Detection of recombinant mouse Fibin expressed in COS-7 cells. (A) COS-7 cells were transfected with the empty vector (Control) or the mouse Fibin-expression vector (Fibin). The cell lysate (a, c) and culture medium (b, d) of the transfected COS-7 cells were separated by SDS–polyacrylamide gel electrophoresis under reducing conditions and transferred onto a nitrocellulose membrane. The protein with the E tag on the membrane was detected using anti-E tag antibody. (B) COS-7 cells transfected with the empty vector (Control) (a–f) or the mouse Fibin-expression vector (Fibin) (g–l) were examined by immunohistochemistry using anti-mannosidase II antibody for the Golgi apparatus (a, d, g, j) and anti-E tag antibody for recombinant Fibin (b, e, h, k). The signals obtained by immunohistochemistry using anti-mannosidase II antibody and anti-E tag antibody were merged (c, f, i, l). Scale bar = 20 μm.

Expression of fibin in zebrafish embryos. (A) The spatiotemporal expression pattern of fibin in zebrafish embryos (10∼24 hpf) was examined by whole mount in situ hybridization. fibin was expressed in the notochord at 10 hpf (a) and in the notochord and somites at 12 and 14 hpf (b, c). fibin was also expressed in the lateral plate mesoderm, tail buds and the forebrain at 14 hpf (c, d). At 18 and 24 hpf, fibin was predominantly expressed in tail buds and presumptive pectoral fin bud regions. In addition, fibin was also expressed in the alar plate (e, f). (B) The expression of fibin in fibin MO-injected zebrafish embryos. The expression of fibin in wild-type (a), antisense five-base mismatched fibin (Control) MO- (b) and antisense fibin MO- (c) injected embryos at 18 hpf was examined by whole mount in situ hybridization. 1, notochord; 2, somites; 3, lateral plate mesoderm or presumptive pectoral fin bud regions; 4, tail; 5, forebrain; 6, alar plate of the hindbrain.

Morphology of pectoral fin buds of fibin knockdown embryos. (A) Embryos were injected with antisense five-base mismatched fibin MO (Control) and fibin MO. The morphology in wild-type (a, b), control (c, d) and fibin knockdown (e, f) embryos was observed at 4 dpf. Arrowheads and asterisks indicate pectoral fins and no pectoral fins, respectively. (B) The morphology of pectoral fin buds was observed in wild-type (a, d, g), control (b, e, h) and fibin knockdown (c, f, i) embryos at 32∼48 hpf.

Expression of tbx5 and fibin in presumptive pectoral fin bud regions of gene knockdown or DEAB-treated embryos. The expression of tbx5 was examined in wild-type (a) and fibin-knockdown (b) presumptive pectoral fin bud regions at 26 hpf by whole mount in situ hybridization. The expression of fibin was examined in wild-type (c), tbx5-knockdown (d), wnt2b-knockdown (e) and DEAB-treated (f) presumptive pectoral fin bud regions at 18 hpf.

Expression of tbx5 and fibin in pectoral fin buds during zebrafish embryonic development. The expression of tbx5 (a–e) and fibin (f–j) was examined in presumptive pectoral fin bud regions during different developmental stages (13∼30 hpf) by whole mount in situ hybridization. The expression of tbx5 and fibin in presumptive pectoral fin bud regions at 22 hpf was also examined at transverse sections (16 μm) (k, l). The expression of pax2.1 was examined in embryos at 14 hpf by whole mount in situ hybridization (m). The expression of pax2.1 and fibin was examined in embryos at 14 hpf by double whole mount in situ hybridization (n). The expression of tbx5 and fibin was also examined in presumptive pectoral fin bud regions at 24 hpf by double whole mount in situ hybridization (o). Arrowheads and arrows indicate the expression of tbx5 and fibin in presumptive pectoral fin bud regions, respectively. S1, S2 and S3 indicate the locations of somite 1, somite 2 and somite 3, respectively.

Expression of Fibin in mouse embryos. The expression of Fibin was examined in mouse embryos at E9.5 (A) and E10.5 (B) by whole mount in situ hybridization. Arrows indicate the expression of Fibin in the forelimb bud regions.