Odd-skipped genes specify the signaling center that triggers retinogenesis in Drosophila

In Drosophila, the eye primordium is specified as a subdomain of the Pax6-expressing cells in the center of the eye disc, by the co-expression of a set of retinal determination genes(Bonini et al., 1993; Cheyette et al., 1994; Dominguez and Casares, 2005; Halder et al., 1998; Mardon et al., 1994; Pappu and Mardon, 2004). Then,retinogenesis is triggered by the hedgehog (hh) and the hh target decapentaplegic (Dpp/Bmp4) signals that are produced by the surrounding posterior margin cells(Fig. 1A), at the so-called`firing point' (Treisman and Heberlein,1998). These margin cells abut the eye primordium and give rise to part of the adult head capsule surrounding the eye(Haynie and Bryant, 1986). Once initiated, retinal differentiation propagates in a posterior-to-anterior wave (Fig. 1B,C), with the differentiation wavefront marked by an epithelial indentation: the morphogenetic furrow (MF) (Treisman and Heberlein, 1998). The gene(s) responsible for this specialization of the posterior margin are unknown.

odd⁵, drm⁶, bowl¹,wg^1-16 (wg^CX3), odd^rK111 (oddZ), hhP30 (hhZ), dppBS3.0 (dppZ), P{en1}wgen11 (wgZ),P{GAL4}hhGal4 (hh-GAL4) are described in FlyBase. Df(2L)drmP2 (Green et al.,2002; Hao et al.,2003) deletes from tim to odd, and uncovers∼30 predicted genes, including drm, sob and odd. UAS strains were UAS-odd(A) and UAS-sob(6)(Hao et al., 2003),UAS-bowl(1.1) (de Celis Ibeas and Bray, 2003), UAS-drm (on the III) and UAS-lines(Green et al., 2002; Hatini et al., 2000), and UAS-Src-GFP(Kaltschmidt et al., 2000). odd-GAL4 faithfully reproduces odd expression (a gift from G. Morata and M. Calleja, CMB, Spain). drm⁶ was recombined onto a FRT40A chromosome.

odd⁵, drm⁶ and bowl¹mitotic clones were induced between 24 and 48 hours after egg laying (AEL) by a 45 minute 37°C heat-shock in larvae from the crosses of odd^*FRT 40A/balancer males to yw hsFLP 122; Ubi-GFP FRT40A females (odd^* represents each of the alleles used). DfdrmP2 cells do not survive unless given a growth advantage, for which we used the `Minute technique'(Morata and Ripoll, 1975). Clones were induced between 24 and 72 hours AEL by a 20 minute 37°C heat-shock in larvae from the crosses of odd^*FRT40A males to yw, hsFLP122; M armZ FRT40A females. In some experiments, we used yw ey-FLP as flipase source(Newsome et al., 2000) to maximize the amount of mutant tissue in eye discs. Mutant cells were identified by the absence of β-galactosidase (armZ).

These clones were induced between 24 and 48 hours AEL (L1 stage) in larvae from the crosses between UAS-odd^* (where odd^* means odd, drm, sob or bowl) or UAS-lines males and y, hsFLP122, actinP>hsCD2>Gal4 females(Basler and Struhl, 1994). Clones were marked negatively by the absence of CD2 (CD2 was induced by a 45 minute 37°C heat-shock, followed by 45 minutes recovery at room temperature). The hhZ, dppZ or oddZ reporters were introduced in the genotypes of some experiments. The overexpression of drm in bowl^- cells was achieved using the MARCM technique (Lee and Luo, 2001). UAS-drm was balanced over TM6B, Tb, so drm-expressing larvae were Tb⁺. Clones were marked positively by expression of GFP.

We used rabbit anti-β-gal (Cappel), mouse anti-β-gal (Sigma),rabbit anti-GFP (Molecular Probes), mouse anti-CD2 (Serotec), guinea pig anti-Odd (Kosman et al., 1998)and mouse ant-Ptc (Nakano et al.,1989). Rat anti-Elav, mouse anti-Wg (4D4) and mouse anti-Eya are from the Iowa University Studies Hybridoma Bank. RNA probes for odd, drm,sob and bowl were as described previously(Hao et al., 2003). Phalloidin-FITC was used to mark filamentous actin. Appropriate fluorescent secondary antibodies were from Molecular Probes. Anti-mouse-HRP (Sigma) was used for immunoperoxidase staining.

