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PvuII-PstI homology 13

Targets of Activity

Given that Pph13 is a transcription factor, it was reasoned that the nature of the signaling defect is due to the loss or misexpression of known phototransduction proteins. To test this hypothesis, presence of phototransduction proteins was examined in adult head extracts isolated from wild-type and Pph13^hazy mutants. Western analysis revealed undetectable levels of four phototransduction proteins -- eye Gß, TRPL, TRPgamma and Arr1 in Pph13^hazy mutants. Eye Gß with Ggamma is responsible for the coupling of Galpha to rhodopsin. In addition, strong hypomorphic alleles of Gß greatly reduce the ability of photoreceptor cells to respond to light. The activation of Galpha ultimately leads to the regulation of light sensitive cation-selective channels. In Pph13^hazy mutants, the cation-selective channels TRPL and TRPgamma are missing. Arr1, which has a role in the deactivation of rhodopsin and termination of the light response is also undetectable. Based on previous genetic and molecular studies of the missing proteins, none of them alone is sufficient to explain the severe loss of light sensitivity in Pph13^hazy mutants. Nonetheless, the defect observed in Pph13^hazy mutants may be a cumulative effect of all the missing proteins identified (Zelhof, 2003).

The majority of the proteins examined are present in Pph13^hazy mutant photoreceptor cells but Western analysis did not address the question of whether these proteins are localized correctly in photoreceptor cells. To check for proper subcellular localization, the expression of proteins was examined via immunofluorescence techniques on frozen thin sections of adult eyes. The results confirm the absence of TPRL and eye Gß in Pph13^hazy mutants. Western blot data suggest an absence of Arr1 expression but immunofluorescent data demonstrates that Arr1 levels are only diminished and not absent in mutant photoreceptors. Arr1 is detected in mutant R7/R8 photoreceptor cells, and in the outer photoreceptor cells (R1-R6) the levels of expression are probably below the level of detection, suggesting Pph13 is only required for full expression of Arr1 in all photoreceptor cells. Molecules that show normal or reduced levels of expression, such as with INAD or TRP, show correct subcellular localization to the malformed rhabdomeres in Pph13^hazy mutants. Overall, these results suggest Pph13 is downstream of the genes required for eye specification and Pph13 transcriptional targets are necessary for the proper detection of light (Zelhof, 2003).

Results suggest that Pph13 exerts its effect on photoreceptor differentiation by regulating transcription. If this is the case, it is predicted that the mRNAs for the missing proteins would not be detectable. RT-PCR reactions confirm that the transcripts for trpl and eye Gß are absent while the transcripts for arr1 are present. It is also predicted that if trpl or eye Gß represents Pph13 transcriptional targets, potential binding sites for Pph13 should exist in their promoter regions. The consensus DNA binding site for a Paired class homeodomain protein containing a glutamine at amino acid position 50 of the homeodomain is a palindrome of TAAT separated by three nucleotides. Scanning the transcriptional units of eye Gß and trpl reveal one element containing strong potential binding sites for Pph13 upstream of the transcriptional start of eye Gß. Within a span of 25 nucleotides, two palindromes are found spaced by three nucleotides and a third overlapping palindrome separated by two nucleotides (Zelhof, 2003).

To demonstrate binding specificity of Pph13 to this element, both a full-length copy of Pph13 and a GST fusion protein containing only the homeodomain of Pph13 were used in electrophoretic mobility shift assays (EMSA). In these assays, both protein forms specifically bind to this element. Mutation of the palindromic sites results in the elimination of Pph13 binding and, as expected, the mutated element could not compete for Pph13 binding. On the other hand, cold competitor of the wild-type element does result in a dose-dependent inhibition of Pph13 binding (Zelhof, 2003).

Based on these findings, it would be predicted that Pph13 acts as an activator and not a repressor of gene transcription. To test whether Pph13 has the ability to activate transcription, a reporter construct was created containing the eye Gß enhancer, nucleotides -323 to -105, upstream of the minimal rat prolactin promoter controlling luciferase expression. In transient transfection assays, upon the co-transfection of Pph13 an average of a thousand fold activation of transcription specifically is seen from the reporter containing the eye Gß enhancer as compared to the parental vector. Mutation of the palindromic binding sites within the response element eliminates the transcriptional activation seen with the addition of Pph13, confirming the role of Pph13 as a potential activator of photoreceptor specific gene expression (Zelhof, 2003).

