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Activation of Ras/MAPK signaling in Drosophila controls cell differentiation, proliferation and size (Friedman and Perrimon, 2006; Prober and Edgar, 2000). Flies with increased Ras/Raf/ERK activity display patterning and proliferation defects and exhibit an increase in the number of photoreceptors per ommatidium, while reducing the activity in this pathway leads to failure of non-neuronal cells to differentiate into neurons (Biggs et al., 1994). Moreover, a tight regulation of Ras/MAPK signaling is required during the embryonic development of numerous structures, including eye and wing (Friedman and Perrimon, 2006; Johnson Hamlet and Perkins, 2001).

To investigate whether DJ-1 interacts with the Ras/ERK pathway, I used constitutively active versions of Ras and the MAPK ERK/rolled (rl), which impair eye development by promoting excessive proliferation and altered cell differentiation (Brunner et al., 1994). Point mutations in the Ras oncogene are found in 30 % of all cancers. The most common mutation is the substitution of glycine 12 with valine (RasG12V, also called RasV12), that leads constitutive association with GTP; Ras is “locked” in this GTP-bound state and is therefore constitutively active (Dhillon et al., 2007). Ras/ERK signaling was found to be particularly important for the development of the seventh photoreceptor neuron (R7) contained in each ommatidium. Overexpression of consitutively active RasV12 in R7 photoreceptor neurons driven by the R7-neuron specific promoter sevenless (that is fused to the coding sequence for RasV12; Sev-RasV12) is sufficient to induce abnormal differentiation of additional cells into R7 photoreceptors; as a result, multiple R7 photoreceptors are induced in each ommatidium (Karim et al., 1996). To test whether DJ-1A/B interacts genetically with activated Ras, I generated flies expressing Sev-RasV12in either a wild-type background or a background of reduced DJ-1A/B activity (Figure 32). As expected, flies expressing Sev-RasV12 in a wild-type background developed with rough eyes (Figure 32B); histological examination of their retina revealed that the total number of photoreceptor neurons in each ommatidium increased to an average of 8,5 photoreceptors /ommatidium (P/O; Figure 32B’,P).

Figure 32. DJ-1A/B interact genetically with Ras signaling in Drosophila. (A-E, P) DJ-1A/B interact

genetically with constitutively active Ras in the fly retina. (A-E) eye images showing a normal control eye (A), the rough eye phenotype of flies carrying a Sev-RasV12 _{construct (B)and the rescue of the}

rough eye in a DJ-1A/DJ-1B heterozygous knockout background (C). GMR-mediated DJ-1A

overexpression further enhances the Ras-induced phenotype (Sev-RasV12_{/GMR/DJ1A, E) as compared}

to control (Sev-RasV12_{/GMR, D). (A’-E’) Photomicrographs of ultrathin eye sections showing normal}

control ommatidia (A’), the Sev-RasV12_{-induced increase in photoreceptors and fused ommatidia (B’,}

stippled lines indicate missing separations due to abnormal fusions of ommatidia) and the rescue after reducing DJ-1A/B function (C’). (E’) Further increase of the number of photoreceptors/ommatidium and worsening of retinal ultrastructural defects after DJ-1A overexpression as compared to control (D’). (P) Quantification of the numbers of photoreceptors/ommatidium (n=4 eyes/group and n>150 ommatidia analyzed per eye; ** p<0.01 and *** p<0.001, t-test). (K-O,Q) No further modulation of constitutively active rolled-induced eye phenotype by DJ-1A or DJ-1B. (K-O) eye images showing a control eye (K), the rough eye phenotype induced by rlSEM _{overexpression (L; GMR-Gal4; UAS-rl}SEM_),

and the lack of modulation after co-overexpression of DJ-1A (M) or DJ-1B (N) or after decreasing DJ- 1B function (O). (K’-O’) Photomicrographs of ultrathin eye sections showing normal control ommatidia (K’), the GMR/rlSEM_{-mediated increase in photoreceptor number/ommatidium (L’) and the}

lack of modulation after co-overexpression of DJ-1A (M’), DJ-1B (N’) or after decreasing DJ-1B levels (O’). (Q) Quantification of the numbers of photoreceptors/ommatidium (n=4 eyes/group and n>150 ommatidia analyzed per eye; *** p<0.001, t-test).

