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Keywords:

  • Drosophila;
  • eye;
  • photoreceptor;
  • development;
  • polarity;
  • crumbs

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The establishment of apicobasal polarity in epithelial cells is a prerequisite for their function. Drosophila photoreceptor cells derive from epithelial cells, and their apical membranes undergo elaborate differentiation during pupal development, forming photosensitive rhabdomeres and associated stalk membranes. Crumbs (Crb), a transmembrane protein involved in the maintenance of epithelial polarity in the embryo, defines the stalk as a subdomain of the apical membrane. Crb organizes a complex composed of several PDZ domain-containing proteins, including DPATJ (formerly known as Discs lost). Taking advantage of a DPATJ mutant line in which only a truncated form of the protein is synthesized, we demonstrate that DPATJ is necessary for the stability of the Crb complex at the stalk membrane and is crucial for stalk membrane development and rhabdomere maintenance during late pupal stages. Moreover, DPATJ protects against light-induced photoreceptor degeneration. Developmental Dynamics 235:895–907, 2006. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The formation of distinct apical and basolateral membrane domains is a crucial requirement for the proper development of epithelial cells. The hallmarks of polarized epithelial cells are various types of intercellular junctions, including the zonula adherens (ZA), a belt-like adhesive structure encircling the apex of the cell, and tight junctions or septate junctions, which form apical or basal to the ZA, respectively, in vertebrates and Drosophila. Several protein complexes have been identified that are required to specify polarity (reviewed in Knust and Bossinger, 2002). In the Drosophila embryonic epithelium, three complexes act in concert to establish and maintain polarity. A complex composed of Bazooka (Baz), DmPar-6, and an atypical protein kinase C (DaPKC) plays an essential role in initial polarization, whereas the Crumbs (Crb)/Stardust (Sdt)/PALS-1-associated tight junction protein (DPATJ) complex acts to maintain the ZA and epithelial tissue structure by counteracting the activity of the laterally localized Discs-large (Dlg)/Lethal giant larvae (Lgl)/Scribble (Scrib) complex (Bilder et al., 2003; Tanentzapf and Tepass, 2003).

Several cell types that derive from epithelial cells adopt a more complex, functionally specialized morphology. One example is the Drosophila photoreceptor cell (PRC), which differentiates from epithelial cells of the eye imaginal disc. The architecture of PRCs is established during pupal development, beginning with a 90-degree shift of the epithelial cell, such that the apical membrane comes to occupy a lateral position. Starting at approximately 37% pupal development, the apical membrane then undergoes a dramatic expansion, progressively forming a highly pleated array of microvilli, the photosensitive rhabdomere, and a supporting stalk membrane, which connects the rhabdomere to the ZA (see Fig. 1 for a schematic summary of this process). This expansion is accompanied by a marked extension of the cell along the proximodistal axis (Longley and Ready, 1995).

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Figure 1. Schematic diagram of photoreceptor cell development during the pupal stage. A–C: Cross-section of an ommatidium with seven photoreceptor cells at 45% pupal development (p.d.; A), and 70% p.d. (B), and at eclosion (C). A: At 45% p.d., the apical membrane of photoreceptor cells is not yet subdivided and consists of a single unit, where future rhabdomeric proteins (green) and future stalk membrane proteins (i.e., Crb, DPATJ, red) colocalize. The ZA marks the apical-most end of the lateral membrane. B: At 70% p.d., the apical membrane is clearly separated into the rhabdomere (green) and the stalk membrane (red). Blue: ZAs. C: At eclosion, the stalk membrane has reached its full length and rhabdomeres are now mature phototransduction units.

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Recent studies have demonstrated that some of the proteins required for epithelial polarization in the embryo also play a role in PRC morphogenesis and polarity. The transmembrane protein Crb was shown to localize specifically to the stalk membrane (Izaddoost et al., 2002; Johnson et al., 2002; Pellikka et al., 2002). Crb, together with Sdt (Hong et al., 2003; Nam and Choi, 2003), controls the length of the stalk membrane, the extension of the rhabdomere, and the correct positioning of the ZA along the proximodistal axis. As in the embryo, baz has a crucial role in the initial establishment of apicobasal polarity in PRCs and in localizing the Crb/Sdt/DPATJ complex in pupal eyes (Hong et al., 2003; Nam and Choi, 2003). In contrast to the case in embryonic epithelia (Tepass and Knust, 1990), loss of crb or sdt does not lead to any obvious polarity defects in PRCs. However, lack of crb in the Drosophila eye leads to progressive light-induced degeneration of the PRCs (Johnson et al., 2002). This observation is especially striking, because mutations in the human crb homolog CRB1 are associated with severe forms of retinal dystrophy, retinitis pigmentosa 12 (RP12), and Leber congenital amaurosis (LCA; den Hollander et al., 1999, 2001; Lotery et al., 2001a, b).

DPATJ, formerly known as Discs lost (Dlt), was initially identified in a screen for binding partners of Neurexin IV, a component of the septate junctions in the Drosophila embryo. DPATJ is maternally expressed and marks the leading edge of the ingrowing plasma membranes during blastoderm cellularization. During gastrulation, it is redistributed to an apical position, where it colocalizes with Crb apical to the ZA (Bhat et al., 1999). In Drosophila eyes, DPATJ colocalizes with Crb at the stalk membrane throughout pupal development (Izaddoost et al., 2002; Hong et al., 2003). crb and sdt are both required for proper localization of DPATJ in postblastoderm embryos (Bhat et al., 1999; Klebes and Knust, 2000; Bachmann et al., 2001; Hong et al., 2001), as well as for its localization at the stalk membrane of PRCs (Izaddoost et al., 2002; Pellikka et al., 2002; Hong et al., 2003; Nam and Choi, 2003). Drosophila PATJ is a scaffold protein, containing an N-terminal L27 (Lin-2, Lin-7) domain, followed by four PDZ (PSD-95/Discs large/ZO-1) domains (Bhat et al., 1999). Several lines of evidence demonstrate that DPATJ is a component of the Crb complex in Drosophila embryos (Klebes and Knust, 2000; Roh et al., 2002b), as well as in adult flies (Pellikka et al., 2002). Its L27 domain binds the N-terminal L27 domain of Sdt (Ö. Kempkens and E. Kunst, unpublished observations), which, in turn, binds to the four C-terminal amino acids of Crb by means of its PDZ domain (Bachmann et al., 2001; Hong et al., 2001; Roh et al., 2002b).

