DEV026435 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The Vlk gene is conserved among vertebrates but does not exist in invertebrates. (A) Protein sequences of mouse, human, rat and zebrafish (DDBJ accession number AB437236) Vlk were aligned using ClustalX. Boxed areas indicate kinase subdomains. (B) Protein sequence alignment using the results of BLAST searches with the sequence of the Vlk kinase domain. Twenty-nine genes were aligned by ClustalX. Phylogenetic trees were drawn using this alignment by MEGA4. The cluster of Vlk orthologs is indicated by yellow shading. This cluster unexpectedly contains one honey bee protein, the distance of which to the Vlk orthologs is artificially small owing to a short sequence alignment, and it is not a Vlk ortholog. (C) Vertebrate Vlk proteins (the 14 shaded proteins in B) were realigned by ClustalX and a tree constructed by MEGA4. Interestingly these trees contain two putative Vlk orthologs from the zebrafish (Danio rerio) genome; however, Ensembl (http://www.ensembl.org/) considers only one of these to be a true ortholog.

• Supplemental Figure S2 -

  Fig. S2. Vlk expression patterns in zebrafish and chick gastrula. (A) At 6 hpf, zebrafish Vlk expression was detected in the organizer. (B) At 12 hpf. zebrafish Vlk is expressed in the anterior ectoderm and mesoderm. (C) In the chick gastrula (stage 4), Vlk is expressed in the anterior endoderm.

• Supplemental Figure S3 -

  Fig. S3. Generation of Vlk Venus knock-in mice. (A) A Venus and floxed PGK-Neo cassette was knocked into the first ATG in exon 1 of the mouse Vlk gene. G418-resistant clones were analyzed by Southern blotting. Correctly targeted ES clones were subjected to transient expression of Cre recombinase. G418-sensitive clones were injected into ICR 8-cell embryos to obtain chimeric mice. (B) Southern blotting of ES clones using 3′ external probe (upper) and internal Neo probe (lower). The 3′ external probe was amplified with the primers 5′-CCAATGGGACCAGAAAAAGA-3′ and 5′-GAACCTGTGGCAGAGAGGAG-3′. (C) The correlation between the Venus gene and endogenous Vlk gene was confirmed by RT-PCR. E12.5 whole heterozygous embryos were trypsinized and Venus-positive cells were sorted by FACS. Only the Venus-positive cells were positive for Vlk.