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. 2014 Jul 3;10(7):e1004484.
doi: 10.1371/journal.pgen.1004484. eCollection 2014 Jul.

Common Transcriptional Mechanisms for Visual Photoreceptor Cell Differentiation Among Pancrustaceans

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Free PMC article

Common Transcriptional Mechanisms for Visual Photoreceptor Cell Differentiation Among Pancrustaceans

Simpla Mahato et al. PLoS Genet. .
Free PMC article

Abstract

A hallmark of visual rhabdomeric photoreceptors is the expression of a rhabdomeric opsin and uniquely associated phototransduction molecules, which are incorporated into a specialized expanded apical membrane, the rhabdomere. Given the extensive utilization of rhabdomeric photoreceptors in the eyes of protostomes, here we address whether a common transcriptional mechanism exists for the differentiation of rhabdomeric photoreceptors. In Drosophila, the transcription factors Pph13 and Orthodenticle (Otd) direct both aspects of differentiation: rhabdomeric opsin transcription and rhabdomere morphogenesis. We demonstrate that the orthologs of both proteins are expressed in the visual systems of the distantly related arthropod species Tribolium castaneum and Daphnia magna and that their functional roles are similar in these species. In particular, we establish that the Pph13 homologs have the ability to bind a subset of Rhodopsin core sequence I sites and that these sites are present in key phototransduction genes of both Tribolium and Daphnia. Furthermore, Pph13 and Otd orthologs are capable of executing deeply conserved functions of photoreceptor differentiation as evidenced by the ability to rescue their respective Drosophila mutant phenotypes. Pph13 homologs are equivalent in their ability to direct both rhabdomere morphogenesis and opsin expression within Drosophila, whereas Otd paralogs demonstrate differential abilities to regulate photoreceptor differentiation. Finally, loss-of-function analyses in Tribolium confirm the conserved requirement of Pph13 and Otd in regulating both rhabdomeric opsin transcription and rhabdomere morphogenesis. Taken together, our data identify components of a regulatory framework for rhabdomeric photoreceptor differentiation in Pancrustaceans, providing a foundation for defining ancestral regulatory modules of rhabdomeric photoreceptor differentiation.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Protein sequence conservation in Otd and Pph13 homologs.
A. Otd homologs. Grey boxes outline paralog Otd subfamilies generated by independent gene duplications. The annotation of the Anopheles gambiae (Agam) Otd1 contains a putative insertion (VSVGE), which has been removed at the position indicated by asterisk. Green background: Leucine residue diagnostic for insect Otd1. Bold font highlights shared residues in each the Daphnia and Parhyale Otd duplicates, which support their independent origins in the two lineages. B. Pph13 homologs. Grey boxes outline invertebrate Arx paralog subfamilies generated by independent gene duplications. Green background: Tyrosine (Y) residue diagnostic for Arx gene family members. Blue background: Phenylalanine (F) residue diagnostic for Pph13 subfamily members. Orange background: Threonine (T) residue diagnostic for the Al subfamily and singleton Arx homologs. Species abbreviations: Acal = Aplysia californica, Agam = Anopheles gambiae, Amel = Apis mellifera, Bflo = Branchiostoma floridae, Ccap = Ceratitis capitata, Cint = Ciona intestinalis, Dmag = Daphnia magna, Dmel = Drosophila melanogaster, Dpul = Daphnia pulex, Mdom = Musca domestica, Mmus = Mus musculus, Pdum = Platynereis dumerilii, Phaw = Parhyale hawaiensis, Skow = Saccoglossus kowalevskii, Smed = Schmidtea mediterranea, Tcas = Tribolium castaneum.
Figure 2
Figure 2. Spatial expression patterns of Pph13, otd1 and otd2 in the developing adult Tribolium visual system.
A–C. Dissected retinas 30–40 hrs APF. A. Pph13 is detected in the developing photoreceptor field and can be observed in all eight photoreceptors of a single ommatidium (A′). B. otd1 expression is limited to all eight photoreceptors of each developing ommatidium (B′). C. otd2 expression like Pph13 and otd1 is detected in developing photoreceptor field. Expression is greater in the two central photoreceptors R7 and R8 (C″) as compared to the outer six photoreceptors (C′). D–F. Dissected retina 24 hrs APF from Tribolium castaneum adult visual system stained for Pph13 protein (green) and 3XP3-RFP (magenta). An antibody recognizing Pph13 (green) demonstrates that Pph13 is located in each photoreceptor nucleus. 3XP3-RFP is a cytoplasmic marker for the developing photoreceptors and labels the cell bodies and axon projections of the eight photoreceptors in each ommatidium . Note the expression of Pph13 appears prior to 3XP3-RFP expression (asterisks). In all panels anterior is to the left. Numbers label the eight photoreceptors of a single ommatidium.
