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In Xenopus, localized factors begin to regionalize embryonic fates prior to the inductive interactions that occur during gastrulation. We previously reported that an animal-to-vegetal signal that occurs prior to gastrulation promotes primary spinal neuron fate in vegetal equatorial (C-tier) blastomere lineages. Herein we demonstrate that maternal mRNA encoding noggin is enriched in animal tiers and at low concentrations in the C-tier, suggesting that the neural fates of C-tier blastomeres may be responsive to early signaling from their neighboring cells. In support of this hypothesis, experimental alteration of the levels of Noggin from animal equatorial (B-tier) or BMP4 from vegetal (D-tier) blastomeres significantly affects the numbers of primary spinal neurons derived from their neighboring C-tier blastomeres. These effects are duplicated in blastomere explants isolated at cleavage stages and cultured in the absence of gastrulation interactions. Co-culture with animal blastomeres enhanced the expression of zygotic neural markers in C-tier blastomere explants, whereas co-culture with vegetal blastomeres repressed them. The expression of these markers in C-tier explants was promoted when Noggin was transiently added to the culture during cleavage/morula stages, and repressed with the transient addition of BMP4. Reduction of Noggin translation in B-tier blastomeres by antisense morpholino oligonucleotides significantly reduced the efficacy of neural marker induction in C-tier explants. These experiments indicate that early anti-BMP signaling from the animal hemisphere recruits vegetal equatorial cells into the neural precursor pool prior to interactions that occur during gastrulation.
Figure 4. Altering Noggin levels in single B-tier or D-tier blastomeres alters the size of the neural plate progenitor pool, indicated by sox2 expression (A-F), and the numbers of nascent primary neurons, indicated by n-tubulin expression (G-T). A: A beta gal mRNA-injected (B-tier) embryo demonstrating that the width of the neural plate (purple) on the injected side (white line) is indistinguishable from the width of the neural plate on the uninjected control side (black line). B: A noggin mRNA-injected (B-tier) embryo has an expanded neural plate on the injected side (white line). C: A bmp4 mRNA-injected (D-tier) embryo has a reduced neural plate on the injected side (white line). D: Injection of the control MO (B-tier) on one side does not alter the width of the neural plate (white line). E: Injection of Noggin MO (B-tier) reduces the width of the neural plate (white line) on the side of injection. F: The entire neural plate is grossly reduced when bmp4 mRNA is injected into a B-tier blastomere. G: Control embryo demonstrating the three stripes of nascent primary neurons on each side of the neural plate. Asterisk indicates side injected with control ( beta -gal) mRNA. H: A noggin mRNA-injected (B-tier) embryo has expanded numbers of cells in the PMN (large arrow) and RBN (small arrow) stripes. I: D-tier expression of BMP4 represses the size of the RBN (small arrow) and interneuron (large arrow) progenitor stripes on the injected side. The PMN stripe is not visible in this neural groove stage embryo. J: Lineage labeling of an A-tier blastomere (red cells) after D-tier bmp4 mRNA injection demonstrates that many A-tier cells continue to express n-tubulin (arrows). J': beta gal mRNA-injected A-tier control. In contrast, many fewer B-tier-derived cells (arrows in K) and no C-tier-derived cells (red in L) express n-tubulin following bmp4 mRNA injection of the D-tier neighbor. K': Many beta Gal-labeled cells (between arrows) in B-tier control; L': Several n-tubulin expressing C-tier cells (arrows) in beta gal mRNA-injected control. M: Injection of a control MO on one side (asterisk) does not alter the three primary neuron progenitor stripes in the neural plate. N: Injection of Noggin MO into a dorsal B-tier blastomere reduces the PMN stripe (large arrow) on the side of the injection. O: Injection of Noggin MO into a ventral B-tier blastomere reduces the RBN stripe (small arrow) on the side of the injection. P,Q: Lineage labeling shows that after nMO injection of a B-tier blastomere, many B-tier-derived cells (between arrows) continue to express n-tubulin (P); in contrast, very few C-tier-derived cells (arrows) express n-tubulin after nMO injection of the B-tier neighbor (Q). R-T: Lineage labeling shows that after nMO injection of a C-tier blastomere, many A-tier-derived cells (R, between arrows) and B-tier-derived cells (S, between arrows) continue to express n-tubulin; in contrast, very few C-tier-derived cells (arrows) express n-tubulin (T). All examples are dorsal views. A-F are stage 14/15 (neural plate); G,H, J-N, P-T are stage 16/17 (neural fold); I, O are stage 19/20 (neural groove/tube).
