1. A microRNA Mediates EGF Receptor Signaling and Promotes Photoreceptor Differentiation in the Drosophila Eye 
Xin Li, Richard W. Carthew 
Cell 
Volume 123, Issue 7, Pages (December 2005)
DOI: /j.cell
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Specific Expression of miR-7 in Photoreceptors
(A–E) Expression of miR-7 RNA in third-instar larval eye discs as detected by in situ hybridization. Posterior is to the right.
(A) A larval eye disc stained for miR-7 RNA. RNA is detected in the morphogenetic furrow (arrow) and cells posterior to the furrow.
(B and C) High-magnification views of a region posterior to the furrow at the focal plane of the photoreceptors (B) and the progenitor cells (C). RNA is detected within the cytoplasm of each photoreceptor neuron and is concentrated in the center of each ommatidium where the R8 photoreceptor resides (arrowheads). The circled ommatidium in (B) has visible photoreceptors labeled.
(D) A GMR-Gal4/UAS-miR-7 disc showing abnormally strong expression posterior to the furrow.
(E) Expression is not detected in a miR-7Δ1 mutant eye disc.
(F–F′′) Double antibody labeling of a tub-EGFP:2x(miR-7') eye disc to detect GFP (F), Elav, which marks all photoreceptors (F′), and color overlap of the two images (F′′).
(G–G′′) Double labeling of a tub-EGFP disc to detect GFP (G) and Elav (G′) proteins and image overlap (G′′). Double labeled cells appear white in image overlaps. Arrows mark the furrow.
Cell  , DOI: ( /j.cell )
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3. Figure 2
Genomic Organization of the miR-7 Gene and Associated Mutations
The miR-7 sequence is embedded within the bancal (bl) gene, oriented in the same direction as the bl transcription unit. Four mature bl transcripts are detected. Transcripts A–C are initiated from a promoter that is different from the promoter used to make transcript D. The EP insertion EP954 is located 52 bp upstream of the D promoter. EP954 was excised by P-transposase activity and excision lines were established. Of the lines, miR-7Δ1 contained the smallest deletion that removed the miR-7 sequence. The mutant is a 6.8 kb deletion that also removes the last two exons of bl and the last two exons of hillarin (Hil), a neighboring gene. For genetic analysis, miR-7Δ1 was placed in trans over the Df(2R)exu1 deletion, which deletes all of the genes shown and additional genes in both directions. The trans-heterozygote exhibited phenotypes similar to the miR-7Δ1/miR-7Δ1 phenotypes. Location of the cluster of conserved Yan binding sites (YBS) is indicated.
Cell  , DOI: ( /j.cell )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
miR-7 Promotes Photoreceptor Differentiation by Repressing Yan
(A and B) Expression of Prospero (green) in the R7 photoreceptor and cone cells, and expression of Elav (red) in all photoreceptors of a GMR-Gal4 (A) or GMR-Gal4/UAS-miR-7 (B) larval eye disc. R7 photoreceptors coexpress Prospero and Elav and therefore appear orange. Arrows denote ectopic R7-like photoreceptors.
(C and D) Anti-Elav stained pupal eyes from wild-type (C) or GMR-Gal4/UAS-miR-7 (D) animals.
(E and F) Expression of Yan protein (green) in progenitor cells of a GMR-Gal4 (E) or GMR-Gal4/UAS-miR-7 (F) larval eye disc. Discs are counterstained for Elav (red) to highlight photoreceptors.
(G) Computational prediction of two conserved miR-7 binding sites in the yan 3′UTR of D. melanogaster and D. pseudoobscura. The position of the 3′ nucleotide in the target site of each 3′UTR is indicated. The seed match is between the 5′ eight bases of the miRNA and its cognate 3′ end of the target mRNA.
(H) Double labeling of a tub-EGFP:YanUTR larval eye disc to detect GFP (cyan) and Elav (red).
(I) A tub-EGFP:Yan(mut)UTR eye disc labeled to detect GFP (cyan) and Elav (red).
(J and K) Expression of tub-EGFP:Yan(mut)UTR (cyan) in progenitor cells of a GMR-Gal4 (J) or GMR-Gal4/UAS-miR-7 (K) eye disc.
(L and M) Expression of tub-EGFP:YanUTR (cyan) in progenitor cells of a GMR-Gal4 (L) or GMR-Gal4/UAS-miR-7 (M) eye disc.
(N and O) Expression of tub-EGFP:YanUTR (cyan) (N) and tub-EGFP:Yan(mut)UTR (cyan) (O) in miR-7Δ1/Df(2R)exu1 mutant eye discs. Discs are counterstained for Elav (red) in all panels. Arrows mark the furrow in (C)–(O).
Cell  , DOI: ( /j.cell )
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5. Figure 4 miR-7 Acts Redundantly to Inhibit Yan
(A and B) Expression of Yan (green) and Elav (red) proteins in larval eye discs counterstained with a dye to highlight all apical nuclei (blue). A wild-type disc section (A) shows that most cells recruited to join ommatidia weakly stain for Yan. In contrast, a miR-7Δ1/Df(2R)exu1 disc section (B) shows an increased number of cells that are recruited within ommatidia and strongly stain for Yan (two examples highlighted by arrows). Note that in both genotypes, Elav-positive cells are predominantly missing nuclear Yan.
