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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Dev Biol. 2013 Jun 20;381(1):50–61. doi: 10.1016/j.ydbio.2013.06.015

optix functions as a link between the retinal determination network and the dpp pathway to control morphogenetic furrow progression in Drosophila

Yumei Li a,e, Yuwei Jiang a, Yiyun Chen a,e, Umesh Karandikar a, Kristi Hoffman f, Abanti Chattopadhyay a, Graeme Mardon a,b,c,d,f, Rui Chen a,d,e,*
PMCID: PMC3742619  NIHMSID: NIHMS497162  PMID: 23792115

Abstract

optix, the Drosophila ortholog of the SIX3/6 gene family in vertebrate, encodes a homeodomain protein with a SIX protein-protein interaction domain. In vertebrates, Six3/6 genes are required for normal eye as well as brain development. However, the normal function of optix in Drosophila remains unknown due to lack of loss-of-function mutation. Previous studies suggest that optix is likely to play important role as part of the retinal determination (RD) network. To elucidate normal optix function during retinal development, multiple null alleles for optix have been generated. Loss-of-function mutations in optix result in lethality at the pupae stage. Surprisingly, close examination of its function during eye development reveals that, unlike other members of the RD network, optix is required only for morphogenetic furrow (MF) progression, but not initiation. The mechanisms by which optix regulates MF progression is likely through regulation of signaling molecules in the furrow. Specifically, although unaffected during MF initiation, expression of dpp in the MF is dramatically reduced in optix mutant clones. In parallel, we find that optix is regulated by sine oculis and eyes absent, key members of the RD network. Furthermore, positive feedback between optix and sine oculis and eyes absent is observed, which is likely mediated through dpp signaling pathway. Together with the observation that optix expression does not depend on hh or dpp, we propose that optix functions together with hh to regulate dpp in the MF, serving as a link between the RD network and the patterning pathways controlling normal retinal development.

Keywords: Optix, SIX domain, dpp, morphogenic furrow, retinal determination network, retinal development

Introduction

Despite the apparent differences in morphology, histology, and physiology, the genetic pathways that underlie organ development in different species often share significant similarities. One prominent example is the development of the visual system across a wide range of metazoans (Oliver and Gruss, 1997). Although it is not clear whether light sensing organs evolved once or multiple times, a shared set of genes controlling eye development, named the retinal determination (RD) network, has been identified in the last two decades. A total of six RD genes have been identified in Drosophila, including twin of eyeless (toy), eyeless (ey), eyes absent (eya), sine oculis (so), dachshund (dac), and optix (Bonini et al., 1993; Cheyette et al., 1994; Czerny et al., 1999; Hanson et al., 1993; Mardon et al., 1994; Quiring et al., 1994; Seimiya and Gehring, 2000). Encoding proteins that function in multiple complexes, these genes form a genetic network that controls the earliest stages of retinal development (Chen et al., 1997; Pignoni et al., 1997). The RD genes share the following features: 1) Loss-of-function mutations block early eye development; 2) misexpression of RD genes can reprogram other imaginal discs to develop as retinal tissue; and 3) these genes are expressed early and anterior to the MF in the eye imaginal disc. Striking parallels have been found among these genes and their vertebrate homologs: Pax6, Eya1/2, Six1/3/6, and Dach1/2 are all expressed in the developing vertebrate retina (Abdelhak et al., 1997; Davis et al., 2001a; Davis et al., 2001b; Hanson, 2001; Jean et al., 1999; Oliver et al., 1995; Quinn et al., 1996; Xu et al., 1997). Moreover, loss of Pax6 function blocks normal eye development in mammals (Hill et al., 1991; Hogan et al., 1986) and Six3/6 play important roles during mammalian retinal differentiation (Bernier et al., 2000; Loosli et al., 1999; Wallis et al., 1999). Furthermore, similar genetic and physical interactions among the RD genes and proteins are found in both insects and vertebrates (Chow et al., 1999; Lengler and Graw, 2001; Loosli et al., 1999; Ohto et al., 1999). Therefore, the RD network genes and their activities have been highly conserved across phylogeny. In addition to these core genes in the RD network, many additional genes that interact closely with the RD network have been identified, such as hth, tsh, tiptop, nemo, dan and danr (Bessa et al., 2002; Curtiss et al., 2007; Datta et al., 2009; Morillo et al., 2012).

