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Three members of the vertebrate Distal-less gene family, Dlx3, 5 and 6, are transcribed in early gastrula embryos of Xenopus laevis. This expression is confined to ectoderm and is excluded from the presumptive neural plate region. Expression of all three genes is dependent upon BMP signaling, with significant differences in how the three genes respond to the BMP antagonist chordin. This correlates with the different expression domain boundaries in vivo for Dlx3 compared to Dlx5 and 6, suggesting that BMP signal attenuation could be the primary factor in determining these different patterns in the gastrulaectoderm.
Fig. 1. Spatial expression patterns of Dlx5 and Dlx6. Embryos were fixed and hybridized under conditions optimized for detection of epidermal transcripts. (A-E), Dlx5. (F-J), Dlx6. (A,F) Mid/ late gastrula (stage 12) showing broad expression in ectoderm, except for the dorsal side (on the right) which is the posterior region of the prospective neural plate. The two black arrowheads indicate the edges of the yolk plugs. (B,G) Early neurula (stage 13). A stripe of relatively intense hybridization has appeared (white arrows in these and subsequent panels) lateral to the epidermal-neural plate boundary (black arrow). The yolk plugs are indicated by black arrowheads. Dorsal is to the right. (C,H) Mid neurula (stage 15). Anterior neural plate hybridization has resolved into anterior neural fold and cement gland (black arrowheads). Anterior is to the right. (D,I) Late neurula (stage 17). The intense hybridization stripes (white arrows) and epidermal-neural plate boundaries (black arrows) continue to be well-resolved. Anterior to right. (E,J) Early tailbud (stage 20). The neural tube has closed at this stage and the epidermal-neural plate boundaries have fused at the dorsal midline. The intense lateral stripe of expression clearly lies outside the neural tube for both Dlx5 and Dlx6 (white arrows). Anterior is to the right.
Fig. 2. Width of dorsal excluded regions for Dlx genes. Stage-matched (st 14) embryos hybridized in situ with probes for chordin (blue/green) and Dlx3, 5 or 6 (purple). (A,D) Dlx3; (B,E) Dlx5; (C,F) Dlx6. The patterns are similar, but the distance between the midline (chordin signal) and Dlx3 is clearly greater than for either Dlx5 or Dlx6. A-C, dorsal view. D-F, lateral view.
Fig. 3. Differential regulation of Dlx gene expression by BMP signaling. Fertilized eggs were injected with 10-1000 pg of RNA encoding chordin, animal caps removed at stage 7- 8, cultured until sibling embryos reached stage 12 and processed for RNA and Northern analysis. (A) Northern blots using probes for Dlx3, Dlx5, Dlx6, XK81 (epidermal keratin) and Zic3 (neural pre-pattern gene). Ethidium bromide staining of the 18S ribosomal RNA band is shown as a control for equal loading of lanes. Dlx3 and XK81 are almost completely repressed by 50 pg chordin RNA while Dlx5 and Dlx6 are much more refractory (see below). (B) Summary of densitometry analysis of multiple exposures of Northern blots from three separate experiments. Dlx5 and 6 require several-fold higher RNA levels, respectively, to achieve the same degree of inhibition as Dlx3 (note the log scale for the x-axis). There is also a significant increase in expression of Dlx5 and Dlx6 in embryos injected with the lowest dose of chordin, relative to uninjected controls.