Like DPATJ, the mammalian PATJ is a scaffolding protein with an N-terminal L27 domain but has 10 PDZ domains; it has been shown to bind Pals1, the vertebrate homologue of Drosophila Sdt, which, in turn, binds the C-terminal amino acids of CRB1 and CRB3 (Lemmers et al., 2002; Roh et al., 2002b; Makarova et al., 2003). PATJ is found at tight junctions, and overexpression of a dominant-negative variant in MDCK cells leads to delocalization of tight junction markers. Reduction of PATJ expression leads to a failure to polarize cells in 3D cultures (Lemmers et al., 2002; Hurd et al., 2003; Shin et al., 2005). In addition, PATJ also interacts by means of its PDZ domains with other tight junction proteins, such as ZO-3 and Claudin (Poliak et al., 2002; Roh et al., 2002a).

Although it was initially reported that mutations in DPATJ lead to polarity defects in the Drosophila embryo, as well as to loss of imaginal discs (hence, the original name Discs lost; Bhat et al., 1999), a recent study has demonstrated that the “discs lost” phenotype is caused by disruption of the adjacent Drosophila codanin-1 homolog and not to mutation of DPATJ (Pielage et al., 2003). DPATJ has been suggested to be required for formation of the follicle epithelium by maintaining Crb in the follicle cells of the Drosophila ovary (Tanentzapf et al., 2000). In addition, DPATJ has recently been shown to be required for the control of planar cell polarity in the eye imaginal disc (Djiane et al., 2005). In this study, we show that DPATJ is necessary for the maintenance of the Crb protein complex in the Drosophila eye. Loss of DPATJ function causes defects in photoreceptor cell morphogenesis and leads to light-induced PRC degeneration.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

DPATJ Is Required to Stabilize the Crb Protein Complex at the Photoreceptor Stalk Membrane

DPATJ was suggested to maintain Crb localization in follicle cells of the Drosophila ovary (Tanentzapf et al., 2000). We wished to analyze whether it has a similar function in the Drosophila eye, where it colocalizes with Crb at the stalk membrane, the part of the apical membrane situated between the rhabdomere and the ZA (Figs. 1C, 2A,D). Clones of crb11A22 cells, which are devoid of Crb protein (data not shown), were induced in the adult eye. In the mutant cells, rhabdomeres are bulky, fused, or absent (compare Fig. 2A with 2B; Izaddoost et al., 2002; Johnson et al., 2002; Pellikka et al., 2002); DPATJ is delocalized and can be found at the rhabdomere base, in the cytoplasm, and at the basolateral membrane (Fig. 2B). This latter finding is consistent with results previously obtained in crb11A22 pupal eyes (Izaddoost et al., 2002; Pellikka et al., 2002; Nam and Choi, 2003). Other proteins reported to be members of the Crb complex in Drosophila epithelia, such as Sdt or βH-spectrin (Bachmann et al., 2001; Medina et al., 2002), are also delocalized from the stalk in a similar manner in crb11A22 mutant PRCs (data not shown). In DPATJ mutant adult eyes (see below and Fig. 5 for a schematic representation of the DPATJ locus), no DPATJ protein was detectable with our antibody (Fig. 2C), which is directed against a portion of the protein containing the four PDZ domains of Drosophila PATJ (see the Experimental Procedures section). DPATJ eyes are slightly disorganized (compare F-actin staining in Fig. 2C and Fig. 2A), and Crb protein is not detectable (Fig. 2E). Similar observations were made for Sdt, another member of the Crb complex (data not shown). These findings led us to conclude that DPATJ is required to stabilize the Crb complex at the stalk membrane in PRCs of the adult Drosophila eye.

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Figure 2. DPATJ is required for maintenance of the Crb complex at the stalk membrane. A–E: Optical cross-sections of adult Drosophila eyes stained with Phalloidin (green) to highlight the F-actin–rich rhabdomeres and anti-DPATJ (purple in A–C) or anti-Crb (purple in D,E). A: In wild-type (w) eyes DPATJ is restricted to the stalk membrane, adjacent to the rhabdomere. B: In crb11A22 eyes, morphological defects in the rhabdomeres are obvious (compare the F-actin staining in A and B). DPATJ is delocalized and can be detected at the rhabdomere base, in the cytoplasm, and at the basolateral membrane. C: DPATJ mutant eyes are slightly disorganized as revealed by F-actin staining (see Fig. 7) and show no DPATJ staining. D: Crb localizes at the stalk membrane in wild-type (w) eyes. E: In DPATJ eyes, Crb is lost from the stalk.