so and optix are two RD network genes that are also members of the Six family of genes that encode homeodomain transcription factors. There are a total of three Six family members in the Drosophila genome: so, optix, and dsix4. dsix4 does not function in the retina but is essential for proper muscle development (Kirby et al., 2001). so is a Six1/2 ortholog in Drosophila and its function during retinal development had been well studied (Cheyette et al., 1994; Pignoni et al., 1997; Serikaku and O'Tousa, 1994). Like other RD genes, so is necessary and sufficient for eye development. so loss-of-function mutants fail to initiate eye development and cells anterior to the MF undergo massive apoptosis, resulting in a reduced or absent adult eye. In addition, so is required posterior to the MF for photoreceptor differentiation (Pignoni et al., 1997). Therefore, so is necessary both anterior and posterior to the MF for normal retinal development. On the other hand, ectopic expression of so in other imaginal discs is sufficient to cause the formation of ectopic eyes, indicating that so is a key gene in the RD network (Pignoni et al., 1997; Weasner et al., 2007).

Unlike so, the normal function of optix, another member of the Six family, is less well-characterized in Drosophila. Based on the predicted Six and homeodomain sequences, Optix belongs to the Six3/6 subgroup (Toy et al., 1998). In vertebrates, Six3/6 genes are required for normal eye development. Mutations in human SIX3 lead to holoprosencephaly and microphthalmia (Wallis et al., 1999). Moreover, homozygous Six3 mutants in mouse have no eyes and severe cranio-facial defects (Lagutin et al., 2003). Furthermore, ectopic expression of mouse Six3 in medaka fish leads to the development of ectopic lens and retina (Loosli et al., 1999). Recent studies indicate that vertebrate Six6, also named Optx2, also plays important roles in retinal development. Deletion of the Six6 gene-containing region leads to bilateral anophthalmia in humans (Gallardo et al., 1999). Consistently, homozygous Six6 mutant mice show retinal hypoplasia (Li et al., 2002). Finally, misexpression of Six6 is sufficient to induce retinal cell development in Xenopus (Bernier et al., 2000). Thus, both Six3 and Six6 are involved in vertebrate retinal development, presumably by directly regulating the expression of downstream targets. Consistent with the function of Six3/6, several lines of evidence suggest that the Drosophila homolog, optix, is also likely to play important roles in retinal determination (Kenyon et al., 2005a; Kenyon et al., 2005b; Seimiya and Gehring, 2000; Weasner et al., 2007). Like other known RD genes, optix is expressed prior to MF initiation and anterior to the MF during furrow progression. In addition, misexpression of optix is sufficient to induce ectopic eye formation, suggesting that it functions high in the genetic hierarchy controlling retinal cell fate determination. However, recent biochemical studies suggest that Optix function is distinct from that of So and their interacting cofactors and DNA binding preferences are different (Kenyon et al., 2005a; Kenyon et al., 2005b; Weasner et al., 2007). Thus, it is important to understand the function of optix during normal eye development and its relationship with the RD network.

In this study, we have generated several optix null mutant alleles and performed detailed phenotypic analysis in the developing eye. In contrast to the function of so, which is required for many steps of retinal development, optix is specifically required for proper progression of the MF. optix controls MF progression in part through regulation of dpp expression in the MF. Functioning in parallel to the hh signaling pathway, expression of optix in the MF is required for induction of dpp. Like other members of the RD network, extensive mutual regulation among members of the RD genes and optix is observed. Expression of optix is depends on Ey, Eya, and So, and positive feedback loops exist between optix and eya and so, likely mediated by dpp. Based on these data, we propose a model where optix functions as a link between the RD network and major patterning pathways, including hh and dpp, to control the synchronized development of retinal cells during Drosophila eye development.

