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First published online January 23, 2009
doi: 10.1242/10.1242/dev.029637

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Definitive hematopoietic stem/progenitor cells manifest distinct differentiation output in the zebrafish VDA and PBI

Hao Jin^1,*, Raman Sood^2,*, Jin Xu¹, Fenghua Zhen¹, Milton A. English², P. Paul Liu^2, and Zilong Wen^1,

¹ Department of Biochemistry, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, P. R. China.
² National Human Genome Research Institute, National Institutes of Health, Building 49, Room 3A26, Bethesda, MD 20892, USA.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Definitive erythroid cells arise in the PBI but not the VDA. (A-K) Whole-mount in situ hybridization to detect e1-globin expression in wild-type embryos (A-E) at 22 hpf (A), 2 dpf (B), 3 dpf (C), 4 dpf (D) and 5 dpf (E); runx1w84x mutant (F,G) and morphant (H) embryos at 22 hpf (F) and 4 dpf (G,H); and vltm651 embryos (I-K) at 22 hpf (I), 2 dpf (J) and 4 dpf (K). The blue arrow in E indicates e1-globin expression in pronephros. The insets are higher magnification (20x) views of the corresponding boxed regions (blue).

View larger version (53K): [in this window] [in a new window]   Fig. 2. VDA originated HSPCs are capable of giving rise to definitive erythroid cells once homed to the PBI. (A) Lateral view of 30 hpf embryo indicates the uncaged position (blue cross) in the anterior part of VDA. (B) Lateral view of 4 dpf embryo. The boxed region (blue) indicates the relative position in the PBI where flu and e1-globin RNA double-positive cells are found after uncaging. (C,D) Confocal images of the boxed region in B show the flu signal and e1-globin RNA staining in the PBI. (E) Merged image of C and D. (F) Superimposed view of E and DIC image. White arrows indicate the co-staining of flu and e1-globin RNA.

View larger version (110K): [in this window] [in a new window]   Fig. 3. Enrichment of definitive myeloid cells in the VDA and the PBI. (A-H) Whole-mount in situ hybridization of l-plastin expression in wild-type embryos at 24 hpf (A), 2 dpf (C), 3 dpf (E) and 5 dpf (G), and runx1w84x mutant embryos at 24 hpf (B), 2 dpf (D), 3 dpf (F) and 5 dpf (H). Insets are high magnification (20X) of the corresponding boxed regions (blue).

View larger version (50K): [in this window] [in a new window]   Fig. 4. Definitive myeloid cells are generated in the VDA. (A) Lateral view of 21 hpf embryo, indicating the uncaging position (blue cross) in the anterior part of the ICM. (B,C) Lateral view of 2 dpf (B) and 3 dpf (C) embryos. The boxed regions indicate the relative positions in the VDA where flu and L-plastin protein double-positive cells were detected after uncaging. (D,E) Confocal images of the boxed region in B showing the flu signal and L-plastin staining in the VDA at 2 dpf after uncaging at cross in A. (D/E) Merged image of D and E. (D'/E') Superimposed view of D/E and DIC image. (F,G) Confocal images of the boxed region in C showing the flu signal and L-plastin staining in the VDA at 3 dpf after uncaging at cross in A. (F/G) Merged image of F and G. (F'/G') Superimposed view of F/G and DIC image. Staining for flu and L-plastin is pseudopainted as green and red, respectively. Arrows indicate co-staining of flu and L-plastin.

View larger version (32K): [in this window] [in a new window]   Fig. 5. HSPCs are found in the VDA in 4 dpf wild-type embryos. (A) Schematic illustration of 4 dpf embryo, indicating blue boxed regions that are magnified in B-E and F-I. (B,C) Double staining of L-plastin protein (B) and cmyb RNA (C) in anterior VDA portion of 4 dpf wild-type embryo. (D) Superimposed image of B and C. (E) Merged view of D with DIC image. (F,G) Double staining of L-plastin protein (F) and cmyb RNA (G) in posterior VDA region of 4 dpf wild-type embryo. (H) Superimposed image of F and G. (I) Merged view of H with DIC image. Arrows indicate HSPCs that are cmyb-positive only, whereas arrowheads represent L-plastin-positive myeloid cells.

View larger version (101K): [in this window] [in a new window]   Fig. 6. Inhibition of HSPC migration from the VDA to PBI blocks their differentiation into erythroid but not myeloid lineages. (A,B) Whole-mount in situ hybridization of runx1 expression in 30 hpf control MO injected vltm651 embryos (A) and sih MO injected vltm651 embryos (B). (C-H) Whole-mount in situ hybridization of cmyb expression in control MO-injected vltm651 embryos at 30 hpf (C), 3 dpf (E) and 4 dpf (G); sih MO injected vltm651 embryos at 30 hpf (D), 3 dpf (F) and 4 dpf (H). (I,J) Whole-mount in situ hybridization of e1-globin expression in 4 dpf control MO injected vltm651 embryos (I) and sih MO-injected vltm651 embryos (J). (K,L) Whole-mount in situ hybridization of lyc expression in 4 dpf control MO-injected vltm651 embryos (K) and sih MO-injected vltm651 embryos (L). Insets are higher magnification (20x) views of the corresponding boxed regions (blue).

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