Possible roles of FGF during development

On the basis of FGF expression patterns it has been suggested that FGFs play a role in a wide range of developmental processes. FGFs have been shown to be potent mesoderm inducers in Xenopus

(Kimelman and Kirschner, I987; Slack et al., 19Ô7; Paterno et al., 1989; Green et al., 1990). Evidence that FGF plays a role during development was shown in Xenopus by overexpressing an FGF-receptor in which the tyrosine kinase domain was deleted and this resulted in embryos that developed defective trunk and tails (Amaya et a l . , 1991» 1993) • It appears that the concentrations of mesoderm inducers (e.g. FGFs and activin) is an important factor in determining the fate of the responding cells (Ruiz i Altba and Melton, 1989; Green and Smith, 1990

,

).

Recent studies in mouse and chick also suggest that FGFs play an important role in limb development. In an in vitro assay it has been shown that after removal of the apical ectodermal ridge of young mouse limb buds, the addition of different members of the FGF-family to the medium can replace the function of the ridge to stimulate mesenchymal proliferation and limb outgrowth (Niswander and Martin, 1993a ) . Limb outgrowth does not occur after removal of the ridge and application of BMP-2, BMP-4, TGF-&, PDGF, EGF, insulin or RA to the culture medium (Niswander and Martin, 1993a)•

In the developing limb bud expression of Evx-1, the vertebrate homolog of the Drosophila segment polarity gene evenskipped, appears to be regulated by FGF-4 (Niswander and Martin, 1993b). Duplicated anterior skeletal limb elements can be formed when chick limb cells are infected with a replication-defective retrovirus expressing FGF-2

and grafted to the anterior margin of a chick wing bud (Riley et al. , 1993)• FGF-3 appears to be required for normal development of the ear and tail (Mansour et al. ^ 1993) • Disruption of the Fgf-5 gene leads to sustained growth of their fur and thus it appears that FGF-5 functions as an inhibitor of hair elongation (Herbert et a l . , 1994).

In vitro studies have shown that FGF-2 can stimulate limb mesenchyme proliferation (e.g. Aono and Ide, I988; Niswander and Martin, 1993a) and to inhibit cartilage differentiation of limb mesenchyme (Anderson et al., 1993)*

1.5*2. Retinoids

It is known that retinoids can affect vertebrate development in that they cause developmental abnormalities. Exposure to high doses of retinoic acid (RA) during pregnancy causes defects depending on the stage of exposure. For example, exposure to RA at the time of limb bud outgrowth causes limb abnormalities in humans.

Local application of RA to the anterior margin of a chick wing bud leads to the development of mirror-image duplicated digits

(Tickle et al., 1982; Summerbell, 1983; Tickle et al., I985), thus mimicking the effect of grafting a polarizing region. In addition, RA can substitute for the polarizing region to allow development of a complete limb (Eichele, 1989; Tamura et al., 1990)* An increase in RA concentration is followed by the development of more posterior digits (Tickle et al., I985). As for the polarizing region, only mesenchymal cells in the subapical progress zone are competent to respond to RA

(Tickle and Crawley, I988). It has been suggested that RA could be the morphogen released by the polarizing region to provide positional information (Tickle et al. , I985) . However, grafts of mesenchyme

located distally to the RA bead at the anterior margin induce

additional digits when grafted to the anterior margin of a chick wing bud (Summerbell and Harvey, 1983; Noji et at., 1991; Wanek et al., 1991 ; Tamura et al., 1993). suggesting that RA might induce a

polarizing region. Moreover, application of RA to the anterior margin induces expression of RAR-^ in anterior limb mesenchyme, whereas a polarizing region graft does not (Noji et al., 1991)* Thus, it appears that RA rather functions to induce a polarizing region than to be the morphogen released by the polarizing region (see Brookes, I99I; Noji et al., 1991; Wanek et al., 1991)* However, there seems to be some controversy about the role of RA during limb development

(reviewed in Tabin, 1991; Tickle, 1991; Tickle and Eichele, 1994). The fact that both RA and RA-receptors (RARs) are present in the developing limb suggests that retinoic acid has a function during limb development. The concentration of RA is higher in posterior part compared to the anterior part of the limb bud (Thaller and Eichele, 1987; Satre and Kochar, I989). The second active retinoid, 3.4-

didehydro RA found in chick limb tissue, is expressed at several fold higher than RA itself and appears to have equivalent polarizing

activity (Thaller and Eichele, 1990), but its distribution in the developing limb has not yet been determined.

Retinoids are small hydrophobic molecules that can diffuse through the cell membrane and then, by binding to cellular receptors, can exert their effects in the nucleus. Cellular cytoplasmatic

retinoic acid binding proteins (CRABP I and CRABP II) have been implicated to play a role in the retinoid signal transduction

pathway, by binding RA with high affinity (reviewed in Mendelsohn et al. , 1992). It is possible that CRABPs are regulators of free RA

concentration, or that they are required for the transport of RA to the nucleus (reviewed in Mendelsohn et al,, 1992). Localised to the nucleus are retinoid receptors which by binding retinoids can act as transcription factors to control expression of several genes (see Evans, I988) . Genes that are known to contain a retinoic acid

response element (RARE) are for example Hoxd-4 (Moroni et a l ., 1993) and CRABP II (Durand et al., 1992).