sall4 is required for pectoral fin outgrowth. (A-C) sall4 expression during zebrafish pectoral fin development. (A) Section through the pectoral fins at 22 hpf. Lateral view of the fin buds at 32 hpf (B) and 48 hpf (C). (D) Schematic of the RT-PCR used to test the efficiency of a sall4 splice-blocking MO. Bands of 255 bp and 345 bp represent spliced and unspliced sall4 mRNA, respectively. Arrows represent primers used in the RT-PCR and the black box represents the sall4 MO. (E) The RT-PCR was performed on wild-type embryos and embryos injected with 5 ng of the sall4 MO. The sall4 MO efficiently blocks sall4 mRNA splicing. There was no inhibition of sall4 splicing in embryos injected with 5 ng of a 5 bp mismatch control sall4 MO (con. MO, control MO; M, DNA molecular weight marker). (F-K) Dorsal views of the pectoral fins of 3 dpf embryos. (F) Wild-type embryo. (G-J) Embryos injected with 5 ng of sall4 MO. The percentages of phenotypes observed at 3 dpf following injection of different concentrations of sall4 MO is listed below each picture. (K) Upturned pectoral fin phenotype produced following injection of 1 ng of tbx5 MO. Arrows highlight the pectoral fin defects in G-K. (L-O) Pectoral fins (5 dpf) stained with Alcian Blue. (L) Wild type. (M-O) The pectoral fins of embryos injected with 4 ng of sall4 MO. Scale bars: 75 µm. cl, cleithrum; sc, scapulocoracoid; pop, postcoracoid process; ed, endoskeletal disc; ac, actinotrichs.

The expression of sall4 in the pectoral fins is regulated by tbx5 but not fgf24. (A) Schematic representation of tbx5, fgf24, sall4 and fgf10 expression during early stages of pectoral fin development. Expression of tbx5 initiates at 17 hpf, fgf24 at 18 hpf, sall4 at 22 hpf and fgf10 at 24 hpf. The genes are all initially expressed throughout the mesenchyme of the pectoral fin primordia. At the time fin bud outgrowth is initiated, fgf24 expression becomes downregulated in the mesenchyme and simultaneously upregulated in the overlying ectoderm. Dorsal (B-D) and dorsolateral (E,F) views of embryos, with red arrows and brackets highlighting the pectoral fin primordia. sall4 is expressed in wild-type pectoral fin primordia at 26 hpf (B), but it is absent in the primordia of embryos injected with 4 ng of tbx5 MO (C). Although sall4 expression is absent in the pectoral fin primordia, it continues to be expressed normally in other regions of tbx5 morphant embryos (black arrowheads in B,C). (D) tbx5 expression in an embryo injected with 4 ng of tbx5 MO at 26 hpf. (E) sall4 is still expressed in the pectoral fin primordia of embryos injected with 9 ng of fgf24 MO, although the domain of expression is more dispersed, consistent with a loss of fgf24 function. This expression pattern is similar to that of tbx5 in embryos injected with 9 ng of fgf24 MO (F).

sall4 is required for FGF signalling in the developing pectoral fins. Comparison of gene expression patterns in wild-type (A,C,E,G,I,K) and sall4 morphant pectoral fins (B,D,F,H,J,L). fgf10 expression in the pectoral fins at 30 hpf (A) is reduced in sall4 morphant embryos (B), as highlighted by the red arrows. fgf10 expression at 40 hpf (C) is reduced in sall4 morphant pectoral fins (D). This reduction is most pronounced in the anterior fin bud. At 40 hpf, fgf24 is detected only in the ectoderm (E) but is absent in sall4 morphant fin buds (F). dlx2 is expressed in the fin bud ectoderm at 40 hpf (G). In sall4 morphant embryos, dlx2 expression is absent in the anterior fin bud ectoderm (H, black arrowhead) but retained in the posterior (red arrowhead). sp9 is also expressed in the fin bud ectoderm at 40 hpf (I) but is downregulated in the anterior ectoderm of sall4 morphant fin buds (J, black arrowhead) and retained in the posterior (red arrowhead). erm is expressed in wild-type pectoral fins (K) but is reduced in sall4 morphant pectoral fins (L).

