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Eyes closed, a Drosophila p47 homolog, is essential for photoreceptor morphogenesis

Tzu-Kang Sang and Donald F. Ready*

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA



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  		Fig. 1. eyc is required for normal Drosophila photoreceptor morphogenesis. (A) In wild-type eyes, the closely packed photosensitive microvilli of the rhabdomere (r) appear as dark circular ovals arranged in a trapezoid. Rhabdomeres are positioned in the inter-rhabdomeral space (IRS) by a surrounding membrane domain, the stalk (s). Together, the rhabdomere and stalk constitute the photoreceptor apical plasma membrane. (B) In eyc1 eyes, abnormal apical membrane contacts, including rhabdomere-rhabdomere and rhabdomere-stalk contacts, disrupt the rhabdomere trapezoid and partition the IRS into irregular chambers. (C) eycP and (D) eycP in trans with Df(2R)Px2 show similar disturbances of rhabdomere topology. (E,F) In 37% p.d. wild-type (E) and eyc1 (F) ommatidia, irregularly infolded photoreceptor apical surfaces, which are bounded by zonula adherens (z.a.) junctions, face each other in a trapped apical cavity. (G) By 55% p.d., wild-type photoreceptor apical surfaces have differentiated well-ordered stalk and rhabdomere subdomains. Face to face contacts have been released and the IRS has opened. Processes of the four cone cells (ccp) appear as small circular profiles ‘behind’ z.a. junctions between photoreceptors. (H) In 55% p.d. eyc1 ommatidia, distinct rhabdomere and stalk membrane can be recognized, but these are irregularly ordered. Face to face apical contacts have not released and the IRS is fragmented into small chambers. Loops of stalk membrane (arrows) are trapped by abnormal adhesions. Scale bar: 2 µm. Anterior is towards the right.

 


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  		Fig. 2. eyc1 R cells inappropriately retain apical adhesions of rhabdomere extension. (A, left) In 37% p.d. wild-type ommatidia, actin-rich R cell apices face each other in a trapped pocket. Bright ‘bars’ of Armadillo staining highlight zonula adherens (z.a.) junctions delimiting R cell apical surfaces. At lesser intensity, Armadillo is detected across the entire apical membrane. Crumbs, an apical membrane protein (Tepass, 1996), is also distributed across R cell apical membranes. R cell apices are relatively small at this stage; the trapped apical pocket is approximately 5 µm deep. By 50% p.d. (A, center), definitive, actin-rich rhabdomere primordia are established and have extended to the retinal floor, a depth of approximately 15 µm. The stereotyped pattern of contacts prefiguring the adult trapezoid is evident along the planes of separation opening the IRS. Armadillo has largely retreated to the z.a. junctions. Light apical face staining is often encountered in R7. Crumbs remains across the entire apical surface. (A, right) In 50% p.d. eyc1 eyes, inappropriate contacts between R cells are evident. Contacts between photoreceptors R2, R4 and R7 are prominent. R5 and R6 often establish strong face to face contact. Arm staining typically marks adhesions (arrowheads). Crumbs staining is normal in the mutant at this stage. (The more open mesh of the pigment cells results from removal of the cornea during dissection.). (B, top) In 55% p.d. wild-type eyes, DE- Cadherin and Armadillo are largely absent from R cell apical surfaces. (bottom) In 55% p.d. eyc1 ommatidia, bars of colocalized Armadillo and DE-cadherin mark sites of contact. Anterior is towards the right, polar is towards the top. Scale bar: 4 µm.

 


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  		Fig. 3. eyc encodes a Drosophila p47 homolog and eyc is misexpressed in early pupae. (A) A Northern blot shows a 2 kb genomic probe detects a 1.1 kb mRNA, the levels of which are elevated at ~30% eyc and P1363/Df(2R) Px2 (P/Df) flies relative to wild type (CS); RP49 was used as loading control. (B) A Western blot shows the anti-Eyc antiserum detects Eyc protein, which is more abundant in ~30% p.d. eyc1 retinal extract compared with wild type at the same developmental stage; ß-Tubulin was used as loading control. (C) Molecular map of eyc locus. 60D1 represents genome project predicted genomic map of eyc locus; predicted genes nearby eyc are denoted as white bars with pointed ends indicating transcript orientation. The eyc locus is shown above, with the white bar indicating the ORF. The eyc allele has two 3' point mutations (arrowheads). The P-element insertion site of eycP allele is shown. The eyc null allele eycl39 is shown below 60D1; brackets mark the determined deletion and broken lines indicate undetermined potential deletion. The DNA fragment used to construct the pUAS-eyc transgene is shown as a hatched bar. (D) ClustalW alignment of the predicted Eyc amino acid sequence with two related proteins, rat p47 (human p47 sequence is 95% identical) and yeast SHP1. Residues identical in all three species are shown on a red background and are indicated by ‘!’; residues identical or with conserved substitutions in two species are shown on black or blue, respectively, and are indicated by an asterisk. The p47/Eyc family shares about 30% protein sequence identity and ~45% similarity. eyes closed sequence data are available from GenBank/EMBL/DDBJ under Accession Number AF170565.

