DEV029637 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Circulating definitive proerythroblasts are present in the bloodstream from 4 dpf onwards. (A,B) May-Grunwald Giemsa staining of proerythroblasts isolated from 5 dpf bloodstream (A) and adult kidney marrow (B). (C) The profile of definitive proerythroblasts present in the blood during various developmental stages. The vertical axis indicates the percentage of definitive proerythroblasts in the blood. The horizontal axis indicates the stage of embryos sacrificed for analysis. The blue bars represent wild-type embryo. The green bars represent runx1 morphant. For counting the number of definitive proerythroblasts, circulating blood cells are pooled from 30 embryos of 30 hpf and 20 embryos of the other stages.

• Supplemental Figure S2 -

  Fig. S2. Characterization of the runx1 null mutation W84X. (A) Schematic representation of Runx1 protein with runt homology domain marked and multispecies alignment of the region containing the mutation W84X shown by red box. A schematic of the predicted mutant protein is depicted underneath. Abbreviations: Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus Musculus. (B-E) Whole-mount in situ hybridization of cmyb expression in 32 hpf wild-type (B), 32 hpf runx1^w84x mutant (C), 5 dpf wild-type (D) and 5 dpf runx1^w84x mutant (E) embryos. The insets are high magnification (20×) of the corresponding boxed regions (blue). Black arrow and arrowheads indicate cmyb expression in pronephros and PBI, respectively.

• Supplemental Figure S3 -

  Fig. S3. Expression of lyc and mpo in wild-type and runx1^w84x mutant embryos. (A-F) Whole-mount in situ hybridization of lyc expression in 24 hpf (A), 3 dpf (B) and 5 dpf (C) wild-type embryos, and 24 hpf (D), 3 dpf (E) and 5 dpf (F) runx1^w84x embryos. (G-L) Whole-mount in situ hybridization of mpo expression in 24 hpf (G), 3 dpf (H) and 5 dpf (I) wild-type embryos, and 24 hpf (J), 3 dpf (K) and 5 dpf (L) runx1^w84x embryos. Inserts are higher magnification views of boxed regions (blue).

• Supplemental Figure S4 -

  Fig. S4. In situ generation of lyc positive myeloid cells in the VDA. (A) Lateral view of 21 hpf embryo indicates the uncaging positions (cross) in the anterior part of the ICM. (B) Lateral view of 2 dpf embryo. The boxed regions show the relative positions in the VDA (region 1) and PBI (region 2) where flu and lyc RNA double-positive cells are found after uncaging. (C,D) Confocal images of the boxed region 1 in B show the flu signal and lyc staining in the VDA at 2 dpf after uncaging at cross A. (E) Merged image of C and D. (F) Superimposed view of E and DIC image. (G,H) Confocal images of the boxed region 2 in B show the flu signal and lyc RNA staining in the PBI at 2 dpf after uncaging at cross in A. (I) Merged image of G and H. (J) Superimposed view of I and DIC image. White arrows indicate the co-staining of flu and lyc RNA.