Materials and Methods

Fly Genetics

Flies were reared at 25°C in standard fly medium. optix deletion alleles were generated using FLP-FRT deletion method described previously (Parks et al., 2004). Imprecise excision was carried out with the P-element insertion line NP2631. optix clones were obtained using the following stocks: yweyflp; FRT42D ubi-GFP/Cyo, ywhsflp;FRT42D P{m-w+;arm-lacZ}M(2)56/Cyo, and w; FRT42D GMR-hid l(3)CLR/Cyo; ey-Gal4 UAS-FLP (Stowers and Schwarz, 1999). Larvae were heat shocked 43 hours AEL. dpp12 clones were created by crossing ywhsflp;ubi-GFP FRT40A/Cyo to w; dpp12 ck FRT40A/CyO. Larvae were heat shocked 43–48 hours AEL. Tkv was overexpressed in optix clones using the MARCM technique (Lee, 2001). hsFLP, UASCD8:GFP/+; FRT42D Gal80; TubGal4/Tm6B was crossed to w; FRT42D optix/CyO; UAS-Tkv{QD}/Tm6B to generate clones. Larvae were heat shocked 43–48 hours AEL. hhts flies were grown at 18°C until early 3rd instar larvae and then shifted to 29°C until late 3rd instar for dissection. eyaCliIID and so3 clones were generated using ywhsflp;FRT40A P{m-w+;armlacZ}M(2)56/Cyo, ywhsflp;FRT42D P{m-w+;arm-lacZ}M(2)56/Cyo respectively and larvae were heat shocked at 72 hours AEL. dac3 clones were generated using ywhsflp;FRT40A P{m-w+;armlacZ}M(2)56/Cyo and larvae were heat shocked at 44 hours AEL.

Immunohistochemistry

The following primary antibodies were used in this study: rabbit anti-Optix (gift of Francesca Pignoni), rat anti-Elav (DSHB), rabbit anti-b-Galactosidase, rabbit-anti-GFP, mouse anti-Hairy (DSHB), guinea pig anti-Ato (Gift of Hugo Bellen), guinea pig Anti-Senseless (Gift of Hugo Bellen), rat anti-Ci (2A1, DSHB), mouse anti-pMad (gift of Peter ten Dijke), mouse anti-Ptc (DSHB), rabbit anti-Ey (gift of Uwe Walldorf), guinea pig anti-So (gift of Ilaria Rebay), mouse anti-Eya (DSHB), mouse anti-Dac (gift of Graeme Mardon). Discs were then processed as described previously (Frankfort et al., 2001). Fluorescent images were captured with a Zeiss LSM 510 confocal microscope. All other images were captured on a Zeiss Axioplan microscope. All images were processed with Adobe Photoshop software and NIH ImageJ software.

Results

optix is required for normal Drosophila eye development

As an essential step toward a more complete understanding of optix function during normal retinal development in Drosophila, loss of function alleles of optix were generated using two independent approaches. First, molecularly defined deletions that remove all or part of the optix genomic locus were obtained using FLP-induced chromosomal recombination (Parks et al., 2004). Three piggyBac elements (f06656, f04738, and f03269) inserted near or within the optix locus were used to generate three distinct mutant alleles (Fig. 1A). Recombination between f06656 and f03269 results in a 70 kb deletion that removes the entire optix locus and several neighboring genes (optixΔF). Deletion between f06656 and f04738 generates the optixΔN allele which removes 38 kb, including the 5' part of the gene that contains both the Six domain and the homeodomain. Finally, deletion between f04738 and f03269 results in the optixΔC allele which removes 32 kb, including the 3' part of optix. Since all three of these deletion alleles also remove other genes in addition to optix, an optix-specific allele, optix1, was obtained through imprecise excision of a nearby P-element insertion (optixNP2631). The optix1 allele removes 5.4 kb, including both the transcription start site and the exons encoding the Six domain and the homeodomain (Fig. 1A). None of these alleles show a dominant phenotype.

Fig. 1.

Fig. 1

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(A) Generation of optix deletion flies. The extent of deletion for optixΔF, optixΔN, optixΔC, and optix1 were depicted. Location of the piggyback elements f06656, f04738, f03269 and the P-element NP2631 were shown. The genomic rescue construct covers both intergenic and introgenic regions of optix. (B) optix is required for normal eye development. EGUF analysis shows that optix mosaic eyes displayed adult eye defects.