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Figure 5. The DPATJ locus. Schematic representation of the chromosomal region at 62B4 (boxed) encompassing the genes α-spectrin, discs lost (dlt), cdc37, CG12020, and DPATJ (arrows; adapted from Pielage et al., 2003). Distal is to the left. Open reading frames (ORFs) of α-spectrin and dlt are shown above the genes. The deficiency Df(3L)My10 used in this study (depicted by a black bar; breakpoints not determined) affects α-spectrin, dlt, cdc37, CG12020, and DPATJ. The DPATJ mutant flies used here are homozygous for the deficiency, whereas the control flies are Df(3L)My10/TM6B. In addition, both control and DPATJ flies carry three transgenes, represented by gray bars at the bottom of the picture: ubi-α-spectrin and ubi-dlt are transgenes expressing the full-length α-spectrin and dlt cDNAs, respectively, under the control of the ubiquitin promoter (empty square); cdc37 is a genomic rescue construct containing the 5′ end of discs lost, the entire cdc37 and CG12020 genes, and the 5′end of DPATJ (Cutforth and Rubin, 1994). Thus, both control and DPATJ flies express a portion of the DPATJ coding sequence.

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Figure 7. DPATJ eyes show morphological defects when kept in the dark. (A–I) Cross-sections of wild-type (A), control (B,H), and DPATJ mutant (C–G,I) eyes from adult flies kept in the dark for 1 day (C,D), 7 days (E,F), or 22 days (B,G,H,I). A,B: Both wild-type (A) and control (B) eyes show the normal number of properly patterned photoreceptor cells (PRCs). C,D: Pigment cells are dilated (blue arrowhead). Individual rhabdomeres are in the process of resorption or completely missing (green arrowheads). E,F: Pigment granules (blue arrowhead) are much more abundant in DPATJ mutant eyes and often appear fused (E). In some cases, pigment cells extend into the inter-rhabdomeral space (F, blue arrowhead). Rhabdomere resorption continues (green arrowheads and inset in E). In F, the red arrowhead indicates remnants of rhabdomeric structures, which are being engulfed by pigment cells. G: Individual photoreceptor cells are missing (green arrowheads), pigment cells are irregular and often contact the interrhabdomeral space (blue arrowheads). I: Zonula adherens (ZA) persist between photoreceptor cells that remain intact (I, blue arrowheads). H,I: Stalk membranes are reduced in length in DPATJ mutants (compare the red line for controls in H and for DPATJ in I). J: Length of stalk membrane in wild-type (n = 77), control (n = 119), and DPATJ (n = 123) photoreceptors. One unit = 1 μm. Scale bar = 1 μm.

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To test whether DPATJ is also required for the establishment of the Crb complex in developing eyes, we analyzed the expression of the different proteins at various times during pupation. At 45% pupal development (p.d.), i.e., before the stalk membrane forms (Longley and Ready, 1995), Crb can be found at the apical membrane (highlighted by F-actin staining) in control and DPATJ mutant eyes (Figs. 1A, 3A,B). Sdt behaves similarly and anti-Sdt also stains the apical membrane in DPATJ mutants at 45% p.d., suggesting that the Crb complex is assembled correctly (Fig. 3C,D). As previously described, Bazooka colocalizes with the ZA at this stage (Hong et al., 2003; Nam and Choi, 2003). No change in Baz distribution was observed in DPATJ PRCs (Fig. 3A,B), suggesting that the ZA is not affected in these eyes. This suggestion is further supported by the correct localization of Armadillo, a component of the ZA (Fig. 3C–F). DPATJ itself is localized to the apical membrane of wild-type eyes (Fig. 3E), as are Crb and Sdt. Surprisingly, we still found staining for DPATJ in mutant PRCs at 45% p.d. (Fig. 3F). This finding can be explained by the fact that the DPATJ mutants still express a truncated form of the DPATJ protein (see below and Fig. 6), which is detectable with the antibodies used here. In summary, DPATJ, Sdt and Crb are colocalized at the forming rhabdomere at 45% p.d. in DPATJ mutant eyes.

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Figure 3. Development of DPATJ mutant eyes before stalk membrane formation. A–F: Face-view of white (w; A,C,E) and DPATJ mutant (B,D,F) Drosophila retina at 45% pupal development (p.d.). At this stage, the stalk membrane cannot yet be distinguished (see Fig. 1A). The incipient rhabdomeres are stained with phalloidin (green). Crb, Sdt, and DPATJ (purple in A,C,E) colocalize with rhabdomere markers at the apical membrane in wild-type eyes. A,C,E: Bazooka (A) and Armadillo (C,E), visible in seven spots, highlight the ZA. B,D: In DPATJ mutant eyes, Crb and Sdt colocalize with the forming rhabdomeres, and ZA markers (Baz and Arm in B and D, respectively) are perfectly localized as in wild-type. F: The mutant DPATJ protein is expressed and correctly localized in DPATJ eyes at 45% p.d.

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Figure 6. A truncated version of DPATJ is unable to stabilize the Crb complex. Western analysis of extracts of adult heads from wild-type flies (first lane), control flies (second lane), and DPATJ mutant flies (third lane). Amounts of protein equivalent to two heads were loaded in each lane, as confirmed by Ponceau staining (see the Experimental Procedures section). The DPATJ protein was detected using an antibody raised against the four PDZ domains. In wild-type or control flies, two bands are detected at approximately 100 kDa, both of which are absent in DPATJ mutants (arrows). The band at the top of the blot is unspecific. In addition, a truncated form of approximately 30 kDa is detected in extracts from DPATJ as well as control fly heads (arrowhead). This truncated form appears as a weaker band in comparison to the 100-kDa forms. Both Crb and Sdt are either strongly reduced or completely absent in DPATJ mutant heads.

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To determine when the complex disintegrates, we stained pupal eyes at 70% p.d., when the forming rhabdomeres become clearly distinct from the stalk membranes (Fig. 1B; Longley and Ready, 1995). At this stage, DPATJ, Crb, and Sdt colocalize at the stalk membrane in wild-type eyes (Fig. 4A,C,E). In the DPATJ mutants however, staining for these proteins, including DPATJ itself, is progressively lost (Fig. 4B,D,F). Of interest, the truncated DPATJ protein seems to delocalize slightly earlier than Sdt and Crb. Ultimately, all proteins of the complex disappear from the stalk membrane in DPATJ mutant adult eyes (Fig. 2E).