To further prove a direct regulation of eye Gß by Pph13, it was asked whether a transgenic construct containing the eye Gß enhancer is expressed in photoreceptor cells and requires Pph13 for expression. As such, GFP was placed immediately downstream of first 424 nucleotides that are prior to the first ATG of eye Gß. Since the expression of GFP was low in all the transgenic lines used and combined with the auto-fluorescence of pigmented eyes, it was not possible to say with absolute certainty that this genomic region of eye Gß limits GFP expression only to photoreceptor cells. However, using Western blot analysis, GFP is detected only in head extracts; more importantly, GFP expression is dependent on the presence of Pph13. When the transgenic construct is recombined into a Pph13^hazy mutant background, GFP expression is greatly diminished (Zelhof, 2003).

Pph13 and orthodenticle define a dual regulatory pathway for photoreceptor cell morphogenesis and function

The function and integrity of photoreceptor cells are dependent upon the creation and maintenance of specialized apical structures: membrane discs/outer segments in vertebrates and rhabdomeres in insects. A molecular and morphological comparison was performed of Drosophila Pph13 and orthodenticle (otd) mutants to investigate the transcriptional network controlling the late stages of rhabdomeric photoreceptor cell development and function. Although Otd and Pph13 have been implicated in rhabdomere morphogenesis, this study demonstrate that it is necessary to remove both factors to completely eliminate rhabdomere formation. Rhabdomere absence is not the result of degeneration or a failure of initiation, but rather the inability of the apical membrane to transform and elaborate into a rhabdomere. Transcriptional profiling revealed that Pph13 plays an integral role in promoting rhabdomeric photoreceptor cell function. Pph13 regulates Rh2 and Rh6, and other phototransduction genes, demonstrating that Pph13 and Otd control a distinct subset of Rhodopsin-encoding genes in adult visual systems. Bioinformatic, DNA binding and transcriptional reporter assays showed that Pph13 can bind and activate transcription via a perfect Pax6 homeodomain palindromic binding site and the Rhodopsin core sequence I (RCSI) found upstream of Drosophila Rhodopsin genes. In vivo studies indicate that Pph13 is necessary and sufficient to mediate the expression of a multimerized RCSI reporter, a marker of photoreceptor cell specificity previously suggested to be regulated by Pax6. These studies define a key transcriptional regulatory pathway that is necessary for late Drosophila photoreceptor development and will serve as a basis for better understanding rhabdomeric photoreceptor cell development and function (Mishra, 2010).

To date, little is known about the transcriptional network required for establishing a rhabdomere. Significantly, these findings emphasize that there are at least two homeodomain transcription factors, Pph13 and Otd, that are required for photoreceptor cell morphogenesis and function. First and foremost, Pph13 and Otd cooperate for rhabdomere elaboration. The loss of either results in poorly formed rhabdomeres, although the rhabdomeres are still present and phototransduction proteins still accumulate within. Only upon the removal of both factors do the rhabdomeres fail to materialize. Moreover, the morphological analyses demonstrate that the failure of rhabdomeres to develop is due neither to degeneration nor to an inability to initiate the process. The data suggest that the roles of Pph13 and Otd are to coordinate and direct the morphological changes of the actin cytoskeleton and apical membrane into the specific and stereotypic structure of a rhabdomere. This dependency on two homeodomain transcription factors is in contrast to vertebrate photoreceptor cells, in which the identical process of expanding the membrane to house the phototransduction machinery is relegated to one protein, Crx, a vertebrate homolog of Otd (Mishra, 2010).

