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First published online September 9, 2004
doi: 10.1242/10.1242/dev.01331

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Sp8 and Sp9, two closely related buttonhead-like transcription factors, regulate Fgf8 expression and limb outgrowth in vertebrate embryos

¹ Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037-1099, USA
² Unidade de Desenvolvimento, Instituto Gulbenkian de Ciencia, Rua Da quinta Grande 6 Aptdo 14. 2780-901 Oeiras, Portugal
³ The Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan

View larger version (70K): [in a new window]   Fig. 1. Phylogenetic analysis and comparison of deduced amino acid sequences of the Sp family. (A) The deduced amino acid sequences of mouse Sp genes together with Drosophila btd and Drosophila Sp1 are analyzed by CLUSTAL-W program. (B) Amino acid sequence alignment of the conserved region of the Sp family, from the btd domain to the Zn-finger domain. Identical residues, when compared with Sp9, are indicated by dots. Gaps in the sequence, indicated by dashes, are introduced to improve sequence alignment. The btd domain and Zn-finger domains are indicated by lines above the amino acid sequences. (C) Alignment of deduced amino acid sequences of Sp8 and Sp9 from human (Homo sapiens, Hs), mouse (Mus musculus, Mm), chick (Gallus gallus, Gg) and zebrafish (Danio rerio, Dr). The btd domain and the Zn-finger domains are indicated by lines.

View larger version (110K): [in a new window]   Fig. 2. Embryonic expression pattern of Sp8 in chick, mouse and zebrafish. (A-M) Chick embryos showing expression of Sp8 from dorsal views (A-E,H-J,M) with the anterior towards the top, a lateral view (G), and transverse sections (F,K,L). (N-R) Mouse embryos showing expression of Sp8 from lateral views (N-Q) and a dorsal view (R). (S-Y) Zebrafish embryos showing sp8 expression from lateral views (S-V,X,Y) and a dorsal view (W) with the anterior towards the left. Pre- and early-somitogenesis stage expression of chick Sp8 was detected at HH stage 5 (A), stage 7 (B), stage 8 (C) and stage 9 (D). (E) Sp8 expression in the surface ectoderm of the limb-forming field (arrowheads) at HH stage 15. (F) A 30 µm section of the embryo in (E) at the forelimb level, showing the expression in the surface ectoderm (arrowheads) and in the neural tube. (G) Head region of the embryo in E showing the expression in the MHB. Sp8 expression in the surface ectoderm in the forelimb field at HH stage 16 (H) and stage 17 (I). (J) Sp8 expression in the AER at HH stage 21. In situ hybridization on 14 µm sections at the forelimb level of a HH stage 21 embryo shows a signal in the AER and the surface ectoderm (K) and signal in the proliferating interneuron (L). Sp8 expression in the forelimb AER at HH stage 28 (M). Mouse Sp8 expression at E8.75 (N) and E9.5 (O). (P,Q) The signal in the forelimb-forming region was detected in the ventral ectoderm at E9.5. The embryonic stage in P is slightly earlier than that of Q. (R) Strong Sp8 expression in the AER at E10.5. Zebrafish sp8 expression at 12 hpf (S), 16 hpf (T) and 20 hpf (U). sp8 expression in pectoral fin buds (arrowhead) at 36 hpf (V) and 48 hpf (W). The expression in the fin bud was observed in the apical fold at 36 hpf (X) and 48 hpf (Y). d, dorsal side; f, forebrain; fl, forelimb; mhb, midbrain/hindbrain boundary; n, Hensen's node; tb, tail bud; v, ventral side.

View larger version (91K): [in a new window]   Fig. 3. Embryonic expression pattern of Sp9 in chick, mouse and zebrafish. (A-I) Chick embryos showing expression of Sp9 from dorsal views (A-C,E,H,I); a lateral view (D) with the anterior towards the top; a frontal view (F) with the dorsal towards the top; and a transverse section (G). (J-L) Mouse embryos showing expression of Sp9 from dorsal views. (M-S) Zebrafish embryos showing sp9 expression from lateral views (M-O,Q,R) and a dorsal view (P) with the anterior towards the left, and a transverse section (S). Chick Sp9 expression at HH stage 8 (A), stage 9 (B), stage 10 (C) and stage 13 (D). (E,F) Sp9 expression in the AER at HH stage 21. (G) Transverse section of the limb in E shows the signal in the AER and surface ectoderm. (H) Sp9 is expressed in a small area in the anterior margin (arrow) in addition to the AER at HH stage 27. Sp9 expression was restricted to the anterior and posterior edges of the autopod at HH stage 31 (I). Mouse Sp9 was detected in the AER and the surface ectoderm at E10.5 (J), E11.5 (K) and E13.0 (L). Zebrafish sp9 expression at 12 hpf (M) and 16 hpf (N). sp9 expression in the pectoral fin (arrowhead) at 36 hpf (O,Q) and 48 hpf (P,R,S). (S) An 8 µm transverse section of the pectoral fin bud of the embryo in P, showing the sp9 expression in the ectoderm. d, dorsal side; mhb, midbrain/hindbrain boundary; v, ventral side.

