Our results highlight the need to control AER size, because syndactyly results if this process fails. As a result of less apoptosis in the AER, there are increased levels of signalling from the hyperplastic ridge and this increase leads to down-regulation of Hoxa13 in the interdigits. This down-regulation in turn leads to lower expression, or lack of expression, of BMPs in cells of the interdigital mesenchyme. Lower levels of BMPs, which normally positively regulate interdigital apoptosis, lead to decreased programmed cell death in this region and subsequent syndactyly.
Application of exogenous sources of fibroblast growth factor (FGF) or BMPs, as well as inhibitors of these signalling pathways to the interdigit of the chick limb, have indicated that FGFs and BMPs control interdigital apoptosis. These observations suggest that both signalling pathways have pro-apoptotic effects (Ganan et al., 1998; Montero et al., 2001). However, it has also been reported that administration of FGFs in the chick limb can antagonize the BMP-induced apoptosis in the limb mesenchyme, resulting in the formation of soft tissue syndactyly (Ganan et al., 1996; Macias et al., 1996; Buckland et al., 1998). These results suggest that, in our mutant hindlimbs, increased FGF signalling from the hyperplastic ridge decreases the level of Hoxa13 expression in the mesenchyme. Consequently, the level of Bmp expression is lowered, leading to a reduction in interdigit-programmed cell death and syndactyly. In the chick, levels of Hoxa13 expression decrease when the AER is removed, which can be restored with an FGF bead. However, when FGF4 is applied to a limb bud, it is unable to activate the expression of Hoxa13 early or expand its domain of expression (Vargesson et al., 2001). Together with our results, these findings suggest there is fine balance of FGF signalling required for the correct expression of Hoxa13. Significantly, we did not observe any difference in the timing of Fgf8 expression in the AER, and expression was down-regulated in the regressing AER at the appropriate time during development (data not shown). This finding indicates that the defects observed result from inappropriate levels of FGF signalling rather than FGF signalling being maintained over an extended period of limb development. Of interest, the phenotypes we observe after disruption of Notch1 signalling in the AER are distinct from those produced when BMP signalling from the ridge is impaired by expressing the BMP antagonist, Noggin, in cells of the AER (Wang et al., 2004) or by conditional deletion of a BMP receptor, BMPR-1A, in the AER (Ahn et al., 2001). Deletion of BMPR-1A early leads to a failure of AER formation and dorsal transformation of ventral limb structures (Ahn et al., 2001). Disruption of BMP signalling from the ridge by the BMP antagonist Noggin leads to severely malformed limbs that have syndactyly similar to the Notch1 mutants but also postaxial polydactyly and dorsal transformation of ventral structures. The domain of Fgf8-expressing cells in the AER in these mutants is expanded in a similar way to that seen in the Notch1 mutant limbs. However, in addition to expansion of the domain of Fgf8, expression of Fgf8 also persists longer in the mutant AER than normal. This finding suggests that the extended period of Fgf8 expression caused by Noggin expression in the AER, rather than perturbation of BMP signalling directly, leads to the postaxial polydactyly. Furthermore, prolonged FGF signalling in the overlying apical ridge produces additional phalanges (Sanz-Ezquerro and Tickle, 2003), which is distinct from the phenotype in the Notch1lox/lox; Brn4-Cre mutant in which the timing of FGF expression is normal. Together, these findings suggest that the defects we observe in our mutant hindlimbs are due to an increase in the level of FGF signalling from the AER rather than an extension of the period of FGF signalling. Our results demonstrate that Notch signalling is required to maintain a delicate balance in the levels of FGF signalling from the AER and that this balance is critical for the correct formation of the interdigital gaps that free the digits of the limbs.
In Drosophila, Notch signalling between the dorsal and ventral compartments of the developing wing specify the position of the wing margin, a line of cells that play a role analogous to that of the AER. Failure of Notch signalling in the wing margin leads to the formation of notches in the wing blade from which the mutant derives its name. We do not see Notch1 playing an analogous role to that of Notch in the Drosophila wing margin because notches do not form in the AER after deletion of the gene. Our results demonstrate that Notch1 signalling is not required to establish the AER but plays an important role in maintaining the correct extent of Fgf8-expressing cells in the AER, and it does so by regulating apoptosis of these cells.