Another intriguing observation from these studies is the difference between the morphological role of Otd and Pph13 in ocelli versus eye photoreceptor cells. The loss of either factor in ocelli does not result in noticeable defects in rhabdomere formation, in stark contrast with the situation for the eye. Why the difference? One possibility is that the lack of an inter-rhabdomeral space decreases the pressure on the microvilli to create a cohesive structure. For example, a defined target of Otd is chaoptin, which encodes a protein that is crucial for proper microvilli adhesion and which is essential to keep the microvilli together during the formation of the inter-rhabdomeral space. As a result, the lack of an extracellular matrix does not interfere with the ability of the apical membrane to form microvilli. Nevertheless, determining exactly how Pph13 and Otd coordinate rhabdomere morphogenesis or why expansion of the photoreceptor membrane in rhabdomeres has been partitioned to two homeodomain transcription factors will require further characterization of many of the Pph13-, Otd- and Pph13-Otd-dependent transcription targets (Mishra, 2010).

The expression of Rhodopsin or other eye-specific phototransduction proteins is a key indicator of when a ciliated or rhabdomeric cell has been designated to act as a photoreceptor cell. With respect to Drosophila, the characterization of Rhodopsin promoters has indicated a bipartite structure (Fortini, 1990) for directing photoreceptor cell expression and subtype specificity. To ensure photoreceptor cell expression there is a common essential element that is found in all Drosophila Rhodopsin promoters: RCSI. RCSI represents one half of the bipartite structure, and mutation of this element eliminates photoreceptor cell expression. Although this element alone is not sufficient for photoreceptor cell expression, when multimerized, such as in the 3XP3 reporter, it is sufficient to drive and limit expression to all photoreceptor cells. Thus, the presence of the RCSI suggests that there is a common factor(s) required in all photoreceptor cells that ensures eye-specific expression of Rhodopsin genes. Based on these observations, it would be expected that an RCSI regulatory factor would have the following characteristics: (1) it should be expressed in all Drosophila photoreceptor cells; (2) it would be a homeodomain transcription factor that is able to form a hetero- or homodimer due to the presence of the palindromic sequence TAAT; and (3) it should be capable of binding the various RCSI elements and, most importantly, be sufficient and necessary for the expression of 3XP3 in vivo. The data presented in this study indicate that Pph13 satisfies all the above criteria. Furthermore, the microarray profiling has identified other known, and yet to be characterized, photoreceptor proteins (Rhodopsins, G?, NinaC, Arr2, Osi18, PIP82) that also share this RCSI sequence in their promoter region and are dependent on Pph13 for expression. Overall, Pph13 is not merely a factor ensuring Rhodopsin expression, but has a greater role in photoreceptor cell function (Mishra, 2010).

One important and conflicting question is, if Pph13 is the general transcription factor binding to the RCSI elements then why is there selective downregulation of specific Rhodopsin promoters in a Pph13 mutant? A model is proposed in which the dependency for expression of Rhodopsins on either the RCSI site or additional elements responsible for subtype-specific expression has shifted between the different Rhodopsin promoters. In other words, Pph13 does bind to every RCSI site but, owing to the bipartite structure of the Rhodopsin promoters, sequence differences in the individual RCSI elements and resultant differences in Pph13 affinity contribute to a situation in which the presence of Pph13 alone is not a limiting factor for expression. There are several observations that support such a model. First, whereas Pph13 is necessary and sufficient for expression of 3XP3, no ectopic expression of any Rhodopsin promoter reporters is observed in tissues outside of photoreceptor cells, confirming the idea that Rhodopsin promoters are coordinately regulated by other factors. Second, each RCSI element is not created equally. Swapping of RCSI domains between the different promoters does not dramatically affect spatial or temporal specificity (Papatsenko, 2001), but does reflect predictable changes in the level of expression based on Pph13 affinities that were observe in this study. For example, when the Rh6 RCSI site is placed into an Rh3 or Rh5 minimal promoter, there is an increase in expression level, and a reciprocal downregulation of expression is observed when the Rh6 RCSI is replaced by the RCSI of Rh3. These results directly correlate with the relative affinity of Pph13 for these RCSI elements, as described in this study. Furthermore, the lower affinity of Pph13 for the Rh3 or Rh5 RCSI element would predict a greater dependency on other elements in these promoters for expression - hence the dependency on Otd. Indeed, the expression of both Rh3 and Rh5 is dependent on Otd binding to, and activating transcription outside the region of, their respective RCSI sequences (Tahayato, 2003). By contrast, as observed in Drosophila, the perfect palindrome/higher affinity binding site contained within the Rh6 RCSI element requires promoter regions outside of the RCSI element to repress expression in every photoreceptor cell (Tahayato, 2003: Mishra, 2010 and references therein). Altogether, these data correlate well with a model in which there is a shifting of dependency between the RCSI element and other upstream photoreceptor subtype-specific elements among the different Rhodopsin promoters. As for the residual Rh1 expression in the otd; Pph13 double mutant, the model predicts that a second element outside of the RCSI site contributes to the expression of Rh1. There are two binding sites for Glass, one inside the minimal promoter of Rh1 and one outside. Glass is expressed in all photoreceptor cells and is required for Rh1 expression. Moreover, like the RCSI element, Glass binding sites when multimerized are sufficient to drive and limit expression to photoreceptor cells (Mishra, 2010).