Figure 3
Figure 3. Spatial expression patterns of Daphnia magna r- opsins during Daphnia eye development.
A–C. Daphnia embryos 50 hours after egg deposition. A. RNA in situ hybridization of a pool containing three antisense probes against potential Daphnia magna long wave (LW) clade A r- opsins. B. RNA in situ hybridization of a pool containing three antisense probes against potential Daphnia magna long wave (LW) clade B r- opsins. C. RNA in situ hybridization of an antisense probe against the single potential Daphnia magna ultra violet (UV) r- opsin. In all three cases, the probes label photoreceptors in the visual system, and the LW clade B probes also mark and label the photoreceptors of the ocellus (arrow). Insets represent a higher magnified view.
Figure 4
Figure 4. Spatial expression patterns of Daphnia magna Otd1, Otd2 and Pph13 during Daphnia eye development.
A–C. Daphnia embryos 40 hours after egg deposition (AED) stained for Otd1 (magenta) and Otd2 (green) protein. The nuclei were countered stained with DAPI (blue). Otd1 expression is limited to the midline and Otd2 can be detected in the ocellus (arrow) and in the visual system (asterisks). Inset in C is a higher magnification of the head region. D–F. Temporal expression profile of Otd2 expression (green). Otd2 is expressed in an increasing number of positive cells in two lateral symmetrical regions of the head during embryogenesis. G. Pph13 RNA in situ pattern. Inset is a higher magnification of the expression of Pph13 in the presumptive visual system. H. Pph13 RNA in situ pattern of the same embryo as in I. I. Double label of Pph13 and Otd2 protein. Note the localization of both Pph13 and Otd2 in the developing eye and ocellus (inset). Asterisks mark the two lateral halves of the developing visual system and arrows mark the ocellus.
Figure 5
Figure 5. DNA binding properties of Pph13 are conserved among Pancrusteceans.
Electrophoretic mobility shift assays of Drosophila (Dmel) and Tribolium (Tcas) and Daphnia magna (Dmag) Pph13 protein on a Pax6 homeodomain binding site (P3) and on endogenous Tribolium and Daphnia RCSI sites. Each homolog has the ability to bind the P3 consensus binding site as well as endogenous sites within its genome. Tribolium Pph13 demonstrates a differential binding to the RCSI sites identified in the cis-regulatory regions of the LW and UV r- opsins and binding is disrupted upon the mutation of the LW RCSI site (LW*). Arrows indicate the specific mobility shift for each protein examined.
Figure 6
Figure 6. In vivo rescue of opsin expression in Pph13 null mutant Drosophila.
A–E. Rh6 (green) and Rh5 (magenta) opsin protein expression in adult Drosophila retinas. A. Wild-type retina. Opsin protein accumulates in the rhabdomeres and thus appears as a tube like structure. Rh6 and Rh5 are expressed a non-overlapping subsets of central R8 photoreceptors. B. Pph13 mutant. Neither Rh6 nor Rh5 opsin can be detected, reflecting the facts that Rh6 is a direct transcriptional target of Pph13 and that the deformed development of rhabdomeres in Pph13 mutants prevents the accumulation of detectable levels of Rh5 opsin. C–E. Rescue of the Drosophila Pph13 mutant with: C. Drosophila (Dmel) Pph13. D. Tribolium (Tcas) Pph13. E. Daphnia magna (Dmag) Pph13. In all rescue experiments we observe the restoration of Rh6 and Rh5 protein expression in the rhabdomeres. Furthermore, the non-overlapping expression of Rh6 and Rh5 in R8 photoreceptors is likewise rescued. Each panel represents a projection of a confocal stack of laser scanning confocal microscope images.
Figure 7
Figure 7. In vivo rescue of rhabdomere morphology in Pph13 null mutant Drosophila.
A–E. Transmission electron microscopy analysis of adult Drosophila eyes. A. Wild-type ommatidium. Seven of the eight photoreceptors and associated rhabdomeres (R1–R7) can be observed per section and each rhabdomere is separated by an inter-rhabdomeral space (IRS). B. Pph13 mutant. The absence of Pph13 activity results in the reduction, malformation or loss of rhabdomeres (arrow). C–E. Rescue of the Drosophila Pph13 mutant with: C. Drosophila (Dmel) Pph13. Rhabdomere morphology is restored. D. Tribolium (Tcas) Pph13. Rhabdomere morphology is restored but occasionally a missing rhabdomere is observed (D′). The arrow denotes a missing rhabdomere of a photoreceptor but the photoreceptor is present. E. Daphnia magna (Dmag) Pph13. Whereas apical sections (E) demonstrate complete restoration of rhabdomere morphology, basal sections (E′) demonstrate that many rhabdomeres do not extend completely or maintain their shape throughout the length of the photoreceptor. Scale bar 2 um.
Figure 8
