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Fig. 6. Inhibition of pronephros development does not result from an effect of Mo3 on the Spemann's organizer. (A) Mo3 does not affect the ability of DMZ explants to induce pronephros markers in VMZ explants. DMZ explants were dissected at the early gastrula stage from embryos previously injected at the 4-cell stage with Cmo and RLDx, or Mo3 and RLDx, in the two dorsal blastomeres. Explanted DMZ were combined with VMZ explants taken from uninjected embryos and combinates were cultured until tailbud stage (NF st. 33/34). In situ hybridization analysis of XSMP30 and XWT1 expression in combinates (left panels) and corresponding RLDx fluorescence (right panels). Induction of XSMP30 and XWT1 expression in the unlabelled VMZ is not affected by Mo3. Undissected DMZ donor embryos injected with Mo3 and cultured until the tailbud stage show head anomalies when compared with Cmo-injected embryos. (B) Mo3 targeted outside of the organizer still inhibits pronephric expression of XPax-8 and XPax-2. Mo3 and RLDx were injected at the 8-cell stage into one ventro-vegetal blastomere. Embryos were cultured until neurula or tailbud stage for the analysis of XPax-8 or XPax-2 expression, respectively. In situ hybridization analysis of XPax-8 and XPax-2 expression (left and up panels, respectively) and corresponding RLDx fluorescence (right and down panels, respectively). Comparison of control and injected sides shows that Mo3 inhibits XPax-8 expression in the mesoderm at the neurula stage. Expression of XPax-2 in the lateral mesoderm is strongly reduced at tailbud stage showing that pronephric development is impaired. However, XPax-8 expression in the otic placode (arrowhead), as well as neural expression of XPax-2 in the hindbrain, optic and otic vesicles are identical on injected and control sides. |
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Fig. 5. Effect of Mix.1 and Mix.2 loss of functions upon later development of the pronephros. In situ hybridization analyses of XPax-2 (A–D) and XWT1 (E–H) in Mo1 or Mo3 injected embryos. Morpholinos were targeted on the right side as described in Fig. 4. On control sides, XPax-2 is expressed in nephrostome, tubules and Wolffian duct. Its expression is totally abolished in response to Mo1 or Mo3. Neural expression of XPax-2 in the hindbrain, optic and otic vesicles is also affected on the injected side. XWT1 is expressed in the glomus part of the pronephros on control sides, while it is totally down-regulated on the injected side both with Mo1 and Mo3. (I) Histological analysis at early tadpole stage (NF st. 38) after injection of Mo3 on the right side. Pronephros tubules differentiated on the control side (pt), are absent on the injected side. They are replaced by a mass of uncharacterized tissue (arrow). n: notochord; nt: neural tube; s: somites. |
