Fig. 3. Multiple regulatory elements are required to determine the anterior boundary of expression in the paraxial mesoderm. (A,B) Whole-mount immunostaining of a 10.5 dpc embryo, using an anti-Hoxb4 antibody. Hoxb4 protein was expressed up to the r6/7 boundary in the neural tube and up to so7 in the paraxial mesoderm. So7 is easily identified at this stage as it lies alongside the second dorsal root ganglion (drg2). Drg2 is the most anterior drg visible at this stage as drg1 has degenerated. (C-J) X-gal staining of 10.5 dpc transgenic embryos. (C,D) Construct CHZ was expressed only up to so14 at this stage. (E,F) Construct Cb4Z had an anterior boundary at so8, indicating that the Hoxb4 promoter can maintain much of the somitic expression established by region C but not the correct anterior boundary. (G,H) Construct b4ZC had identical boundaries of expression to Cb4Z, demonstrating that the position of region C relative to the promoter does not affect the expression pattern. (I,J) Construct CBb4Z did recapitulate the somitic boundary of Hoxb4 expression, identifying a requirement for region B in the regulation of Hoxb4 in the paraxial mesoderm.