Rfx is a Ch-enriched gene that is directly regulated by ato.
(A–C) Rfx is expressed in a ‘Ch-enriched’ pattern despite being required for both Ch and ES ciliary differentiation. (A) Early in neurogenesis, Rfx protein is present in Ch precursors but not ES precursors. (B) Later expression is strong in Ch lineages and weak in ES lineages. (C) During differentiation, Rfx protein is largely confined to Ch neurons. (D) Co-expression of Rfx mRNA and ato protein in Ch precursor cells. (E) Three segments from embryo stained to detect Rfx (magenta) and sc (green) proteins. There is no expression of Rfx in sc-expressing ES precursor cells. (F) Schematic of first three exons of Rfx gene, showing the location of separate Ch and ES enhancers; the tested E box is indicated (‘E’); lines indicate fragments tested in GFP reporter assay, with a summary of their expression. (G) GFP driven by RfxA enhancer is expressed early in Ch lineages. GFP mRNA is coexpressed with ato protein. (H) Mutation of an EATO box in RfxA abolishes the early Ch expression of GFP; Ch cells are marked by ato expression. (I,J) RfxA-GFP is ectopically expressed in response to ato misexpression. (I) Expression of RfxA-GFP in scaGal4 driver background (wild type). (J) Ectopic expression of RfxA-GFP in embryo in which ato protein and its dimerisation partner, daughterless (da), are jointly misexpressed in the ectoderm. (It has been shown that proneural factor activity in embryos is limited by da levels such that misexpression of a proneural factor alone has little effect ). (K,L) GFP driven by RfxB is not expressed at stage 11 (when ES and Ch precursors are present) (K) but is expressed later in ES lineages (L). We note that the RfxB enhancer also contains an EATO motif even though the enhancer is not active in Ch lineages; however, we cannot rule out the possibility that this motif is a functional ATO binding site in the context of the intact Rfx locus.