To assess the phenotypic nature of these alleles, we first conducted complementation tests. All four alleles (optixΔF, optixΔN, optixΔC, optix1) are homozygous lethal as early larvae and fail to complement each other. We then assessed the mutant phenotype in the adult eye of each optix allele. Using the EGUF system (see Materials and Methods), we found that these alleles show a range of defects in the adult eye. Three of the four alleles (optixΔF and optixΔN, optix1) are most severe and show similar phenotypes, resulting in small, kidney-shaped eyes (Fig. 1B, 1C, 1E). Since optixΔF completely removes the entire optix gene, these results suggest that optixΔN and optix1 are phenotypic null alleles of optix. In contrast, the optixΔC allele shows a milded phenotype and therefore is likely to be a partial loss-of-function allele of optix (Fig. 1D). Consistent with the genetic data, optix transcripts are not detected in optix1 mutant clones induced by hs-FLP in eye discs by RNA in situ hybridization (data not shown). Finally, to test if the eye phenotypes we observe are indeed due to loss of optix function, rescue experiments were performed. A 40 kb genomic construct that contains only the optix genomic locus (Fig. 1A) was introduced into an optix1 mutant background and both the lethality and eye phenotypes are fully rescued (data not shown). Therefore, we conclude that optix1 is a clean null optix allele. All the subsequent experiments described below were conducted using the optix1 mutant allele.

optix is required for normal MF progression

Since loss of optix function causes early larval lethality, clonal analysis induced by ey-FLP was used to assess its function during retinal development. In third instar larvae eye discs, optix clones that span the MF show defects in MF progression compared to surrounding wild type tissues (Fig. 2A). Consistent with the results, cells in optix mutant clones continue to divide as undifferentiated cells anterior to MF (Fig. 2B). Furthermore, delay in photoreceptor differentiation is observed in optix mutant clones (Fig. 2C). To further examine the MF progression defect, we examined markers that are expressed immediately anterior to or in the MF. First we examined atonal (ato), which is expressed in all cells in the anterior part of the MF and then restricted to R8 cells immediately posterior to the MF. In optix mutant clones, expression of Ato is greatly reduced (Fig. 2D, open arrow), including the early strip, intermediate cluster, and later R8 specific expression. This result is consistent with the MF progression defect observed. Expression of ato anterior to the MF is under complex regulation by the RD genes, dpp, and Notch signaling and is important for priming cells for subsequent neuronal differentiation. To further identify which of these signaling pathways are affected in optix mutant cells, expression of hairy was examined. hairy, encoding a bHLH protein that functions as negative regulator to prevent premature MF progression, is normally expressed in a stripe within and just anterior to the MF (Baonza and Freeman, 2001; Brown et al., 1991; Brown et al., 1995). Expression of hairy is tightly regulated by signals from the MF with dpp and hh as a positive and negative regulator respectively (Fu and Baker, 2003; Greenwood and Struhl, 1999). In optix mutant clones, expression of hairy anterior to the MF is greatly reduced (Fig. 2D, open arrow). Given that both ato and hairy are regulated by dpp and hh, we hypothesize that optix may be required for the proper production or function of these signals during MF progression.

Fig. 2.

Fig. 2

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optix is required for MF progression. (A) optix mutant clones generated by eyflp spanning the MF (marked by the absence of anti-GFP staining, open arrow) show defects in MF progression as indicated by phalloidin staining (red) that marks cells at and posterior to the MF by detecting cell apical shape changes. (B) Expression of CyclinB in optix clones generated by hsflp. (C) The expression of Elav and Senseless, makers for all photoreceptor cells and R8 respectively, is delayed in optix mutant clones. However, optix is not required for cells inside of the mutant clones which eventually differentiate (asterisk). (D) optix mutant clones generated by hsflp spanning the MF (marked by the absence of anti-GFP staining, open arrow) show defects in the expression of Atonal and Hairy. Clones that are close to lateral margin of the disc shows less severe defect (closed arrow). (E) optix is not required for MF initiation. Posterior margin clones generated by eyflp (marked by the absence of anti-GFP staining, arrowhead), stained for Elav and Senseless, show that the MF initiation starts normally. MF is marked by triangle.

Interestingly, although MF progression is delayed in optix mutant clones, cells inside the clone can eventually develop into photoreceptors (Fig. 2C, asterisk). As shown in figure 2, specification of R8 cells and recruitment of the remaining photoreceptor neurons is observed in clones posterior to the MF as indicated by Senseless and Elav staining (Fig. 2C). In addition, all eight photoreceptors and the accessory cells are observed in optix mutant clones in adults (data not shown). Therefore, optix is not essential for proper differentiation of photoreceptor cells. In contrast, although optix is expressed prior to MF initiation, it does not appear to be required for MF initiation. MF initiates successfully even in large optix1 mutant clones that include the posterior margin of the eye disc (Fig. 2E, arrowhead). It is also worth noting that the clonal phenotype varies depending on the position of the mutant cells within discs, with the strongest effects observed for clones at the center of the disc and little or no phenotype in clones close to the lateral margins (Fig. 2C, closed arrow).