sall1a is essential for pectoral fin development. (A,B) sall1a expression during wild type pectoral fin development. (A) Dorsal view of a 26 hpf embryo with red arrows indicating the pectoral fin primordia. (B) Lateral view of a 40 hpf pectoral fin showing sall1a expression in the fin bud. (C) Dorsal view of a 3 dpf embryo injected with 5 ng of sall1a MO. The pectoral fins are truncated as highlighted by red arrows (compare with wild type in Fig. 1F). The percentages of phenotypes observed at 3 dpf following injection of 5 ng and 10 ng of sall1a MO are shown in the table. (D,E) Alcian Blue stained pectoral fins of 5 dpf embryos injected with 2 ng of sall1a MO show that pectoral fin development is disrupted in sall1a morphant embryos (compare with wild-type pectoral fin in Fig. 1L). (F) A lateral view of fgf10 expression at 40 hpf in an embryo injected with 5 ng of sall1a MO. The expression of fgf10 is most profoundly affected in the posterior fin bud (compare with Fig. 3C). (G) sall1a is not expressed in the pectoral fin primordia (red arrows) of embryos injected with 4 ng of tbx5 MO. (H,I) Dorsolateral views show that sall1a (red brackets) continues to be expressed in the pectoral fin primordia of embryos injected 9 ng of fgf24 MO (H), but is in a diffuse pattern when compared with the expression of sall1a in wild-type primordia (I). (J,K) Dorsal views of fgf10 expression at 26 hpf in the fin primordia (as indicated with red arrows) of a wild-type embryo (J) and an embryo injected with 5 ng of sall1a MO (K). (L,M) Expression of the ectodermal fin bud markers sp9 (L) and dlx2 (M) are downregulated in the posterior fin bud ectoderm of embryos injected with 5 ng of sall1a MO (black arrowheads) but continue to be expressed in the anterior fin bud (red arrowheads; compare L with wild-type expression in Fig. 5C, and M with Fig. 3G).

Pectoral fin development is severely disrupted in embryos injected with both sall1a and sall4 MO. As shown in the table, coinjection of sall1a and sall4 MOs leads to a significant increase in the most severe phenotype (no fins). Methylene Blue stained sections of the pectoral fin forming region in a wild-type embryo (A) and an embryo injected with 4 ng sall1a + 4 ng sall4 MO (B) at 48 hpf shows that a fin bud initially forms in sall1a/sall4 double morphant embryos. (C-F) Lateral views of 32 hpf pectoral fin buds in wild-type embryos (C,E) and embryos injected with 4 ng of sall1a + 4 ng of sall4 MO (D,F). At this time point, sp9 is expressed in the ectoderm of wild-type fin buds (C) but is absent in sall1a/sall4 double morphant fin buds (D). Likewise, dlx2 is present in wild-type fin bud ectoderm (E) but is absent in sall1a/sall4 double morphant fins (F). At 26 hpf (G), fgf10 expression fails to initiate in the pectoral fin primordia of sall1a/sall4 double morphant embryos (compare G with Fig. 4J).

sall1a and sall4 are required for fgfr2 expression in the pectoral fin primordia. A section of a 23 hpf embryo shows that fgfr2 is expressed in the pectoral fin primordia mesenchyme but is excluded from the ectoderm (A). Dorsal views highlight fgfr2 expression in a wild-type embryo at 24 hpf (B). At the same time point, fgfr2 expression is absent in the pectoral fin primordia of embryos injected with 4 ng sall1a MO + 4 ng sall4 MO (C), but continues to be expressed in the primordia of embryos injected with 9 ng of fgf24 MO (D). At this time point, fgfr2 is also downregulated in the trunk of sall1a/sall4 double morphant embryos (C and data not shown). The red arrows indicate the pectoral fin primordia in B-D. (E) A schematic representation of the regulatory relationships between genes required for pectoral fin development. Green arrows signify relationships between FGF receptors and ligands, while black arrows represent transcriptional regulatory relationships.