Fig. 3. Dorsocross expression along the dorsal midline of blastoderm stage embryos requires dpp and zen. (A-D) Double-fluorescent staining for Doc3 RNA and nuclear Phospho-Mad (PMad) protein. An antibody specific for the activated (phosphorylated) form of the Dpp effector Mad allows monitoring Dpp activity. All views are dorsal, except the lateral view in D. The longitudinal stripe is absent from homozygous dpp^H46 embryos (B) when compared with the wild type (A). (C,D) Upon induction of ectopic Dpp via P{GAL4-nanos.NGT}40 and UAS-dpp, nuclear PMad and expression of Doc3 (as well as of Doc1 and Doc2, not shown) appear in a significantly broader longitudinal stripe when compared with A. PMad, but not Doc3, is also detected in ventral cells (D). (E) Double in situ hybridization for Doc3 mRNA (green) and zen mRNA (red) shows that the dorsal stripe of Doc expression appears during the time when refined zen expression is detected on top of the weaker broad zen pattern. (F) During mid stage 5, when zen transcripts in the broad dorsal domain have disappeared, and dorsal Doc3 and zen are expressed at peak levels, the widths of the Doc3 and zen domains are identical. (G) Dorsolateral view of heterozygous zen⁷/CyO embryo and (H) homozygous zen^- mutant (zen⁷, also known as zen^W36) stained with anti-Doc3+2 antibody. Very little Dorsocross protein is detectable along the dorsal AP axis in zen mutants. By contrast, expression is maintained in the head stripe and at the termini (foregut and hindgut primordia, more prominently at later stages). Embryos in A,B,G,H were analyzed at the beginning of gastrulation, when wild-type embryos have a fully formed dorsal stripe.