optix is required for dpp expression but not its function during MF progression

Two major signaling molecules that are produced within and posterior to the MF and required for MF progression are Dpp and Hh (Baker, 2007). hh is normally expressed in all differentiated photoreceptor cells posterior to the furrow while dpp is expressed in the MF. As dpp signaling is involved in the regulation of both ato and hairy expression, we first looked at the relationship between dpp and optix using a dpp-lacZ reporter (Blackman et al., 1991). In the early third instar larvae eye disc prior to MF initiation, Optix is mainly detected interior of the eye field with little protein detected along the posterior and lateral margin of the discs where dpp is highly expressed (Fig. 3A). During MF progression, Optix is mainly observed anterior to the MF. However, reduced level of Optix protein is detected in dpp expression cells at the anterior part of the MF (Fig. 3B). The expression of dpp is examined in optix mutant clones. Consistent with clonal phenotype, expression of dpp during MF progression but not initiation is affected by optix mutant. As shown in figure 3C, posterior and lateral eye disc margin dpp expression prior to MF initiation remains largely unaffected in optix margin clones. In contrast, expression of dpp-lacZ in the MF is dramatically reduced in internal optix mutant clones in early third instar larvae (Fig. 3D). These results indicate that the initial expression of dpp is optix independent, but continued expression of dpp in the furrow during MF progression is optix dependent.

Fig. 3.

Fig. 3

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optix is required for normal Dpp pathway signaling within the MF. (A, B) Relative position of Optix and Dpp in early and late 3rd instar larvae. (C, D) Dpp-lacZ is expressed in second instar (C) optix clones, but not in third instar clones (D) generated by eyflp. MF is marked by triangle.

The activated form of Mad, phospho-Mad (pMad), which is the downstream effector of the dpp signaling pathway, was also examined (Fig. 4A). In the wild-type developing eye, pMad accumulates in the MF where dpp is expressed at the highest levels. Consistent with the observation that dpp expression is abolished in optix mutant cells, pMad levels are greatly reduced within optix1 mutant clones (Fig. 4A). To further test if optix is required for proper dpp signal transduction, we overexpressed a constitutively active form of thick veins (TkvQD) (Wiersdorff et al., 1996), the Dpp receptor to mimic dpp activation using the MARCM system. Consistent with previous reports, ectopic MF progression is observed and high levels of pMad are induced in cells expressing TkvQD in the wild type background (Fig. 4B). Similarly, high levels of pMad are also detected in optix mutant clones when TkvQD is over-expressed anterior to and at the MF, indicating that optix is not required for proper dpp signal transduction downstream of the receptor (Fig. 4C). Therefore, the reduced level of dpp signaling in MF when optix is mutated is likely due to the reduction of dpp expression.

Fig. 4.

Fig. 4

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optix is required for normal Dpp pathway signaling within the MF. (A) optix mutant clones generated by eyflp spanning the MF (marked by the absence of anti-GFP staining) show defects in the expression of phospho-Mad. (B–C) pMad expression in optix clones is rescued by expression of active form of Tkv (clones marked by the presence of anti-GFP staining and generated using MARCM system). MF is marked by triangle.

optix is not required for hh pathway signaling

Expression of dpp in the MF is under the control of the Hh signal secreted posterior to the furrow. Although optix expression does not overlap with hh, since hh is a diffusible signal molecule, it is possible that reduction of dpp expression in optix mutant clones could result from defects in Hh signaling at MF. To test this possibility, we first checked hh expression in optix mutant clones. When optix mutant cells are stained for Sens and a hh-lacZ reporter (hhP30) (Lee et al., 1992), all cells that are Sens positive also express hh-lacZ, indicating that hh expression does not depend on optix (Fig. 5A). During MF progression, the major effect of the Hh signal is to prevent the formation of the 75 kd repressor form of the Ci protein by blocking cleavage of full-length Ci (Ci155) (Dahmann and Basler, 2000; Pappu et al., 2003). As a result, Ci155 protein accumulates in cells in the MF upon receiving the Hh signal. In optix mutant clones, a modest reduction and broad band of Ci155 accumulation is observed, which is likely linked to a delay of MF progression. Since Ci155 still accumulates at the MF, indicating that responding to Hh signal does not dependent on optix (Fig. 5B). This hypothesis was further tested by examining Patched (Ptc) expression in optix mutant clones. Ptc is a Hh receptor and also a direct downstream target of Ci (Alexandre et al., 1996). During MF progression, Ptc is expressed in the MF in response to Hh signaling. In optix mutant clones, although lower than the adjacent wild type tissue, Ptc expression is still elevated in the MF compared to cells anterior to the MF, further indicating that Hh signal transduction is largely intact in optix mutant cells (Fig. 5C). Therefore, optix plays an essential role to regulate dpp expression during MF progression either in parallel to or downstream of the hh pathway.

