ways during mid-embryonic development. Early progenitor cells and stem cells tend to generate more neuronal progeny, while later progenitor cells and stem cells tend to generate more glial progeny. Early and late progenitor cells also differ in their competence to generate deep layer neurons. The developmental change in the latter property also appears to be controlled by cell-cell signaling. Studies using viral transduction of EGFRs suggest that mitotic stimulation of progenitor cells with EGF does not control developmental changes in the ratio of neuronal and glial progeny. It will be interesting to determine whether the signals that have been identified as regulators of EGFR expression and responsiveness also control other properties of progenitor cells that change during embryonic development, or whether these properties are controlled by distinct mechanisms (Lillien, 2000).

Multipotent stem cells that generate both neurons and glia are widespread components of the early neuroepithelium. During CNS development, neurogenesis largely precedes gliogenesis: how is this timing achieved? Using clonal cell culture combined with long-term time-lapse video microscopy, it has been shown that isolated stem cells from the embryonic mouse cerebral cortex exhibit a distinct order of cell-type production: neuroblasts first and glioblasts later. This is accompanied by changes in their capacity to make neurons versus glia and in their response to the mitogen EGF. Hence, multipotent stem cells alter their properties over time and undergo distinct phases of development that play a key role in scheduling production of diverse CNS cells (Qian, 2000).

To examine the gliogenic potential of stem cells, the composition of E10 and E12 cortical progenitor cell clones, exposed to different concentrations of FGF2 during the culture period, were compared. Changes in the concentration of FGF2 can regulate the appearance of glia from E10 cortical stem cells. When grown in 0.1 ng/ml FGF2, E10 stem cells generate neurons, but most do not generate glia. In contrast, in 1 or 10 ng/ml FGF2, E10 stem cells generate the same number of neurons as in 0.1 ng/ml FGF2, but they also generate glia. In each FGF2 concentration tested, the percentage of clones containing glia is higher for E12 cells than for E10 cells. Moreover, the percentage of glia-only clones increases dramatically with age. E10 cells grown in 0.1 ng/ml FGF2 have 0% glia-only clones. This increased to 15% when clones are grown in 10 ng/ml FGF2. In contrast, the percentage of glia-only clones seen in cultures made from E12 cells is significantly higher -- 20% in 0.1 ng/ml FGF2 and 75% in 10 ng/ml FGF2. This suggests that more cells are committed to making solely glia in the E12 cortex than in the E10 cortex. Why the incidence of glia-only clones increases with increasing FGF2 concentration is not clear. Perhaps there is a subpopulation of glial progenitor cells that requires a higher level of FGF2 to divide. Alternatively, perhaps some of the cells that at low concentrations of FGF2 give neurons as well as glia are switched to generate solely glia at the higher FGF2 levels. If this were the case, then these data suggest that E12 cells can be pushed more easily to produce solely glia than E10 cells (Qian, 2000).

One of the defining features of stem cells is self-renewal. This concept was established largely through studies on adults, in which the key function of stem cells is homeostasis, and maintaining their potential to make a specific range of cell types makes sense. The major function of stem cells in developing systems, however, is not homeostasis, but cell production, and in the CNS, it is diverse cell production at defined times. Consequently, stem cells in developing systems may not be adapted to make exactly the same range of progeny over their sequence of divisions. In invertebrates, neuroblasts change as they divide, helping them produce a series of diverse ganglion mother cells. In vertebrates, there are few studies of stem cells in development. However, evidence indicates that stem cells in the developing blood system are distinct from adult blood stem cells. These data suggest that stem cells in the developing nervous system also change over time. Acutely isolated stem cells at various embryonic ages differ in their neurogenic and gliogenic capacities even when grown under identical conditions, indicating changes occur during development. Perhaps changes in growth factor receptor systems seen in the cortical ventricular zone occur within the stem cell population. Indeed, early embryonic stem cells allowed to develop in culture eventually acquire the EGF responsiveness characteristic of late embryonic and postnatal stem cells. These data indicate that cortical stem cells are not immutable and that, as suggested for invertebrate neuroblasts, changes in their properties may have important consequences for normal CNS development and lead eventually to a form adapted to the adult (Qian, 2000).

