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During early patterning of the vertebrate neuraxis, the expression of the paired-domain transcription factor Pax-3 is induced in the lateral portions of the posterior neural plate via posteriorizing signals emanating from the late organizer and posterior nonaxial mesoderm. Using a dominant-negative approach, we show in explant assays that Pax-3 inductive activities from the organizer do not depend on FGF, retinoic acid, or XWnt-8, either alone or in combination, suggesting that the organizer may produce an unknown posteriorizing factor. However, Pax-3 inductive signals from posterior nonaxial mesoderm are Wnt-dependent. We show that Pax-3 expression in the lateral neural plate expands in XWnt-8-injected embryos and is blocked by dominant-negative XWnt-8. Similarly, we show that the homeodomain transcription factor Msx-1, which like Pax-3 is an early marker of the lateral neural plate, is induced by posterior nonaxial mesoderm and blocked by dominant-negative XWnt-8. Finally, we show that Rohon-Beard primary neurons, a cell type that develops within the lateral neural plate, are also blocked in vivo by dominant-negative Xwnt-8. Together these data support a model in which patterning of the lateral neural plate by Wnt-mediated signals is an early event that establishes a posteriolateral domain, marked by Pax-3 and Msx-1 expression, from which Rohon-Beard cells and neural crest will subsequently arise.
FIG. 2. Whole-m ount in situ hybridization analysis of Pax-3 expression in Xenopus em bryos. (A) Dorsovegetal view at stage 11.5 showing that Pax-3 is expressed in distinct lateral domains of the presumptive neural plate. (B) Dorsal view at stage 13 showing the refinement of Pax-3 expression to lateral dom ains of the neural plate during convergence and extension. (C) Dorsovegetal view at stage 11.5 showing that XWnt-8 is expressed in involuting ventral-lateralmesoderm. ingsof involuted mesoderm that underlie the prospective lateral neural plate are indicated by arrows. (D) Dorsal view at stage 13 showing XWnt-8 expression in the lateral neural plate.
FIG. 3. Effects of gain and loss of Wnt function on the expression of Pax-3 and Msx-1. Embryos were injected with beta-galactosidase RNA alone (A) or co-injected with beta-galactosidase and Xwnt-8 DNA (B and C), or beta-galactosidase and dn Xwnt-8 RN A (D). Dorsal views are shown in all images (anterior is at the top) with the injected side always on the left. Injected embryos were analyzed by whole-mount in situ hybridization for expression of Pax-3 (A), Msx-1 (G, H), or myoD (I).
FIG. 4. Effects of loss of Wnt function on the expression of elrC, beta-tubulin, and Xnrgr-1. Embryos were co-injected with beta-galactosidase and dn Xwnt-8 RN A (A) or injected with beta-galactosidase RN A alone (G, H). Dorsal views are shown in all images (anterior is at the top) with the injected side always on the left. 10-um transverse paraffin sections showing that the beta-galactosidase tracer colocalizes with the affected domains of primary neurons are shown immediately beneath the corresponding whole-mounted embryo. Injected embryos were analyzed by whole-mount in situ hybridization for expression of beta-tubulin (A, B), elrC (C, D, H), and Xnrgr-1 (E). The lateral (l), intermediate (i), and medial (m ) domains of elrC-expressing primary neurons are indicated by arrows in H.
FIG. 5. (A) XWnt-8 upregulates beta-tubulin expression in noggin animal caps. Animal caps from Xenopus blastulae were injected with noggin RN A (lanes 2 and 6) or with XWnt-8 DNA (lanes 3 and 7) or were co-injected with noggin RN A and XWnt-8 DNA (lanes 4 and 8). Lanes 4 and 8 show that Xwnt-8 upregulates beta-tubulin expression in noggin animal caps. Note that in lane 4 NCAM expression is decreased (see also Fig. 1B, lane 2, 1C, lane 8, and 1D, lane 3). Animal caps were analyzed by RPA when sibling controls were stage 16 (neurula) and stage 25 (early tadpole).ect,ectoderm;n,noggin;N-tub,neural specific beta-tubulin.(B)Whole-mount in situ hybridization analysis of a stage 12.5 embryo showing that the Rohon-Beard cells, marked by elrC (arrow, punctuate staining), lie outside the central portion of the neural plate, marked by NCAM (diffuse staining). Both probes were detected with NBT/BCIP.
FIG. 6. Model for induction of Pax-3 expression in the lateral neural plate. A Wnt-mediated posteriorizing activity arising from PNM would act to initiate and/or maintain Pax-3 expression in the posteriolateral neural plate. The secreted inhibitors of Wnt signaling, frzB and szl, which pattern the mesoderm, could also play a role in restricting the activity of Wnt signaling in the neural plate, leading to a refinement of the Pax-3 expression domain. Furthermore, the Wnt-dependent induction of a Pax-3-positive domain would establish spatial inform ation necessary for appropriate specification of later arising cell types such as Rohoneard prim ary neurons and neural crest.