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Figure 4. Development of DPATJ mutant eyes during stalk membrane formation. A–F: Face-view of control (A,C,E) and DPATJ (B,D,F) mutant retina at 70% pupal development (p.d.; see Fig. 1 for a schematic representation). Genotypes of control and DPATJ flies are described in detail in the legend to Figure 5. By this stage, stalk membranes have become distinct from the rhabdomeres, which are highlighted by F-actin (blue). A,C,E: Crb, Sdt, and DPATJ (green) localize at the forming stalk membrane in control eyes. B,D,F: In DPATJ mutant eyes, staining of these proteins is less prominent (B,D,F), as is particularly obvious for DPATJ (B). The right column shows a close-up of the merged images.

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A Truncated Version of DPATJ Is Not Sufficient to Stabilize the Crb Complex

The DPATJ mutant flies used throughout this work carry a deficiency, complemented with several transgenes, because no DPATJ single point mutation that results in a complete loss of function is currently available. To obtain flies that affect only DPATJ, we used a line that is homozygous for Df(3L)My10, which removes the coding regions of DPATJ and several adjacent genes and carries three transgenic inserts (Fig. 5; Pielage et al., 2003). Two of these encode the open reading frames of α-spectrin and dlt (the codanin-1 homolog), respectively, under the control of a ubiquitously expressed promoter. The third one is a genomic fragment that comprises the 5′ end of dlt, cdc37, CG12020, and the 5′ portion of DPATJ, encoding its L27 domain and the first PDZ domain (amino acids 1 to 261; Cutforth and Rubin, 1994; Pielage et al., 2003; Djiane et al., 2005). The control flies used throughout this work were heterozygous for the deficiency and carried the three rescue constructs. Flies homozygous for the deficiency are embryonic lethal (Pielage et al., 2003). When the α-spectrin and cdc37 constructs were added, pupal lethality was observed. When the dlt rescue construct was then added, viable and fertile females were obtained, which lacked the DPATJ gene product, as determined by polymerase chain reaction (PCR) and antibody staining (Pielage et al., 2003). However, the anti-DPATJ antibody used by these authors is directed against the most C-terminal portion of DPATJ (Tanentzapf et al., 2000) and would not be expected to recognize the truncated form of the DPATJ protein encoded by the cdc37 genomic rescue construct.

To definitively resolve the discrepancy between the results reported by Pielage et al. (2003) and the DPATJ staining we observed in pupal eyes of flies deficient for DPATJ, we performed Western analysis on protein extracts from adult heads. As shown in Figure 6, two forms of DPATJ were detected. The wild-type isoforms of the DPATJ protein of approximately 100 kDa (Bhat et al., 1999) are present in wild-type heads and in heads of control flies heterozygous for the deficiency and containing the three transgenes. In addition, a shorter form of DPATJ of approximately 30 kDa can be detected in the heads of DPATJ mutant and control flies but not in extracts of wild-type heads. Because our antibody was raised against a fusion protein containing the four PDZ domains of DPATJ, the presence of the small 30-kDa protein is consistent with the assumption that the cdc37 transgene (see Fig. 5) encodes a truncated DPATJ protein, including the L27 domain and the first PDZ domain. Hence, our results strongly suggest that the DPATJ flies used here produce a truncated form of DPATJ and, thus, have to be considered as hypomorphic and not amorphic. In light of the high sensitivity of the Western blot relative to antibody staining of tissues, it is not surprising that we did not see any DPATJ staining in DPATJ adult eyes (Fig. 2C). The results further suggest that this truncated, 30-kDa form of DPATJ is stable during early pupal development (Fig. 3). The Western blots also confirmed that Crb and Sdt levels are virtually absent in DPATJ mutant adult heads (Fig. 6). Taken together, the data suggest that full-length DPATJ is required to stabilize Crb and Sdt, which is consistent with the antibody staining patterns in adult eyes (Fig. 2E and data not shown for Sdt). The 30-kDa form of DPATJ present in the DPATJ flies, which contains the most N-terminal PDZ domain of the protein, remains properly localized until approximately 70% p.d. and is apparently capable of stabilizing the other members of the complex. However, this truncated form of DPATJ is subsequently unable to maintain the Crb complex at the stalk membrane.

Morphogenetic Defects in DPATJ Mutant Eyes

Loss of crb has been shown to affect the morphogenesis of PRCs. This finding becomes manifest as a shortening of the stalk membrane and defects in photoreceptor elongation (Izaddoost et al., 2002; Johnson et al., 2002; Pellikka et al., 2002). The observation that DPATJ adult eyes fail to maintain the Crb complex at the stalk membrane prompted us to analyze the morphology of these eyes in more detail. We observed no defects in pupal eyes until approximately 70% p.d.. The ZAs were correctly formed and rhabdomeric structures seemed not to be affected (Fig. 4). However, in newly eclosed flies, the eyes showed variable defects. In contrast to wild-type and control eyes, in which pigment cells are very thin and only recognizable by the presence of pigment granules (Fig. 7A,B), pigment cells of DPATJ mutant eyes were strongly dilated (Fig. 7C). Many PRCs exhibited dramatic changes (Fig. 7C): some of the rhabdomeres were undergoing resorption or had already been lost (see Fig. 7D for a close-up). Note, however, that ZAs are still correctly positioned and structured at this time, suggesting that apicobasal polarity is not affected.

