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Meis-family homeobox proteins have been shown to regulate cell fate specification in vertebrate and invertebrate embryos. Ectopic expression of RNA encoding the Xenopus Meis3 (XMeis3) protein caused anterior neural truncations with a concomitant expansion of hindbrain and spinal cord markers in Xenopus embryos. In naïve animal cap explants, XMeis3 activated expression of posterior neural markers in the absence of pan-neural markers. Supporting its role as a neural caudalizer, XMeis3 is expressed in the hindbrain and spinal cord. We show that XMeis3 acts like a transcriptional activator, and its caudalizing effects can be mimicked by injecting RNA encoding a VP16-XMeis3 fusion protein. To address the role of endogenous XMeis3 protein in neural patterning, XMeis3 activity was antagonized by injecting RNA encoding an Engrailed-XMeis3 antimorph fusion protein or XMeis3 antisense morpholino oligonucleotides. In these embryos, anterior neural structures were expanded and posterior neural tissues from the midbrain-hindbrain junction through the hindbrain were perturbed. In neuralized animal cap explants, XMeis3-antimorph protein modified caudalization by basic fibroblast growth factor and Wnt3a. XMeis3-antimorph protein did not inhibit caudalization per se, but re-directed posterior neural marker expression to more anterior levels; it reduced expression of spinal cord and hindbrain markers, yet increased expression of the more rostral En2 marker. These results provide evidence that XMeis3 protein in the hindbrain is required to modify anterior neural-inducing activity, thus, enabling the transformation of these cells to posterior fates.
Fig. 3. Expression pattern of neural markers in embryos injected with XMeis3-AM RNA. Two-cell albino embryos were injected unilaterally into the animal hemisphere of one blastomere with 50-100 pg of XMeis3−AM RNA. The red arrow delineates the dorsal midline. In all embryos, XMeis3-AM injection is on the left side. In all cases (except Fig. 3M), embryos are viewed dorsally; embryos are oriented anterior (top) to posterior (bottom). (A) In situ hybridization with otx2. Expression is expanded posteriorly on the XMeis3-AM injected side. The red lines delineate the AP extent of otx2 expression on the uninjected versus injected side. (B) In situ hybridization with otx2 and Krox20. otx2 expression is expanded posteriorly and Krox20 expression (blue arrows) is lost on the XMeis3-AM-injected side. The red lines delineate the AP extent of otx2 expression on the uninjected versus injected side. (C) In situ hybridization with otx2 and En2 (red). Expression of otx2 and En2 (blue arrows) is expanded posteriorly. (D) In situ hybridization with cpl-1 and En2 (red). cpl-1 expression is expanded posteriorly and En2 expression (blue arrow/uninjected side) is lost on the XMeis3-AM-injected side. The red lines delineate the AP extent of cpl-1 expression on the uninjected versus injected side. (E) In situ hybridization with XAG1; expression is expanded posteriorly and laterally on the XMeis3-AM injected side. (F) In situ hybridization with nrp1; expression is unchanged on the XMeis3-AM injected side. (G) In situ hybridization with Krox20 and HoxB9; Krox20 expression (blue arrows/uninjected side) is eliminated on the XMeis3-AM-injected side. HoxB9 expression is unchanged on the XMeis3-AM injected side. (H) In situ hybridization with XE10. XE10 expression is eliminated on the XMeis3-AM-injected side. (I) In situ hybridization with HoxB1. HoxB1 expression is eliminated on the XMeis3-AM injected side. (J) In situ hybridization with HoxB3. HoxB3 expression is eliminated on the XMeis3-AM-injected side. (K) In situ hybridization with En2 (red) and Krox20. En2 expression is pushed posteriorly to the r3/r4 boarder and Krox20 expression is pushed posteriorly to the r5/r7 boarder on the XMeis3-AM-injected side. (L) In situ hybridization with En2 and Krox20. En2 expression is pushed posteriorly to r3 on the XMeis3-AM-injected side. The blue arrow delineates the reduced Krox20 expression (fused stripes) on the injected side. (M) In situ hybridization with En2 (red) and Krox20. Expression of En2 and Krox20 (blue arrows/uninjected side) is eliminated on the XMeis3-AM-injected side. An anterior view of the embryo: dorsal (top) to ventral (bottom). (N) In situ hybridization with n-tubulin. n-tubulin expression is eliminated on the XMeis3-AM-injected side. The trigeminal neuron is marked by blue arrows on both sides.
Fig. 6. XMeis3 antisense morpholino oligonucleotide eliminates hindbrain marker expression. Two-cell albino embryos were injected unilaterally into the animal hemisphere of one blastomere with 6-7.5 ng of the XMeis3 AMO. In situ hybridization was performed in late neurula stage embryos. In all cases, embryos are viewed dorsally; embryos are oriented anterior (top) to posterior (bottom). The red arrow delineates the dorsal midline. Embryos were injected on the right side. (A) In situ hybridization with Krox20 and HoxB9; Krox20 expression is eliminated on the AMO-injected side. HoxB9 expression is unchanged on the AM- injected side. (B) In situ hybridization with XE10; expression is eliminated on the AMO-injected side. (C) In situ hybridization with HoxB3 and HoxB9; HoxB3 expression is eliminated on the AMO-injected side. HoxB9 expression is unchanged on the AMO-injected side. (D) In situ hybridization with XMeis3 and En2 (red); expression of both markers is posteriorized on the AMO-injected side. (E) In situ hybridization with XMeis3; expression is inhibited on the AMO-injected side. The XMeis3 expression in r2 is indicated by arrows on both sides.
