Pros and dPax2 are sufficient to recruit cone cells, but from different cell populations. (A,B,E,F,I,J,M) Retinas from 45% pupa (A,B,E,F,I,J,M) were immunostained for CC nuclei (Cut, green in (A,A',E,E',I,I',M,M')), PR nuclei (Otd, magenta in (A,A'E,E'I,I',M,M')), and PPC nuclei (BarH1, green in (B,F,J) or white in (B',F',J')), and cell outlines were revealed by Dlg staining (black in (B,F,J)). (C,D,G,H,K,L) Adult eyes were stained with toluidine blue (C,G,K) or immunostained (D,H,L) with the R7 opsins Rh3 (magenta) and Rh4 (blue) and fluorescently labeled phalloidin was used to mark the actin-rich apical surfaces of photoreceptors (green). (N) Quantification of PRs (magenta), CCs (green), and PPCs (blue). (O) Summary of phenotypes observed with the gain-of-function (GOF) experiments (top) and wild-type Pros and dPax2 expression patterns (bottom). MC, mystery cells , a potential source of ectopic CCs in the double GOF experiments. (A-D) Control pupal eyes form eight PRs (A,A'), four CCs (A,A'), and two PPCs (B,B'), and adult eyes form a trapezoid of six large outer PR rhabdomeres surrounding a smaller, central R7 PR (C) expressing Rh3 and Rh4 (D). (E,F) In sev> Pros pupal retinas, ectopic Cut-positive CCs are observed in ommatidia with a full complement of PRs (E, circle), and only one PPC is commonly observed (F,F', circle). Ommatidia with a complement of four CCs occasionally form an extra R7 (E, dotted circle; G,H). Ectopic CCs are also observed in sev>dPax2 pupal retinas (I,I'). PPCs are disorganized in sev>dPax2 retinas, but do not change significantly in number (J,J',N). PR number is reduced by one in sev>dPax2 pupal retinas (I,I') and R7s are frequently absent from adult eyes (K,L). (M) In sev>pros+dPax2 pupal eyes, one ectopic CC per ommatidia is frequently observed (circle), but PR and PPC numbers remain unchanged (N) (also see Additional file 1C,D). *P < 0.05, **P < 0.001. Error bars represent standard deviation.