With increasing age, pigment cells in DPATJ mutant eyes accumulate large numbers of pigment granules, which often fuse (Fig. 7E). Rhabdomeres continue to be resorbed (Fig. 7E, inset) and individual PRCs are lost in some ommatidia. In those ommatidia that contain only remnants of PRCs or lack some of them completely (Fig. 7F), pigment cells often localize at the margin of the interrhabdomeral space, between the photoreceptor cells. In these cases, PRCs lose their ZAs (Fig. 7F). After 22 days in the dark, control flies looked like wild-type (compare Fig. 7A and Fig. 7B). In DPATJ mutant ommatidia, PRCs were sometimes lost (we did not observe a systematic disappearance of any particular PRC; Fig. 7G). Pigment cells were irregularly arranged and often contacted the interrhabdomeral space (Fig. 7G). Of interest, ZAs were retained between the PRCs that remained intact (Fig. 7I). The penetrance of the PRC mutant phenotype (loss of rhabdomeres or PRCs) is incomplete (40.3 ± 17.22% of PRCs; see the Experimental Procedures section), probably because we are dealing with a hypomorphic allele of DPATJ. In contrast, all pigment cells of an eye were similarly affected at a given time point. The PRC and pigment cell phenotypes were not enhanced in flies that lacked both zygotic and maternal DPATJ expression (see the Experimental Procedures section).

In DPATJ mutant PRCs, the length of the stalk membrane is reduced by approximately 40% in comparison to controls (compare Fig. 7H,I; Fig. 7J), which is similar to what was observed in sdtXP96 mutants but less than in crb mutant PRCs (Pellikka et al., 2002; Hong et al., 2003). However, and in contrast to what has been reported for crb or sdt mutant eyes (Izaddoost et al., 2002; Johnson et al., 2002; Pellikka et al., 2002; Nam and Choi, 2003), we did not observe any defects in PRC elongation in DPATJ mutant eyes: rhabdomeres always reached the basal lamina as revealed in longitudinal sections (data not shown) and did not appear bulkier than usual in cross-section (Fig. 7C–G). Taken together, the observations suggest that DPATJ is essential for stabilization of the Crb complex during late pupal development. This stabilization, in turn, is crucial for the proper differentiation of the apical membrane into stalk and rhabdomere and the stabilization of the rhabdomeric structure.

DPATJ Mutant Eyes Show Light-Dependent Retinal Degeneration

Mutations in human CRB1 are implicated in RP12 and LCA, two heritable forms of retinal degeneration (den Hollander et al., 1999, 2001; Lotery et al., 2001a, b). In Drosophila, crb PRCs also degenerate upon long-term exposure to intense light, a feature that resembles the phenotype observed in the human diseases (Johnson et al., 2002). The relationship between Crb and DPATJ in the eye raised the question whether DPATJ flies might also display PRC degeneration when exposed to high-intensity light. To test this, newly eclosed DPATJ or control flies were kept under constant light for 7 days before analyzing the eyes. Whereas the retina of control flies was unaffected (Fig. 8A,B), DPATJ mutant eyes showed clear signs of degeneration, in that individual ommatidia were no longer distinguishable (Fig. 8C). Remnants of dying PRCs, characterized by their darker cell bodies and degenerating rhabdomeres (Fig. 8C,D), were observed in DPATJ ommatidia. The pigment cells, which can be recognized by their glycogen-rich cytoplasm, appeared to survive (Fig. 8D) and were very frequently seen to replace the missing photoreceptor cells, extending into the interrhabdomeral space (Fig. 8C,D). Although progressive loss of rhabdomeres and PRCs was also observed in DPATJ mutant eyes kept in the dark, the defects were always mild, even after 22 days in the dark, in comparison to the massive degeneration seen in flies kept for 7 days in the light. These observations, thus, suggest that the Crb complex is required to protect against light-induced PRC degeneration.

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Figure 8. DPATJ eyes show degeneration in the light. A–D: Cross-sections of control (A,B) and DPATJ mutant (C,D) adult eyes kept in the light for 7 days. A,B: Control eyes show no sign of degeneration. C,D: In DPATJ mutant eyes, dying photoreceptor cells can be recognized by darker staining and degenerating rhabdomeres (green arrowheads). D: Pigment cells with their glycogen-rich cytoplasm (blue arrowheads) are disorganized but persist. They frequently make contact with the inter-rhabdomeral space. Scale bar = 1 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

It is now well established that the evolutionarily conserved Crumbs complex is involved in the control of epithelial cell polarity. Some of its members are also necessary for the morphogenesis of PRCs in the eye, both in flies and in vertebrates (Izaddoost et al., 2002; Pellikka et al., 2002; Hong et al., 2003; Nam and Choi, 2003; van de Pavert et al., 2004). In addition, Drosophila crb and mouse CRB1 are needed for cell survival when PRCs are exposed to light (Johnson et al., 2002; van de Pavert et al., 2004). Whereas work performed in Drosophila and mammals has provided deeper insight into the functions of Crb/CRB1 and Sdt/Pals-1, the role of the scaffolding protein DPATJ/PATJ is less well understood, in particular with respect to its role in PRC development and survival. In this work, we present evidence to show that DPATJ plays an important role in stabilizing the Crb complex in PRCs. We demonstrate here that a truncated form of DPATJ, consisting of the N-terminal L27 and the first PDZ domain, is produced in DPATJ mutant eyes. This result contrasts those published recently by Nam and Choi (2003). These authors also used the Df(3L)My10 deficiency but introduced only the cdc37 genomic rescue construct for complementation. Although these pupae should express the truncated DPATJ protein (encoded by the cdc37 genomic rescue construct), they did not observe any DPATJ expression with antibodies directed against either the full-length or the N-terminal portion of DPATJ (probably containing the first two PDZ domains; Bhat et al., 1999), which should recognize the 30-kDa DPATJ protein. Because the pupae they analyzed lack α-spectrin and dlt, the absence of either of these proteins might have had an adverse effect on the expression and/or stability of DPATJ. As shown recently, the absence of dlt has no effect on the expression of DPATJ in larval imaginal discs or in the follicular epithelium (Pielage et al., 2003). Lack of α-spectrin, on the other hand, leads to delocalization of βH-spectrin from the apical membrane in follicle cells (Lee et al., 1997). Because βH-spectrin is known to interact with the Crb complex (Medina et al., 2002; Pellikka et al., 2002), it is conceivable that the lack of α-spectrin in the DPATJ clones studied by Nam and Choi could indirectly lead to loss of DPATJ. Clones in follicle cells with a corresponding genotype also fail to maintain Crb at the apical membrane (Tanentzapf et al., 2000).