Given that the cerebral cortex produces an array of distinct types of neuronal cells in a predictable temporal order, it is tempting to speculate that changes in stem cells are involved in this process. Neurons arise from asymmetrically dividing neuroblasts and from stem cells making neurons and glia, both of which are isolated from E10 cortex. Whether these progenitor cells are lineally related via an earlier stem cell or represent discrete cell types is not clear at this stage. However, mechanisms of generating diverse neuronal types by altering the properties of an asymmetrically dividing progenitor might well operate in both progenitor types. Changes in stem cell behavior may involve both cell-intrinsic events and environmental signals. Feedback from early-born cortical cells influences the fate of later-born cells, perhaps acting through a multipotent progenitor population. However, older cells are unable to generate as many types of neurons as younger cells, a restriction that may occur at the stem cell level (Qian, 2000).

A model incorporating these observations and current hypotheses to explain the role of stem cells in timing cortical development is proposed. The early embryonic cortical stem cell (Se) undergoes asymmetric cell divisions, each generating a different restricted neuroblast and another, asymmetrically dividing stem cell (S1, S2, S3...Sn). The neurogenic potential of the stem cell declines so that it generates fewer neurons with age. The neurogenic potential may be enhanced or decreased by environmental factors, but at a certain point (the switch point), the stem cell stops making neurons and produces a highly proliferative progenitor cell that makes glial cells, provided that a high level of FGF2 or another suitable environmental signal is present. During this process, the stem cell matures to a form (Sm) that has a different response to growth factors than the early embryonic stem cell. An alternative model consistent with the current data is that at the switch point the stem cell changes into a glioblast that generates glial progeny, some of which have the potential to revert to a mature stem cell phenotype. The observation that isolated stem cells make neurons before glia focuses attention on these cells for continued exploration of cellular and molecular mechanisms that control the stereotyped schedule of neural cell generation in the developing CNS (Qian, 2000).

Secreted peptide growth factors are critical extracellular signals that interact to promote the proliferation, differentiation, and survival of progenitor cells in developing tissues. IGF-I signaling through the IGF type I receptor provides a mitogenic signal for numerous cell types, including stem and progenitor cells. The O-2A oligodendrocyte progenitor has been used to study the mechanism of IGF-I mitogenic actions since these progenitors respond to IGF-I in vitro, and gene targeting studies in mice have demonstrated that IGF-I is essential for normal oligodendrocyte development in vivo. The goal of this study was to elucidate the mechanism by which IGF-I promotes the proliferation of oligodendrocyte progenitors in the context of other mitogens critical for their proliferation. IGF-I significantly amplifies the actions of FGF-2 and PDGF to promote DNA synthesis in O-2A progenitors. Investigation of cell cycle kinetics revealsthat IGF-I has no significant effect on the rate of cell cycle progression. Instead, IGF-I promotes increased recruitment of O-2A progenitors into the S phase of the cell cycle. These studies support a role for IGF-I as a cell cycle progression factor for progenitor cells (Jiang, 2001).

The studies presented here demonstrate that IGF-I significantly enhances the ability of FGF-2 and PDGF to promote DNA synthesis in O-2A progenitors. Of greatest significance is the finding that IGF-I synergizes with FGF-2 in promoting the incorporation of thymidine into these cells and that the mechanism for this synergism includes increased recruitment of progenitor cells into the cell cycle. These results are consistent with a competence/progression factor model for the O-2A progenitors. The concept of competence and progression factors in promoting progression of cells past G1 checkpoints and into the S phase of the cell cycle was first proposed for fibroblasts. In fibroblasts, PDGF acts as a competence factor to promote fibroblast cell entry into G1 but the presence of a progression factor (such as platelet-poor plasma or IGF-I) is also required for the cells to pass the G1 restriction point and enter S phase. Based on data presented here, a similar model is proposed for the proliferation of O-2A progenitors where FGF-2 and PDGF are competence factors and IGF-I is a progression factor for the O-2A progenitors (Jiang, 2001).