Figure 8. In vivo rescue of rhabdomere phenotype of Drosophila orthodenticle mutant background.
A–G. Transmission electron microscopy analysis of adult Drosophila eyes. A. Wild-type ommatidium. B. otd mutant. The absence of Otd activity results in smaller malformed or even absent rhabdomeres (arrow). C–G. Rescue of otd mutant with: C. Drosophila (Dmel) otd. D. Tribolium (Tcas) otd1. E. Tribolium (Tcas) otd2. The arrow denotes a missing rhabdomere of a photoreceptor. F. Daphnia magna (Dmag) otd1. G. Daphnia magna otd2. In all cases, except Dmag otd2, rhabdomere morphology is restored. The rhabdomere phenotype observed with Dmag otd2 differs from both wild type and the otd mutant phenotype. Scale bar 2 um.
Figure 9
Figure 9. In vivo rescue of Rh3 opsin expression in Drosophila orthodenticle mutant background.
A–E. Rh3 opsin protein expression in adult Drosophila retinas. A. Wild-type retina. Rh3 opsin protein accumulates in the rhabdomeres of approximately 30% of the R7 photoreceptor cells. B. otd mutant. Consistent with the requirement of Otd for the transcriptional activation of rh3, no protein is detected. C–G. Rescue of otd mutant with: C. Drosophila (Dmel) otd. D. Tribolium (Tcas) otd1. E. Tribolium (Tcas) otd2. F. Daphnia magna (Dmag) otd1. G. Daphnia magna otd2. All Otd orthologs are capable of restoring Rh3 expression in an otd mutant except Dmag otd2. Insets represent a magnified view of a region from each panel. Each panel represents a projection of a confocal stack.
Figure 10
Figure 10. Knockdown of Tribolium Pph13, otd1 and otd2 affects rhabdomere biogenesis.
Scanning and transmission electron microscopy analyses of Tribolium adult eyes. A,F. Mock injection. B,G. Pph13 RNAi. C,H. otd1 RNAi. D,I. otd2 RNAi. E,J. otd1 and otd2 RNAi. The green shading marks the cell body of one of the eight photoreceptors (PC) found in each ommatidium. Each photoreceptor produces a rhabdomere (R1–R8) that remains juxtaposed to the others. The knockdown of Pph13 and orthodenticle paralogs did not affect the overall structure of the adult eye (A–E). However, the knockdown of Pph13 (G) and the combinatorial removal of both otd paralogs result in a complete absence of rhabdomeres (J). Photoreceptors are detected but the microvillar rhabdomere structures are absent. The knockdown of otd1 generates smaller rhabdomeres (H) and the removal of otd2 (I) alone results in rhabdomeres in an active state of degeneration (arrow).
Figure 11
Figure 11. Tribolium Pph13 and Otd2 are required for r-opsin expression.
A–E. 3XP3-RFP expression in RNAi knockdown animals. Photoreceptor specific RFP fluorescence can be detected through the pupal case in the developing adult eyes (brackets). Only the knockdown of Pph13 results in the loss of the transcriptional reporter 3XP3-RFP fluorescence (arrows). The asterisks mark the non-visual enhancer trap expression of RFP that is present in each RNAi knockdown animal. F. RT-PCR analysis of r-opsin transcription in Tribolium (Tcas) Pph13, otd1, otd2, and otd1/2 RNAi knockdown animals. The transcriptional levels of LW r-opsin are only reduced/absent with the reduction of Pph13 and transcription levels of UV r- opsin are only affected by the reduction of otd2. The transcription of Tribolium ribosomal protein subunit three (RpS3) was used as a control for comparisons. G–H. RNA-seq quantification of r- opsin expression levels, LW (G) and UV (H) in the knockdown animals. The RNA-seq data confirms the requirement of Pph13 and Otd2 for transcription of r- opsin LW (TC013765) and UV (TC000118), respectively. Wild type (wt) are represented by the control mock injections. P-Values have been adjusted for multiple testing.

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References

    1. Eakin RM (1965) Evolution of photoreceptors. Cold Spring Harbor Symposia on Quantitative Biology 30: 363–370. - PubMed
    1. Arendt D, Wittbrodt J (2001) Reconstructing the eyes of Urbilateria. Philos Trans R Soc Lond B Biol Sci 356: 1545–1563. - PMC - PubMed
    1. Arendt D (2008) The evolution of cell types in animals: emerging principles from molecular studies. Nat Rev Genet 9: 868–882. - PubMed
    1. Lamb TD, Arendt D, Collin SP (2009) The evolution of phototransduction and eyes. Philosophical transactions of the Royal Society of London Series B, Biological Sciences 364: 2791–2793. - PMC - PubMed
    1. Nilsson DE, Arendt D (2008) Eye evolution: the blurry beginning. Current Biology: CB 18: R1096–1098. - PubMed

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This work was supported by funds from a grant from the Ministry of Education, Culture, Sports, Science and Technology (YS), NSF proposal IOS-0951886 (MF) and Indiana University (ACZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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