Antibody-staining of P(en/lac) transgenic embryos show that regulatory sequences controlling establishment of even-numbered en stripes are functionally distinct from those controlling establishment of odd-numbered stripes. Anterior left and ventral down in all panels, unless indicated otherwise, a, P(en/lac) transformant stained with rabbit anti-β-galactosidase antibody to visualize the expression from P(en/lac), 3.5–4h after egg laying (AEL). Seven ectodermal stripes are evident (indicated by dots), corresponding to en stripes 2, 4, 6, 8, 10, 12 and 14, which mark the posterior portions of the maxillary, first and third thoracic, and second, fourth, sixth and eighth abdominal primordia. Variable expression seen in the anterior (amg) and posterior (pmg) midgut primordia is not part of the normal en expression program. This expression may be due to sequences from en, or may be spurious, since expression from similar vectors in mesodermal and gut primordia has been reported. b, c: Magnified (ventral) view of a doubly-labelled embryo (mouse anti -en and rabbit anti-β-gal) reveals expression of endogenous en (b) and of P(en/lac) (c), 4.5 h AEL. The stripes of en expression are 1(Mn), 2(Max), 3(La) and 4(T1). Expression of β-gal is coincident with even-numbered en stripes 2(Max) and 4(T1); arrows indicate the same cells in b and c. There is no detectable β-gal expression at positions corresponding to odd-numbered en stripes. In even-numbered stripes there is virtually one-to-one correspondence between β-gal and en expressing cells. The occasional cell containing low levels of β-gal antigen but no detectable en is indicated (open arrow). The β-gal signal is not as discrete as the en signal since the en-β-gal fusion protein is not restricted to the nucleus, d, e: Embryos were doubly-labelled for en expression (d) and β-gal expression (e) at the onset of gastrulation, 3 h AEL. Expression from P(en/lac) is induced at about the same developmental stage as en, although there may be a slight lag in induction or accumulation. Arrows point to co-expression in 2(Max), and in 4(T1), where β-gal is just detectable.
Methods. Preparation of embryos for immunocytochemistry was as in refs , . Rabbit anti-β-gal was from Cappel, rabbit anti-en, (ref. ); mouse monoclonal anti-en (and inv), a gift of K. Coleman, C. Goodman and T. Kornberg. Final magnification was ×130, except for b and c which were ×520.