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First published online 24 July 2008
doi: 10.1242/dev.022194

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Self-modulation of Notch signaling during ommatidial development via the Roughened eye transcriptional repressor

David del Alamo^* and Marek Mlodzik

Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.

View larger version (123K): [in this window] [in a new window]   Fig. 1. roe loss-of-function causes defective ommatidial cell recruitment. (A-C) Tangential eye sections with anterior leftwards and dorsal upwards. (A) Wild-type adult eye. Outer photoreceptors (R1-6) and the inner R7 are found in every ommatidium (R8 is in a different plane) surrounded by pigment cells (rhabdomeres are numbered in inset). (B) rn16/rn20 mutant eye with most ommatidia containing fewer R-cells and irregular spacing. (C) Mosaic eye bearing rn16 clones (absence of pigment). roe- ommatidia show a reduced number of R1-R6 cells and often multiple R7s (examples indicated with yellow arrowheads; see Fig. S1 in the supplementary material). There are also defects in spacing (example marked by asterisk). (D-E') Areas posterior to the MF in 3rd instar eye discs (anterior is leftwards) stained for -Elav (red, marking all R-cells), -Boss (turquoise in D, marking R8) and -Pros (blue in E,E'; marking R7). (D) rn16 mutant disc; (E,E') mosaic discs with wild-type tissue marked by arm-lacZ (green in E, outlined in E'). Fewer R cells are recruited per ommatidium. Spacing defects result from the presence of fewer R8 founder cells (examples highlighted by asterisks, see also Fig. 2A,A'). -Pros labels R7 cells (and cone cells in a different plane, which are Elav negative). The multiple R7 phenotype is observed inside mutant tissue (example indicated by yellow arrowhead; wild-type clusters with a single R7 are indicated by arrows). Only the nuclei of R7, R3 and R4 are visible in this focal plane in most clusters. (F) Graph showing the frequency of individual mutant R cells in mosaic ommatidia with a wild-type wt phenotype (random frequency reflecting no requirement is 0.5; n=152 in nine eyes analyzed). R1 and R6 show a significantly reduced frequency, indicating a functional requirement of roe in these cells. (G,H) Pupal retinae stained with -Arm, which delineates cell membranes: (G) wild type (`c' marking cone cells and `1' primary pigment cells in example) and (H) rn16 are shown. The quasi-crystalline lattice of wild-type pupal eyes is severely disturbed in roe mutants. Cone cell, secondary and tertiary pigment cell, and interommatidial bristle numbers are reduced compared with wild type.

View larger version (95K): [in this window] [in a new window]   Fig. 2. roe loss-of-function affects the expression of N-signaling targets. Panels show third instar mosaic imaginal discs bearing rn16 clones, marked by absence of arm-lacZ (red in A,E,F) or Ubi-GFP (red in B-D) and outlined in white in right panels. -Elav is green in B-E. (A,A') -Ato (blue) is detected as a continuous band at the MF and resolves into individual cells (R8) posterior to it. -Ato is reduced in single cells inside the mutant clone (arrows) when compared with surrounding wild-type tissue (arrowheads). (B,B') The Ato reporter line ato5'F9.3-Z (blue), which is repressed by N-signaling during R8 lateral inhibition, is not detected inside mutant tissue. (C,C') The E(spl)mβ1.5-Z reporter line (blue), which is regulated by N signaling, shows higher expression levels inside mutant clones when compared with surrounding wild-type cells. (D,D') The E(spl)m1.1-Z reporter (blue), which is activated by N signaling, is expressed in isolated cells posterior to the MF. Inside mutant tissue, more positive cells are observed when compared with surrounding wild-type tissue. (E-F') Neither N (E,E', blue and monochrome, detected by -Nintra) nor Dl expression levels (F,F', turquoise and monochrome) are affected inside mutant tissue.

View larger version (127K): [in this window] [in a new window]   Fig. 3. Overexpression of Roe phenocopies N loss-of-function phenotypes and represses N target genes during eye development. (A,A') roe overexpression clones (marked by GFP in green, outlined in A') in third instar eye disc cause recruitment of supernumerary photoreceptors (anti-Elav, magenta), similar to N pathway loss-of-function clones (compare with Fig. 5E). (B) Tangential section of adult eye of the genotype sepGAL4, UAS-roe with schematic shown on the right (black and red arrows represent the two chiral forms; green arrows represent symmetrical clusters; black dot shows loss of R cells). Increased levels of Roe expression in R3/R4 precursors often cause the formation of symmetrical R3/R3 type ommatidia (some R4/R4 type are also observed; quantified in C). (C) Quantification of the phenotypes of sev-driven expression of roe, NECD and together. Ectopic expression of NECD causes a high number of R4/R4-type symmetrical ommatidia and R-cell loss, while Roe causes mostly R3/R3-type and occasional R-cell loss. The NECD phenotype is antagonized by Roe co-expression and a reversion to a significant percentage of R3/R3-type ommatidia typical of Roe overexpression is observed (the `loss of R-cell' phenotype is enhanced, see text for details). Total number of ommatidia scored was 391 (roe), 545 (NECD) and 329 (roe + NECD) with at least three eyes per genotype analyzed. (D,D') Third instar eye disc bearing clones of cells expressing two copies of sepGAL4, UAS-roe (marked by absence of Ubi-GFP in green, see Materials and methods). Expression of the R4-specific N reporter m-lacZ (red) is largely suppressed inside the Roe-overexpressing tissue. Cells outside the clone can be either wild type or have one copy of sepGAL4, UAS-roe, which can also suppress m-lacZ to a lower extent (not shown). -Elav is in blue.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Roe represses N signaling targets during leg disc patterning. Panels show leg discs, with anterior leftwards and dorsal upwards. Wg (blue, negative control) is expressed in a ventral wedge at the A/P boundary in a N-independent manner. (A,A') Bib (red, monochrome in A') is expressed in concentric rings in leg discs, corresponding to the presumptive leg segment joints defined by N signaling. (B,B') dppGAL4-driven NECD ectopic expression (marked by GFP, green) causes a cell-autonomous expansion of Bib expression inside the dppGAL4 domain. (C,C') Ectopic expression of Roe under the control of dppGAL4 (green) causes cell-autonomous repression (arrowheads) of Bib (red, monochrome in C'). (D,D') The E(spl)mβ1.5-Z reporter line (mβ-lacZ red and monochrome) is regulated by N signaling and expressed in concentric rings corresponding to presumptive cells of the leg joints. (E,E') dppGAL4-driven ectopic expression of Roe (green) causes cell-autonomous repression (arrowheads) of mβ-lacZ (red, monochrome in E').

