DEV02540 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Organization of the ZBP-89 gene and its encoded protein. (A) Schematic of the protein domains of ZBP-89. The genomic structure of human ZBP-89 is shown below, with the coding exons in yellow, noncoding exons in cyan and introns represented by thick lines. The 24 kb intron 7 is interrupted in the figure. atgMO and spliceMO (indicated by red lines) represent the positions of the morpholino oligonucleotides used in the zebrafish knockdown experiments. (B) Amino acid sequence of human and zebrafish ZBP-89. Identical amino acids are in red. The protein domains are boxed or overlined. Putative nuclear localization signals in the basic domains (BD) are overlined. Vertical blue lines indicate exon boundaries. The conserved cysteine-histidine (CH) linkers are underlined.

• Supplemental Figure 2 -

  Fig. S2. Expression of ZBP-89 in early hematopoietic progenitors and in angioblasts in day 4 EB cultures. FACS sorting was carried out using FLK1 and SCL (human CD4) markers. (Inset) Semi-quantitative RT-PCR showing the ZBP-89 expression (white arrow) profile in hemangioblasts FLK1⁺SCL⁺ (F+/S+), angioblasts FLK1⁺ SCL⁻ (F+S−) and hematopoietic progenitors FLK1⁻SCL⁺ (F-S+), as defined by the expression of FLK1 (F) and SCL (S). β-actin expression (black arrow) was used as internal control. M, markers. H2O, negative control lane.

• Movie 1
• Movie 2 -

  Movies 1 and 2. zbp-89 gene knockdown results in a bloodless phenotype in zebrafish. The translation blocking morpholino atgMO was microinjected into zebrafish embryos at the one- to two-cell stage. Live embryos at 48 hpf were anesthetized and videos taken under a Zeiss Microscope with a high-speed digital camera. Uninjected wild-type (Movie 1) and atgMO-injected mutant (Movie 2) embryos are shown. Video images are presented at 60 frames per second. Anterior is to the right. The heart and trunk are shown in wild type but only the heart is shown in the zbp-89 morphant, where the absence of circulating blood in the heart chambers is obvious.