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To increase our understanding of haematopoiesis during early vertebrate development, we have studied the expression pattern of the transcription factor GATA-2 in Xenopus embryos, and asked how this is regulated. We show that the blood island precursors of the ventral mesoderm express GATA-2 RNA at neural tube stages, some 5 hours before globin RNA is detected in their derivatives. Prior to this however, GATA-2 is expressed much more widely within the embryo. Maternal transcripts are uniformly distributed, and zygotic transcription is activated during gastrulation throughout ventral and lateral regions of the embryo, with expression highest in the sensorial ectoderm and only weak in the ventral mesoderm. The domain of GATA-2 expression in neurulae outlines the region of the neural plate and suggests a possible wider role in dorsoventral patterning. To identify the signals involved in regulating this pattern of expression, we performed experiments with embryo explants. GATA-2 is activated autonomously in isolated animal caps and this activation is suppressed by the mesoderm-inducing factor activin, but not by FGF. Thus, the down-regulation of GATA-2 observed in the region of the Spemann organiser may be a response to an activin-like signal emanating from the dorsal-vegetal region or Nieuwkoop centre. GATA-2 activation in animal caps and ventral marginal zones was suppressed by co-culturing with dorsal marginal zones, suggesting that a signal from the Spemann organiser is involved in suppression of GATA-2 in the dorsal region of the embryo. Expression of a candidate for this signal, noggin, had the same effect. Taken together, the observations presented here suggest that GATA-2 activation occurs by default in the absence of signals, that the restriction of its expression within the early embryo is controlled by negative signals emanating from the Nieuwkoop centre and the organiser, and that noggin and activin-like molecules play a role in these signalling pathways.
Fig. 1.(A-D) αT4 globin expression in mid to late tailbud stage embryos and expression patterns of GATA-2 in mid-neurula stages. (A,B) Albino embryos probed for αT4 globin mRNA by whole-mount in situ hybridisation at stage 35 (A) and stages 24-26 (B). The foregut staining at the rostral (left) end of the mid tail bud embryos (B) is non-specific. The V- shape of the ventralblood islands at mid-tailbud (B) extends caudally as the embryo matures (A). (C-E) In situ hybridisations of mid-neural fold (stage 15) non- albino embryos hybridised with an antisense GATA- 2 probe. (C) Low power magnification showing strong ventral (v) and lateral but not dorsal (d) labelling. Arrowheads define the boundary between the GATA-2 positive and GATA-2 negative regions. (D) Higher power magnification showing strong peripheral labelling in the sensorial (inner) ectoderm (arrow) and weaker labelling in the mesoderm
(double arrows) and endodermal cells (arrowhead). [NOTE shown as separte image XB-IMG- ](E) Stage 15 non-albino embryos probed for GATA-2 RNA (purple) by whole-mount in situ hybridisation, before clearing. Staining is apparent throughout the ventral surface up to the edges (dashed line) of the developing neural plate (np, brown due to pigmentation) giving rise to a ennis ballpattern. The embryo in the centre (V) is viewed from the ventral aspect.
Fig 1 (E) Stage 15 non-albino embryos probed for GATA-2 RNA (purple) by whole-mount in situ hybridisation, before clearing. Staining is apparent throughout the ventral surface up to the edges (dashed line) of the developing neural plate (np, brown due to pigmentation) giving rise to a ennis ballpattern. The embryo in the centre (V) is viewed from the ventral aspect.
Fig. 2. GATA-2 and globin mRNAs are co-localised in the ventral blood islands. Serial sections taken from stage 25/26 non-albino embryos and hybridised with either GATA-2 (A,C) or αT4 globin (B,D) riboprobes. The orientation of the embryos is ventral side down. Note the co-localisation of GATA-2 and αT4 globin mRNAs. Anteriorly, both transcripts can be detected in the arms of the of the ventral blood islands (A,B). Co-expression can also be detected in a broad band more posteriorly (C,D). Sections were taken from the levels indicated in the schematic ventral view of a stage 25/26 embryo shown in (E) where GATA-2 expression (stippled areas) is shown. For orientation, the cement gland (hatched area, ANT) and blastopore (curve, POST) are shown.
Fig. 3. GATA-2 is localised in presumptive haematopoietic cells before globin expression is established. Late neural tube (stage 21) non-albino embryos were hybridised with an antisense GATA-2 probe. (A,B,D,E) The orientation of the embryos is ventral side down. (A) Transverse section through the anterior end of the presumptive blood islands showing ventral position and two discrete expression domains (arrows). (B) Section taken just posterior to (A) showing the two discrete expression domains closer together. (C) Schematic diagram of the ventral aspect of a stage 21 embryo showing the mesodermal expression of GATA-2 (stippled area) reconstructed from examination of serial sections. Note the of the future blood islands. For orientation, the cement gland (hatched area, ANT) and blastopore (curve, POST) are shown.
(D) Section from the same region as in (A), at higher power, stained with brilliant cresyl blue for histological detail. (E) A schematic illustration of the section shown in (D) to indicate the three primary germ layers. Note that in this position along the anteroposterior axis of the embryo, the mesoderm does not extend to the ventral midline. Areas of GATA-2 expression (shown in black) are restricted to the medial edges of the mesoderm in this region.
