The zebrafish orthologs of syk and zap-70 mediate angioblast migration to form the ISVs. (A) Zebrafish syk and zap-70 have similar domain structures with N-terminal SH2 domains, and a C-terminal tyrosine kinase domain (PTK). (B) Alignment of the nucleotide sequences of syk and zap-70 with orthologs in other species shows a clear grouping of zebrafish syk with the other syk orthologs, and zap-70 with the other zap-70 orthologs. (C) At 30 hpf, ISVs in Tg(fli1a:EGFP)^y1 embryos have reached the dorsal aspect of the somite and are contributing to the DLAV in wild-type embryos. (D–E) Injection of a low dose of syk morpholino (syk^LO, D) or zap-70 morpholino (zap-70^LO, E) results in delayed (arrows) or completely suppressed (asterisk) angioblast migration to form the ISVs. (F) Co-injection of low doses of both morpholinos results in a more severe stalling and suppression of angioblast migration (syk^LO:zap-70^LO). (G–H) Tg(fli1a:EGFP)^y7 embryos with endothelial-expressed nuclear GFP show fewer cells in the ISVs of syk^LO:zap-70^LO embryos. Anterior is to the left in all embryos. DA: dorsal aorta; PCV: posterior cardinal vein; ISV: intersegmental vessel; DLAV: dorsal longitudinal anastomotic vessel; hpf: hours post fertilization.

syk^LO and zap-70^LO morphants display late stage ISV patterning defects. (A) The trunk of 5 dpf wild-type Tg(fli1a:EGFP)^y1 embryos display regularly patterned ISVs between somites, with the dorsal-most ISV endothelial cell forming a clear T shape and contributing to the DLAV. In contrast, 5 dpf syk^LO (B), zap-70^LO (C), and syk^LO:zap-70^LO (D) morphants display abnormal ISV vasculature with mild patterning defects. The ventral stalk ISV endothelial cell is relatively normal, and the dorsal T-cell still contributes to the DLAV, but the branching of the cell into the T shape is abnormal (asterisks) and can occur as ventrally as midsomite (arrows). These dorsal patterning defects are more pronounced in the syk^LO:zap-70^LO morphants.

syk^HI:zap-70^HI morphants resemble vegfa morphants and lack activated ERK. (A) syk^HI:zap-70^HI Tg(fli1a:EGFP)^y1morphants have a complete suppression of ISV sprouting which is phenotypically similar to the ISV stalling observed in high dose vegfa morphants (B). While the artery of 20s wildtype (WT) embryos (arrow, C′) shows activated ERK (arrow, C″) comparable staged syk^HI:zap-70^HI morphants (D) show a strong downregulation of activated ERK (arrow D′ = artery, D″). The level of downregulation of activated ERK in the artery (arrow, E′) is similar to vegfa^HI morphants (E, E″). C′, D′, E′ are enlargements of GFP signal in the boxed region of C, D, E, respectively. C″, D″, E″ are enlargements of the αERK-P immunohistochemistry in the boxed regions of C, D, and E respectively.

syk rescues angioblast migration defects after syk knockdown. (A–D) syk and zap-70 are expressed in a ubiquitous manner and have overlapping expression profiles. At 16 somites (16s) syk (A) and zap-70 (C) are highly expressed in the head, with low levels of expression throughout the rest of the embryo. (B, D) By 21s increased expression is seen in the ventral trunk. (E–F) By 30 hpf, the ISVs of wildtype Tg(fli1:egfp)^y1 embryos have reached the dorsal portion of the embryo to form the DLAV (E) while the ISVs of syk morphants are delayed at the mid-trunk (F). (G–H) Expression of a C-terminal myc-tagged syk in the vasculature (G, fli1:syk:myc) or blood (H, gata1:syk:myc) partially rescues the number of ISVs that have reached the DLAV (black arrow).

Endothelial and vascular expression of syk rescues angioblasts migration in vegfa morphants. (A–B) Compared to 30 hpf wildtype Tg(fli1:egfp)^y1 embryos with complete ISVs (A), vegfa morphants lack any ISV sprouting (B). (C–D) Expression of a C-terminal tagged syk under the control of a vascular fli1 promoter (fli1:syk:myc, C) or a blood gata1 promoter (gata1:syk:myc, D) in single celled embryos injected with 4 ng of vegfa morpholino results in increased number of ISV sprouting (white arrow). Expression of the fli1:syk:myc construct is detected by immunohistochemistry against the myc tag (red, C, D). The large myc-expressing cells visible in the dorsal and mid-trunk of gata:syk injected embryos are superficial and do not represent expression of the transgene in blood vessels, while ventral myc expression cells are within the axial vessels.

Quantification of morpholino efficiency
8.5ng of Syk^e2i2 morpholino, 11.3ng of Syk^e7i7, 4ng of Zap70^e3i3 and 5.6ng of Zap70^i1e2 were individually injected into early stage Tg(kdra:EGFP)^la116 embryos. All morpholinos target splice sites within the transcripts. The efficiency of morpholino inhibition of proper splicing was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) with gene specific primers surrounding the morpholino target site (sykFe1/sykRe5 for Syk^e2i2; sykFe6/sykRe8 for Syk^e7i7; and Zap70Fe2a/zap70Re5a for both Zap70^e3i3 and Zap70^i1e2). Effective knockdown of transcript in syk and zap-70 morphants (MO) was observed as compared to uninjected controls in all experimental groups, with negligible recovery at 48 hpf. All primer sequences are listed in Supplementary Table 1. Equal loading of products was confirmed by RT-PCR with primers specific to elongation factor 1α (eF1-αF/eF1-αR).

Phenotype of syk and zap-70 morphants at 24 hpf.
The phenotype of the morphants shows no major developmental delay.

syk^LO:zap-70^LO double morphants have fewer endothelial cells in their axial vessels at 30 hpf. The number of endothelial cells in axial vessels was enumerated in the region below 10 ISVs of the trunk of Tg(fli1a:nEGFP^y7) uninjected (n= 50 ISVs) or syk^LO:zap-70^LO double morphants (n= 70 ISVs) at 30 hpf. Error bars represent standard deviation.

Acridine orange shows low off-target cell death in syk^LO: zap-70^LO double morphants. (A) Acridine orange staining of 30 hpf wildtype embryos shows few acridine orange positive cells. (B) Staining of 30 hpf syk^LO: zap-70^LO double morphants shows a small increase in acridine orange positive cells (arrows), though not in regions associated with endothelial cells.

Syk and Zap morphants lack a thoracic duct. Angiography in wild type Tg(fli1a:EGFP)^y1 embryos shows a clear separation between the dorsal aorta and posterior cardinal vein, and the presence of the thoracic duct lacking dye between the two vessels (A, A′). Conversely, angiography in syk^LO (B, B′), zap-70^LO (C, C′), and syk^LO:zap-70^LO double morphants (D, D′) demonstrates the clear lack of a thoracic duct between the artery and vein. In syk^LO:zap-70^LO double morphants, some ISVs also appear to have restricted or lack blood flow.