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Fig. 6. The TSG-MO and Chd-MOs cooperate to reduce expression of dorsal-specific genes at the expense of expanded ventral-specific genes. (A-H) Embryos at early to mid-gastrula stages; uninjected control (A,C,E G) or injected with 5 ng TSG-MO into each blastomere at the four-cell stage (B,D,F,H). Expression of the dorsal markers goosecoid (gsc, A-D) and otx2 (E-H) were examined at early (A,B,E,F) or mid-(C,D,G,H) gastrula stages by whole-mount in situ hybridization. While no changes in expression of these dorsal markers were observed at early gastrula stages, by midgastrula both gsc and otx2 expression was reduced in TSG-MO embryos. A, B, E and F are viewed from the vegetal side with the dorsal region to the top. C, D, G and H are viewed from the dorsal side with the anterior to the top. (I-P) In situ hybridization with the ventral marker sizzled (szl) at early neurula (stage 15, I,J,M,N) and tailbud (K,L,O,P) stages. szl expression is expanded in TSG-MO embryos. Anterior is to the left in these embryos. (Q-Z) Neurula stage embryos injected with 5 ng TSG-MO (R,W), 5 ng Chd-MOs (S,X), 5 ng TSG-MO + 5 ng each Chd-MOs (T,Y), or 5 ng TSG-MO + 5 ng each Chd-MOs + 30 pg BM40-XTSG (a modified Xenopus TSG which is not inhibited by the TSG-MO; U,Z) into the marginal zone of each blastomere at the four-cell stage. The dorsal markers myoD (for paraxial mesoderm) and sox2 (for neural tissues) were examined. While TSG-MO and Chd-MOs alone had minor effects on the expression of these markers (R,S,W,X), the MOs cooperate to greatly reduce their expression (T,Y). BM40-XTSG partially rescued the inhibitory effect by the MO combination (U,Z). The embryos are viewed from the dorsal side with anterior to the left. (a-h) TSG-MO and Chd-MOs cooperate to reduce anterior neural markers. 5 ng TSG-MO (b,f) or 5 ng each of Chd-MOs (c,g) were microinjected into each of the four-cell stage blastomeres, alone or in combination (d,h), and the embryos were examined at tailbud stages for expression (arrowheads) of the anterior and posterior neural markers otx2 (fore- and midbrain), Krox20 (hindbrain) and HoxB9 (spinal cord) by in situ hybridization. While the TSG-MO and Chd-MOs mildly reduce the expression of otx2 and krox20 by themselves (b,c,f,g), they cooperate to further inhibit the expression of krox20 and otx2 (d,h). The embryos are shown in lateral view (a-d) or dorsal view (e-h), with the anterior towards the left. |