The results form the basis for a better understanding of all rhabdomeric photoreceptor cell development and function. Interestingly, the activity of the 3XP3 reporter is a common marker for transgenic constructs in many invertebrate species and thus its activity is not limited to Drosophila. Given the relationship between Pph13 and Otd in determining rhabdomere morphogenesis and the ability of both factors to ensure photoreceptor cell function, it will be crucial to determine whether these same relationships exists in other invertebrate rhabdomeric photoreceptor cells (Mishra, 2010).

DEVELOPMENTAL BIOLOGY

Pph13, alternatively named Munster (Mu), is a homeobox-containing gene of the Paired-class which is specifically expressed in the developing Bolwig organs, the Drosophila larval eyes. This expression is first detected during early germ band retraction stage (stage 12 from 7 h 20 min at 25°C) and persists until the end of embryogenesis. Mu homeodomain is most similar to that of Aristaless and Drosophila Goosecoid. Strikingly, the Munster gene maps within 6 kb of goosecoid, in the same genomic region as aristaless, suggesting that these genes are part of a homeobox gene cluster (Goriely, 1999).

In situ hybridization reveals that embryonic expression is limited to the Bolwig organs. Expression is confined to two small clusters of epidermal cells on each side of the embryonic head and is first detectable during early stage 12 at the germ band retraction stage. At stage 13, the Mu-expressing cluster adopts a rosettelike shape and contains a total of 12 cells, seven of which are superficial, while the five others are located deeper. After head invagination (stage 17), the expressing clusters appear more compact and elongated. These cell clusters clearly correspond to the Bolwig organs (BO), the primitive larval eyes. In Drosophila, the BO is typically composed of 12 light-sensitive neurons whose axons fasciculate and extend as a nerve towards the brain. Mu expression is unusual in that it is limited to the BO, unlike other genes such as Kruppel, disconnected or orthodenticle that are also involved in other developmental processes. Its expression at stage 12 makes Mu the earliest gene so far described to be expressed in BO. Given the universal conservation of the Pax-6 genetic cascade in eye morphogenesis, it would be of interest to determine whether Mu is a target of Pax-6 during embryonic development. However, the role of Pax-6 in BO development has yet to be documented and no vertebrate homologs of Mu have been reported to date. Mu expression is not detected in the eye imaginal disc. However, Mu is found in the adult eye, where it is expressed in most of the cells of the retina as well as in the lamina and the medulla, parts of the optic lobes (Goriely, 1999).

Using an antibody raised against the C-terminal region of Pph13, the spatial and temporal expression of Pph13 was examined throughout Drosophila development. In agreement with Pph13 mRNA localization (Goriely, 1999), Pph13 expression during embryogenesis is limited to the cells of Bolwig's organ, the larval photoreceptor center. The adult photoreceptor cells are specified during the third larval instar. However, no Pph13 staining is detected in the eye imaginal disc or any other imaginal tissue. Pph13 expression is first detected at 36 hours APF prior to the detection of the membrane folds that will give rise to the rhabdomeres. Expression is limited to only photoreceptor cells and expression is maintained during adult life. As expected, Pph13 localization is nuclear as compared to a nuclear lacZ marker and immunoreactivity is not detected in Pph13^hazy mutant embryos or photoreceptor cells (Zelhof, 2003).