Fig. 5.

Fig. 5

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optix is independent of the Hh pathway. Expression of Hh (A), Ci155 (B), and Ptc (C) are largely normal in optix mutant hsflp clones (marked by the absence of anti-GFP staining, arrows). MF is marked by triangle.

optix expression is independent of hh and dpp

Based on the results described above, it is likely that optix plays a crucial role during MF progression through modulation of the dpp signaling pathway. Previous studies have suggested complex regulatory interactions among the patterning signals and the RD gene network (Chen et al., 1999; Pappu et al., 2003). We sought to test if optix expression is also in turn regulated by dpp and hh during MF progression. We first examined optix expression in dpp12 clones. During MF progression, optix is normally expressed in a broad stripe just anterior to the MF and the same expression pattern is observed in dpp clones (Fig. 6A). optix expression is also independent of Hh signaling. Although MF progression is arrested in flies homozygous for a hhts allele at the non-permissive temperature, optix expression anterior to the MF is largely normal in these discs (Fig. 6B). Consistent with this result, optix expression is still present in cells that are mutated in smoothened (smoD16), the receptor of hh (Fig. 6C) (van den Heuvel and Ingham, 1996). Furthermore, smo3mad1–2 double mutant clones were used to test if optix is redundantly regulated by Hh and Dpp signaling. smo3mad1–2 double mutant clones abolish both the dpp and hh signaling pathways and thereby block MF progression and photoreceptor differentiation (Curtiss and Mlodzik, 2000; Greenwood and Struhl, 1999). We found that optix continues to be expressed in smo3mad1–2 double mutant clones, suggesting that optix expression does not rely on either hh or dpp signaling (Fig. 6D). It is worth noting that optix expression in the posterior portion of the double mutant clone is properly shut down, presumably due to MF progression across these clones.

Fig. 6.

Fig. 6

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optix expression is independent of the dpp and hh pathways. (A) Optix expression is present in mutant clones for dpp12 (marked by the absence of anti-GFP staining, arrow). (B) Optix expression is normal in hhts mutant when the larvae were shifted to 29°C. (C, D) Mutant clones for smoD16, and smo3mad1–2 (marked by the absence of anti-GFP staining, arrows) show Optix expression. MF is marked by triangle.

Regulation of optix by the RD genes

Previous studies indicate that optix functions as a member of the RD network as it is sufficient to induce ectopic eye formation (Seimiya and Gehring, 2000). To test the relationship between optix and other members of the RD network, we first determined if normal optix expression depends on other RD genes in developing eye discs using clonal analysis. optix is normally expressed in undifferentiated retinal cells starting at the late second instar larvae stage and expression is restricted to cells anterior to the MF in third instar larvae (Fig. 7A) (Seimiya and Gehring, 2000). optix expression in the retinal field is abolished in eya or so mutant clones, indicating that optix is downstream of eya and so (Fig. 7E,F,G,H). In contrast, optix expression is still present in dac mutant clones, suggesting that optix is either upstream of or in parallel to dac (Fig. 7C, D, arrow). We further tested the relationship between optix and other RD genes by examining the expression of ey, eya, so, and dac in optix mutant clones. Consistent with the idea that optix is a downstream target of ey, expression of ey is not affected in optix mutant clones (Fig. 7I, J). In contrast, unlike Ey, the level of both Eya and So anterior to and at the MF is reduced in the optix mutant clones, suggesting positive feedback regulation between optix, eya, and so in undifferentiated retinal cells (Fig. 7K–N). Similar to Eya and So, expression of Dac anterior to and at the MF is present but reduced in optix mutant clones (Fig. 7O, P). As it has been shown that dpp regulates eya and so expression, we test if reduction of eya and so expression in the optix mutant clones at the MF is due to defects in dpp expression. As shown in Fig 7, overexpressing a constitutively active form of thick veins (TkvQD) in optix mutant clones is sufficient to restore normal so expression (Fig. 7R, S). Together with previous results that optix is sufficient to induce ectopic eye formation and RD gene expression, it is likely that optix functions as an integral part of the RD network by establishing positive feedback between RD genes and the dpp signal pathway (Fig 8 model).

