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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00651

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Mutation of the photoreceptor specific homeodomain gene Pph13 results in defects in phototransduction and rhabdomere morphogenesis

Andrew C. Zelhof*, Edmund Koundakjian, Audra L. Scully{dagger}, Robert W. Hardy and Linda Pounds

Division of Biology, University of California, San Diego. La Jolla, CA 92093, USA



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  		Fig. 1. Pph13hazy mutants have malformed rhabdomeres and a dramatic loss in light sensitivity. (A,B) Transmission electron microscopy analysis of cn bw (A) and Pph13hazy (B) ommatidium. Mutant rhabdomeres are small, malformed and individual microvilli are misaligned. (C) Electroretinograms of cn bw, Pph13hazy and Pph13hazy; hs-Pph13 (a Pph13 cDNA under the control of a heat shock promoter). Each consecutive 520 nm light pulse represents a 10-fold increase in light intensity relative to the previous one. The last light pulse is a 10 second pulse of 570 nm light at equal intensity to the last 1 second pulse. The reintroduction of the Pph13 cDNA rescues all Pph13hazy mutant phenotypes.

 


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  		Fig. 2. Pph13hazy genetic interval and homeodomain protein comparisons. (A) Recombination and deficiency mapping placed Pph13hazy in a small genetic interval 21C7-21D1 on the left arm of the second chromosome. All deficiency and duplication stocks were obtained from the Bloomington Stock Center, except Df(2L)4989, which represents an X-ray induced allele of Pph13hazy (A.C.Z., unpublished). (B) Comparison of the Pph13 homeodomain to Drosophila Aristaless (Al) and Aristaless-related homeobox protein (Arx). The red asterisk represents the amino acid change in Pph13 (W to Stop).

 


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  		Fig. 3. Pph13 is expressed in Drosophila photoreceptor cells. Spatial and temporal expression profile of Pph13. (A) Stage 16 embryo. Pph13 is only detected in the bilateral cluster of cells that constitute Bolwig's organ. The box represents a higher magnification view. The staining of the embryonic trachea is non-specific. (B-D) Pph13 (green) and F-actin (red) staining of developing photoreceptor cells: (B) 36 hours APF; (C) 48 hours APF; (D) 72 hours APF. Pph13 expression is detected in all eight photoreceptor cells. At 72 hours APF, not all of the photoreceptor nuclei are shown in this optical section. (E-G) Pph13 (red) and Arr2-nuclear lacZ (green) expression in a cryostat-section of a 3-day-old adult head. Pph13 localization is maintained in adult Drosophila photoreceptor cells.

 


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  		Fig. 4. Pph13hazy mutants are stalled in development at 72 hours APF. (A-C) Immunofluorescence of rhabdomeric proteins in cn bw ommatidia at 72 hours APF. (D) Actin and 21A6 staining in cn bw ommatidium at 96 hours APF. (E-G) Immunofluorescence of rhabdomeric proteins in Pph13hazy ommatidia at 72 hours APF. (H) Actin and 21A6 staining in a Pph13hazy ommatidium at 96 hours APF.

 


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  		Fig. 5. Transmission electron microscopy of a cn bw (A,C) and Pph13hazy (B,D) ommatidium at 60 hours APF (A,B) and at 72 hours APF (C,D). Arrows denote irregular shape and size of the mutant rhabdomeres.

 


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  		Fig. 6. Rac1 expression and localization in wild-type and Pph13hazy mutant photoreceptor cells. (A-D) Wild-type (A,C) and mutant (B,D) photoreceptor cells expressing Rac1 (green) and F-actin (red) at 72 hours APF (A,B) and at 96 hours APF (C,D). The preparations were scanned at the same intensity of light. Arrows indicate the ectopic and grossly abnormal accumulation sites of Rac1 localization in mutant photoreceptor cells.

 


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  		Fig. 7. Pph13 is required for the expression of photoreceptor specific genes. (A-C) Western analysis of photoreceptor specific proteins in cellular extracts isolated from cn bw and Pph13hazy adult heads. There is no detection of TRPL, Arr1, TRP{gamma} and eye Gß in Pph13hazy mutant extracts. Both NinaC isoforms (p132 and p174) are shown. (D) Immunofluorescence of photoreceptor specific proteins of 1 µm frozen sections of light-exposed cn bw and Pph13hazy eyes. INAD and TRP localize to the rhabdomeres in both wild-type and mutant cells. TRPL and eye Gß are not detected in Pph13hazy mutants. Arr1 expression is only detected in the inner photoreceptor cells (R7, R8, arrow) and rhodopsin (Rh1) is detected only in the six outer photoreceptor cells (R1-R6).

 


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  		Fig. 8. eye Gß is a transcriptional target of Pph13. (A) Reverse transcriptase reactions coupled to PCR for the detection of photoreceptor specific mRNAs. Transcripts for trpl and eye Gß are not detected in Pph13hazy mutants. (B) Potential Pph13 binding sites in the eye Gß enhancer and electrophoretic mobility shift assay. Full-length Pph13 binds specifically to the palindromic sites located in the eye Gß enhancer. Arrow indicates shifted complex and asterisks denote the mutated base pairs. W, wild-type enhancer; M, mutated enhancer. (C) Transient transfection assay. The bar graph shows the data from one experiment that is representative of all the transient transfection experiments performed in this study. Each point represents the average of 16 transfected wells (error bar indicates s.d. of each data set). (D) Western analysis of eye Gß-GFP expression in wild-type and mutant Pph13hazy flies. Protein extracts were isolated from both heads and bodies of wild-type and Pph13hazy mutant flies that contained two copies of the eye Gß enhancer driving GFP expression. Antibodies against G{alpha} were used as a loading control for the head extract lanes.

 

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© The Company of Biologists Ltd 2003