DEV022194 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Roe restricts the number of R7 internal photoreceptors. (A) Tangential section of an adult mosaic eye bearing rn¹⁶ homozygous clones. Together with the reduced number of photoreceptors per ommatidium (see Results), loss of roe also causes the appearance of multiple R7 inner photoreceptors (e.g. red arrowhead). (B) Graph showing the quantification of the number of R7 photoreceptors per ommatidium with respect to the number of external photoreceptors. We failed to find any ommatidia with a normal complement of six external photoreceptors and multiple R7 internal photoreceptors. This suggests that extra-R7 photoreceptors are being produced at the expense of external photoreceptors, most likely R1 and R6 (see Results). Sixty-seven ommatidia with multiple R7 photoreceptors from nine mosaic adult eyes were analyzed.

• Supplemental Figure S2 -

  Fig. S2. Roe binds specifically to E(spl)-C regulatory sequences in vitro. (A) Representative EMSA showing Su(H) binding to a 40 bp fragment from the E(spl)m8 regulatory region (see Materials and methods). Su(H) is used as a positive control of this probe. No shift was detected with Roe, indicative that Roe does not bind nonspecifically to DNA. Lanes were as follows: (a) free probe (FP), (b) FP+200 ng SuH), (c) FP+500 ng Su(H), (d) FP+1 µg Su(H), (e) FP+2 µg Su(H), (f) FP+500 ng Su(H)+500 ng Roe, (g) FP+200 ng Roe, (h) FP+500 ng Roe, and (i) FP+1 µg Roe. Addition of Roe does not affect Su(H) binding ability (compare lane f with lane c). (B) Representative EMSA showing Roe binding to 60 bp fragment P1 (see Results and Fig. 5). Lanes are as follows: (1) FP, (2) FP+2.5 µg Roe, (3) FP+5 µg Roe, (4) FP+2.5 µg Roe+Excess cold probe, and (5) FP+2.5 µg Roe+Excess cold non-competitor. Roe shift can be supressed by an excess of cold probe, but not by an excess of the cold 40 bp fragment depicted in A (non-competitor), suggesting that Roe binding to DNA is specific. Results are representative of experiments carried out with fragments P2-P9 (not shown). This suggests that multiple binding sites are present in the original 0.3 kb regulatory fragment (see Results).