The eye disc is a flat epithelial sac. By early third larval stage (L3),columnar cells in the bottom (disc proper: Dp) layer are separated by a crease from the surrounding rim of cuboidal margin cells. Margin cells continue seamlessly into the upper (peripodial; Pe) layer of squamous cells(Fig. 1C-G). The Dp will differentiate into the eye, while the margin and Pe will form the head capsule(Haynie and Bryant, 1986). In addition, the posterior margin produces retinal-inducing signals(Treisman and Heberlein,1998).

By examining gene reporters we found that the zinc-finger gene odd-skipped (odd) is expressed restricted to the posterior margin and Pe of L3 eye discs (Fig. 1). As the odd family members drumstick (drm), brother of odd with entrails limited (bowl) and sister of odd and bowl (sob) are similarly expressed in leg discs(de Celis Ibeas and Bray,2003; Hao et al.,2003), we examined them in eye discs. In L2, before retinogenesis has started, odd and drm are transcribed in the posterior Pe-margin (Fig. 1H,I), and this continues within the posterior margin after MF initiation(Fig. 1L,M). bowl is transcribed in all eye disc Pe-margin cells of L2 discs(Fig. 1J), but retracts anteriorly along the margins and Pe after the MF passes(Fig. 1N). In addition, bowl is expressed weakly in the Dp anterior to the furrow. sob expression in L2 and L3 is mostly seen along the lateral disc margins (Fig. 1K,O). Therefore drm, odd and bowl are co-expressed at the posterior margin prior to retinal differentiation initiation.

Odd family genes regulate diverse embryonic processes, as well as imaginal leg segmentation (de Celis Ibeas and Bray,2003; Green et al.,2002; Hao et al.,2003; Hatini et al.,2005; Johansen et al.,2003). Bowl is required for all these processes(Green et al., 2002; Hao et al., 2003). In embryos,the product of the gene lines(Bokor and DiNardo, 1996) binds to Bowl and represses its activity, while Drm relieves this repression in drm-expressing cells (Hatini et al., 2005). As drm/odd/bowl expression coincides along the posterior margin around the time retinal induction is triggered, we asked whether they controlled this triggering. First, we removed bowlfunction in marked cell clones induced in L1. bowl^- clones spanning the margin, but not those in the DP, cause either a delay in, or the inhibition of, retinal initiation (Fig. 2A,B) and the autonomous loss of hh-Z expression(Fig. 2C,E). Correspondingly,there is a reduction in expression of the hh-target patched(ptc) (Fig. 2D). These effects on hh and ptc are not due to the loss of margin cells, as drm is still expressed in the bowl^-cells (not shown). The requirement of Bowl for hh expression is margin specific, as other hh-expressing domains within the disc(Royet and Finkelstein, 1997)are not affected by the loss of bowl (not shown). As expected from the bowl-repressing function of lines(Green et al., 2002; Hatini et al., 2005), the overexpression of lines along the margin phenocopies the loss of bowl (Fig. 2F). Nevertheless, the overexpression of bowl in other eye disc regions is not sufficient to induce hh (not shown). This suggests that, in regions other than the margin, either the levels of lines are too high to be overcome by bowl or bowl requires other factors to induce hh, or both.

drm and odd are expressed together along the posterior disc margin-Pe (Fig. 1), and drm (at least) is required for Bowl stabilization in leg discs(Hatini et al., 2005). Nevertheless, the removal of neither drm(Fig. 3A) nor odd (not shown) function alone results in retinal defects. odd and drm may act redundantly during leg segmentation(Hao et al., 2003) and this may also be the case in the eye margin. To test this, we induced clones of DfdrmP2, a deficiency that deletes drm, sob and odd, plus other genes (Green et al., 2002). When DfdrmP2 clones affect the margin, the adjacent retina fails to differentiate, suggesting that drm and odd (and perhaps sob, for which no single mutation is available) act redundantly to promote bowl activity at the margin(Fig. 3B,C) (although we cannot exclude that other genes uncovered by this deficiency also contribute to the phenotype). To test the function of each of these genes, we expressed drm,odd and sob in cell clones elsewhere in the eye disc. Only the overexpression of drm or odd induced ectopic retinogenesis(Fig. 3D and not shown), and this was restricted to the region immediately anterior to the MF, which is already eye committed. Interestingly, bowl is also expressed in this region of L3 discs (Fig. 3E). The retina-inducing ability of drm requires bowl, because retinogenesis is no longer induced in drm-expressing clones that simultaneously lack bowl function(Fig. 3F). Therefore, it seems that in the eye, drm (and very likely also odd) also promotes bowl function.

