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First published online 31 October 2007
doi: 10.1242/dev.012781

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Senseless functions as a molecular switch for color photoreceptor differentiation in Drosophila

Department of Pediatric Ophthalmology, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Inner photoreceptors exhibit differential gene expression during Drosophila eye development. (A-C) Adult photoreceptor (PR) subtypes and relevant cell-specific factors. (A) Inner photoreceptor cells (IPRs) versus outer photoreceptor cells (OPRs): R7 and R8 IPRs express Salm and are surrounded by R1-R6 OPRs. (B) Polarization versus color-sensitive IPRs: all R7s express Pros (pink nucleus), whereas only pale/yellow R8 PRs express Sens (green nucleus). (C) Ommatidial subtypes: DRA ommatidia (two dorsal rows) express Rh3 in both R7 and R8, 30% ommatidia (termed pale) express Rh3 and Rh5 in R7 and R8, respectively, and 70% ommatidia (termed yellow) express Rh4 and Rh6 in R7 and R8, respectively. (D) Transcription factor expression timeline during PR development. From R8 specification at the morphogenetic furrow (MF) onward, Sens expression is R8-specific. Early sens expression is ato-dependent, whereas later expression is Sal gene-dependent. R7 recruitment occurs more posteriorly than R8 specification and coincides with Pros and Salm expression (Domingos et al., 2004; Kauffmann et al., 1996). Otd expression begins at the end of PR recruitment and is expressed in all PRs throughout development (Vandendries et al., 1996). (E-G) Larval (E), 50% pupal (F) and adult (G) eye co-staining with Otd (purple) and Sens (green). E is oriented anterior to posterior, F is oriented dorsal right and includes the DRA (not indicated), and G is oriented dorsal up, distal right, with the DRA boxed. From R8 specification at the MF onward, Sens expression is R8-specific. Sens is expressed in all R8 cells during larval and 50% pupation (E,F), but is absent from adult DRA (G). It should be noted that Sens and Hth are coexpressed in DRA R8 cells from 15-50% pupation (data not shown; and M. Wernet, personal communication) (Wernet et al., 2003).

View larger version (81K): [in this window] [in a new window]   Fig. 2. Transient sens expression rescues inner photoreceptor specification. (A) Coronal section of 55% pupal and 70% pupal heads from sca109.68-GAL4, UAS-nuclear GFP (sca>nGFP) Drosophila. All PRs are marked with Elav (purple). GFP (green) is present in R8 cells at 55% APF, but not after 70% APF. (B) Summary of sens expression in full LOF and late LOF experiments. (C-E) Adult retinal sections stained for Salm (green); dorsal left, distal up. (C) UAS-sens/+, sensE1/TM6B: the R7 and R8 layers of Salm-positive nuclei are labeled. (D) sensE1 full LOF eyes (see Materials and methods) rarely develop IPRs (arrowhead). (E) sensE1 mutant eyes with sca109.68>sens (sens late LOF) develop two Salm-positive layers of IPRs (arrows). (F-H'') Adult cryosections of control and sensE2.1 late LOF retinas stained for Salm (green, F-H,F'-H'), Pros (purple, F'-H'), or Sens (white, F''-H''). F-F'', G-G'', and H-H'' represent eyes co-stained for all three factors. Pros stains distal Salm-positive cells in both control and sensE2.1 late LOF retinas, and the majority of Salm-positive cells in sensE2.1 full LOF retinas. Sens is expressed in R8 cells only in control retinas (F'').

View larger version (107K): [in this window] [in a new window]   Fig. 3. Sens is required for inner photoreceptor differentiation. Adult retinas from wild-type (A-C,G,I,K,M) or sensE2.1 late LOF (D-F,H,J,L,N) Drosophila. (A-J) Cryosections (10 µm); dorsal left, distal up. (K,L) Plastic sections (1 µm). (M,N) Optical sections from retinal whole-mounts. Samples were stained for Rh3 (purple, A,B,D,E,H-J; red, M,N), Rh4 (green, A,D,G,H,M,N), Rh5 (green, B,E), Rh6 (green, C,F; purple, G), Elav (purple, C,F; green, I,J), or the actin/rhabdomere marker, phalloidin (blue, M,N). R7 and R8 layers of WT rhabdomeres are clearly separated within the retina (A-C,G,I), but are ambiguously positioned in sens late LOF eyes (D,E,H,J). Wild-type control R8 nuclei (I, Elav, green) lie along the base of the retina (dotted white line), whereas sens late LOF R8 nuclei (F,J) are often located midway in the retina (purple, F or green, J). Wild-type control plastic sections (K) show a single small IPR rhabdomere (arrow); OPR rhabdomeres are circled in black; single ommatidia are outlined with a dotted blue line. In sens late LOF eyes (L), two small rhabdomeres are observed in the R7 and R8 positions (arrows). Rh3 and Rh4 staining reveals a single opsin-expressing cell (the R7) in wild-type ommatidia (M), whereas two opsin-positive cells (R7 and sens-negative `R8') are observed in sens late LOF ommatidia (N). Phalloidin marks OPR rhabdomeres. Inset in N is higher magnification of a single ommatidium with Rh4 expression in R7, and Rh3 expression in R8. (O) Summary of control, full sens LOF, or late sens LOF phenotypes. `R2' represents the pre-R8 cell that transforms into an R2/R5 OPR; `R8' represents sens-negative R8s. See Fig. 1 for color code.

