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The early embryonic events involved in the commitment of mesoderm to form blood have not been studied in detail for lack of molecular markers. We have studied the expression of the hematopoietic transcription factors GATA-1 and GATA-2 during Xenopus embryogenesis. During development GATA-1 expression is localized to the ventral region of the embryo and precedes the expression of embryonic globins. GATA-2 is highly expressed in the ventral region of the embryo by the end of gastrulation and later is expressed in the blood island region and the central nervous system. Lithium-induced dorsalization of embryos abrogates GATA-2 expression, and uv-induced ventralization of embryos leads to a radially symmetrical expression of GATA-2. Therefore, GATA-2 expression reflects the ventral character of the embryo. The expression of the GATA-binding proteins and globin in ventral marginal zone explants demonstrates that hematopoiesis is programmed as early as the blastula stage. GATA-1 and GATA-2 are also expressed in cultured animal cap explants, suggesting that these cells have hematopoietic potential. The developmental expression of GATA-1 and GATA-2 is consistent with their role in hematopoiesis in higher organisms and defines the ventral regions of the early embryo that give rise to hematopoietic progenitors. Our studies indicate that these genes will be useful in defining the inductive events that lead to the formation of hematopoietic mesoderm.
FIG. 1. (A) Whole embryo in 8itu analysis using an antisense digoxigenin-labeled larval a-globin RNA probe. a-Globin is not detected at stage
22 ( t.op embryo). The second and third embryos are ventral and lateral views, respectively, at stage 26 showing a-globin RNA to be expressed in
the ventral region in a characteristic V shape. The fourth embryo. stage 32, demonstrates the boundat·ies of the blood island-expressing C~-globin
RNA. At stage 41 globin is seen throughout the circulation. (B) Whole embryo immunohistochemistry using monoclonal antibody L4-27 against
larval globins; stage 32 embryo. (C) Cross section of stage 32embryo immunohistochemically stained with the L4-27 antibody. (D) Whole embryo
(stage 32} i11 situ analysis using an antisense digoxigenin-labeled GATA-1 RNA probe.
FIG. 2. Whole embryo in situ analysis using an ant isense digoxigenin-labeled GATA·2 RNA probe. (A) Embryos include stages 9, 13, 20, 24, 34,
and 39. Note the ventral expression of GATA-2 RNA at stage 13 and localization of GATA-2 to the ventralblood island region by stage 24. In
stage 34 and 40 embryos, GATA-2 is also expressed in the central nervous system. (B-E) Tissue sections of whole embryo in situ analysis with a
GATA-2 RNA probe. (B) sagittal section showing GATA-2 expression in animal pole (arrows) of stage 11 embryo. (C) Ventral expression
(arrows) at stage 13; sagittal section. (D) Expression is more prominen t in inner layer by stage 20 (ventral region of transverse section). (E)
GATA·2 expression in mesodermal layer of transverse midsection at stage 26 (arrow points to most ventral region).
FIG. 3. Expression of GATA-2 in embryos with perturbed dorsal-ventral patterning. (A) Whole embryo in s£tu analysis shows that GATA-2
expression at stage 11 is suppressed in embryos which have been hyperdorsalizcd by exposure to lithium at the 32-cell stage. The upper embryos
are untreated wi ld-type embryos (WT a, animal cap view; WT v, vegetal view), and the lower embryos have been treated with lithium chloride
(Li v, vegetal view; Li a, animal cal' view). (B) In situ analysis comparing GATA-2 expression in normal and uv-ventralized stage 13 embryos.
The four wild-type embryos on the top row exhibit staining in the ventral/lateral half of the embryo. Anterior axis faces right; dorsal axis faces
top, except the third embryo, showing the dorsal view where no G ATA-2 staining is seen. The four uv-trcated embryos (bottom row) demonstrate
radially symmetrical expression of GATA-2 in the outer cell layers of the embryo except the region around the remaining blastopore (viewed in
first three embryos).
FIG. 4. RT-PCR analysis of expression of GAT A-binding factors in animal cap explants. (A) Animal caps dissected from stage 8 blastula were
cultured for 3 hr (stage 10), 18 hr (stage 20), 25 hr (stage 24), 4:3 hr (stage 34), or 49 hr (stage 36). EF-la is used as a control for RNA levels. On
the left, the expression of GATA-1 and larval a-globin in animal cap explants is compared to the expression of MyoD and cardiac actin. The
expression of t hese genes can be compared to equivalent stages in the whole embryo (right). (B) RT-PCR analysis of GATA-2 express ion in
isolated animal caps from the stage 8 blastula. Lane 1, 3 hr (stage 10); lane 2, 5 hr (stage 11); lane 3, 11 hr (stage 2Q); lane 4, 24 hr (stage 25); lane
5, 47 hr (stage 36).
FIG. 5. GATA-1 and GATA -2 expression in isolated ventral and dorsal marginal zones. (A) RNA samples were pr~pared from explants isolated
at stage 10", as shown, and cultured until the stages indicated. For compat·ison, the level of expression in isolated animal caps (CAP), ventral
marginal zone (VMZ), dorsal marginal zone (DMZ), and whole embryos is also shown. By stage 17, GATA-1 is localized to the ventral half of the
marginal wne; however, it is expressed at higher levels in animal cap explants. As GATA·l expression in the VMZ increases, it decreases in cap
explants. (B) GAT A- I RNA expression in stage 10' ventral (right) and dorsal (left) marginal zones explants cultured until stage 35. (C) GATA-
2 is expressed at similar levels in ventral (V) and dorsal (D) cxplnnts. GATA-2 expression is highest in animal cap (A) explants cultured until
stage 17 and the level of expression decreases in all explants with further culture. E, whole embryo; NT, no template control.
FIG. 6. (A) Western blot analysis of larval a-globin (arrow) expressed in ventral (VMZ) but not dorsal (DMZ) marginal zones isolated from
stage 8 blastula. (B) mRNA expression of GATA-1, GATA-2, larval a-globin, and EF-la in ventral (V) and dorsal (D) marginal zones isolated
from stage 8 blastula, as determined by RT-PCR. E, whole embryo RNA and NT, no template as control.
