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The expression of GATA-1, which encodes for a hemopoietic transcription factor, initiates at gastrula stage in the Xenopus embryo (1). In order to examine a possible function of GATA-1 in dorso-ventral patterning of mesoderm and ectoderm derivatives, the synthesized RNA of GATA-1 was overexpressed in embryonic cells to assess its biological effects. In the embryos injected with GATA-1 RNA in the dorsal marginal zone at 4-cell stage, dorsal epidermis did not cover the vegetal cells so that the gastrulation was not completed. The same dose of GATA-1 RNA injected into ventral marginal zone did not influence the development, and GATA-2 RNA transcribed from the same vector had little effect, suggesting that this phenomenon is physiologically important. The morphological and immunohistochemical studies revealed that notochord and neural tissue were mostly eliminated in the embryos or the dorsal marginal zone explants after injection of GATA-1 RNA. GATA-1 also inhibited neurogenesis in animal cap explants, which was induced by the injection with noggin RNA. Northern blot analysis using dorsal marginal zone explants showed, however, that only a slight amount of alpha-globin message was induced, and cardiac alpha-actin message was retained. Therefore, GATA-1 did not convert completely the dorsal phenotype to the ventral one. Furthermore, the injection of GATA-1 RNA didnot alter the expression of early dorsal and ventral markers at the onset of gastrulation. These results suggest that GATA-1 is an potential inhibitor of the dorsalization and the neurogenesis, but it affects on the specification of dorsal tissues in relatively later steps.
FIG. 1. Morphological views of GATA-1 RNA-injected embryos. Dorsal two blastomeres at 4-cell stage were injected with GATA-1 RNA
(1ng/embryo), and the injected (A-D) and uninjected (E-H) embryos were fixed at early gastrula (stage 101, A and E) (38), late gastrula (stage
13, B and F), neurula (stage 15, C and G), and late neurula (stage 20, D and H). GATA-1-injected embryos appear normal at the onset of
gastrulation (A), but the delay of invagination process is obvious during gastrulation stages (B and C). Finally as they start to elongate at
late neurula stage, the endoderm cells are exposed to outside without any structures of neural tissue differentiation (D). A, E; vegetal view.
B-D; dorsal-vegetal view. F-H; dorsal view. Bar in A indicates 0.5 mm.
FIG. 2. Histological analyses of GATA-1 RNA-injected embryos. Dorsal two blastomeres at 4-cell stage were injected with 1ng GATA-1 RNA. A-E, Sagittal views of injected (A, stage 101; B, stage 15; C, stage 20) and uninjected control embryos (D, stage 15; E, stage 20). Note that GATA-1 RNA injection arrests of the invagination of mesoderm, and disturbs the differentiation of notochord. Arrowheads in A-D indicate the edge of involuting cells. Dorsal to the left of figures in A-C. Anterior to the left of figures in D and E. Bar in A indicates 100 mm. nc, notochord; nt, neural tube. F-G, Transverse sections of injected (F) and uninjected (G) embryos at a trunk level. The sections were stained by anti-notochord antibody, MZ-15, indicating that the injected embryos lack the differentiated notochord. A trace of positive staining was observed in an embryo exceptionally (white arrowhead in F). Bar in F indicates 100 mm. H-J, Effect of increased doses of injected GATA-1 RNA. Dorsal two blastomeres at 4-cell stage were injected with 5ng (H) or 1ng (I) GATA-1 RNA, and the embryos were cultured until stage 32 (2 days old). High dose of RNA injection causes loss of eye capsules and cement gland, suggesting that neural tissues are completely eliminated in these embryos. J shows uninjected control embryo. Bar in H indicates 1 mm.
FIG. 3. Injection of GATA-1 RNA inhibits the differentiation of notochord and neural tissue in dorsal marginal zone (DMZ) explants.
Dorsal two blastomeres at 4-cell stage were injected with GATA-1 RNA (1ng/embryo), and the DMZ explants (B, D) or control uninjected
explants (A, C) were allowed to develop to stage 35/36. The explants were immunostained with anti-N-CAM antibody, 4d ( A, B) or with
anti-notochord antibody, MZ-15 (C, D), showing significant reduction of both markers in injected embryos (B, D). Arrowheads show the
positively stained sites. Arrows show pigments of cement gland. Bar in A indicates 0.5 mm.
FIG. 4. Injection of GATA-1 RNA inhibits the differentiation of
neural tissue in animal cap explants. Two balstomeres at 2-cell stage
were injected with noggin RNA, or coinjected with noggin and
GATA-1 RNAs, and the animal cap (AP) explants were cultured for
2 days. The explants were harvested, and the lysate from 3 explants
was separated by 7.5% SDS-PAGE. The expression of N-CAM antigen
was detected by Western blot analysis as described in Materials
and Methods. The 55 kd major band, which is detected nonspecifically
but in all the embryonic tissues is shown for a protein
loading control. In lanes S1 and S2, lysates from 25 and 12.5 mg of
adult brain were run.
FIG. 5. Expression of late markers in dorsal marginal zone explants.
Two dorsal blastomeres of 4-cell stage embryo were injected
with either BMP-4 RNA (5 ng/embryo) or GATA-1 RNA (5 ng/embryo),
and explanted DMZ tissues were cultured for 2 days. Two mg total RNA
from each sample was electrophoresed in each lane for Northern blot
analysis to detect a larval a-globin and muscle a-actin. For RNA loading
control, the same blot was rehybridized with EF-1a probe.
FIG. 6. Expression of early markers in GATA-1-injected embryos.
A–C. The expression of goosecoid in dorsal lip region at stage
101 embryos was examined by whole-mount in situ hybridization
after injection of BMP-4 (B)orGATA-1 (C) RNA (5ng/embryo) into
dorsal marginal zone at 4-cell stage. A indicates uninjected control
embryos. All figures are vegetal view, and dorsal to up. Dorsal to up.
Bar in A indicates 1 mm. D. The RT-PCR anaysis showing the
expression of goosecoid, chordin, Xwnt8, and EF1a in dorsal marginal
zone (DMZ) or control ventral marginal zone (VMZ). The dorsal
blastomeres of 4-cell stage embryo were injected with designated
amounts of GATA-1 RNA, and DMZ or VMZ was excised at stage
101 early gastrula, and 0.5 mg total RNA from these tissues was
used for subsequent RT-PCR analysis. Note that no significant
change was observed in the expression of early dorsal and ventral
marker genes by injection of GATA-1 RNA.