We further show that the truncated DPATJ protein fails to stabilize Crb and Sdt at the stalk membrane during late pupal development and in adult eyes. It has already been demonstrated that Crb is required for proper localization of Sdt and DPATJ at the stalk membrane of PRCs and that, in sdtXP96, the maintenance of Crb and DPATJ is compromised (Izaddoost et al., 2002; Pellikka et al., 2002; Hong et al., 2003; Nam and Choi, 2003). These data together with the results presented here support the view that lack of any component of the Crb complex leads to mislocalization and/or dysfunction of the whole complex in the Drosophila eye.

To understand how the Crb complex may ensure a proper morphogenesis of photoreceptor cells, two major events during photoreceptor development have to be considered. In the first half of pupal development, stabilization of the ZAs is essential to maintain adhesion between PRCs during the tremendous cell shape changes that take place later when the cells undergo elongation. Furthermore, from 37% p.d. onward, the apical membrane differentiates into the rhabdomere and the stalk.

In crb and sdt mutants, the rhabdomeres are shorter and thicker, suggesting a failure to stabilize adhesion in early stages of pupal development, which in turn prevents proper elongation. This interpretation is consistent with the observation that, in eyes lacking crb function, the continuity of the ZA is interrupted at early stages of pupal development (Izaddoost et al., 2002; Johnson et al., 2002; Pellikka et al., 2002; Hong et al., 2003; Nam and Choi, 2003). DPATJ mutants do not exhibit any obvious defects in ZA development or PRC elongation, which can be explained by the fact that components of the Crb complex are still correctly localized until 70% p.d. This timing contrasts with crb or sdt mutant PRCs, in which the integrity of the Crb complex is lost at an early stage of pupal development. Several explanations may account for this different behavior of DPATJ mutant PRCs. First, the N-terminal portion of DPATJ may still retain some function during early pupal development, which stabilizes the Crb complex and, hence, the ZA. Alternatively, DPATJ does not play a major function for the stability of the complex at early stages. Finally, additional factors may interact with DPATJ and stabilize the Crb complex at early stages of development, and these interactions still occur with a truncated DPATJ. In fact, recent in vitro studies have suggested direct interactions between DPATJ and either DmPar-6 (Nam and Choi, 2003) or DaPKC (Sotillos et al., 2004), two members of the other apically localized protein complex, which is essential for epithelial cell polarity in the embryo. However, we can exclude that the suggested interaction between the third PDZ domain of DPATJ and the N-terminal domain of DmPar-6 (Nam and Choi, 2003) plays any role in the stabilization of the complex during the first half of pupal development. The truncated DPATJ protein studied here lacks the third PDZ domain, yet it remains localized at the apical membrane at this stage (see Fig. 3).

The other major aspect of PRC maturation—the differentiation of the apical membrane into rhabdomere and stalk—is affected in crb, sdtXP96, and DPATJ mutant eyes, suggesting that all three components are necessary for this process. These mutations result in a shortening of the stalk membrane. The weaker phenotype of the DPATJ mutant relative to that of crb mutants is probably due to the hypomorphic nature of the former. Separation of the apical membrane of PRCs into two distinct domains, the rhabdomere and the stalk, becomes manifest at approximately 55% p.d. (Longley and Ready, 1995) and coincides with the restriction of Crb and its associated proteins to this region. No other mutant affecting the length of stalk membrane has been described to date, although some mutants affect individual aspects of the crb or DPATJ morphogenetic phenotype, displaying thicker (bifocal; DSec61β), malformed (Glued; WASp) or missing (overexpression of amphiphysin) rhabdomeres (Bahri et al., 1997; Fan and Ready, 1997; Valcarcel et al., 1999; Zelhof et al., 2001; Zelhof and Hardy, 2004). Thus, the regulation of stalk membrane development seems to be a unique function for members of the Crb complex.

One phenotype of DPATJ mutants observed here, the progressive resorption of rhabdomeric microvilli, has not been described to date for any other mutant of the Crb complex. This raises the question whether DPATJ is involved in other processes in addition to those that are controlled by crb and sdt. The rhabdomere is composed of microvilli, each of which is supported by actin filaments (Arikawa et al., 1990). Rhabdomere morphogenesis and integrity depend on constant renewal of the membrane and on a highly organized actin cytoskeleton. Thus, it is not surprising that mutations in proteins involved in endo- or exocytosis, such as dynamin, Rab1, Rab6, Rab11, Sec6, Sec61β, or Sunglasses, affect the integrity of the rhabdomere (Dietrich and Campos-Ortega, 1980; Satoh et al., 1997, 2005; Shetty et al., 1998; Valcarcel et al., 1999; Xu et al., 2004; Beronja et al., 2005). It has been suggested that the addition of new membrane occurs at the base of the rhabdomere in Drosophila (Stark et al., 1988), while shedding occurs at the distal tip in tipulids (Williams and Blest, 1980; reviewed in Williams, 1991). The further analysis of the function of these genes, the subcellular distribution of the respective proteins and their possible interactions with members of the Crb complex will be required to determine any involvement of the Crb complex in these processes. Rhabdomere integrity is also affected in eyes lacking proteins involved in actin structure and remodeling, such as NinaC, Chaoptin, Glued, Moesin or Rac1 (Matsumoto et al., 1987; Reinke et al., 1988; Van Vactor et al., 1988; Kumar and Ready, 1995; Fan and Ready, 1997; Chang and Ready, 2000; Karagiosis and Ready, 2004) but also in mutants for rhodopsin itself, which plays a structural role in addition to its function in signal transduction (Kumar and Ready, 1995). In this scenario, DPATJ could help to stabilize the cytoskeleton and thereby maintain the integrity of the rhabdomere. Alternatively, the four PDZ domains in DPATJ may mediate the assembly of additional proteins. The identification and functional characterization of these proteins will shed light on the process by which DPATJ controls the stability of PRCs.