The CNS is thought to develop from self-renewing stem cells that generate neurons, astrocytes, and oligodendrocytes. Other data, however, have suggested that astrocytes and oligodendrocytes are generated from separate progenitor populations. To reconcile these observations, progenitors have been prospectively isolated that do or do not express Olig2, an oligodendrocyte bHLH determination factor. Both Olig2 non-expressing and Olig2 expressing progenitors can behave as tripotential CNS stem cells (CNS-SCs) in vitro. Growth in FGF-2 causes induction of Olig2 in the former population, permitting oligodendrocyte differentiation; extinction of Olig2 in the latter cells permits astrocyte differentiation. The induction of Olig2 by FGF-2 is mediated, in part, via endogenous Sonic Hedgehog. These data indicate that clonogenic competence to generate neurons, astrocytes, and oligodendrocytes reflects a deregulation of dorsoventral patterning during expansion in vitro, raising the question of whether such trifatent cells actually exist in vivo (Gabay, 2003).

Mice expressing green fluorescent protein (GFP) in the Olig2 expression domain were used to prospectively isolate Olig2-expressing and nonexpressing progenitor cells by fluorescence-activated cell sorting (FACS). Both Olig2+ and Olig2- progenitors were found to be able to form neurospheres and can behave in vitro as tripotential CNS-SCs. In the case of initially Olig2- cells, the acquisition of oligodendrocyte capacity is caused by an unexpected effect of FGF-2, a mitogen commonly used to expand CNS-SCs, to induce expression of Olig2. This induction is mediated, at least in part, by endogenous Sonic Hedgehog (Shh) and reflects a ventralization of positional identity. Conversely, the ability of initially Olig2+ cells to generate astrocytes in vitro reflects the extinction of Olig2 expression. These data indicate that competence to clonogenically generate neurons, astrocytes, and oligodendrocytes, an identifying hallmark of CNS-SCs, reflects a deregulation of dorsoventral patterning caused by expansion in FGF-2 (Gabay, 2003).

During development, spinal cord oligodendrocyte precursors (OPCs) originate from the ventral, but not dorsal, neuroepithelium. Sonic hedgehog (SHH) has crucial effects on oligodendrocyte production in the ventral region of the spinal cord; however, less is known regarding SHH signalling and oligodendrocyte generation from neural stem cells (NSCs). NSCs isolated from the dorsal spinal cord can generate oligodendrocytes following FGF2 treatment, a MAP kinase dependent phenomenon that is associated with induction of the obligate oligogenic gene Olig2. Cyclopamine, a potent inhibitor of hedgehog signalling, does not block the formation of oligodendrocytes from FGF2-treated neurosphere cultures. Furthermore, neurospheres generated from SHH null mice also produced oligodendrocytes, even in the presence of cyclopamine. These findings are compatible with the idea of a hedgehog independent pathway for oligodendrocyte generation from neural stem cells (Chandran, 2003).

FGF responsiveness of neural cells

Epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) induce the proliferation of neural precursor cells isolated from specific regions of the embryonic and adult brain. However, the lineage relationship between the EGF- and FGF-2-responsive cells is unknown. Phosphorylation of the transcription factor cAMP response element-binding protein was used as a functional readout to identify cells responding to EGF and FGF-2. In primary cultures of mouse embryonic day 14 (E14) striatum, maintained in vitro for 24 hr, 12% of the cells respond to FGF-2, whereas no response to EGF can be detected. Seventy-five percent of these FGF-2-responsive cells are beta tubulin III (TuJ1)-positive neurons; 25% express nestin, a marker for neuroepithelial precursors. After growth factor treatment for 6 days, a population of nestin-positive cells responding to both EGF and FGF-2 were identified. The 6 day old cultures also contain a small number of TuJ1-positive cells that respond to FGF-2 only. Priming of striatal cells for 24 hr with FGF-2 but not with EGF is sufficient to induce the appearance of EGF- and FGF-2 responsive cells after only 2 days in vitro. Thus, neural precursor cells from the mouse E14 striatum initially responding to FGF-2 only acquire EGF responsiveness later during in vitro development. At this stage, EGF and FGF-2 act on the same cells. The acquisition of EGF responsiveness is promoted by FGF-2 (Ciccolini, 1998).