 


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  		Fig. 4. Expression of Eyc in pupae produces an eyc-like eye phenotype. (A) Electron micrograph of an adult eye of a Hs-GAL4/UAS-eyc fly exposed to six pulses of 45 minutes heat shock starting at 30% p.d. Approximately 23% of ommatidia in which Eyc was transgenically misexpressed show abnormal contact between rhabdomeres. (B) Heat shocks from 30% to 100% p.d. resulted in reduced rhabdomeres with abnormal contacts. Unshocked animals or parallel heat shocks to control flies did not produce rhabdomere defects. (C) Confocal micrographs of phalloidin-stained GMR-GAL4/UAS-eyc eyes show abnormal contacts of rhabdomeres (arrows). (D) Confocal micrograph of an Hs-GAL4/UAS-eyc eye whole-mount double-labeled with rhodamine-phalloidin (red) and 4C5 anti-Rh1 antibody (green). This animal received the same heat shock regimen as that in B. It shows abnormal rhabdomere adhesion in some photoreceptors (arrows). Deficient delivery of rhodopsin to the rhabdomere is also evident. Arrowheads indicate normal rhodopsin localization in some photoreceptors. Hs-GAL4 flies that received the same heat shock treatment do not show an eye phenotype. Scale bars: in A, 1 µm in A,B; in C, 5 µm in C,D.

 


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  		Fig. 5. Eyc misexpression increases R cell ER and inhibits rhodopsin delivery to the rhabdomere. In parallel confocal (A,B) and electron microscope (C,D) preparations, Hs-GAL4/UAS-eyc flies (A,C) and Hs-GAL4/+ (B,D) were given three pulses of 1 hour 37°C heat shock with 5 hours 25°C recovery starting at ~70% p.d. After the last recovery, animals were dissected and processed for parallel experimental preparations. For confocal microscopy, retinal whole-mounts were stained using rhodamine-phalloidin (red) and 4C5 anti-Rh1 antibody (green). (A) Eyc overexpression results in rhodopsin accumulation in the cytoplasm and diminished delivery to the rhabdomere. (B) Rhodopsin localization is normal in heat-shocked controls; the central R7 rhabdomere does not express Rh1 and consequently only stains with phalloidin. (C) ER accumulates in photoreceptors overexpressing Eyc. (D) ER is normal in parallel heat-shocked Hs-GAL4/+ controls. Scale bars: in A, 5 µm in A,B; in D, 2 µm in C,D.

 


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  		Fig. 6. Eyc overexpression causes ER proliferation and inhibition of Rh1 maturation. Quantitation of ER stacks in R cells from the experiment shown in Fig. 5C,D. (A) In Hs-GAL4/+ control, the majority of R cells show two to three ER stacks per cross section, while heat shocked Hs-GAL4/UAS-eyc R cells typically show greater numbers of stacks. (B) Western blot from the parallel experiment shows the overexpression of Eyc inhibits Rh1 maturation. Protein samples were extracted from two eyes; ß-Tubulin was used as a loading control. 4C5 anti-Rh1 antibody detects a 35 kDa mature form of Rh1, levels of which are significantly reduced in Hs-GAL4/UAS-eyc fly eyes compared with Hs-GAL4/+. Additionally, higher molecular weight, immature Rh1 is more abundant in Hs-GAL4/UAS-eyc eyes than in the Hs-GAL4/+ control.

 


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  		Fig. 7. Inhibition of p47/Eyc disrupts nuclear envelope assembly. Wild-type, eycl39 (1.5-2 hours at 25°C), and microinjected syncytial blastoderm embryos triple labeled with phalloidin (red), anti-Lamin (green) and YOYO-1 (blue). Confocal images focus on the cortex of the syncytial blastoderm. (A) In wild-type M phase embryos, Lamin staining shows disassembled nuclear envelope while nuclear vesicles congregate the separated chromosomes. (B) Interphase embryos show Lamin staining of nuclear envelopes and a normal hexagonal array of phalloidin staining. Two examples of eycl39 embryos show abnormal Lamin staining pattern. (C) A commonly observed eycl39 phenotype shows loss of normal staining pattern for the three markers. (D) An optical section of a mutant embryo in the same focal plane as in B shows a cloudy Lamin staining, possibly owing to the aggregation of Lamin-coated nuclear vesicles. (E-H) Microinjection of wild-type stage 2-3 syncytial blastoderm embryos with pre-immune serum (E,G) or anti-Eyc antiserum (F,H). Equal volumes of serum were microinjected at the posterior (right) end of the embryo. (E,F) Projection views of nine sections, 4 µm apart from the cortex to the embryo core. (E) Embryos injected with pre-immune serum show a normal hexagonal array of cortical nuclei (the blue patch at the posterior is a YOYO-1 crystal). (F) Anti-Eyc antiserum-injected embryo shows fewer nuclei in the posterior region with abnormal nuclear aggregates. (G) An optical section through the embryo shows normal, densely-packed cortical nuclei surrounding embryo in a pre-immune serum injected embryo. (H) A corresponding side view of an embryo injected posteriorly with anti-Eyc shows loosely-distributed, spherical nuclei at the posterior. Scale bar: 20 µm in A-D; 100 µm in E-H.

 

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© The Company of Biologists Ltd 2002