Dpp directly regulates FoxK in the endoderm. (A) Transheterozygous combinations of dpp^+/− and FoxK^+/− mutant alleles result in lethality. (B and C) A control embryo (stage 15) shows normal Lab (arrowhead) and FoxK (green; arrows) accumulation in the endoderm. (D–F) dpp overexpression in the visceral mesoderm (24B-Gal4) induces ectopic Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in F. (G–I) Expression of tkv^DN in the endoderm (48Y-Gal4) eliminates both Lab (arrowhead) and FoxK (arrow) in the endoderm. Merged panel is shown in I. (J–L) Homozygous FoxK¹⁶ embryos that also overexpress dpp in the visceral mesoderm lack Lab expression in the endoderm (arrowhead). K shows negative FoxK staining and the merged image is in L. (M) EMSA performed with a genomic-derived probe (Oligo-Mad) containing multiple Mad-binding sites (right) and protein extracts from S2 cells transfected with combinations of Mad, Med, and tkv^act constructs. The cellular extracts from nontransfected cells (S2) or cells transfected with Mad and Med constructs resulted in a small shift of the Oligo-Mad (arrow). Extracts expressing tkv^act produced a stronger binding, but extracts expressing all three constructs resulted in the strongest binding to the Oligo-Mad probe. The lanes with no cell extract (−) and the use of an unspecific probe (GAS) produced no shift. The free oligonucleotides and oligonucleotide complexes are indicated by the arrowhead. (N) The 24-B-Gal4 strain induces GFP expression in the mesoderm (arrow).