Cerebellar granule cells are the most abundant type of neuron in the brain, but the molecular mechanisms that control their generation are incompletely understood. Sonic hedgehog (Shh), which is made by Purkinje cells, regulates the division of granule cell precursors (GCPs). Treatment of GCPs with Shh prevents differentiation and induces a potent, long-lasting proliferative response. This response can be inhibited by basic fibroblast growth factor or by activation of protein kinase A. Blocking Shh function in vivo dramatically reduces GCP proliferation. These findings provide insight into the mechanisms of normal growth and tumorigenesis in the cerebellum (Wechsler-Reya, 1999).

FGF and axon guidance

Antibody perturbation experiments on cultured cockroach embryos demonstrate that a localized source of an FGF-2-like immunoreactive molecule in the head is required for the proper growth of pioneer axons in the leg. The study of axon growth in various fragments of cultured embryos and in the presence of various conditioned media showed that FGF-2 is needed to counteract the effects of an inhibitor of axon growth produced in the body trunk of the embryo. Endogenous heparan sulfate proteoglycans mediate these effects of FGF-2 on axon growth. The results of experiments with FGF-2 and/or body trunk axon growth inhibitor added to the culture medium indicate that more globally and uniformly distributed molecules may play as important a role in axon guidance as the more spatially restricted guidance cues. The results are interpreted in terms of a model that is consistent with a role for the FGF-2 receptor in axon growth (Nyhus, 1998).

The growth cones of Xenopus retinal ganglion cells express fibroblast growth factor receptors. bFGF stimulates neurite extension from cultured retinal neurons and is abundant in the developing optic tract, but is reduced in the optic tectum. To test whether FGF signaling plays a role in axonal guidance in vivo, bFGF was exogenously applied to the developing optic pathway in "exposed brain" preparations. FGF-treated retinal axons navigate normally through the optic tract, but the majority veer aberrantly at the tectal border and bypass the target. These results implicate FGF signaling in target recognition and suggest that diminished levels of bFGF in the tectum cause arriving axons to slow their growth (McFarlane, 1995).

Astrocytes are heterogeneous in expression of the ECM molecule tenascin (See Drosophila Tenascin-major). High-tenascin astrocytes have a reduced ability to support neurite outgrowth. In addition, astrocytes treated with exogenous basic fibroblast growth factor (bFGF) support reduced neuronal growth and adhesion. Basic FGF was added to cultures of rat cerebral cortical astrocytes at concentrations of up to 30 ng/ml, concentrations shown to have a significant effect on neuronal adhesion. Tenascin levels begin to increase after 24-48 hr and continued to increase throughout 8 days in culture. The increase in tenascin is concentration-dependent, with the largest increase seen at 5 ng/ml bFGF. Tenascin production increases approximately 5.5-fold in serum-containing medium but only about 2-fold in serum-free medium. When heparin is included along with bFGF in serum-free medium, tenascin production is further enhanced. The bFGF treatment was discontinued after 8 days, and the cells were maintained for an additional 8 days in culture. Tenascin levels returned to control values, demonstrating that the bFGF effect is transient. The action of bFGF during injury may evoke the induction of tenascin on astrocytes, thereby reducing regeneration in the central nervous system (Meiners, 1993).

Fibroblast growth factor 14 (FGF14) belongs to a distinct subclass of FGFs that is expressed in the developing and adult CNS. The Fgf14 gene was disrupted and an Fgf14N-ß-Gal allele was introduced that abolishes Fgf14 expression and generates a fusion protein (FGF14N-ß-gal) containing the first exon of FGF14 and ß-galactosidase. Fgf14-deficient mice are viable, fertile, and anatomically normal, but develop ataxia and a paroxysmal hyperkinetic movement disorder. Neuropharmacological studies showed that Fgf14-deficient mice have reduced responses to dopamine agonists. The paroxysmal hyperkinetic movement disorder phenocopies a form of dystonia, a disease often associated with dysfunction of the putamen. Strikingly, the FGF14N-ß-gal chimeric protein is efficiently transported into neuronal processes in the basal ganglia and cerebellum. Together, these studies identify a novel function for FGF14 in neuronal signaling and implicate FGF14 in axonal trafficking and synaptosomal function (Wang, 2002).

