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First published online 15 November 2006
doi: 10.1242/dev.02669

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Direct control of neurogenesis by selector factors in the fly eye: regulation of atonal by Ey and So

Ophthalmology Department, Harvard Medical School/MEEI, Boston, MA 02114, USA.

View larger version (61K): [in this window] [in a new window]   Fig. 1. Independent regulation of ato in the eye and other sensory organs. (A) Schematic summarizing neuronal morphogenesis in the eye primordium, which begins early in the third larval stage (L3), when eye progenitor cells initiate transcription of ato. Development of the 800 single eyes or ommatidia occurs progressively, as consecutive rows of eye progenitors from posterior to anterior (horizontal arrow) begin to differentiate. This transition is marked by a visible indentation in the epithelium called the morphogenetic furrow (MF). Thus, in the L3 eye disc, one can visualize cells at various stages of development. Anterior to the Ato-positive cells lie retinal progenitors expressing the eye-specification or RD factors Ey, So and Eya. Posterior to the ato domain, one finds differentiating neuronal clusters and accessory cells in progressively more advanced stages of development (Wolff and Ready, 1993). The enlarged diagram (upper right) shows the early (blue and dark blue) and late (yellow) phases of ato transcription. The position of ato expression relative to the Senseless and Eya domains is indicated by the green (Senseless) and red (Eya) bars. (B) Construct maps and relationship to the genomic ato region. The 3'atoM'- ßgal (2.1 kb) and 3'atoM"-ßgal (2 kb) constructs, which overlap by 1.5 kb but differ at both ends, drive expression in essentially the same pattern. (C-F') In situ hybridization to LacZ mRNA (except for inset in C). In all images, discs are oriented posterior to the left and dorsal up. (C,C') 3'atoFL- ßgal mRNA reflects ato expression in the eye (C), JO (C and C inset) and CH (C'). The inset in C shows overnight staining for ß-gal activity. Arrows point to JO in the antenna (C) and femoral CH in the leg (C'). Expression in the antenna occurs at a much lower level than in the eye. (D,D') 3'atoL- ßgal is expressed in JO (D) and femoral CH (D'). Diffuse low level expression can also be detected upon longer staining anterior to the MF and throughout the antennal disc, but not at the sites of endogenous ato expression by the MF or in the ocellar regions. (E,E') 3'atoM'- ßgal drives expression in the eye (E), but not in antenna (E) or leg (E'). Expression is also observed in the ocellar region (arrowhead). (F,F') 3'atoR- ßgal does not drive expression in eye, antennal or leg discs.

View larger version (75K): [in this window] [in a new window]   Fig. 2. A 348 bp fragment contains a core module for ato activation in eye progenitors. (A) Schematic showing the DNA fragments driving reporter gene expression in the different constructs. The green bars IC1, IC2, A1 and A2 mark the relative positions of the evolutionarily conserved DNA sequences investigated in this work. (B-F') All panels (except D',E',F') show in situ hybridization to LacZ mRNA. Staining times differ and were optimized for visualization of either the entire stripe (large panels) or the initial clusters (small panels). For a summary of relative levels of expression for all constructs see Fig. S1 in the supplementary material. Panels D',E',F' show confocal images of triple stainings for the Sens (green), ß-gal (blue), and Eya (red) proteins. (B) Early phase of ato expression as reported by 3'atoM"- ßgal. (C) Loss of eye disc expression in 3'atoM"-348-ßgal. A faint signal could be detected along the margins of the eye disc after overnight ß-gal staining (not shown). (D-D') 3'ato1.2- ßgal is sufficient to drive high level reporter gene expression in the early ato pattern including the initial clusters (D,D'). The anterior margin of ß-gal protein expression (blue) lies anterior to Sens (green) but posterior to the anterior margin of the Eya domain (red) (D'). (E-E') 3'ato348- ßgal drives reporter gene expression in a stripe (E). However, the initial clusters do not form (E'). Activation of the reporter gene (blue) occurs in more anterior progenitors as shown by the shift of the anterior margin of ß-gal protein expression (blue) farther away from Sens (green) and closer to the anterior border of the Eya domain (red) (E'). (F-F') 3'ato1.2-298- ßgal drives reporter gene expression in a stripe (F). However, the initial clusters do not form (F'). The anterior margin of ß-gal protein expression (blue) appears to be minimally affected (F').

