3.27 Cell Differentiation

[Bulah Landaker]

Ahallmark 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.

Visual systems are populated by one of two fundamental types of photoreceptors, ciliary and rhabdomeric. Each photoreceptor type is defined by the opsin molecule expressed and the final morphological form adapted to house the phototransduction machinery. Here we address whether a common transcriptional mechanisms exists for the differentiation of rhabdomeric photoreceptors. We demonstrate that orthologs of two Drosophila (fruit fly) transcription factors, Pph13 and Orthodenticle, are expressed in photoreceptors of Pancrustaceans, Tribolium (red flour beetle) and Daphnia (water flea), and are capable of executing conserved functions of rhabdomeric photoreceptor differentiation. In particular, Tribolium and Daphnia orthologs are capable of substituting and rescuing the photoreceptor differentiation defects observed in their corresponding Drosophila mutants. Furthermore, loss of function analysis in Tribolium of both Pph13 and orthodenticle genes demonstrate they regulate opsin transcription and morphogenesis of the photoreceptor apical membrane. Our data illuminate a framework for rhabdomeric photoreceptor differentiation and provide the foundation for defining the ancestral regulatory modules for rhabdomeric differentiation and potential modifications that underlie the functional diversity observed in rhabdomeric photoreceptors.

Copyright: 2014 Mahato et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In Drosophila, two homeodomain proteins have been identified that are critical for regulating r- photoreceptor differentiation. The first, Orthodenticle (Otd), is the Drosophila ortholog of a conserved family of Otd/Otx homeodomain transcription factors, which are essential for head and brain development across species [11], [12]. In the r- photoreceptors of Drosophila eyes, otd is required for both aspects of differentiation [13]. Otd promotes the proper morphogenesis of rhabdomeres and directs multiple aspects of the differential expression of r- opsin paralogs, which characterizes the complex visual organization of the Drosophila retinas (for a review see [14]). In particular, Otd is required for the expression of rh3, an ultra-violet (UV) sensitive r- opsin, and rh5, a blue (B) sensitive r- opsin in the two inner photoreceptors of Drosophila ommatidia [15], [16]. In addition, Otd is critical for repressing rh6, the Drosophila ancestral long-wave (LW) opsin [17] in the six outer photoreceptors of the Drosophila ommatidium [16].

To examine whether Pph13 and Otd could represent a common set of transcription factors required for r- visual photoreceptor differentiation, we chose to investigate their orthologs from two key nodal species, Tribolium castaneum (red flour beetle), a second insect, and Daphnia (water flea), a crustacean. Together, insects and crustaceans define the superclade Pancrustacea within the Arthropoda [26], [27] and any similarities between Daphnia, Tribolium, and Drosophila r- photoreceptor differentiation would indicate a pathway common to the ancestor that generated both lineages, at least 500 million years ago [28], [29]. First, we demonstrate that Otd and Pph13 orthologs are present and expressed in the visual systems of both species. Consistent with conservation of Pph13 mediated r- opsin regulation, the Pph13 RCSI binding site is conserved in the promoters of the r-opsin genes of both Tribolium and Daphnia and found only within LW r- opsins. Further, the Tribolium and Daphnia Pph13 homologs have retained similar DNA binding capabilities to their respective endogenous RCSI sites and we confirmed their functional equivalency to direct photoreceptor differentiation in Drosophila photoreceptors by transgenic rescue. The Otd paralogs of Tribolium and Daphnia are comparable in their ability to direct rhabdomere morphogenesis but exhibit differential abilities with respect to r- opsin regulation in Drosophila. Lastly, functional analyses in Tribolium reveal that both Pph13 and Otd homologs are essential for both aspects of photoreceptor differentiation, rhabdomere creation and r-opsin expression. In particular, Pph13 is a critical factor for LW r-opsin expression and Otd2 is necessary for the transcription of UV sensitive r-opsin. In summary, our data identify common components for rhabdomeric photoreceptor differentiation among Pancrustaceans, providing a foundation for defining the ancestral transcriptional mechanisms for rhabdomeric photoreceptor differentiation throughout Bilateria.

As a first step towards examining whether the role of Pph13 and Otd in Drosophila r- visual photoreceptor differentiation was conserved, we investigated the conservation of orthologs in the genome sequences of the distantly related arthropod species, Tribolium castaneum and Daphnia pulex [30], [31]. Tribolium had been previously shown to possess two paralogs of Otd: Otd1 and Otd2 [32]. The same state was described in the Crustacean Parhyale hawaiensis [33]. However, the relationships of the crustacean and coleopteran Otd homologs to the singleton homolog of Drosophila were previously considered unresolved due to the low level of sequence conservation outside the homeodomain; within Diptera there has been a reduction to only one otd paralog (Figure 1A, S1 and [33]). As in Parhyale, our search in Daphnia pulex as well as Daphnia magna identified two Otd homologs. Protein sequence alignment of an expanded set of Otd homologs (Figure 1A) revealed a highly conserved leucine (L) residue at the C-terminal end of the Otd1 homeodomain, which was unique for Paired-class homeodomain proteins in general [34], distinguished the insect representatives of the Otd1 subfamily, including all dipteran homologs. This finding established Drosophila Otd as a member of the insect Otd1 subfamily, implying the loss of insect Otd2 in the evolutionary lineage to Diptera. This conclusion was tentatively supported in a molecular phylogenetic analysis of the relationships between Otd homologs (Figure S1). The latter approach and amino acid residues in the homeodomain that were unique to each of the Parhyale and Daphnia Otd sequences further suggested that the latter duplicates represented the results of independent gene duplications in crustacean and insect lineages. Thus, the use of the previously introduced acronyms Otd1 and Otd2 for Parhyale and Daphnia paralogs do not imply specific orthology to insect Otd1 and Otd2.

The presence of potential RCSI sites in photoreceptor-expressed genes of all three species suggested that these sites could serve as Pph13 binding sites. To test this possibility, we investigated whether the Pph13 homologs could bind the putative endogenous RCSI sequences with electrophoretic mobility shift assays (EMSAs). These experiments revealed that Tribolium and Daphnia Pph13 have similar binding abilities to a consensus RCSI site (P3) [24], [43] as well as specific Drosophila RCSI sites (Figure 5 and data not shown). Furthermore, each has the capability to bind to their endogenous RCSI sites (Figure 5 and Figure S7). Interestingly, in Tribolium like Drosophila [20], we observed a differential affinity of Pph13 to the identified RCSI sites of UV and LW r-opsins. Tribolium Pph13 bound efficiently to the LW opsin RCSI site but binding was barely detectable on the UV opsin RCSI element, suggesting that the simple presence of a correctly spaced palindromic sequence of TAAT was not sufficient to bind Pph13.

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.

3a8082e126