Formation of the trochlear nerve within the anterior hindbrain provides a model system to study a simple axonal projection within the vertebrate central nervous system. Trochlear motor neurons are born within the isthmic organizer and also immediately posterior to it in anterior rhombomere 1. Axons of the most anterior cells follow a dorsal projection, which circumnavigates the isthmus, while those of more posterior trochlear neurons project anterodorsally to enter the isthmus. Once within the isthmus, axons form large fascicles that extend to a dorsal exit point. The possibility that the projection of trochlear axons towards the isthmus and their subsequent growth within that tissue might depend upon chemoattraction was investigate. Both isthmic tissue and Fgf8 protein are attractants for trochlear axons in vitro, while ectopic Fgf8 causes turning of these axons away from their normal routes in vivo. Both inhibition of FGF receptor activation and inhibition of Fgf8 function in vitro affect formation of the trochlear projection within explants in a manner consistent with a guidance function of Fgf8 during trochlear axon navigation (Irving, 2002).

FGFs and synaptic organization

Target-derived cues promote local differentiation of axons into nerve terminals at sites of synaptic contact. Using clustering of synaptic vesicles in cultured neurons as an assay, putative target-derived presynaptic organizing molecules were purified from mouse brain and FGF22 was identified as a major active species. FGF7 and FGF10, the closest relatives of FGF22, share this activity; other FGFs have distinct effects. FGF22 is expressed by cerebellar granule cells during the period when they receive synapses. Its receptor, FGFR2, is expressed by pontine and vestibular neurons when their axons (mossy fibers) are making synapses on granule cells. Neutralization of FGF7, -10, and -22 inhibits presynaptic differentiation of mossy fibers at sites of contact with granule cells in vivo. Inactivation of FGFR2 has similar effects. These results indicate that FGF22 and its relatives are presynaptic organizing molecules in the mammalian brain and suggest new functions for this family of signaling molecules (Umemori, 2004).

FGFs and apoptosis

The role of FGFs in the control of programmed cell death during limb development has been investigated by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered to be the signals responsible for cell death. FGF signaling is also necessary for apoptosis and the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. Cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and cell death mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together these findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although evidence is provided for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. MSX2 and Snail have been identified as candidate genes associated with apoptosis and their expression requires the combined action of FGFs and BMPs (Montero, 2001).

Unregulated FGF receptor signaling results in bone malformations that affect both endochondral and intramembranous ossification, and is the basis for several genetic forms of human dwarfism. FGF signaling inhibits chondrocyte proliferation and the transcription factor STAT1 mediates the growth inhibitory effect of FGF in vitro. Genetic evidence suggests that STAT1 is a modulator of the negative regulation of bone growth by FGF in vivo. Stat1-/- mice were crossed with a transgenic mouse line overexpressing human FGF2 (TgFGF). TgFGF mice exhibit phenotypes characterized by chondrodysplasia and macrocephaly, both of which affect endochondral and intramembranous ossification. The chondrodysplasic phenotype of these mice results both from reduced proliferation and increased apoptosis of growth plate chondrocytes. Loss of STAT1 function in TgFGF mice leads to a significant correction of the chondrodysplasic phenotype, but does not affect the skull malformations. The reduced proliferation of TgFGF growth plate chondrocytes, as well as their excessive apoptosis, is restored to near-normal levels in the absence of STAT1 function. Unregulated FGF signaling in TgFGF mice also induces apoptosis in calvarial osteoblasts that is not, however, corrected by the absence of STAT1. Detailed analysis of Stat1-/- growth plates has uncovered a transient phenotype, characterized by an expansion of the proliferative zone and by acceleration of longitudinal bone growth, which attenuates as the animals grow older. These results document an essential role for STAT1 in FGF-mediated regulation of cell growth that is specific to the epiphyseal growth plate (Sahni, 2001).

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