View larger version (53K): [in a new window]   Fig. 4. Expression of sp8 and sp9 in the fin bud was downregulated in the zebrafish mutants with fin defects. (A-C) Dorsal views of the expression pattern of sp8 at 40 hpf. (D-F) Dorsal views of the expression pattern of sp9 at 40 hpf. (A) Expression of sp8 in the pectoral fin bud of a wild type embryo (black arrowheads). The expression in the pectoral fin bud was downregulated (red arrowheads) in hst (B) and dak (C) mutants, while a normal expression in the neural tube was still detected. (D) Expression of sp9 in the pectoral fin bud (black arrowhead) of a wild-type embryo. The expression in the pectoral fin bud was downregulated (red arrowheads) in hst (E) and dak (F) mutants, while a normal expression in the brain was still detected.

View larger version (88K): [in a new window]   Fig. 5. Fgf10 signaling regulates Sp8 and Sp9. Dorsal views of Sp8 expression (A,B) and Sp9 expression (C,D) in E9.5 mouse embryos. (E-H) Dorsal views of Sp8 expression in chick limb buds. (I-L) Dorsal views (I,J) and ventral views (K,L) of Sp9 expression in chick limb buds. All panels are with the anterior towards the top. (A-D) Sp8 and Sp9 expression detected in wild-type mouse embryos (A,C, black arrowheads), was significantly downregulated in Fgf10–/– embryos (B,D, red arrowheads). Implantation of Fgf10-soaked beads upregulated chick Sp8 expression in the surface ectoderm and in the AER after 6 hours (F; compare with the contralateral control, E). Implantation of SU5402-beads downregulated expression of chick Sp8 in the AER after 24 hours (red arrowhead in H; compare with the contralateral control, G). Implantation of Fgf10-beads upregulated chick Sp9 expression in the surface ectoderm and in the AER after 6 hours (J; compare with the contralateral control, I). Implantation of SU5402-beads downregulated expression of chick Sp9 in the AER after 24 hours (red arrowhead in L; compare with the contralateral control, K). Arrowheads in E,F and I,J indicate the anterior margin of the expression domain. The asterisks in F,H,J,L indicate implanted beads.

View larger version (72K): [in a new window]   Fig. 6. Wnt signaling regulates Sp8 expression. (A) Implantation of a control cell pellet had no effect on Sp8 expression in the chick limb bud. (B) Implantation of a Wnt3a-expressing cell pellet induced Sp8 expression in the ectoderm (arrowheads). Asterisks indicate pellets. (C) Injection of RCAS dominant-active ß-catenin induced Sp8 expression in the wide region of ectoderm. (D) Electron microscopic analysis of RCAS dominant-active ß-catenin-injected limb. ß-Catenin activity induced ectopic ridge-like spikes. The inset in D is a closer view of the ectoderm. (E,F) Sp8 expression in the forelimb bud in wild-type (E) and Lrp6–/– (F) embryos at E10.5. The Sp8 expression domain in E and F are labeled with black and red arrowheads, respectively. (G,H) Sp8 expression in the forelimb bud in wild-type (G) and Dkk1–/– (H) embryos at E12.5. In Dkk1–/– embryos, the Sp8 expression is stronger in the AER (blue arrow) and ectopically induced outside the AER (blue arrowhead).

View larger version (85K): [in a new window]   Fig. 7. Sp8 and Sp9 regulate Fgf8 expression and limb outgrowth. Frontal views (A-C,E,F) and a dorsal view (D) of Fgf8 expression in chick limb buds with the anterior towards the top. (G,H) Dorsal views of chick forelimbs with the anterior towards the top. (I-L) Dorsal views of chick forelimb skeletons. (M-R) Dorsal views of zebrafish embryos with the anterior towards the left. (A-F) Injection of virus was made on the right side; the left side serves as the contralateral control. Injection of RCAS-Sp8 (A) and RCAS-Sp9 (B) resulted in an expansion (arrowheads) of the Fgf8 expression domain in the developing limb. Injection of RCAS-VP16-N-Sp8 (C) and RCAS-VP16-N-Sp9 (D) resulted in the elongation of the AER marked by the elongated expression domain (arrowheads) of Fgf8. Injection of RCAS-EnR-N-Sp8 (E) and RCAS-EnR-N-Sp9 (F) resulted in downregulation of Fgf8 expression (arrowheads) in the limb. (G,H) Injection of RCAS-EnR-N-Sp8 caused an indentation (H, arrowhead), whereas control injection had no effect (G). (I,J) Injection of RCAS-VP16-N-Sp8 caused ectopic digit formation in the anterior domain (J, blue arrowheads) when compared with the control (I). (K,L) Injection of RCAS-EnR-N-Sp8 and RCAS-EnR-N-Sp9 caused hypoplasia of the limb, such as the formation of a small spike with unidentifiable elements (K, red arrowhead) and loss of cartilage elements (L, red arrowheads). (M-O) fgf8 expression at 36 hpf injected with a control morpholino (M), sp8 morpholino (N) and sp9 morpholino (O). sp8 and sp9 morpholinos downregulated fgf8 expression in the fin buds (red arrowheads). The embryos represent the typical `faint signal' phenotype. (P-R) prx1 expression in zebrafish injected with a control morpholino (P), sp8 morpholino (Q) and sp9 morpholino (R) at 36 hpf. sp8 morpholino and sp9 morpholino perturbed fin outgrowth, as visualized by prx1 expression in the fin mesenchyme (red arrowheads).