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Fig. 4. Pho is directly involved in maintaining eve CNS expression. (A) GFP expression driven by the eve RR regulatory region in the wandering third instar larval CNS, in a wild-type background. When a region including PRE300 was removed from the RR transgene construct (to generate RN, see text), GFP expression was not maintained to the third larval instar (not shown). (B) Mutation of a single Pho-binding site decreased the intensity and penetrance of GFP expression (sequences shown in Fig. 3). This line is representative of five out of the eight lines with normal embryonic expression; the other three showed essentially complete loss of expression at this stage, similar to F. (C,D) The same GFP-RR transgenic line shown in A in either a doubly homozygous phol^81A, pho¹ (C) or a homozygous pho¹ (D) mutant background. (A-D) Arrows/arrowheads indicate eve-positive aCC/RP2 neurons in two adjacent hemisegments. (B-D) Note that GFP expression is decreased in both aCC and RP2 neurons, and the effect tends to be more pronounced in RP2 neurons. (E,F) Two GFP-RR lines inserted at the same chromosomal location (site 25C) (Bateman et al., 2006) using the RMCE system, one with an unaltered PRE300 (E), the other with the single Pho site mutated (F). Mutation of the Pho site at this chromosomal location abolishes specific expression, as well as increasing background expression throughout the CNS.

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