Drosophila Aop imposes a delay on E(spl)-mediated repression of Ato during R8 specification

Drosophila retinal patterning requires the expression of Atonal (Ato) through coordinated regulation of 5’ and 3’ enhancer modules. ato-3’ directs initial expression of Ato which then directs autoregulation via 5’-ato. Notch (N) signaling also regulates 5’-ato, first enhancing Ato expression and later repressing Ato by inducing E(spl) bHLHs. N signaling balances these opposing functions by directing its obligate nuclear transcription factor, Suppressor of Hairless (Su(H)), only in repressing 5’-ato. In this study, we reveal a novel and more nuanced role for Su(H) in its regulation of 5’-ato. During retinal patterning, Su(H) is required for the expression Anterior open (Aop), which, in turn, promotes 5’-ato activity. We demonstrate that Aop is induced early in retinal patterning via N pathway activity, wherein Aop is required cell-autonomously for robust Ato expression during photoreceptor specification. In aop mutants, expression from both ato enhancers is perturbed, suggesting that Aop promotes the Ato autoregulation through maintenance of ato-3’ activity. Clonal analysis indicates that Aop indirectly opposes E(spl)-mediated repression of Ato. In the absence of both Aop and E(spl), Ato expression is restored and the founding ommatidial photoreceptors, R8s, are specified. These findings suggest that N signaling, through a potentially conserved relationship with Aop, imposes a delay on ato repression, thus permitting autoregulation and retinogenesis. Author Summary The eye of the fruit fly has served as a paradigm to understand tissue patterning. Complex intercellular signaling networks cooperate during retinal development to allow cells to become specialized visual-system precursor neurons at a specific time and place. These neurons are precisely spaced within the developing retina and later recruit other cells to form the repeated units that comprise insect eyes. The exact placement of each precursor cell precipitates from the precise regulation of the atonal gene, which is first expressed in a cluster of (10-20) cells before becoming restricted to only one cell from each cluster. The Notch signaling pathway is required for both aspects of atonal regulation, first permitting up-regulation within each cluster, and then the subsequent down-regulation to a single cell. However, the connection between these two modes of Notch signaling had remained unclear. In this report, we have identified that the anterior open gene is required to impose a delay on the restrictive mode of Notch signaling, permitting the initial up-regulation of atonal to occur freely. In flies mutant for anterior open, atonal bypasses its own up-regulation and proceeds directly to its singled-out pattern but with significantly diminished robustness than occurs normally.

5 81 MAPK activation is cell-autonomously dependent upon Ato, and may, therefore, be dependent 82 upon N signaling as well (Chen and Chien, 1999;Lim and Choi, 2004). Thus, the correlation 83 between MAPK activity and Ato repression bears further investigation for possible nodes of N-84 EGFR crosstalk with regard to Ato expression.

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To date, only E(spl)M8 (referred to as M8) has emerged as an apparent genetic link

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Anterior open (Aop, also known as Pokkuri or Yan), an ortholog of human TEL1/ETV6, 100 may also link N and EGFR signaling during R8 specification. Aop, an ETS repressor, is 101 expressed throughout a variety of developmental contexts (Rebay and Rubin, 1995). In its  Rubin, 1992; Rogge et al., 1995). aop flies were originally characterized with respect to eye 108 development for having supernumerary R7 photoreceptors, which are produced in a process 109 that was later shown to be both N and Ras-MAPK-dependent (Tei et al., 1992

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To explore whether Aop regulates ato, we observed Ato expression in mitotic clones that 133 were derived using the FLP-FRT recombination system (Xu and Rubin, 1993). aop clones 134 feature a striking, novel Ato patterning defect in which Ato is present, but with reduced range 135 when compared to WT ( Fig. 2A,B). Despite seemingly normal initiation at the anterior margin of

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Ci ACT accumulates ahead of the MF as is WT and is abruptly lost with the passage of the MF, 157 suggesting that Hh signal response is largely unperturbed (Fig. 3B, arrows).

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The morphogen Dpp is also employed in eye development. Hh stimulates the secretion

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In light of the stage-specific aop phenotype, we reasoned that there might be an 285 enhancer-specific effect on ato. Unsurprisingly, 5'-ato report was highly aberrant in aop mutant 286 eyes. ato-3' was also affected such that although the enhancer report initiated appropriately, it 287 failed to build to the same high levels detected in WT eyes. Given that Aop colocalizes with Ato 288 during early stage-2 (when Ato is solely derived from ato-3') and that ato-3' fails to strongly 289 report from aop mutants, it is plausible that Aop exerts its neuroprotective effect toward ato-3' classic E(spl) gain-of-function phenotype (Fig 9).

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Stage-2 is a critical inflection point in Ato patterning. At this time, Ato successfully 297 switches from strict ato-3' dependence to primary dependence upon 5'-ato (Fig 10). As 298 discussed previously, stage-2 is best considered as divided into discrete sub-stages. Early  Thus, data support a mechanism of Ato regulation in which Aop and E(spl) respectively 311 regulate ato-3' and 5'-ato in parallel (Fig. 10B). In the absence of MAPK activity, Aop maintains 312 a protective function toward the 3' enhancer. However, in the presence of active MAPK, Aop 313 would be relieved of this duty. In support of this mechanism, in situ hybridization against ato-3' 314 reveals that the 3' enhancer is restricted into clusters that resemble early stage-2 IGs prior to

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To clarify, Aop's involvement in IG formation and R8 selection suggests that EGFR-332 MAPK is likely integrated into R8 selection through regulation of ato-3'. To maintain ato-3' 333 activity during early stage-2, Aop must likely repress one or more genes that antagonize Ato.

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However, native Aop function would be impaired during stage-3 given by the activation of MAPK 335 at that time. Thus, we expect Aop may regulate one or more genes that are 1) antagonistic to Ato expression, and 2) induced in or near the MF by MAPK signaling such as the 337 homeorepressors Rough (Ro) and Bar (Dominguez et al., 1998;Lim and Choi, 2003).

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The mechanism described herein bears homology to the signal-regulated events that

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The role for ETV6 within T-cell specification is consistent with our proposed model for