Sema6D Is highly conserved during evolution. (A) Alignment of the amino acid sequences of the sema domain of sema6D/sema6Dl in Danio rerio, Bostaurus, Gallus gallus, Homo sapiens, Musmusculus, Oryctolaguscuniculus, Oryziaslatipes, Rattusnorvegicus, Sus scrofa, and Xenopus tropicalis. Consensus values indicate the similarity of amino acid sequence, and the greater the similarity. These protein sequences were aligned using MEGA7 software and edited by TBtools. (B) The amino acid sequence similarity (%) of sema domain in above species. (C) Phylogenetic tree of the sema6D protein.

Figure 2. Expression of sema6D gene in embryonic zebrafish at different stages. (A) QRT-PCR analysis of sema6D expression in embryonic zebrafish at different stages (n = 3). One-way ANOVA, values with ** and *** above the bars are significantly different (P < 0.01 and P < 0.001, respectively); value with “ns” above the bars indicated no significantly different. (B–D) Whole mount in situ hybridization analysis of sema6D in zebrafish embryos at different stages, blue arrowheads indicate blood vessels. (E) The procedure of the endothelial cells sorting and RT-PCR. (F) The agarose gel electrophoresis results of RT-PCR on fli1a-EGFP sorted cells.

Primary motor neuron morphogenesis defects in the sema6D knockout zebrafish. (A) Confocal imaging analysis of PMNs in control and sema6D knockout groups at 48 and 72 hpf. (B) The statistical analysis of the length of each cap axon in the control and sema6D morphants at 48 and 72 hpf (n = 7). The length of five cap axon was measured in each zebrafish and the average was used. Mann-Whitney test, P = 0.0012. Values with ** above the bars are significantly different (P < 0.01). (C) The statistical analysis of the ratio of Caps across different segments in the control and sema6D morphants at 48 and 72 hpf (n = 8). Mann-Whitney test, P = 0.0007. Values with *** above the bars are significantly different (P < 0.001). (D) The statistical analysis of the number of branches in one cap axon in the control and sema6D morphants at 48 and 72 hpf. The number of branches in each cap axon was measured in five axon of each zebrafish and the average was used. Mann-Whitney test, 48 hpf: P = 0.0017; 72 hpf: P = 0.0012. Values with ** above the bars are significantly different (P < 0.01).

Figure 4. Deficiency of sema6D caused aberrant vascular networks. (A) Confocal imaging analysis of ISVs in the control and sema6D-MO embryos at 48 and 72 hpf. (B) The statistical analysis of the ratio of vessels that span different segments in the control and sema6D morphants at 48 and 72 hpf (n = 7). About 9–12 ISVs were used for statistics per zebrafish. Mann-Whitney test, P = 0.0012.Values with ** above the bars are significantly different (P < 0.01).(C) The statistical analysis of the ratio of shorter vessels in the control and sema6D morphants at 48 and 72 hpf (n = 7). About 9–12 ISVs were used for statistics per zebrafish. Mann-Whitney test, 48 hpf: P = 0.0006; 72 hpf: P = 0.0012. Values with ** and *** above the bars are significantly different (P < 0.01 and P < 0.001, respectively).

Knockout of sema6D caused both aberrant phenotypes in PMNs and ISVs. (A) Schematic diagram showing the targeting site of the gRNA on the sema6D gene. Starting codon (ATG) site is indicated by arrow. (B) The statistics of the ratio of wildtype to mutant sequences (n = 50). (C) Knockout patterns of sema6D-gRNA/cas9-injecting embryos by sequencing. (D) Confocal imaging analysis of primary motor neurons and intersegmental vessels in control and the F0 generation of the injected Tg(mnx1:EGFP::kdrl:ras-mCherry) zebrafish at 48 hpf, blue arrowheads indicate aberrant PMN and ISV. (E) The statistical analysis of the ratio of aberrant axonal projection of Caps (P = 0.0006) and short Caps (P = 0.0006) in the wild, F0 knockouts, and cas9 negative control at 48 hpf (n = 7). About 9–11caps were used for statistics per zebrafish. Mann-Whitney test. Values with *** above the bars are significantly different (P < 0.001). (F) The statistical analysis of the ratio of vessels that span different segments (P = 0.0006) and the ratio of proliferated vessels (P = 0.006) in the wild, F0 knockouts, and cas9 negative control at 48 hpf (n = 7). About 9–12 ISVs were used for statistics per zebrafish. Mann-Whitney test. Values with *** above the bars are significantly different(P < 0.001).

Overexpressing sema6D partially restored the defects of PMNs and ISVs in sema6D-deficient embryos. (A) Confocal imaging analysis of PMNs and ISVs in control, sema6D-MO, and sema6D-mRNA injected embryos at 48 hpf. Blue arrowheads indicate aberrant PMNs and ISVs. (B) The statistical analysis of the ratio of aberrant axonal projection of Caps (P = 0.0006) and short Caps (P = 0.0006) in sema6D-MO, m-MO+sema6D-mRNA, and sema6D-mRNA injected embryos at 48 hpf (n = 7). About 9–11 caps were used for statistics per zebrafish. Mann-Whitney test. Values with *** above the bars are significantly different (P < 0.001). (C) The statistical analysis of the ratio of vessel that span different segments (***P = 0.0006; *P = 0.0291) and the ratio of ectopic vessels (P = 0.0006) in the sema6D-MO, m-MO+sema6D-mRNA, and sema6D-mRNA injected embryos at 48 hpf (n = 7). About 9–12 ISVs were used for statistics per zebrafish. Mann-Whitney test. Values with *** above the bars are significantly different (P < 0.001). Value with “ns” above the bars indicated no significantly different. (D) Confocal imaging analysis of PMNs and ISVs in control, sema6D morphants, and sema6D morphants with fli1a:sema6D plasmid at 48 hpf. (E) The statistical analysis of the ratio of aberrant axonal projection of Caps (P = 0.0012) and the length of Caps (P = 0.0047) in the sema6D morphants and sema6D morphants with fli1a:sema6D plasmid at 48 hpf (n = 7). About 9–11 caps were used for statistics per zebrafish. Mann-Whitney test. Values with ** above the bars are significantly different (P < 0.01). (F) The statistical analysis of the ratio of vessels that span different segments (P = 0.0006) and the ratio of ectopic vessels (P = 0.0006) in the sema6D morphants and sema6D morphants with fli1a:sema6D plasmid at 48 hpf (n = 7). About 9–12 ISVs were used for statistics per zebrafish. Mann-Whitney test. Values with *** above the bars are significantly different (P < 0.001).