FoxK regulates Lab expression in midgut endoderm. (A and B) Lab (red; arrowhead) and FoxK (green; arrow) partially colocalize in midgut endoderm in a wild-type embryo. (C) Lab does not accumulate in the endoderm in FoxK¹⁶ homozygous embryos (arrowhead). (D) GFP accumulates in the endoderm under the control of 48Y-Gal4 (arrow). (E and F) Silencing of FoxK transcripts in the endoderm with an RNA interference construct (48Y-Gal4/UAS-FoxKi) also eliminates Lab expression (E, arrowhead). (G–I) Overexpression of FoxK in the endoderm (arrow) does not induce ectopic Lab accumulation (red; arrowhead). Anterior is always to the left. (J) The lab regulatory region contains multiple consensus FH-binding sites (open circles), five verified FoxK-binding sites (black circles), a cluster of Smad/Mad-binding sites (diamonds), and Dfos/AP1-binding sites (open squares). The lab550 regulatory element and a 678-bp element containing five FH-binding sites are indicated. The coordinates with respect to lab ATG are shown in red. (K) EMSA performed with an oligonucleotide containing the ATAAATA sequence and GST-FoxK[414–654]. FoxK strongly and specifically binds to this sequence (arrow) as indicated by the effective competition of the cold probe. (L) Transactivation assays in cell extracts expressing FoxK-L and FoxK-S show that a single copy of the lab678 element robustly responds to FoxK in vitro. The error bars correspond to the standard deviation of three independent experiments. This experiment was conducted as described in Fig. 2.