At present, we cannot exclude the possibility that the defects observed in pigment cells in DPATJ mutant eyes contribute to the mutant phenotype observed in PRCs. In vertebrate eyes, the pigment epithelium plays an active role in the renewal of rhodopsin, and defects in the pigment epithelium can lead to degeneration of PRCs (reviewed in Marmorstein et al., 1998). It is not yet known whether pigment cells in the Drosophila eye have a comparable function, although they certainly serve to insulate the PRCs of individual ommatidia from the light impinging on their neighbors. The accumulation and fusion of pigment granules in DPATJ mutant eyes may point to a defect in vesicular biogenesis and/or secretion. Whether this defect also affects interactions with the PRCs, in other words, whether pigment cells play an active role in the maintenance of the rhabdomeres or PRC function, and if so, whether DPATJ is involved in this process, is not known.

Finally, the results presented here demonstrate that DPATJ, like crb, protects PRCs from the deleterious effects of excess light. The degeneration of PRCs observed in DPATJ mutant eyes may be a direct consequence of the failure to stabilize the components of the Crb complex at the stalk membrane. Previously published data have shown that the absence of crb in Drosophila eyes leads to retinal degeneration under similar lighting conditions. Similarly, mice deficient for CRB1 display signs of retinal degeneration upon exposure to light (van de Pavert et al., 2004), which are reminiscent of defects seen in patients bearing mutations in the CRB1 gene (den Hollander et al., 1999, 2001; Lotery et al., 2001a, b). However, the penetrance of degeneration observed in crb eyes is much higher than that observed in DPATJ eyes, although the cellular features of degeneration observed in both mutants are similar. Taking into account that the crb clones were produced in a white background (Johnson et al., 2002), whereas DPATJ eyes are red (due the transgenes that were introduced), it is not unlikely that the pigments could play a protective role in the latter case, as also shown previously for white mutants (Lee and Montell, 2004). Preliminary experiments suggest that the presence of pigments in crb mutant ommatidia indeed slows down the light-dependent degeneration (M.R., unpublished observations).

Taken together, our results extend our knowledge of the genes involved in controlling retinal morphogenesis and preventing light-dependent PRC degeneration in the fly. Mutations in human CRB1 lead to RP12 and LCA, two severe forms of retinal dystrophy, raising the question whether mutations in the homologues of the other members of the complex might result in similar phenotypes. Understanding the molecular mechanisms leading to the mutant phenotype in the fly will certainly contribute unraveling the pathogenesis of these retinal dystrophies in humans.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Fly Strains and Lighting Conditions

Flies were kept at 25°C. To obtain DPATJ mutant flies, the following crosses were performed. Virgin w P[ubi-spec] / w P[ubi-spec]; P[cdc37] /P[cdc37]; Df(3L)My10/TM6 females were crossed with w/Y; P[ubi-dlt]/P[ubi-dlt]; Df(3L)My10/TM6 males (Pielage et al., 2003). The DPATJ progeny are w P[ubi-spec]/w or Y; P[cdc37]/P[ubi-dlt]; Df(3L)My10/Df(3L)My10 and the controls w P[ubi-spec]/w or Y; P[cdc37]/P[ubi-dlt]; Df(3L)My10/TM6. P[ubi-spec] and P[ubi-dlt] indicate the corresponding full-length cDNAs under the control of the ubiquitin promoter (Lee et al., 1993; Pielage et al., 2003). P[cdc37] is a genomic rescue construct encompassing the entire cdc37 and CG12020 genes, the 5′ end of discs lost (encoding 851 of its 1,240 amino acids), and the 5′ end of DPATJ, encoding the L27 domain and the first of the four PDZ domains (Cutforth and Rubin, 1994; Pielage et al., 2003). Df(3L)My10 was described by Bhat et al. (1999).

To remove the maternally contributed full-length DPATJ, virgin w P[ubi-spec]/w; P[cdc37]/P[ubi-dlt]; Df(3L)My10/Df(3L)My10 females were crossed with w/Y; P[ubi-dlt]/P[ubi-dlt]; Df(3L)My10/TM6 males, and adult progeny lacking TM6 were analyzed.

Eyes mosaic for crumbs were generated by crossing yw eyFLP;;FRT82B w+cl3R3/TM6B females (Newsome et al., 2000) to w;;FRT82Bcrb11A22/TM6B males. Light-induced retinal degeneration was analyzed according to Johnson et al. (2002).

