Response of a Foxg1-dependent cell-survival pathway to different levels of Fgf8 expression. (A) Schematic representation of procedure for isolating E10.5 telencephalic explants. (Left) Surface ectoderm (orange) and craniofacial mesenchyme (yellow), which were dissected away from the neural tube (white). (Center and Right) Forebrain region that was subsequently isolated (purple) and then flattened on a Nucleopore filter. The caudal and rostroventral limits of the explant were the approximate telencephalic/diencephalic border and the optic stalks, respectively. (B and C) RNA in situ hybridization of E10.5 explants using a probe, Fgf8-ex2,3, that hybridizes to sequences deleted by Cre. (D–F and P) TUNEL assays for apoptosis in horizontal sections of E10.5 forebrains of the genotypes indicated. (Lower Left) Low magnification view of the telencephalon, with a dotted box demarcating the area shown at higher magnification. The plane of section is indicated by the dashed line in A (Left). (H–J, L–N, Q, and R) Analysis by whole-mount RNA in situ hybridization of Foxg1 and Bmp4 expression in telencephalic explants from E10.5 embryos of the genotypes indicated. (G, K, and O) Explants from E10.5 wild-type embryos were electroporated with lacZ (control) or Fgf8 expression vectors. Near-adjacent sections of control and of +Fgf8 explants were assayed for TUNEL (G), Foxg1 RNA (K), and lacZ or Fgf8 RNA (O). The representative plane of the section is illustrated by the dashed line in A (Right). C, caudal; ey, eye; L, lateral; M, medial; os, optic stalk; R, rostral.

Effect of ectopic expression of Spry1 on apoptosis and Foxg1 expression. In parallel with the experiments shown in Fig. 2 G, K, and O, explants from E10.5 wild-type embryos were electroporated with a Spry1 expression vector. Near-adjacent sections of each explant were assayed for Spry1 expression (A), TUNEL (B), or Foxg1 expression (C). Arrows in A indicate regions in which cells expressed the electroporated cDNA. Spry1 expression resulted in an increase in TUNEL-positive cells (B, compare with control explant in Fig. 2G) and a decrease in Foxg1 expression (C, compare with control explant in Fig. 2K).

Crosses used to generate mutant embryos. In these crosses, the Fgf8^flox allele has wild-type activity, Fgf8^neo is a hypomorphic allele, and Fgf8⁻ is a null allele described as Fgf8^Δ2,3 (14). Foxg1^cre and Foxg1^lacZ are both null alleles.

Response of Spry1 and Emx2 to different levels of Fgf8 expression. Whole-mount RNA in situ hybridization assays for Fgf8, Spry1, and Emx2 expression in telencephalic explants from E10.5 embryos of the genotypes indicated. The full-length Fgf8 probe (Fgf8-FL) hybridizes to Fgf8 sequences not deleted by Cre. The level of Fgf8 RNA in hypomorphs is lower than in Tel-KO mutants, presumably because the Fgf8-neo fusion RNA, which constitutes ≈60% of transcripts produced from the Fgf8^neo allele carried by hypomorphs, is unstable (ref. 14; E. Meyers and G.R.M., unpublished results). Black asterisk and open arrowhead (I) indicate regions in which Emx2 expression is low. White asterisks and filled arrowheads (J, K, and O) indicate regions in which Emx2 expression is increased. To increase Fgf8 expression, telencephalic explants from E10.5 wild-type embryos were electroporated with a 1:1 mixture of Fgf8 and Gfp (22) expression vectors. GFP fluorescence was monitored to detect regions in which Fgf8 was ectopically expressed (G and G′). The effects of ectopic Fgf8 expression (H and L) were assayed by using probes for Spry1 and Emx2 (H′ and L′). Note that in some regions these effects extend beyond the cells that express Fgf8, presumably because FGF8 is secreted. In control experiments (Gfp only, data not shown), the expression patterns of Fgf8, Spry1, and Emx2 were similar to those observed in normal embryos (A, D, and I).

A model to explain the effects of varying Fgf8 dosage on cell survival. Our data identify two classes of response to variations in Fgf8 expression in the developing mouse forebrain, schematically illustrated in A. The x and y axes represent FGF8 concentration and pathway(s) activity, respectively. One response is proportionate with the level of Fgf8 expression (Right), and the other, a Foxg1 pathway, is not (Left). We suggest that genes, expressed as part of the proportionate response, such as Spry1, encode inhibitors of the Foxg1 cell-survival pathway and that their expression is not subject to negative feedback. (B) Schematic diagram illustrating the combined responses to variation in FGF8 concentration, in which it is assumed that the proportionate response is activated at a higher level of FGF8 than is the Foxg1-dependent response. In the absence of FGF8 (Tel-KO midline), the Foxg1 pathway is inactive and cell survival is reduced. At very low FGF8 concentrations, there is little or no inhibitor activity, so Foxg1 pathway activity is not antagonized and cell survival is high. We suggest that the FGF8 concentration is within this range in the midline of hypomorphs. As FGF8 concentration rises, the inhibitor(s) produced as part of the proportionate response becomes active and begins to inhibit the Foxg1 pathway. Further increases result in more inhibitor activity and decreased cell survival. We suggest that the FGF8 concentration is within this range in the normal midline and in our gain-of-function experiments.