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Fig. 6. Dazap2 has potent posteriorizing activities in neuralized tissues. (A) Both cells of 2-cell stage embryos were injected with the following mRNAs: NI, non-injected; dazap2 (DZ2, 250 pg/embryo); fgf8a (F8a, 100 pg/embryo); noggin (12.5 pg/embryo); or combination as indicated above lanes. RT-PCR analysis shows expression of a complete range of anterior and posterior markers in whole embryo (WE, line 1) but none in the NI caps (lane 3). Dazap2 and fgf8a both induce posterior genes hoxd1 and hoxb9, but Dazap2 also activates otx2 and en2 while fgf8 activates cdx1, 2 and 4 (lanes 4, 5). Upon neuralization by noggin (indicated by sox2 expression), animal caps express anterior markers only (otx2, lane 6). Dazap2 and noggin co-injections (lane 7) induce a complete range of anterior and posterior markers (but not cdx1, 2 or 4), while Fgf8 and Noggin co-injections only partially posteriorize the explants (en2 induction, lane 8). (B) A range of dazap2 mRNA (125 pg/cell, 50 pg/cell, 10 pg/cell or 2 pg/cell) alone or in combination with noggin (12.5 pg/cell) was injected into both cells of 2-cell stage embryos. Explants were dissected at stage 9 from these embryos and aged until stage 18. Decreasing levels of dazap2, alone or in combination, show a decrease level of induction of en2, hoxd1 or hoxb9. This is the opposite effect for otx2, which shows a decrease of induction alone but an increase induction in combination with noggin. Additionally, sox2 induction requires noggin. For in vivo analysis, embryos were injected at 4-cell stage with lacZ (100 pg/cell, C�E), dazap2 (250 pg) and lacZ (G�I), noggin (25 pg) and lacZ (K�M) or a combination of dazap2, noggin and lacZ (O�Q). Dazap2 injected embryos show a reduction of otx2 and krox20 expression (arrow) and ectopic expression of hoxb9 (arrowhead) in the anterior neural plate at stages 16�18 (G�I). Noggin-injected embryos show an increase in both otx2 and krox20 (arrow), and a decrease in hoxb9 expression (arrowhead) (K�M). The combination of dazap2 and noggin results in rescued otx2 and krox20 expression in injected cells (arrows) and hoxb9 expression is activated ectopically in the anterior neural plate (arrowhead) (O�Q). |