Fig. 7.

Fig. 7

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The interaction between optix and RD network. (A, B) Normal expression of Optix in 2nd and 3rd instar larvae. (C, D, E, F) optix expression in wild-tyep, eya2, so1 and dac3 mutants dicses. (G, H, I, J). Expression of RD network proteins in optix mutant clones. Ey is unaffected (G), while the expression of Eya, So, and Dac are reduced (H, I, J). (R, S) Expression of So is rescued by overexpression of active form of Tkv. MF is marked by triangle.

Fig. 8.

Fig. 8

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Model of optix function during MF progression and its regulation. Independent of Hh, Optix regulates the expression of Dpp and the progression of morphogenetic furrow. optix expression is under the control of ey, eya and so. It also feeds back on the expression on the RD network genes (Eya, So and Dac). Arrow reflects genetic interactions with bold reflect evidence provided by this paper.

Discussion

optix is required for normal MF progression during Drosophila retinal development

One interesting evolutionary comparison is the function of the Six family members during retinal development in Drosophila and vertebrates. In mouse, a total of six Six genes have been identified, named Six1–6. Two members, Six3 and Six6, are essential for retinal development while the other members are involved in development of other organs, such as kidney and muscle. In Drosophila, a total of three Six family members have been identified, including so (a Six1/2 homolog), optix (a Six3/6 homolog), and dSix4 (a Six4/5 homolog). Unlike vertebrate Six1/2, which is not required for retinal development, so is required for multiple stages of retinal development (Pignoni et al., 1997). Although Six3/6 are required for normal eye development in mammals, the role of the Drosophila homolog optix in retinal development is not known. In this report, we have generated multiple null alleles of optix using a combination of deletion, excision, and genomic rescue. Interestingly, optix shows a much more restricted role during retinal development compared to that of so. Consistent with its expression in a narrow region anterior to the MF, loss of optix function results in loss of expression of many MF markers and a dramatic delay of MF progression. However, optix is not absolutely required for either MF initiation or retinal cell differentiation. These results suggest significant differences for the function of Six family members during retinal development in Drosophila and vertebrates as the Six1/2 homolog so plays a more prominent role in Drosophila retinal development than the Six3/6 homolog optix. Therefore, despite the high conservation of protein sequence and the requirement of Six family members in controlling retinal development, detailed involvement of these members has diverged among different species. Recent studies of Six1/2/So and Six3/6/Optix proteins indicate that their DNA binding properties and interacting partners are distinct (Weasner and Kumar, 2009). As a result, although so mutant phenotypes can be rescued by vertebrate SIX1/2, they cannot be rescued by either optix or SIX3/6. Therefore, it is likely that Six family proteins regulate distinct downstream targets in Drosophila and vertebrates during retinal development. Further studies of the differential usage of Six family members and their downstream targets will aid our understanding of the retinal development program.

A feedback loop between optix and the RD gene network

In developing eye imaginal discs, expression of optix is restricted to undifferentiated cells anterior to the morphogenetic furrow (MF). It has been suggested that optix expression and function in the eye is independent of eyeless (ey) (Seimiya and Gehring, 2000). However, our previous studies suggest that optix is regulated directly by Ey. To resolve this inconsistency and uncover the mechanisms of optix regulation, we have examined optix expression in RD gene mutant eye discs. Our results show that optix expression is abolished in eya and so mutant eye discs but not in dac mutants. In addition, expression of Ey, So, Eya, and Dac are detected in optix mutant tissue. Given that Ey expression is not affected while the expression of eya, so, and dac is reduced in optix mutant clones, our results support the idea that optix acts downstream of ey in the RD gene regulatory hierarchy and at a similar level as eya, so and dac. It is worth noting that optix expression can be detected in ey mutant discs, suggesting that optix is parallel to ey. However, this observation can be potentially explained by the redundant function of toy. Indeed, the eye phenotype of ey mutant is variable. To fully resolve the issue, it is essential to investigate optix expression in an ey and toy double mutant background.

