1. by Tara L. Huber, Yi Zhou, Paul E. Mead, and Leonard I. Zon
Cooperative Effects of Growth Factors Involved in the Induction of Hematopoietic Mesoderm
by Tara L. Huber, Yi Zhou, Paul E. Mead, and Leonard I. Zon
Blood
Volume 92(11):
December 1, 1998
©1998 by American Society of Hematology

2. An assay for the induction of erythroid cells from animal caps.
An assay for the induction of erythroid cells from animal caps. (A) One-cell stage embryos are injected with RNA or plasmids. Animal caps are excised at stage 8 and cultured for 2 days under defined conditions and then dispersed by collagenase. The cells are incubated with o-dianisidine and immobilized onto a glass slide for analysis by microscopy. (B) Protein factors can be tested on dispersed animal caps cells. At stage 8, animal caps are excised and dispersed in CMFM. The outer pigmented layer is discarded. The dispersed inner layer cells are incubated with or without factors. After 1 to 2 hours the cells are washed and reaggregated in calcium/magnesium containing media and cultured for 2 days.o-dianisidine cells are detected as described in (A).
Tara L. Huber et al. Blood 1998;92:
©1998 by American Society of Hematology

3. o-Dianisidine staining of whole embryos and erythroid cells.
o-Dianisidine staining of whole embryos and erythroid cells. (A) Stage-35 whole embryo; the arrow points to the blood island that is stained orange-brown. (B) pcDNA3–BMP-4 (200 pg)– injected embryos; the ventralized embryos have diffuse orange-brown staining of the mesoderm. The arrow points to the enlarged blood island of a partially ventralized embryo. (C, D)o-dianisidine–positive cells from a dispersed stage-33 tadpole VBI. The cells in (C) were photographed at 10× original magnification, whereas the cells in (D) were photographed at 20× original magnification.
Tara L. Huber et al. Blood 1998;92:
©1998 by American Society of Hematology

4. Erythroid cells are detected in animal caps treated with BMP-4 and mesoderm inducers.
Erythroid cells are detected in animal caps treated with BMP-4 and mesoderm inducers. (A) For experiments using plasmid BMP-4 embryos are injected with control vector or pcDNA3–BMP-4 (200 pg) and treated with activin (12 ng/mL) or FGF (200 ng/mL). (B) For experiments using BMP-4 protein, animal caps are excised at stage 8 and dispersed in CMFM. The cell dispersion is treated with BMP-4 (1 μg/mL) with and without activin (12 ng/mL) in CMFM for 2 hours before the cells are washed and allowed to reaggregate in the calcium and magnesium containing cap culturing solution. In both experiments the cap cells are cultured until control stage 35 and analyzed foro-dianisidine–positive cells. The arrowhead points to an erythroid cell. The black arrow indicates a nonerythroid pigmented cell. The panels in (A) were photographed at 20× original magnification and the panels in (B) were photographed at 10× original magnification.
Tara L. Huber et al. Blood 1998;92:
©1998 by American Society of Hematology

5. BMP-4 and activin, or BMP-4 and FGF treatment of animal caps induce more of o-dianisidine–positive cells than BMP-4 treatment alone.
BMP-4 and activin, or BMP-4 and FGF treatment of animal caps induce more of o-dianisidine–positive cells than BMP-4 treatment alone. The animal caps were treated as described in Fig 3. The o-dianisidine–positive cells were counted from two cytospins for each experimental condition in a representative experiment. As each cytospin represents three caps, the sum ofo-dianisidine–positive cells from two cytospins was divided by six to give the number of erythroid cells per cap.
Tara L. Huber et al. Blood 1998;92:
©1998 by American Society of Hematology

6. Comparison of morphology of erythroid cells induced in animal caps to those found in tadpoles and adult frogs.
Comparison of morphology of erythroid cells induced in animal caps to those found in tadpoles and adult frogs. (A) An erythroid cell from an animal cap treated with BMP-4 and FGF; (B) circulating erythroid cells in a 3-day-old tadpole (stage 41); (C) adult erythroid cells. The erythroid cells are stained with Wright Giemsa. The primitive erythroid cells in (B) are 1 day older than and look slightly more differentiated than the cell shown in (A). Primitive erythroid cells are larger and more circular than definitive erythroid cells; they contain circular and less condensed nuclei and still possess yolk granules (compare B with C). The panels are photographed at 40× original magnification.
Tara L. Huber et al. Blood 1998;92:
©1998 by American Society of Hematology

7. Dose response of BMP-4 plasmid for globin protein induction.
Dose response of BMP-4 plasmid for globin protein induction. Western blot analysis for globin expression in animal cap explants. Embryos were injected with 5, 20, and 200 pg of pcDNA3–BMP-4, and animal caps were explanted and cultured with or without activin (12 ng/mL) or bFGF (200 ng/mL) until sibling embryos were at stage 35. Protein extracts were made from the animal caps and separated on a SDS polyacrylamide gel. Proteins were transferred to nitrocellulose and incubated with a larval -globin monoclonal antibody. Each lane is loaded with one cap equivalence of protein extract. The finding that 5 pg of pcDNA3–BMP-4–loaded caps treated with activin (lane 2) induces more globin protein than FGF treatment (lane 3) is variable.
Tara L. Huber et al. Blood 1998;92:
©1998 by American Society of Hematology

8. Tara L. Huber et al. Blood 1998;92:4128-4137
©1998 by American Society of Hematology

9. Tara L. Huber et al. Blood 1998;92:4128-4137
©1998 by American Society of Hematology

10. Tara L. Huber et al. Blood 1998;92:4128-4137
©1998 by American Society of Hematology

11. Tara L. Huber et al. Blood 1998;92:4128-4137
©1998 by American Society of Hematology

12. Tara L. Huber et al. Blood 1998;92:4128-4137
©1998 by American Society of Hematology

13. Tara L. Huber et al. Blood 1998;92:4128-4137
©1998 by American Society of Hematology