View larger version (126K): [in this window] [in a new window]   Fig. 5. roe expression is under the control of N activity. Panels show third instar eye disc regions around the MF; anterior is upwards. (A) In situ hybridization staining: roe is expressed in the MF (yellow arrowhead) and posterior to it. (B,B') -Roe staining (turquoise, monochrome in B'). Roe is a nuclear protein detected at high levels in the MF and at lower levels posterior to it. Roe is higher in interommatidial (progenitor) cells and shows only low levels in photoreceptors (Elav, red). (C) Third instar eye disc posterior to the MF showing -Roe (turquoise) and -Elav (red) staining. Note higher levels of Roe in R1/R6 precursors (examples marked with asterisks). (D,D') High magnification of an ommatidial precluster showing -Elav (red), -Roe (blue) and sevGAL4 UAS-GFP (green) staining (D) and schematic cartoon (D'). Note that sevGAL4 and Roe are co-expressed in R1/R6 precursors prior to Elav detection. (E,E') N loss-of-function clones (marked by absence of Ubi-GFP, green) cause reduction in Roe expression levels (blue, monochrome in E'), both within the MF (compare to B') and posterior to it. Photoreceptors are marked with -Elav (red in E). (F-F'') Clones expressing NECD (marked by co-expression of GFP, green, outlined in F') cause ectopic MF initiation in cells anterior to endogenous MF and ectopic cell-autonomous upregulation of Roe (blue, monochrome in F') posterior to MF. Clones are as expected associated with loss of photoreceptors (note reduction in -Elav, red and monochrome in F''). Close to and within the MF, NECD-expressing clones cause a precocious initiation of the MF state with associated non-autonomous Roe activation (see also Results).

View larger version (55K): [in this window] [in a new window]   Fig. 6. Roe binds E(spl)-C regulatory DNA sequences both in vitro and in vivo. (A) Schematic presentation of the relative position of the 0.3 kb fragment used as a probe for EMSA or amplified in in vivo chromatin immunoprecipitation (ChIP) assays with respect to the E(spl)m gene. Coordinates are based upon `Release 5' of the Drosophila genome sequence (BDGP, http://www.fruitfly.org/). 60 bp fragments denoted as P1-P9 correspond to the probes used in EMSAs described in Fig. S2 in the supplementary material. (B) Representative EMSA experiment using the 0.3 kb fragment (A) as a probe. Lanes are as follows: (1) Free probe (FP) +2.5 µg Su(H), (2) FP + 5 µg Su(H), (3) FP+2.5 µg Su(H) + 2.5 µg Roe, (4) FP + 2.5 µg Su(H) + 2.5 µg Roe + excess cold probe (ECP) as competitor, (5) FP + 2.5 µg Roe, (6) FP + 5 µg Roe, and (7) FP + 2.5 µg Roe + ECP. Shifts caused by Su(H) and Roe (lanes 1,2 and 5,6, respectively) are unaffected when both proteins are simultaneously present (lane 3). (C) In vivo ChIP from a wild-type eye imaginal discs. A band corresponding to the 0.3 kb fragment is amplified when the sample is immunoprecipitated with anti-Roe antibody (Roe-ChIP lane, compare with INPUT lane) but not when preimmune NGS was used for IP (mock-ChIP lane). As a control for specificity, anti-Roe did not co-immunoprecipitate DNA from the AttacinA (AttA) promoter. (D) In vivo ChIP from homozygous roe null (rn16) eye discs. No specific ChIP band was amplified when the sample was immunoprecipitated either with anti-Roe antibody or preimmune NGS (mock-IP). Same results were obtained with DNA from the AttA promoter. In both C and D, weak bands can be observed occasionally, owing to nonspecific binding of DNA to the resin used during the IP process. (E) Model for Roe action on N targets. In the absence of N-signaling activity, N target genes are repressed by DNA-bound Su(H) together with transcriptional co-repressors. Upon N activation, Nintra translocates to the nucleus and, together with Mam and other transcriptional co-activators (not shown), binds to Su(H) to turn ON the expression of target genes. We propose a third scenario in which N self-modulates its transcriptional activity by upregulating Roe. Roe binds to regulatory sequences of N target genes independently of Su(H), leading to an attenuated transcriptional activity.

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