View larger version (89K): [in this window] [in a new window]   Fig. 3. Genetic control of 3'ato348- ßgal expression by RD factors.(A-C) Wild-type L3 eye discs (A) stained for ß-gal (blue) to detect expression of the 3'ato348- ßgal reporter, and for the pan-neural marker Elav (brown) to detect developing neuronal clusters. The 3'ato348- ßgal reporter is not expressed and neurons do not form in so1 (B) or eya2 (C) mutant discs. (D-F) dpp-Gal4-driven expression of Ey (D) or So+Eya (E) in the wing disc activates expression of the 3'ato348-ßgal reporter. A negative control (dpp-gal4 alone) is shown in F; the inset shows the pattern of Gal4 expression in a dpp-Gal4 UAS-lacZ wing disc. (G) dpp-Gal4-driven expression of Ey (D) is not sufficient to induce the 3'ato348- ßgal reporter in the so1 mutant.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Ey and So bind specifically to the core eye-enhancer.(A) Sequence of two potential binding sites for So (red) and Pax6 (blue) present in probe III. Consensus sequences are shown above. The sequence changes introduced to disrupt protein-DNA interactions are underlined. (B) Schematic showing the six overlapping probes (I-VI) used in EMSA. Green bars mark the conserved sequences A1 and A2. The relative position of the So and Pax6 sites within A1 is indicated. (C) EMSA with Ey protein. The position of the shifted Ey-III complex is marked by an arrow. Ey shifts probe III (lane 4) but not other probes (lanes 2,3,5,6,7). The binding of Ey to probe III can be competed by unlabeled III DNA (lane 10) but not by unlabeled Pax6MUT-III DNA (lane 11). Probes (I-VI), protein (Ey) and/or competitor DNA (S,NS) added to each reaction are listed above each lane. S, specific: non-labeled probe III DNA. NS, non-specific: non-labeled probe III with the Pax6MUT site. Reticulocyte lysate (L) only was added in the negative controls (lanes 1,8). The lower band (arrowhead) reflects a non-specific shift due to the lysate. (D) EMSA with So protein (GSTSoSixHD). The position of the shifted GSTSoSixHD-III complex is marked by an arrow. GSTSoSixHD shifts probe III (lane 4) but not the other probes (lanes 2,3,5,6,7). The binding of So to probe III can be competed by unlabeled DNA with a So-binding site, but not by unlabeled SoMUT-III DNA. Probes (I-VI), protein (GSTSoSixHD) and/or competitor DNA (S,NS1,NS2) added to each reaction are listed above each lane. GST was added to the negative controls (lanes 1,8). S, unlabeled soAE oligo [So-binding site from Pauli et al. (Pauli et al., 2005)]; NS1, unlabeled probe II DNA; NS2, unlabeled probe III with the SoMUT site.

View larger version (93K): [in this window] [in a new window]   Fig. 5. In vivo requirement for cis sites and direct interaction between Ey and So. (A-D,F,G) Detection of LacZ mRNA by in situ hybridization; in all cases, the staining reaction was carried out for 3 hours. (A) 3'ato348-ßgal reporter gene expression. (B) The introduction of several base-pair changes in the Ey-binding site results in loss of reporter gene expression in 3'ato348Pax6MUT-ßgal. (C) The introduction of several base-pair changes in the So-binding site results in loss of reporter gene expression in 3'ato348SoMUT- ßgal. (D) The introduction of multiple base-pair changes in both So- and Ey-binding sites results in loss of reporter gene expression in 3'ato1.2Pax6-SoMUT-ßgal. A faint signal as a stripe and/or along the margins of the eye disc was detected in some of the transgenic lines after overnight ß-gal staining (not shown). (E) Ey and So can directly interact at the protein level. A GST-SoSixHD fusion protein (containing the Six and homeobox domains) binds full-length Ey protein. GST alone does not bind Ey, and GST-SoSixHD does not bind luciferase. 10% of the 35S-labelled proteins and 100% of the pulled-down yields are shown. (F) Insertion of three As between the So- and Ey/Pax6-binding sites in 3'ato348+3A- ßgal results in a reduction in reporter gene expression. (G) Insertion of six As between the So- and Ey/Pax6-binding sites in 3'ato348+6A-ßgal severely affects reporter gene expression. A weak signal was detected in some of the transgenic lines after overnight ß-gal staining (not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Regulation of ato and integration of the ey/Pax6 and ato pathways. (A) The regulatory elements controlling the early phase of ato expression in the fly eye lie within a 1.2 kb region located 3.1 kb downstream of the ato transcription unit (t.u.). The early-ato stripe and its intermediate clusters result from the integration of multiple regulatory inputs through separate enhancers. A likely model for the regulation of ato by the RD network involves the formation of an Ey-So (or Ey-So-Eya) complex onto adjacent Pax6 and So cis-regulatory sites present within the evolutionarily conserved A1 box. (B) Proposed integration of eye specification and neurogenic inputs during eye development. The RD network directly controls ato transcription in eye progenitor cells and, most likely indirectly, modifies the neurogenetic program downstream of ato. Black arrows indicate regulatory interactions based on genetic and molecular evidence (solid) or genetic evidence only (dashed).