Like other members of the RD gene network, extensive cross regulations are observed between optix and other RD gene members. First, expression levels of Eya, So, and Dac are reduced anterior to the MF in optix mutant clones, suggesting that optix is required for maintaining high levels of expression of these genes in undifferentiated retinal cells. Second, ectopic expression of optix is sufficient to induce ectopic eye formation and presumably expression of the RD genes (Weasner et al., 2007, Seimiya and Gehring, 2000). Positive feedback loops between optix and other members of the RD network can serve as a ”lock in” mechanism to ensure a robust switch of cell fate. It has been postulated that to ensure the synchronized cell differentiation process anterior to the MF, coordinated high levels of expression of the RD genes as well as other patterning genes, such as Notch, dpp, and hh, are required.

optix acts to link the RD network and patterning pathways

Retinal development is tightly controlled by both the RD network and the patterning pathways, including dpp, hh, and wg. Prior to MF initiation, ey functions together with hh and dpp to initiate the entire RD gene network (Chen et al., 1999; Curtiss and Mlodzik, 2000). In contrast, during MF progression, these two pathways seem to function in parallel as the RD gene network is required to lock in retinal cell fates while the dpp, hh, and wg pathways are required for synchronized progression of the MF (Firth and Baker, 2009). Consistent with this model, expression of each RD gene is largely normal when the hh and/or dpp pathways are blocked during MF progression (Curtiss and Mlodzik, 2000). However, whether hh or dpp expression is affected by mutations in other RD genes is not entirely clear. Clonal analysis has not been reported for ey or toy. Hh and Dpp expression do not depend on dac while eya and so are not required for initiation but they are required for maintenance of dpp (Pignoni 1997). However, as both eya and so are involved in multiple steps of retinal development and cell survival, interpretation of the data is not clear. As large optix mutant clones can be obtained, we were able to test if hh or dpp expression during MF progression depends on optix function. Our results suggest that optix regulates MF progression through modulate the level of dpp expression. Although hh expression is unaffected, dpp expression in the MF is largely abolished in optix mutant clones. Interestingly, this defect is specific to MF progression inside of the disc as dpp expression during early development and at the posterior margin of eye discs is not affected by optix mutant clones. This may explain the observation that less severe defect is observed for optix mutant clones located close to the eye disc margin. Since optix expression does not depend on either hh or dpp signaling during MF progression, it is likely that optix functions in parallel with or downstream of the hh signal to either directly or indirectly regulate dpp transcription during MF progression. Consistent with this model, both the hh and dpp signal transduction pathways are functionally intact in the absence of optix function. For example, accumulation of full length Ci is observed in optix mutant clones in response to Hh. Similarly, expression of constitutive form of Tkv induces accumulation of activated Smad in optix mutant clones. However, whether dpp expression in the MF is directly regulated by optix needs further investigation.

In summary, we report that optix serves as a direct key connection between the RD gene network and patterning pathways during MF progression (Fig. 8). The mechanisms by which optix interacts with these two pathways are different. In the case of regulating dpp, optix is involved in regulating its expression. Functioning in parallel with or downstream of Hh signals from differentiated photoreceptor cells, optix is required for induction of dpp in the MF during progression. In contrast, positive feedback loops from optix to eya, so, and dac is utilized to maintain high levels of expression of other RD genes anterior to the MF. The positively feedbacks between optix and the RD network is likely through regulation of the patterning pathways, thereby facilitating synchronized, high level expression of dpp and the RD genes at the MF to ensure proper furrow progression across the entire eye disc (Fig. 8).

Highlights

  • optix is required only for morphogenetic furrow (MF) progression, but not initiation.

  • Expression of dpp in the MF is dramatically reduced in optix mutant clones

  • optix is regulated by sine oculis and eyes absent

  • optix expression does not depend on hh or dpp.

  • Generation and characterization of optix loss-of-function mutant phenotype

  • optix regulates dpp expression

  • optix serves as a link between the RD network and the patterning pathway during MF progression

Acknowledgments

We thank Francesca Pignoni, Hugo Bellen, Peter ten Dijke, Uwe Walldorf, Ilaria Rebay, the Bloomington Stock Center and the Developmental Studies Hybridoma Bank for stocks and antibodies used in this study. This work is supported by supported by funds from the National Eye Institute (R01EY016853) and the Retina Research Foundation Research to R.C. and the National Eye Institute (EY011232) to G.M.

Footnotes

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