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Fig. 5. Mutation of a conserved Pax3-binding site causes upregulation of an Msx2 transgene in the postotic hindbrain. (A) Alignment of homologous regions of human and murine Msx2 5' flanking sequence. A stretch of 520 bp within the Pax3 responsive region matched closely (87% identity) with a region in the 5' flanking sequence of the human MSX2 gene. This stretch included a single, highly conserved Pax3 binding site (Pax site 1). We show the nucleotide sequence of Pax site 1 and surrounding DNA aligned with the human MSX2 locus. Also shown is a Pax3 site in the Tyrp1 promoter (Galibert et al., 1999), a high-affinity Pax3-binding site, Nf3' (Epstein et al., 1995) and the Pax3 consensus (Chalepakis and Gruss, 1995; Epstein et al., 1996). Pax site 1M is a base substitution mutant of Pax site 1. Bold letters show identity with the consensus sequence. (B) Protein titration EMSA analysis of Pax3 binding to Nf3', Pax site 1 and Pax site 1M. In vitro synthesized Pax3 protein (0.5, 1, 2 and 3 µl) was incubated with radiolabeled Nf3' (lanes 1-4), Pax site 1 (lanes 6-9) or Pax site 1M (lanes 12-14). Mock transcription-translation lysate (no DNA added) (1 µl) was used as a background control (lanes 5,10,11). Protein-DNA complexes were visualized by autoradiography. (C-F) Effect of Pax site 1 mutation on {Delta}4Msx2-hsplacZ transgene expression. Embryos carrying either the {Delta}4 or {Delta}4 Pax site 1M transgene were stained at E9.5 for ß-gal expression. (C,D) Dorsal views of whole mounts. The region of the neural tube from which the cardiac neural crest originates is indicated by brackets. The position of rhombomere 5 (r5) is shown to the left, the otic vesicle (o) to the right. Note elevated X-gal staining in the postotic neural tube of the Pax site 1 mutant embryo compared with the wild type. (E,F) Sections through the postotic neural tubes (arrows) of the embryos in (C,D). Note ventral and lateral expansion of X-gal stain in mutant (F) relative to wild-type (E) (arrows). Also note ectopic stain in ventromedial neural tube of mutant (F) (arrowhead). Scale bar: 100 µm.