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First published online February 20, 2009
doi: 10.1242/10.1242/dev.027318

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Hindsight modulates Delta expression during Drosophila cone cell induction

¹ Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, 101 College Street, Toronto, Ontario, Canada M5G 1L7.
² Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Model for cone-cell induction. A schematic of the cone-cell induction pathway, based on published data (Canon and Banerjee, 2003; Flores et al., 2000; Tsuda et al., 2006; Tsuda et al., 2002; Xu et al., 2000), showing how the combination of the Notch (N) ligand Delta (DL) and the EGFR ligand Spitz (SPI), emanating from the R1/R6 precursor cell, leads to the activation of the cone-cell determinant D-Pax2 in the adjacent cone-cell precursor. Delta transcription is derepressed in a precise window of time by the activation of the EGFR in the R1/R6 photoreceptor precursor cells. The Lozenge (LZ) transcription factor is required in both cells to activate Bar and to co-activate D-Pax2 and Prospero (Pros) in the cone-cell precursor. From the results in this paper, we hypothesize that Hindsight (HNT) is required in the R1/R6 precursors to regulate Dl transcription in a pathway independent of Charlatan (CHN).

View larger version (82K): [in this window] [in a new window]   Fig. 2. hnt mRNAi knockdown eye phenotypes. (A-D') Apical views of third-instar larval eye discs stained with anti-HNT antibody (red) and (A',B' and inset) anti-ELAV antibody (green) and (D') anti-SENS (green) and anti-BAR (blue). Anterior is to the right. (A,A') A no-driver control disc: UAS-hnt mRNAi2A/+; UAS-hnt mRNAi2B/+. (A) Developing ommatidial clusters express HNT posterior to the furrow. (A') All of the cells expressing HNT also express ELAV, demonstrating that they are photoreceptor (R) precursor cells. No other apical cells stain for HNT. (B,B' and inset) An hnt RNAi mutant disc (UAS-hnt mRNAi2A/pGMR-Gal4; hnt mRNAi2B/+). (B) HNT is expressed in the R8, R2 and R5 cells, at low levels initially in, but then absent from, the R3 and R4 cells. HNT is very reduced, or absent from, the more-posterior R1, R6 and R7 cells. (B' and inset) Cells that lack HNT expression still express ELAV (arrowheads in B' inset), demonstrating that these cells are neuronally determined. (C) Wild-type eye disc stained with anti-HNT antibody to show the normal configuration of the R precursor cells in a developing cluster. (D,D') An hnt mRNAi mutant disc (UAS-hnt mRNAi2A/pGMR-Gal4; hnt mRNAi2B/+). (D) HNT expression is reduced after knockdown. (D') Specifically, HNT expression is retained in three cell nuclei: the SENS-positive R8 nucleus and the neighboring R2 and R5 nuclei. HNT is reduced/absent in the BAR-positive R1/R6 pair and later absent from the R3/R4 pair and the R7 nucleus. In more-posterior rows HNT expression is occasionally seen late in the R7 nuclei (arrowheads). (E-G) Light-microscope pictures of the external adult eye. Anterior is to the right. (E) A UAS-hnt mRNAi2A/pGMR-Gal4 fly eye. The eye of this escaper fly has several defects. The surface of the eye is glossy and most facets lack intact lenses. The red pigmentation is patchy and there is a large necrotic scar (arrowheads) and smaller black pocks, which may indicate ruptured lenses. (F) A lz-Gal4/Y; UAS-hnt mRNAi2A/+ fly eye. The eye of this fly has a gradient of pigmentation lessening towards the posterior side. The eye has facets, but is somewhat smoothened compared with a wild-type eye. (G) The eye of this spa-Gal4/+; UAS-hnt mRNAi2A/+ fly has a wild-type appearance. The pigmentation is normal and the facets all have intact lenses.

View larger version (113K): [in this window] [in a new window]   Fig. 3. Cone precursor cell phenotypes in hnt mutant eye discs. Third-instar larval eye discs. Anterior is to the right. (A-B') Antibody staining with anti-CUT (green) and anti-ELAV (red). (A,A') A no-driver control disc: UAS-hnt mRNAi2A/+;UAS-hnt mRNAi2B/+ shows a wild-type pattern of CUT expression in the cone precursor cells beginning at about row 7 of ELAV expression. (B,B') In an hnt mRNAi mutant disc (UAS-hnt mRNAi2A/pGMR-Gal4;hnt mRNAi2B/+) of the same age, there is delayed and reduced expression of the cone-cell precursor marker CUT. (C-C'') Antibody staining with anti-CUT (green) and anti-HNT (red). A clone of the allele hnt1142 (bounded by arrowheads) is marked by a lack of HNT and CUT expression (green). (D,D') Antibody staining with anti-CUT (red) and anti-BAR (blue), and in D also with anti-HNT (green). In this hnt1142 clone, within the ommatidium where only R1/R6 and R7 cells are mutant for hnt (dotted line), the levels of CUT staining are significantly reduced relative to the cone-cell staining in adjacent wild-type ommatidia (asterisk in D'). The disc has been rotated to clearly show the R1/R6 cells, but has been serially reconstructed to score the remaining HNT+ R cells. An adjacent mosaic ommatidium (arrowheads) has only one CUT-positive cone cell, but is not counted because it has only one BAR-positive cell. (E-E'') Antibody staining with anti-D-PAX2 (green) and anti-HNT (red). Larval eye discs with several clones of hnt1142 (bounded by arrowheads and identified by their lack of HNT expression) have no D-PAX2 expression (green) compared with the adjacent wild-type tissue.

