Fig. 3. The PH and P/M sites are both essential for Phox2b enhancer regulation in cell culture and ventral r4. (A,B) Fold activation of luciferase activity assayed from P19 cells transiently co-transfected with combinations of Hoxb1, HOXB2, Pbx1a or Prep1 vectors along with P2b_0.38/Luc (A) or mutant P2b_0.38mPH/Luc (B) reporter constructs. The box in B shows the nucleotide changes in the PH site of P2b_0.38mPH/Luc. Note that Hox, Pbx and Prep synergistic activity depends on an intact PH site. (C-J) Dorsolateral views (anterior towards the left) of stage 17-18 chick embryo hindbrains electroporated with P2b_0.38/lacZ (C), P2b_0.38mPH/lacZ (D), P2b_0.38PM/lacZ (H) or P2b_0.38mPM/lacZ (J) constructs. P2b_0.38mPH/lacZ carries the same mutation as P2b_0.38mPH/Luc. P2b_0.38PM/lacZ and P2b_0.38mPM/lacZ carry P/M site mutations shown in H and J, respectively. (C) High reporter expression is restricted to r4 and, to a lesser extent, to r2. (D,H,J) Overall ß-gal levels decrease and ventral r4 expression is lost (arrows). Thus, both PH and P/M sites are required for ventral r4 expression. Co-electroporation of Hoxb1 (E), HOXB2 (G) or Hoxa2 (I) vectors significantly enhances expression from wild type P2b_0.38/lacZ but not mutated P2b_0.38mPH/lacZ (F). ov, otic vesicle.