Fig. 6. Both high-affinity Pax2-binding sites are essential for the initiation of mid-hindbrain-specific Brn1 expression. (A) Expression of the 6.2wt-lacZ transgene at E9.5. A 6.2 kb AflII-NotI fragment from the 5' region of Brn1 (–6267/–59 relative to the start codon) directs expression of a lacZ reporter gene in the posterior forebrain (fb), mid-hindbrain boundary (arrowhead) region and spinal cord (sc), which correspond to a subset of the endogenous Brn1 expression domains at E9.5 (see Fig. 2F). (B,C) Inactivation of the Pax2-binding site Dd or Pc prevents mid-hindbrain-specific expression of the 6.2Ddm-lacZ or 6.2Pcm-lacZ transgene, respectively, while leaving the forebrain and spinal cord expression unaffected. The expression of all transgenes was analyzed by X-gal staining of injected founder (G₀) embryos at E9.5. Each transgenic construct gave rise to three lacZ-expressing embryos with a similar ß-galactosidase staining pattern. The embryo shown in B revealed ectopic ß-galactosidase expression in the epidermis (ep) from the forebrain to the hindbrain (hb), which was not seen with other embryos carrying the same 6.2Ddm-lacZ gene. Arrowheads in B,C indicate the midbrain-hindbrain boundary (mhb). (D) Brn1 promoter sequence. The mouse (m) DNA sequence of promoter element P is shown together with the transcription initiation sites, two conserved CCAAT boxes and the functional Pax2/5/8-binding site Pc. Only the divergent nucleotides of the corresponding human (h) Brn1 sequence are indicated.