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. 2008 Feb 12;111(9):4500–4510. doi: 10.1182/blood-2007-09-110569

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© 2008 by The American Society of Hematology

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Analysis of etsrp, pu.1, and scl expression by the 2-color in situ hybridization. Flat-mounted embryos, anterior is to the left. (A, B) Etsrp (red) and pu.1 (blue) expression in the anterior region of a flat-mounted embryo at the 6-somite stage. Panel B is a higher magnification of panel A. Note that pu.1-expressing cells lie immediately adjacent to etsrp-expressing cells but expression of the 2 markers does not overlap. (C,D) Etsrp (blue) and scl (red) expression in the anterior (C) and posterior (D) regions of a flat-mounted embryo at the 6-somite stage. Note that the 2 markers completely overlap in panel C while in panel D the trunk region contains only etsrp-expressing cells (); scl expression partially overlaps with etsrp in the tail region where scl is restricted to erythroid cells during later stages ( and the right ). (E,F) Etsrp RNA expression expands into the myeloid region in etsrp morphants injected with Etsrp translation-blocking MOs. Etsrp expression in control uninjected embryos (E) and etsrp morphants (F) at the 9-somite stage. Note the more intense and expanded etsrp expression in panel F. (G-J) scl RNA restores pu.1 expression in etsrp morphants with pu.1 and etsrp-expressing cells intermingled. Two-color in situ hybridization analysis for pu.1 (blue) and etsrp (red) expression at the 9- to 10-somite stage. Only the anterior part of an embryo is shown. (G) Control uninjected embryo; (H) etsrp MO-injected embryo; (I) scl RNA-injected embryo; (J) etsrp MOs and scl RNA coinjected embryo. Etsrp staining is very weak in the control embryos because of the short staining time, which was the same for all experimental batches. Note that etsrp morphants in panel H have absent pu.1 expression and strongly up-regulated and expanded etsrp expression. scl RNA-injected embryos (I) display up-regulated etsrp expression. pu.1-expressing cells are intermingled with etsrp-expressing cells in panel J but they do not overlap. Images were taken using Axioskop2 and 10×/0.30 NA (A; C-J) or 20×/0.50 NA (B) dry objectives (Zeiss), Axiocam color camera (Zeiss, model 412-312) and Openlab 4.0 software (Improvision). Magnification: 100× (A,C,D,G-J); 200× (B); 75× (E,F).

Inline graphic — Analysis of etsrp, pu.1, and scl expression by the 2-color in situ hybridization. Flat-mounted embryos, anterior is to the left. (A, B) Etsrp (red) and pu.1 (blue) expression in the anterior region of a flat-mounted embryo at the 6-somite stage. Panel B is a higher magnification of panel A. Note that pu.1-expressing cells lie immediately adjacent to etsrp-expressing cells but expression of the 2 markers does not overlap. (C,D) Etsrp (blue) and scl (red) expression in the anterior (C) and posterior (D) regions of a flat-mounted embryo at the 6-somite stage. Note that the 2 markers completely overlap in panel C while in panel D the trunk region contains only etsrp-expressing cells (); scl expression partially overlaps with etsrp in the tail region where scl is restricted to erythroid cells during later stages ( and the right ). (E,F) Etsrp RNA expression expands into the myeloid region in etsrp morphants injected with Etsrp translation-blocking MOs. Etsrp expression in control uninjected embryos (E) and etsrp morphants (F) at the 9-somite stage. Note the more intense and expanded etsrp expression in panel F. (G-J) scl RNA restores pu.1 expression in etsrp morphants with pu.1 and etsrp-expressing cells intermingled. Two-color in situ hybridization analysis for pu.1 (blue) and etsrp (red) expression at the 9- to 10-somite stage. Only the anterior part of an embryo is shown. (G) Control uninjected embryo; (H) etsrp MO-injected embryo; (I) scl RNA-injected embryo; (J) etsrp MOs and scl RNA coinjected embryo. Etsrp staining is very weak in the control embryos because of the short staining time, which was the same for all experimental batches. Note that etsrp morphants in panel H have absent pu.1 expression and strongly up-regulated and expanded etsrp expression. scl RNA-injected embryos (I) display up-regulated etsrp expression. pu.1-expressing cells are intermingled with etsrp-expressing cells in panel J but they do not overlap. Images were taken using Axioskop2 and 10×/0.30 NA (A; C-J) or 20×/0.50 NA (B) dry objectives (Zeiss), Axiocam color camera (Zeiss, model 412-312) and Openlab 4.0 software (Improvision). Magnification: 100× (A,C,D,G-J); 200× (B); 75× (E,F).