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During and after gastrulation, the neural axis in vertebrates is patterned along the antero-posterior axis by the combined activity of signaling factors secreted in the neural ectoderm and the underlying mesoderm. These signals divide the neural axis into four major divisions: the forebrain, midbrain, hindbrain and spinal chord. Among the signals that pattern the neural axis, Wnts play a prominent role and many patterning genes have been found to be direct Wnt/beta-catenin target genes, including several homeobox domain-containing transcription factors. Here we show that HoxD1 and Irx3 are transcriptionally induced by the Wnt pathway during neurulation. Using induction in the presence of the translation blocking drug cycloheximide and chromatin immunoprecipitation assays, we confirm that HoxD1 and Irx3 are both direct Wnt target genes. In addition, we identified Crabp2 (cellular retinoic acid binding protein 2) as an indirect target that potentially links the activities of Wnt and retinoic acid during antero-posterior patterning.
Fig 1. Overview of the vectors used to generate transgenic Xenopus embryos for microarray analysis and validation of the identified target genes. (A) Constructs for the Wnt pathway activating (pVP16) and repressing (pEnR) system and a control construct (pControl). EF1, Xenopus EF1 promoter; LEFN, mouse LEF-1 DNA binding domain; VP16, transactivation domain of the Herpes simplex virus VP16 protein; EnR; transrepression domain of Drosophila Engrailed; GR, hormone binding domain of human glucocorticoid receptor; GR755A, GR with a Glu to Ala substitution at posi- tion 755; GAL4, DNA binding domain of GAL4 transactivator; 14xUAS, 14 repeats of Upstream Activating Sequences; E1b, carp basal E1b promoter; hEcpr, minimal human E-cadherin promoter; eGFP, enhanced Green Fluorescent Protein; HA, haemagglutinin epitope. , unique sites to linearize the vectors for transgenesis. (B) Validation of microarray gene expression results by real-time qPCR analysis for Msx1, Hoxd1, Irx3 and Crabp2. The data are represented as relative gene expres- sion levels in Dex-induced Xenopus embryos transgenic for pVP16, pEnR and pControl. (C) Whole-mount in situ hybridization with probes for Msx1, Hoxd1, Irx3 and Crabp2. All embryos are shown from the anterior side, dorsal up. Two-cell stage embryos were injected in one of their blastomeres with 15 pg RNA for the Dex inducible Wnt activating construct (LefN-VP16-GR) and 300 pg for the Dex inducible Wnt repressing construct (EnR-LefN-GR755A) together with 50 pg of -galactosidase RNA as a tracer. The injected side is indicated by an arrowhead. All embryos were treated with Dex at late gastrula (st12.5) until end-neurula (st17). Upregulation in LefN-VP16-GR injected embryos is seen for Msx1 (71%, n=14), Irx3 (67%, n=12) and Hoxd1 (76%, n=21). An expanded expression domain was frequently observed for Crabp (33%, n=15). Reduced expression is seen in EnR-LefN-GR755A injected embryos for Msx1 (87%, n=24), Irx3 (79%, n=14), Hoxd1 (92%, n=25) and Crabp (47%, n=15).
Fig. 4. Wnt repression during neurulation can induce a posterior shift in Krox20 and Engrailed-2 expression. Embryos were injected unilaterally with 300 pg Dex-inducible Wnt-repressing RNA (EnR-LefN-GR755A) at the four-cell stage in two blastomeres (B,D,F). As a tracer, 50 pg of -gal RNA was co-injected. Non-injected (NI) embryos were included as a control (A,C,E). Both injected and control embryos were treated with Dex from late gastrula (st12.5) until st20. The most anterior stripe (*) represents En2 expression, the other 2 show expression of Krox20 in rhombomeres 3 and 5. Expression of Krox20 and Engrailed-2 was frequently shifted to a more posterior position. In contrast, expression of Rx2a (shown in Fast Red staining) was not shifted. Bright field (A-D) and red fluorescent images (E, F) are shown. The injected side is indicated by an arrowhead. The dotted line indicates the dorsal midline.