View larger version (92K): [in this window] [in a new window]   Fig. 4. R1/R6- and R7-precursor-cell specification in hnt mutant eye discs. Third-instar larval eye discs stained with antibodies for (A,B) BARH1 (green) and (C,D) for Prospero (PROS, red). The insets also show staining for the pan-neural marker ELAV (blue). (A) A no-driver control disc: UAS-hnt mRNAi2A/+;UAS-hnt mRNAi2B/+ shows wild-type anti-BAR antibody staining in the determined R1/R6 precursor nuclei. (B) In a hnt mRNAi mutant disc (UAS-hnt mRNAi2A/pGMR-Gal4;hnt mRNAi2B/+) the anti-BAR antibody staining looks very similar to the control. There is slightly elevated staining in the peripodial membrane compared with the control (arrow), only some of which is shown in this image. (C) A no-driver control disc: UAS-hnt mRNAi2A/+;UAS-hnt mRNAi2B/+ shows wild-type anti-PROS antibody staining in determined R7 precursor cell nuclei and in the cone-cell precursor nuclei. (D) In a hnt mRNAi disc (UAS-hnt mRNAi2A/pGMR-Gal4;hnt mRNAi2B/+), the anti-PROS antibody staining in the R7 cells looks like that in the control. In the inset we confirm that these PROS-positive nuclei are the R7 precursor cell nuclei because they express the neural marker for ELAV (blue), which does not stain cone-cell precursor nuclei. In this knockdown disc, there is reduced anti-PROS staining in the cone-cell nuclei compared with the control disc shown in C and C inset. These nuclei do not stain with anti-ELAV (blue) in the D inset.

View larger version (132K): [in this window] [in a new window]   Fig. 5. hnt RNAi affects Delta expression. Basal views of third-instar larval eye discs stained with (A,B) anti-HNT antibody (red) and anti-BARH1 antibody (blue) to mark the R1/R6 precursor nuclei or (C-D') anti-β-galactosidase antibody (green) to detect Dl05151 enhancer trap expression and anti-BARH1 antibody. (A) A no-driver control disc: UAS-hnt mRNAi2A/+; UAS-hnt mRNAi2B/Dl-lacZ shows HNT expression (red) in all the R1/R6 precursor cells (blue). (C,C') These same cells (blue and dotted lines in C') express high levels of Dl (green) all the way to the posterior of the disc (left). (B)A lz-Gal4/Y; UAS-hnt mRNAi2A/+; UAS-hnt mRNAi2B/Dl-lacZ eye disc lacks HNT expression (red) in almost all of the R1/R6 precursor cells (blue). (D,D') There is reduced or absent Dl expression (green) in 37% of the R1 and R6 precursor nuclei (blue and dotted lines in D'). (E-H) Light-microscope pictures of the external adult eye. Anterior is to the right. (E) A lz-Gal4/+; UAS-hnt mRNAi2A/+ fly eye, as described in detail in Fig. 2F. (F) A control lz-Gal4/UAS-Dl::GFP fly eye looks like a wild-type eye externally. (G) A lz-Gal4/+; UAS-hnt mRNAi2A/UAS-Dl::GFP fly eye is partially rescued relative to the hnt knockdown phenotype shown in E. The eye has almost normal pigmentation, no glossy surface, but is still slightly rough compared with a wild-type eye. (H) A control lz-Gal4/+; UAS-hnt mRNAi2A/UAS-lacZ fly eye is not rescued relative to the HNT knockdown phenotype shown in E. (I-J') Basal view of third-instar eye discs stained with anti-CHN antibody (red) and anti-BAR antibody (blue in inset). (I) A no-driver control disc: UAS-hnt mRNAi2A/+; UAS-hnt mRNAi2B/+ shows CHN expression in a band of basal nuclei around the furrow and in a very few late R precursor cell clusters at the posterior edge of the disc (arrowhead). (J,J') In UAS-hnt mRNAi2A/pGMR-Gal4; hnt mRNAi2B/+ eye discs, the early CHN expression is unaffected. In the late-developing R-cell clusters, where HNT expression is knocked down, there is novel CHN expression in some of the R-cell precursors (arrows in J'), but not in the BAR-positive R1 and R6 precursor cells (blue in J') that emit the DL signal required for cone-cell induction.

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