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The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10

Jennifer K. Ng^1,*, Yasuhiko Kawakami^1,*, Dirk Büscher^1,*, Ángel Raya^1,*, Tohru Itoh¹, Christopher M. Koth¹, Concepción Rodríguez Esteban¹, Joaquín Rodríguez-León², Deborah M. Garrity³, Mark C. Fishman³ and Juan Carlos Izpisúa Belmonte^1,

¹ The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037-1099. USA
² Instituto Gulbenkian de Ciencia, Rua Da Quinta Grande n 6, 2780-901 Oeiras, Portugal
³ Cardiovascular Research Center, Massachusetts General Hospital, 149 13^th Street, Charlestown, MA 02129, USA

View larger version (43K): [in a new window]   Fig. 1. Zebrafish tbx5 is required for pectoral fin initiation and outgrowth. All panels show the dorsal view of zebrafish embryos with anterior to the top. (A-E) Five dpf zebrafish embryos injected with control (A,C) or tbx5 (B,D,E) morpholinos. Embryos with 10 ng of tbx5 morpholino lack pectoral fins (B, asterisk) and Alcian Blue cartilage staining (E, asterisk), unlike control-injected embryos (A,C, arrows). Injection of a lower dose (2 ng, as in D) results in a milder fin phenotype such as a reduced pectoral fin blade (arrow in D).

View larger version (43K): [in a new window]   Fig. 2. Amino acid sequence alignment of FGF10 proteins and comparison of fgf10 and tbx5 expression in the zebrafish embryo. (A) Alignment of deduced FGF10 protein sequences from zebrafish (Danio rerio, Dr), chick (Gallus gallus, Gg), mouse (Mus musculus, Mm), and human (Homo sapiens, Hs). Identical amino acids are indicated by dots, and gaps by dashes. The zebrafish sequence shows homology to that of chick (44.7%), mouse (42.7%) and human (41.7%). The GenBank accession number for zebrafish fgf10 is AF544025. (B-G) All panels show the dorsal view of zebrafish embryos with anterior to the top, excluding F and G. Whole-mount in situ hybridization staining of wild-type zebrafish embryos during pectoral fin development. fgf10 (B,D,F) and tbx5 (C,E,G) appear to be expressed in a similar spatial and temporal pattern during pectoral fin initiation and outgrowth. (B,C) fgf10 and tbx5 are expressed in the LPM of the presumptive pectoral fin bud (arrowheads) at 24 hpf. (D,E) At 30 hpf fgf10 is expressed in the branchial arches, otic vesicle, and pectoral fin bud (arrowheads), while tbx5 is expressed in the dorsal eye, heart tube and pectoral fin bud (arrowheads). (F,G) Lateral view of pectoral fin buds of 36 hpf embryos showing fgf10 and tbx5 expression throughout the mesenchyme. Anterior is to the left.

View larger version (38K): [in a new window]   Fig. 3. Zebrafish tbx5 is necessary for fgf10 and fgf8 expression in the pectoral fin budding region. All panels show the dorsal view of zebrafish embryos with anterior to the top. (A-D) Comparison of fgf10 and fgf8 expression patterns in embryos injected with 10 ng of control (A,C) or tbx5 (B and D) morpholino. (A and B) fgf10 expression in the pectoral fin bud region of 26 hpf (A, arrowheads) was not detected in the tbx5-morpholino injected embryo (B, asterisks). Note that expression remains unchanged in other regions of the embryo. (C and D) In 36 hpf tbx5-morpholino-injected embryos, fgf8 expression could not be detected in the region where the pectoral fin buds develop (D, asterisks), as compared to the control embryos (C, arrowheads point to the expression in the apical fold). fgf8 expression in the midbrain-hindbrain boundary remains unaltered in the injected embryos, as noted by the arrows. (E) fgf10 was not detected in the pectoral fin bud region of 24 hpf hst mutants (asterisks), but remained unaltered in other regions.

View larger version (66K): [in a new window]   Fig. 4. Amino acid sequence alignment of WNT2b proteins and expression of zebrafish wnt2b in the zebrafish embryo. (A) Alignment of zebrafish (Danio rerio, Dr), chick (Gallus gallus, Gg), mouse (Mus musculus, Mm), and human (Homo sapiens, Hs) deduced WNT2b amino acid sequences, in addition to that of human WNT2. Identical amino acids are indicated by dots, and gaps by dashes. Asterisks indicate conserved cysteine residues among the WNT family members. The zebrafish Wnt2b sequence shows homology to that of chick (71.4%), mouse (61.2%), and human WNT2b (61.0%) and human WNT2 (63.0%). The presence of a 17 amino acid stretch just after the initiation methionine residue, which is characteristic of WNT2b but not WNT2, indicates that our clone encodes zebrafish wnt2b. The GenBank accession number for zebrafish wnt2b is AF544026. (B and C) Whole-mount in situ hybridization of 22 hpf zebrafish embryos showing wnt2b expression from a dorsal view (B) and a lateral view (C). Expression can be detected in the tissue medial to the LPM at the somite level where the pectoral fin buds will form (arrowheads) and the developing eye. Anterior to the left. (D) Cross section of 22 hpf zebrafish embryo at the pectoral fin bud forming level. wnt2b is expressed ventral to the somites and medial to the pectoral fin-budding region of the LPM. nt, neural tube; s, somite; nc, notochord.

