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Blood, Vol. 113, Issue 5, 1192-1199, January 29, 2009
Snrk-1 is involved in multiple steps of angioblast development and acts via notch signaling pathway in artery-vein specification in vertebrates
Blood Chun et al.
113: 1192
Supplemental materials for: Chun et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 97.2 KB)
- Figure S1. Sequence analysis of the zebrafish Snrk-1 (JPG, 137 KB)
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(A) Shows alignment of human, mouse and zebrafish snrk-1. ATP binding domain, Ser/Thr kinase active site, ubiquitin associated domain and bipartite nuclear localization signal of Snrk-1 are conserved from zebrafish to human. (B) Genome of zsnrk-1 has 5 exons (E1 to E5). Two morpholinos, MO1 and MO2 were generated. MO1 targets the splicing junction between exon 2 and exon 3 while MO2 targets the translation initiation site (ATG). (C) Efficacy of MO1 and MO2 was examined by RT-PCR and shows that the target gene, snrk-1 was knocked down in both MOs (8 and 16 ng) injected embryos. Injection of increased MOs (16 ng) shows complete knock down of snrk-1. (D) RT-PCR analysis for snrk-1 and actin across embryonic zebrafish development, 0.5, 3, 5, 9, and 30 hpf. (E) RT-PCR analysis on human hemangioma (1, dT and 2, RP) and placenta (3, dT and 4, RP) samples. dT: oligo dT primer; RP: random primer.
- Figure S2. Snrk-1, Gata-1, and Scl whole mount in situ and kinase activity in vitro (JPG, 63.5 KB)
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Starting at 10 hpf (A) until 20 hpf (D), snrk-1 is expressed ubiquitously. (A) to (C) In situ hybridization with gata-1 antisense RNA probe at 20 hpf. (D) to (F) In situ hybridization with scl antisense RNA probe at 6-somite. (G) is a cartoon depiction of the snrk-1 wild type (WT) and point mutant snrk-1 kinase mutant (KM) construct design. ATP: ATP binding domain, K: Serine/Threonine kinase active domain, UB: ubiquitin associated domain, NLS: bipartite nuclear localization signal, V5: V protein of simian virus 5. Black asterisk in K domain indicates the Pro→Ala substitution. A V5 western blot (WB) is shown on 293 lysates from cells transfected with snrk-1 WT, and snrk-1 KM cDNA constructs or untransfected (UT) cells. The protein migrates at the expected 82 kDa size. (H) shows the kinase assay plotted in a graphical format with the luciferase activity on the y-axis corresponding to the different samples on the x-axis. In the Kinase Glo (Promega) assay, luciferase activity is inversely proportional to ATP consumption. Luciferase activity on the y-axis was corrected for equal amount of V5 tagged snrk-1 protein in the lysates. Histone substrate was used for the kinase assay. Controls included: no substrate or no ATP. + indicates addition and – indicates no addition.
- Figure S3. Etsrp whole mount in situ hybridization (JPG, 60.6 KB)
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(A) to (J) A montage of in situ hybridization with etsrp antisense RNA probe from 11 hpf to 30 hpf. (A) We initially noticed the two lateral stripes of angioblasts cells evenly distributed on either side of the midline at 11 hpf. (B) At 13 hpf, an asymmetric break is noticed in the linear distribution of cells between the anterior and posterior populations. (C) The anterior population stays in place but the posterior population moves laterally to create space in the middle for downstream events. (C) Beginning at 15 hpf, a new inner population of etsrp+ cells arises in the trunk region from the posterior angioblasts. These new cells are closer to the midline than the lateral angioblast cells in 13 hpf and continue to move towards the midline presumably making the axial vessels. (F) and (G) At 18–19 hpf, the anterior trunk angioblasts and the midline cells fuse at the site of future lateral dorsal aorta. (I) and (H) Over the course of next few hours in development, the posterior trunk angioblasts eventually disappear after contributing several waves of angioblasts to the midline.
- Figure S4. High-power images of etsrp in situ for snrk-1 gain and loss-of-function embryos (JPG, 54 KB)
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High power trunk-tail etsrp whole mount in situ images from panels 3A, 3B and 3C are shown here in A, B and C respectively. Black asterisks are included to indicate expanded etsrp stained region at LPM in (B) compared to (A). Panels (D) – (I) indicate etsrp whole mount in situ at 19 hpf for uninjected wild type (WT) embryo (D and G), snrk-1 MO1 injected embryo (E and H), and snrk-1 mRNA injected embryo (F and I). (G), (H) and (I) are high power images of white boxed regions in panels (D), (E) and (F) respectively.
- Figure S5. Quantification of Snrk-1 knockdown defective embryos for artery vs. vein markers (JPG, 71.3 KB)
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(A) Graphical representation of percentage of embryos showing reduced grl expression. MO1 and MO2 injected embryos show reduction of grl expression at 18 hpf. (B) Graphical representation of percentage embryos of notch1- and notch2-ICD injected embryos and notch2-ICD and snrk-1 MO1 co-injected embryos showing induced/reduced flt-4 or ephrin-B2a marker. (C) Graphical representation of percentage embryos of uninjected (UI), MO1 and MO2 (morpholino injected), snrk-1 and snrk-1KM mRNA injected embryos showing ephrin-B2a induction (eph-ind.), blue bars; ephrin-B2a reduction (eph-red.), white bars; flt-4 induction (flt-4 ind.), black bars; or flt-4 reduction (flt-4 red.), green bars; at time points indicated in parenthesis. Ephrin-B2a is an artery marker while flt-4 is venous marker.
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