Antibodies and Immunofluorescence Analyses

Antisera against DPATJ were obtained by repeated immunization of rabbits with an affinity-purified GST-DPATJ fusion protein (including the PDZ domains 1 to 4; Eurogentec, Belgium). Antiserum against DPATJ was used for immunofluorescence analyses at a 1:1,000 dilution. Rat anti-Crb antiserum was obtained by immunizing animals with a GST-fusion protein coding for amino acids 737–1703 of the full-length Drosophila Crb and corresponding to the protein encoded by the p2.8 construct described by Tepass and Knust (1990; Eurogentec, Belgium). Rat anti-Crb was used at 1:500. Other primary antibodies used were mouse monoclonal anti-ArmN2-7A1 (1:50; Developmental Studies Hybridoma Bank), rabbit anti-Baz (1: 500; Wodarz et al., 1999) and rabbit anti-SdtMPDZ (1:500; S. Berger and E.K., unpublished observations). Fluorescence-labeled secondary antibodies (1:200) were purchased from Jackson ImmunoResearch Laboratories (Cy2 or Cy3-conjugates) or Molecular Probes (Alexa-647). Rhabdomeres were visualized by labeling with fluorescein isothiocyanate (FITC) -phalloidin or Alexa-660-phalloidin at 1:40 (Molecular Probes).

Pupal eyes were dissected from staged pupae. Third-instar wandering larvae were collected and allowed to develop at 25°C for 46 hr or 72 hr after puparium formation (corresponding to 45% and 70% p.d., respectively). The retina–brain complex was dissected in PBT (phosphate buffered saline [PBS] with 0.1% Triton X-100), fixed for 1 hr in 4% paraformaldehyde, and stained in PBT–0.1% bovine serum albumin (BSA). After overnight incubation with primary antibodies, eyes were washed with PBT and incubated for 2 hr with fluorescence-coupled secondary antibodies and FITC–phalloidin. Immunostained eyes were embedded in glycerol/propylgallate and analyzed with a Leica TCS NT confocal microscope. Samples designated for direct comparison were processed in parallel under the same conditions and with identical confocal settings.

For staining of adult retina, cryosections were prepared. Adult fly heads were bisected and fixed in Stefanini's fixative (8% formaldehyde and 15% picric acid in 75 mM PIPES pH 7.4) for 25 min on ice. The heads were washed in PBS and cryopreserved in 10% sucrose for 30 min, followed by an overnight incubation in 25% sucrose. Heads were immersed in tissue-freezing medium (GSV1, Slee Technik, Germany) and rapidly frozen on dry ice. Cryosections (approximately 10 μm thick) were collected on Superfrost slides (Menzel Glaeser, Germany). The sections were permeabilized in 0.3% Triton X-100 in PBS. Samples were then incubated overnight with the primary antibody in PBT–0.1% BSA, washed with PBT, and incubated with the secondary antibody for 2 hr. The samples were washed again, mounted in Mowiol (Polysciences, PA) containing DABCO (Sigma) as an anti-fading agent, and examined under the confocal microscope. All images were processed and mounted using Adobe Photoshop 7.0 and Deneba Canvas 9.0.

Western Analysis

Protein extracts from adult heads were prepared on ice. Heads from 25 adult flies were collected and frozen in liquid nitrogen. Heads were homogenized in 50 μl of lysis buffer containing 50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% Triton, 1 mM MgCl2, and protease inhibitor cocktail (1 μM Pefabloc, 5 μM leupeptin, 1 μM pepstatin, 0.3 μM aprotinin from Roche Diagnostics). After a 90-min extraction on a shaker, the homogenate was cleared by centrifugation for 5 min at 3,000 × g. Extract amounts equivalent to two heads were loaded in each lane, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto nitrocellulose membrane. Protein levels were checked by Ponceau S staining (Sigma). After blockage in TBST/3% dry milk/1%BSA, the membrane was incubated overnight with mouse anti-Crb Cq4 (1:3; Tepass and Knust, 1993), rabbit anti-SdtMPDZ (1:2,000; S. Berger and E.K., unpublished observations), or rabbit anti-DPATJ (1:4,000; this work). Peroxidase-conjugated secondary antibodies in combination with the ECL system (Amersham Pharmacia Biotech) were used to detect immunoreactive bands. The truncated form of DPATJ was weakly expressed in comparison to the 100-kDa forms. Therefore, blots were exposed for different times to detect the bands (see Fig. 6).

Transmission Electron Microscopy

Sections were prepared according to (Tepass and Hartenstein, 1994) with modifications. In brief, 0.1 M phosphate buffer (pH 7.4) was used to fix bisected heads in 25% glutaraldehyde, followed by simultaneous fixation in 1% osmium tetroxide/2% glutaraldehyde, followed by 2% OsO4. After dehydration, eyes were embedded in Araldite and semithin (2.5 μm) sections were cut on a Reichert OM U2 microtome and stained with toluidine. Ultrathin sections (0.1 μm thick) were contrasted and analyzed with a Zeiss EM9 S2.

To measure the penetrance of the PRC mutant phenotype in DPATJ mutant eyes, we analyzed 1114 ommatidia, independently of fly age, for missing rhabdomeres or lost PRCs on semithin sections. For stalk membrane measurements, electron micrograph negatives were digitized by scanning and stalk membranes were hand-traced with IMAGEJ software (http://rsb.info.nih.gov/ij/) at 200–300% magnification to reduce tracing errors. Only stalk membranes from R1–R6 with clearly identifiable ZAs were measured. Pixel measurements were transformed into microns using Adobe Photoshop 7.0. Statistical analyses were performed using Microsoft Excel.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Tobias Stork and Christian Klämbt for generously providing fly stocks, Andreas Wodarz for anti-Bazooka antiserum, Eva Theilenberg for anti-Crb and anti-DPATJ antibody production, Otto Baumann for help with cryosections, and André Bachmann and Paul Hardy for critical reading of the manuscript. The work was supported by an EMBO long-term fellowship to M.R.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES