The ikarus (ika) mutant phenotype. (A) Wild-type larva at 3 days of development. Note the prominent pectoral fins protruding from the flanks. (B) ika mutant larva at 3 days of development. Note the complete absence of pectoral fin structures (asterisks). (C) Alcian Blue staining of the endoskeleton of a wild-type larval pectoral fin. cl, cleithrum; sco, scapulocoracoid; pc, postcoracoid process; ed, endodermal disc. (D) Alcian Blue staining of the endoskeleton of an ika mutant larval pectoral fin. Note the complete absence of the fin endoskeleton, including the scapulocoracoid, or shoulder girdle. (E) Transverse section through a wild-type embryo at the level of the pectoral fin buds at 36 hpf. (F) Transverse section through an ika mutant embryo at the level of the pectoral fin buds at 36 hpf. Note the absence of pectoral fin buds (asterisks). Scale bars: 100 µm.

ika encodes fgf24. (A) Mapping data for ika with the SSLP marker z5435. The upper gel shows the PCR products amplified from 24 siblings, while the lower gel shows the products amplified from 24 ika mutant embryos. The mutant embryos only show the (upper) Tuebingen band, except for a single embryo, which also shows the (lower) WIK band (arrow). This is the only recombination event we observed with this marker out of 938 embryos tested. (B) Schematic representation of the region of linkage group 14 to which ika and fgf24 map, depicting the genetic map on the left, and the radiation hybrid (RH) map on the right. (C) Schematic representation of the alterations to the Fgf24 protein found by sequencing fgf24 in the two ika alleles, ika^hx118 and ika^tm127c. (D) The phenotype generated by injecting a morpholino targeted against the translation start site of fgf24. These embryos lack pectoral fins, but otherwise appear normal, thus phenocopying ika (compare with Fig. 1A,B). Scale bar: 100µm.

The expression pattern of fgf24 in the pectoral fin primordia. (A) Transverse section through a wild-type embryo at 18 hpf at the level of the pectoral fin primordia. Note the prominent expression in the mesenchyme. (B) Transverse section through the pectoral fin primordium of a wild-type embryo at 24 hpf. Note that fgf24 is still strongly expressed in the mesenchyme, but not the ectoderm. (C) A wild-type pectoral fin bud at 32 hpf. fgf24 has been downregulated in the mesenchyme, and is now expressed in the AER. Scale bars: 50 µm.

Marker gene expression in the fin mesenchyme and ectoderm of 24 hpf wild-type and fgf24^-/- embryos. (A) Wild-type tbx5 expression in the pectoral fin primordia (arrows). (B) tbx5 expression in the absence of fgf24 activity. (C) Wild-type msxc expression in the pectoral fin primordia. (D) msxc expression in the absence of fgf24 activity. (E) Wild-type dlx2 expression in the pectoral fin primordia. (F) dlx2 expression in the absence of fgf24 activity. (G) Wild-type shh expression in the pectoral fin primordia. (H) shh expression in the absence of fgf24 activity. Scale bar: 50 µm. All photos are at the same magnification.

Marker gene expression in the fin mesenchyme and ectoderm of 30 hpf wild-type and fgf24^-/- embryos. (A) Wild-type pea3 expression in the pectoral fin bud. Note expression both in the mesenchyme and the ectoderm. (B) pea3 expression in the absence of fgf24 activity. Note there is expression in the mesenchyme, but not the ectoderm. (C) Wild-type expression of erm1 in the pectoral fin bud. (D) erm1 expression in the absence of fgf24 activity. (E) Wild-type fgf4 expression in the pectoral fin bud. (F) fgf4 expression in the absence of fgf24 activity. (G) Wild-type dlx2 expression in the pectoral fin bud. (H) dlx2 expression in the absence of fgf24 activity. (I) Wild-type fgf8 expression in the pectoral fin bud. (J) fgf8 expression in the absence of fgf24 activity. Scale bar: 50 µm. All photos are at the same magnification.

fgf24 acts downstream of wnt2b and tbx5, and upstream of fgf10. (A) Wild-type fgf24 expression at 24 hpf. (B) fgf24 expression in a tbx5-morpholino knockdown embryo (tbx5mo) at 24 hpf. (C) fgf24 expression in a wnt2b-morpholino knockdown embryo (wnt2bmo) at 24 hpf. (D) Wild-type fgf10 expression at 24 hpf. (E) fgf10 expression in the absence of fgf24 activity at 24 hpf. (F) Schematic representation of the hierarchy of genes involved in triggering limb development. Scale bar: 50 µm. All photos are at the same magnification.

Failure of tbx5-expressing cells to move towards the pectoral fin buds in the absence of fgf24 activity. Shown is a time course of tbx5 expression in the pectoral fin region (arrowheads) from 18 hpf to 32 hpf in wild-type and fgf24 mutant embryos. Pictures were taken as oblique side views of embryos, with anterior towards the left. (A) Wild-type tbx5 expression at 18 hpf. (B) tbx5 expression at 18 hpf in the absence of fgf24 activity. (C) Wild-type tbx5 expression at 27 hpf. (D) tbx5 expression at 27 hpf in the absence of fgf24 activity. (E) Wild-type tbx5 expression at 32 hpf. (F) tbx5 expression at 32 hpf in the absence of fgf24 activity. Scale bar: 100 µm. All photos are at the same magnification.

Expression of marker genes in the fin anterior/posterior axis of 30 hpf wild-type and fgf24^-/- embryos. Anterior is towards the left. (A) Wild-type shh expression in the pectoral fin bud. (B) shh expression in the absence of fgf24 activity. (C) Wild-type msxc expression in the pectoral fin bud. (D) msxc expression in the absence of fgf24 activity. Note that msxc is expressed in the mesenchyme, but not the ectoderm. Note also that msxc fails to be downregulated in the posterior mesenchyme. (E) Wild-type bmp2 expression in the pectoral fin bud. (F) bmp2 expression in the absence of fgf24 activity. Note that bmp2 is expressed in the posterior mesenchyme, but not in the ectoderm. (G) Wild-type hand2 expression in the pectoral fin bud. (H) hand2 expression in the absence of fgf24 expression. Scale bar: 50 µm. All photos are at the same magnification.