88 A’ B’ C’ D’ E’ P L’ K’ Q O’ N’ M’ 0 4 8 12 R c e lls /o m m a tid iu m Sev -Ras V12;D J1A +/-;D J1B +/- Sev -Ras V12 CTR L Sev -Ras V12/G MR Sev -Ras V12/G MR /DJ1 A R c ells /o m m a tid iu m GM R/rl SEM /DJ1 A GM R/rl SEM GM R GM R/rl SEM /DJ1 B GM R/rl SEM ;DJ1 B+/ - 0 4 8 12 GMR/rlSEM DJ1A+/-DJ1B+/- + GMR +GMR/+DJ1A *** *** *** *** n.s. GMR + DJ1B + DJ1A WT WT DJ1B+/- WT WT GMR/rlSEM GMR/rlSEM GMR/rlSEM A B C D E L K M N O

When reducing the DJ-1A/B levels (Sev-RasV12;DJ-1A+/-;DJ-1B+/-), this phenotype was significantly rescued (Figure 32C) the Sev-RasV12;DJ-1A+/-;DJ-1B+/-flies displaying an improved retinal morphology while the number of P/O was restored (Figure 32C’,P). In addition, while in Sev-RasV12 flies 67 % of ommatidia were abnormally fused with their neighbors, only 5 % abnormally fused ommatidia were detected in the rescued Sev-RasV12;DJ-1A+/-;DJ-1B+/- flies (Figure 32 C’and data not shown). Thus, DJ-1A/B activity is required for the full manifestation of the RasV12_-

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To test whether DJ-1 is sufficient to modulate the RasV12-mediated eye phenotype, I co-overexpressed RasV12 in R7 neurons (Sev-RasV12) and DJ-1A in all photoreceptor neurons (using the GMR driver). While Sev-RasV12and Sev-RasV12/GMR-Gal4 flies displayed the same moderate eye phenotype (Figure 32D,D’,P),Sev-RasV12/GMR/DJ- 1A eyes were severely affected and very rough (Figure 32E). At the histological level, Sev-RasV12/GMR/DJ-1A eyes displayeda strong increase in the number of P/O (11 on average) compared to Sev-RasV12 and Sev-RasV12/GMR-Gal4 retinas (Figure 32E’,P). Thus, DJ-1 activity appears sufficient to modulate the RasV12-mediated eye development.

The use of a constitutively active version of Ras also allows us to determine whether the novel Ras interactor DJ-1 functions upstream, downstream or in parallel to Ras in controlling photoreceptor neuron development. Constitutively active RasV12 cannot be further regulated by any upstream interactor that normally regulates the wild-type Ras. In contrast, the phenotype mediated by RasV12 could still be modulated by interactors acting downstream of Ras or in a parallel pathway. Since modulation of DJ-1A/B levels caused a modification of the RasV12-mediated eye phenotype, DJ- 1A/B functions either downstream of Ras or in a parallel pathway that cooperates with Ras signaling to control Drosophila eye development.

I then assessed themodulation of constitutively active rolled(Erk) signaling by DJ-1. A substitution of a single conserved amino acid in the kinase domain of rolled (called sevenmaker) is sufficient to constitutively activate its Ser/Tyr kinase activity; the mutation was named sevenmaker (SEM) because its targetting to the developing retina leads to the formation of multiple R7 photoreceoptor neurons in each ommatidium (Brunner et al., 1994). I overexpressed rlSEM in all photoreceptor neurons using the GMR driver. The resulting GMR/rlSEM _{eyes were rough, slightly reduced in size and}

relatively disorganized compared to control GMR-Gal4 eyes (Figure 32L). When analyzing the retinal ultrastructure, I found an increase in the number of P/O (9,5 P/O on average; Figure 32Q). I then generated flies that co-overexpress rlSEM and DJ-1A (GMR/rlSEM_{/DJ-1A) or DJ-1B (GMR/rl}SEM_{/DJ-1B); I also generated flies}

overexpressing rlSEM in a DJ-1B heterozygous knockout background (GMR/rlSEM;DJ- 1B+/-_{). The rough eye phenotype induced by rl}SEM _{overexpression was not further}

enhanced after DJ-1A/B overexpression (Figure 32M,N) nor did a decrease in DJ-1B activity exert a suppressing effect on the rlSEM_{-mediated phenotype (Figure 32O); at}

the histological level, the increase in the number of P/O caused by rlSEM overexpression was not further modulated after modifying the levels of DJ-1A/B (Figure 32M’-O’,Q). Because DJ-1A/B activity failed to further modulate the constitutively active rolled signaling, DJ-1A/B function either upstream of Rolled/ERK or in parallel to the Rolled/ERK signaling pathway to control cell differentiation and proliferation in the retina.

Taken together, these results indicate that DJ-1A/B interacts with Ras-mediated signaling and DJ-1A/B function either between Ras and ERK or in parallel to Ras/ERK signaling to control eye development induced by overactive Ras/ERK signaling.

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2.16. DJ-1 and ERK interaction controls Drosophila eye and wing development