View larger version (63K): [in a new window]   Fig. 5. Zebrafish wnt2b is required for tbx5 and tgf10 expression and pectoral fin initiation and outgrowth. (A-F) Zebrafish embryos injected at the one-cell stage with a control morpholino (A,D), wnt2b morpholino (B,E) or wnt2b morpholino + tbx5 RNA (C,F), were allowed to develop for 5 days. Normal pectoral fin development (A, arrows) is abrogated after wnt2b morpholino injection (B, asterisks), and rescued by co-injection of tbx5 RNA (C, arrows). Other defects caused by tbx5 RNA injections were observed (arrowhead in C). (D-F) Cartilage staining confirms normal pectoral fin development in control injections (D, arrows) and after rescue with tbx5 RNA (F, arrows), and lack of pectoral fins after wnt2b morpholino alone (E, asterisks). Dorsal view of zebrafish embryos, anterior to the top. (G-K) Effect of wnt2b morpholino injections on tbx5 expression in whole embryos at 28 hpf (G,H) and in pectoral fin buds at 36 hpf (I-K). tbx5 is expressed in control-injected embryos in the presumptive pectoral fin bud (arrowhead) and in the eye (G). After injection of wnt2b morpholino, tbx5 expression was no longer detected in the presumptive fin field (arrowhead), but remained in the eye at 28 hpf (H). In 36 hpf embryos, expression was seen in the developing fin bud of control injected embryos (I), but was reduced after wnt2b morpholino injections of low dosage (2ng, J) or absent after injections of higher dosage (10 ng, K). Lateral view, anterior to the left. (L,M) Comparison of fgf10 expression patterns in embryos injected with 10 ng of control (L) or tbx5 (M) morpholino. After injection of wnt2b morpholino, expression of fgf10 in the presumptive fin bud forming area at 24 hpf can no longer be detected (M, asterisks), compared to the control-injected embryo (L, arrowheads). Dorsal view, anterior to the top. (N) wnt2b expression in embryos injected with 10 ng of tbx5 morpholino. In injected embryos, wnt2b expression remains unchanged in the LPM at 22 hpf (arrowheads). Dorsal view, anterior to the top. See Fig. 4B for comparison.

View larger version (71K): [in a new window]   Fig. 6. Tbx5 expression depends on WNT/ß-catenin signaling and is sufficient for limb induction in chick embryos. All panels show anterior to the top. (A,B) Axin- and EGFP-expressing adenoviruses were co-injected into the LPM of stage 8 chick embryos. At stage 15, Tbx5 expression was downregulated in the injected side (A, arrow). EGFP expression marks the spatial distribution of the adenovirus and the integrity of the injected tissue (B, arrow). A and B are images of the same embryo. (C-F) tbx5 regulates Fgf10 expression and mediates limb outgrowth in chick embryos. RCAS expressing an N-terminal truncated mutant of Tbx5 (Tbx5N; C,E,F) or control RCAS (D) was injected into the presumptive wing field of stage 8 embryos. In stage 16 embryos injected with RCAS-Tbx5N, Fgf10 expression is downregulated on the injected side (C, arrow). Cartilage staining of stage 36 injected embryos revealed that truncations occurred in the zeugopodal and autopodal structures of the wing, and consisted of hypoplasia of the radius and ulna as well as the complete absence of anterior digits (E,F, arrows). Control RCAS infection caused no obvious wing phenotypes (D). (G,H) Tbx5 is sufficient for limb induction in chick embryos. RCAS expressing full length Tbx5 was injected into stage 7 chick embryos. Five days after injection, cartilagenous elements of the embryos were visualized by Alcian Blue staining. Ectopic limb-like structures were induced (arrows), and cartilage staining revealed additional autopod- and zeugopod-like elements.

View larger version (46K): [in a new window]   Fig. 7. Fgf10 maintains Tbx5 expression during limb initiation. (A) Beads soaked in SU5402 were applied to the LPM of stage 15 chick embryos. At stage 17, Tbx5 expression was strongly reduced on the manipulated side (arrow). An asterisk indicates the position of the implanted bead. (B-D) fgf10 maintains tbx5 expression in the presumptive pectoral fin bud field of zebrafish. In the hst mutants, tbx5 expression is downregulated at 26 hpf. In addition, the few remaining tbx5-expressing cells fail to condense into circular areas of the forming fin buds. (C, compared to the wild-type embryo in panel B). The expression is no longer detected at 36 hpf (not shown). (D) fgf10 RNA was injected into hst mutant embryos at the one-cell stage, resulting in the maintenance of tbx5 expression in the pectoral fin bud region at 36 hpf, although the fin buds were not formed. Magnified view of the pectoral fin region, anterior to the left.