View larger version (49K): [in this window] [in a new window]   Fig. 4. Sens alone can both repress R7 and activate R8 features. Adult cryosections from control and sens GOF Drosophila. (A) IP-GAL4; UAS-lacZ; (B,D) yw67; UAS-sens/CyO; TM2/TM6B; (C,I) IP-GAL4; Sp/CyO; TM2/TM6B; (E-G,J-L) IP-GAL4; UAS-sens (H) IP-GAL4; UAS-sens/CyO; TM2/TM6B. All retinas oriented dorsal left, distal up; R7 and R8 layers are bracketed. Sections were stained for ß-gal (purple, A), Rh3 (purple, B,C,F,G), Rh4 (green, B,F), Rh5 (green, C,G; purple, D,H), Rh6 (green, D,H; purple, L), Sens (green, E,I-L) and Pros (purple, I-K). Note that many sens-misexpressing R7 cells exhibit an R8-like proximal nucleus (E,J-L). (M) Summary of control or sens GOF phenotypes. `R7' represents sens-misexpressing R7 cells in sens GOF eyes.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Sens directly represses Rh3 and Rh4 promoter activity. (A) Position-weighted matrix (PWM) of Gfi1 binding sites (top), table of two known Sens binding sites, R21 and S-box (Jafar-Nejad et al., 2003), and sites within the Rh3 and Rh4 promoters with >70% homology to the consensus. Corresponding PWM scores are listed. Nucleotides that are present >20% in the PWM data set are highlighted green. Rh3 and Rh4 promoter diagrams represent the conserved RCSI/Pax6 binding site present in all Rh promoters (Papatsenko et al., 2001; Sheng et al., 1997) (gray), K50 Otd-binding sites (Tahayato et al., 2003) (blue) and potential Sens binding sites (green). (B) EMSAs with Rh3 (-247 to +18) and Rh4 (-159 to +85) promoters and 0, 50 or 500 ng His-SensZF. (C) Relative luciferase activity in Drosophila S2 cells transfected with pAc5.1 or pAc-Sens and pGL3 with or without Rh3 (-247 to +18), Rh4 (-159 to +85), Rh5 (-236 to +50) or Rh6 (-555 to +121) promoters. **, P<0.01 compared with pAc alone. (D) Relative luciferase activity of Rh3 or Rh4-containing pGL3 reporters with mutated Sens binding sites (AATC core GGTC). Sites correspond to those in A. *, P<0.05 compared with pAc alone. (E,F) X-Gal staining of cryosections from transgenic lacZ reporter lines carrying wild-type or Sens mutant binding sites in the Rh3 (E) or Rh4 (F) promoters. R7 and R8 layers are bracketed.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Sens and Otd regulate R8 opsin genes in vitro. (A) Table of potential Sens binding sites in Rh5 (-239 to +50) and Rh6 (-555 to +121) promoters. Underlined sequences represent overlapping Otd-binding K50 sites (Tahayato et al., 2003). Promoter diagrams represent binding sites for Otd (blue), Pros (pink) (Cook et al., 2003), or Pax6 (gray) (Papatsenko et al., 2001). (B) EMSA with Rh5 (-239 to +50) and Rh6 (-555 to +121) promoters and 0, 50 and 500 ng His-SensZF. (C-E) Relative luciferase activity of pGL3 or pGL3 with Rh3, Rh4, Rh5 or Rh6 promoters in S2 cells transfected with pAc, pAc-Otd and/or pAc-Sens. (C) *, P<0.01 and **, P<0.001, compared with pAc alone. (D) *, P<0.01 and **, P<0.001, compared with Otd alone. (E) *, P<0.005 and **, P<0.001, compared with Otd alone.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Sens and Pros oppositely regulate R8 opsin genes in vitro. (A) Relative luciferase activity of pGL3 or pGL3 with Rh3, Rh4, Rh5 or Rh6 promoters in S2 cells transfected with pAc, pAc-Otd and/or pAc-Pros. (B) Otd+Sens (green) versus Otd+Pros (purple) regulation of IPR opsin expression in S2 cells. Values are normalized to cells transfected with Otd alone, and represent the average of two experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Model for R7 versus R8 color PR differentiation in Drosophila. (A) Summary of phenotypes observed in inner PRs from wild type, late sens LOF (see Figs 2, 3), sens GOF (see Fig. 4) and pros LOF (Cook et al., 2003) eye-specific mutants. (B) Model for pros, sens and otd function in wild-type R7s and R8s.

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