Fig. 7. HFGa13 interferes with the cellular functions of Hoxa13 and Hoxd13. (A) Transcriptional transactivation by wild-type Hoxa13 and HFGa13 proteins in a GAL4-fusion assay in COS-7 cells. Relative luciferase activities were normalized to the empty GAL4 DNA-binding domain expression vector. Fusion protein of the GAL4 DNA-binding domain and Hoxa13 shows transcriptional activation of the synthetic reporter. By contrast, fusion protein of the GAL4 DNA-binding domain and HFGa13 fails to activate transcription of the same promoter and is able to decrease the basal activity. Luciferase assays were performed after two independent transfections, each done in triplicate (A,B). (B) Perturbation of the transcriptional transactivation of wild-type Hoxa13 and Hoxd13 by HFGa13 in a GAL4-fusion assay in COS-7 cells. In this assay, we monitored GAL4 transcriptional activity induced by GAL4 DBD fusion proteins without or with pcDNA3-HFGa13 construct. In a same molar ratio, the HFGa13 form specifically decreases Hoxa13 and Hoxd13 transcriptional activation. (C,D) Intracellular localization of Hoxa13 and HFGa13 proteins. Immunostaining of transfected N-flag tagged Hoxa13 (C) and HFGa13 (D) constructs in COS-7 cells with specific N-flag antibody shows that both have nuclear localization. Note an additive cytoplasmic signal with the HFGa13 construct. (E-K) Hematoxylin and Eosin stained sections of E18 control (E,F) or infected (G-K) guts. (E) Normal midgut with thin and long villi. (F) Normal hindgut with flat and short villi. (G) HFGa13 mesodermally infected midgut has wild-type midgut epithelium. Hoxa13 (H) and Hoxd13 (J) mesodermally infected midgut shows hindgut-like epithelial transformation (as shown by arrows). HFGa13 midguts co-infected with either Hoxa13 (I) or Hoxd13 (K) show rescue of the epithelial hindgut phenotype. (L) Hoxd13 and HFGa13 mesodermal midgut co-infection show presence of virus (detected by 3C2-Ab, L1), and ectopic HFGa13 (detected with Hoxa13 probe, L2) and Hoxd13 (L3) co-expression, which is associated with normal epithelial phenotype.