(C) Anti-Elav stained pupal eye from a miR-7Δ1/Df(2R)exu1 animal. Shown are several ommatidia with the normal number of eight Elav-positive cells forming characteristic ring-like structures. Only 2% of scored ommatidia showed a defect in photoreceptor number at this stage.
(D and E) Anti-Yan stained larval eye discs from GMR-YanAct (D) and GMR-YanAct miR-7Δ1/Df(2R)exu1 (E) animals.
(F and G) Expression of Elav (red) in larval eye discs counterstained with a dye to highlight apical nuclei (blue). Shown are confocal optical sections of GMR-YanAct (F) and GMR-YanAct miR-7Δ1/Df(2R)exu1 (G) discs.
(H and I) Anti-Elav stained pupal eyes from GMR-YanAct (H) and GMR-YanAct miR-7Δ1/Df(2R)exu1 (I) animals. Shown are several ommatidia in each panel exhibiting incomplete photoreceptor composition.
(J–N) SEM views of adult eyes from wild-type (J), miR-7Δ1/Df(2R)exu1 (K), GMR-YanAct (L), GMR-YanAct miR-7Δ1/Df(2R)exu1 (M), and GMR-YanAct miR-7Δ1/Df(2R)exu1 ; GMR-Gal4/UAS-dsRED:miR-7 (N).
Cell  , DOI: ( /j.cell )
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6. Figure 5 Expression of miR-7 Is Regulated by EGFR and Yan
(A and B) miR-7 expression detected by in situ hybridization in GMR-Gal4 (A) and GMR-Gal4/UAS-EGFR.λtop (B) larval eye discs. Circles highlight individual ommatidia in (A).
(C and D) Eye discs stained for Elav from GMR-Gal4/UAS-EGFR.DN (C) and GMR-Gal4/UAS-EGFR.DN UAS-miR-7 (D) mutants. White circles outline representative ommatidia. Ommatidia in (C) recruit two (occasionally three) Elav-positive cells, which likely represent R8 and R2 or R5. No further recruitment occurs. In contrast, ommatidia in (D) recruit additional Elav-positive cells in positions that suggest they are R3 and R4. Older ommatidia are disorganized, and identification of other additional Elav-positive cells becomes ambiguous.
(E) miR-7 expression in a yan1 mutant eye disc. Each circle outlines a representative ommatidium with extra miR-7-positive cells.
(F) A GMR-Gal4/UAS-YanAct larval eye disc showing reduced staining for miR-7 RNA.
(G) A GMR-Gal4/UAS-Pointed-P1 larval eye disc showing enhanced staining for miR-7 RNA.
Cell  , DOI: ( /j.cell )
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7. Figure 6
Yan Binds Specifically to Conserved DNA Sites upstream of miR-7
(A) CISTER and COMET identified six conserved ETS binding sites clustered approximately 2 kb upstream of miR-7 (see Figure 2 for genomic position). The cluster encompasses 286 base pairs in the D. melanogaster genome and 268 base pairs in the D. pseudoobscura genome. The locations and orientations of the six sites, named E-1 to E-6, are indicated. Below, an alignment of the six sites from the D. melanogaster genome. Highlighted bases are shared between a majority of the sites. The sequences of the E-1 to E-5 sites are identical in both D. melanogaster and D. pseudoobscura. The D. pseudoobscura E-6 sequence is not identical to its D. melanogaster counterpart and is listed in parentheses. E-M is a mutated Ets binding site that is defective for Yan binding (Xu et al., 2000).
(B) Electrophoresis mobility shift complexes of GST or GST-Yan bound to labeled prospero enhancer DNA. The unbound enhancer DNA is shown on the far right as free probe. Indicated binding reactions also contained unlabeled DNA oligonucleotides. Each oligonucleotide contained one of the E-1 to E-6 sites or the E-M site.
Cell  , DOI: ( /j.cell )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7
Model for the Role of Yan and miR-7 in Photoreceptor Differentiation
In the absence of a signal through EGFR, Yan is active and represses the expression of the miR-7 gene. When an extracellular signal is received by EGFR, activated ERK phosphorylates Yan and downregulates its activity, and so it is no longer capable of repressing transcription of miR-7 RNA. In contrast, Pointed-P1 is able to activate miR-7 transcription. The synthesized miR-7 RNA binds to the 3′UTR of yan transcripts, thereby repressing protein synthesis of Yan. This reciprocal negative feedback loop between miR-7 and Yan exerts output upon the differentiation state of cells through the action of Yan upon downstream genes. It is also possible that miR-7 regulates other downstream genes outside of the feedback loop.
Cell  , DOI: ( /j.cell )
Copyright © 2005 Elsevier Inc. Terms and Conditions