The expression of hh(Heberlein et al., 1995) or activation of its pathway (Chanut and Heberlein, 1995; Dominguez and Hafen, 1997; Ma and Moses,1995; Pan and Rubin,1995; Strutt and Mlodzik,1995; Wehrli and Tomlinson,1995) anterior to the furrow is sufficient to generate ectopic retinal differentiation. As (1) bowl is required for hhexpression at the margin, (2) this hh expression is largely coincident with that of odd and drm(Fig. 3G), and (3) drm(and possibly odd) functionally interacts with bowl, we checked whether drm- and odd-expressing clones induced the expression of hh. In both types of clones hh expression is turned on autonomously, as detected with hh-Z (shown for drmin Fig. 3H), which would thus be responsible for the ectopic retinogenesis observed. That the normal drm/odd/bowl-expressing margin does not differentiate as eye could be explained if margin cells lack certain eye primordium-specific factors.

Our results indicate that the expression of odd and drmdefines during L2 the region of the bowl-expressing margin that is competent to induce retinogenesis. How is their expression controlled?wingless (wg) is expressed in the anterior margin, where it prevents the start of retinal differentiation(Ma and Moses, 1995; Treisman and Rubin, 1995). drm/odd are complementary to wg (monitored by wgZ)during early L3, when retinal differentiation is about to start, and also during later stages (Fig. 4A,C,E). In addition, when wg expression is reduced during larval life in wgCX3 mutants, drm transcription is extended all the way anteriorly (Fig. 4B,D). This extension precedes and prefigures the ectopic retinal differentiation that, in these mutants, occurs along the dorsal margin(Fig. 4B,D,F). Therefore, wg could repress anterior retinal differentiation by blocking the expression of odd genes in the anterior disc margin, in addition to its known role in repressing dpp expression and signaling(Hazelett et al., 1998; Treisman and Rubin, 1995).

Interestingly, the onset of retinogenesis in L3 is delayed relative to the initiation of the expression of drm/odd (this work) and hh(Cavodeassi et al., 1999; Cho et al., 2000) in L1-2. This delay can be explained in three, not mutually exclusive, ways. First, the relevant margin factors (i.e. drm/odd, hh) might be in place early,but the eye primordium might become competent to respond to them later. In fact, wg expression domain has to retract anteriorly as the eye disc grows, under Notch signaling influence, to allow the expression of eye-competence factors (Kenyon et al.,2003). Second, building up a concentration of margin factors sufficient to trigger retinogenesis might require some time. In fact, the activity of the Notch pathway along the prospective dorsoventral border is required to reinforce hh transcription at the firing point(Cavodeassi et al., 1999). Third, other limiting factors might exist whose activity becomes available only during L3. Such a factor might be the EGF receptor pathway, which is involved in the triggering and reincarnation of the furrow along the margins during L3 (Kumar and Moses,2001).

In addition to hh, other genes are required for retinal triggering, including dpp (Burke and Basler, 1996; Pignoni and Zipursky, 1997; Wiersdorff et al., 1996), eyes absent (eya)(Bonini et al., 1993) and the target of eya dachshund (dac)(Mardon et al., 1994; Pignoni et al., 1997). These genes are expressed in both the posterior region of the eye primordium and the posterior margin. In addition to their role in eye specification, they might also specify the margin. Although the regulatory relationships between hh and dpp, or dpp and eya are obscured by cross-regulatory interactions (Borod and Heberlein, 1998; Chen et al.,1999; Curtiss and Mlodzik,2000; Hazelett et al.,1998; Pignoni and Zipursky,1997), recent functional data indicate that dpp and eya are functionally downstream of hh(Pappu et al., 2003). The possibility that the odd genes control the expression or function of dpp and eya at the margin remains to be tested.

We are grateful to A. Casali, S. Bray, M. Calleja, I. Guerrero, V. Hatini,G. Morata, C. Rauskolb, J Reinitz and I. Rodríguez for reagents, and to J. L. Gomez-Skarmeta, F. Pichaud and C. Rauskolb and members of the laboratory for comments. This work has been funded through grants BMC2003-06248(Ministerio de Educación y Ciencia, Spain) and POCTI/BIA-BCM/56043/2004[Fundação para a Ciência e a Tecnologia (FCT), Portugal],which are co-funded by FEDER, to F.C. C.B-P. and J.B. are funded by FCT.