Effects of Mutation or Deletion

As a first step in understanding the molecular mechanisms leading to the severe decrease in light sensitivity and rhabdomeric defects in hazy mutants, the responsible gene was isolated. Recombination and deficiency mapping places hazy in a small genomic interval of 70 kB of DNA at 21C7-21D1 on the left arm of the second chromosome. Sequencing of candidate genes from this interval in both mutant and isogenic wild-type flies reveals a single base change in the open reading frame of the previously identified homeodomain gene Pph13 (Dessain, 1993). The nucleotide change results in the introduction of a premature termination codon at amino acid position 58 (W to Stop). The premature stop is within the homeodomain and therefore would also effectively eliminate any DNA binding capacity. More importantly, reintroduction of a wild-type full-length Pph13 cDNA, under the control of a heat shock promoter, by P-element mediated germ line transformation fully rescues all Pph13^hazy phenotypes. Rescue is obtained without heat shock, suggesting only a basal level of expression is required for proper photoreceptor cell morphogenesis (Zelhof, 2003).

How does Pph13 control the morphogenesis of rhabdomeres? The small malformed rhabdomeres observed in Pph13^hazy mutants may either be the result of retinal degeneration or a flaw in their biogenesis. EM ultrastructural analysis of mutant photoreceptor cells did not reveal any clear signs of degeneration. The rhabdomere terminal web, which is responsible for maintaining the separation and support of the rhabdomere from the cell cytoplasm, forms in Pph13^hazy flies. No large concentration of vesicles or multivesicular bodies (MVBs) was observed in the cell cytoplasm associated with degenerating rhabdomeres; neither was the characteristic involuting of the rhabdomere membrane, as seen in ninaE mutants (Zelhof, 2003).

If the malformed rhabdomeres were the result of improper morphogenesis, an examination of the spatial and temporal appearance of rhabdomeric proteins during the steps of microvilli formation would not only define the developmental time of the defect but also identify possible Pph13 transcriptional targets. The exact mechanism of microvilli initiation and elongation are not known but there are a few protein markers that can be used to gauge the progression and structure of the developing rhabdomere. At 48 hrs after pupariation (APF), when the initial microvilli folds are forming, the accumulation of rhabdomeric proteins (Chaoptin, Amphiphysin, 21A6), in mutants is indistinguishable from wild type. At 72 hrs APF, the progression of rhabdomere development and localization of rhabdomeric proteins appears to be normal in Pph13^hazy mutants. However, at 96 hours APF, the staining of F-actin clearly reveals a severe lack of growth and elongation of the rhabdomere microvilli in mutants. Staining for F-actin demonstrates that the mutant rhabdomeres are not full and round when compared with their wild-type counterparts. In agreement with immunofluorescent studies, EM ultrastructure analysis reveals rhabdomere biogenesis is not proceeding correctly by 72 hours APF. The microvilli of mutant photoreceptor cells are misaligned and loosely packed, as seen in adult mutant rhabdomeres. Whereas at 60 hours APF no discernible ultrastructure defects are observed between wild-type and mutant photoreceptor cells, suggesting Pph13 function is crucial between 60 and 72 hours APF for proper rhabdomere formation (Zelhof, 2003).

Little is known about the molecular events that occur during this 12 hr period of development. Nevertheless, at this time Rac1 is expressed. Rac1 has been implicated in the formation of the rhabdomere terminal web and Rac, as seen in lamellipodia formation, has the ability to reorganize the actin cytoskeleton to create and support membrane protrusions. Since the terminal web forms in Pph13^hazy mutants, the appearance of normal Rac1 expression and localization was expected in Pph13^hazy mutants. The data indicate that the temporal expression of Rac1 is normal in Pph13^hazy mutants but surprisingly, the accumulation of Rac1 at the interface between the rhabdomere and the cell cytoplasm is very irregular in mutants. As early as 72 hours APF, ectopic spots of Rac1 are seen in the photoreceptor cell body and by 96 hours APF, there is a grossly abnormal amount of Rac1 accumulating at the rhabdomere cell cytoplasm interface and within Pph13^hazy photoreceptor cells. Interestingly, this misregulation of Rac1 activity or accumulation may be contributing to the rhabdomere defects observed in Pph13^hazy photoreceptor cells (Zelhof, 2003).

Does Pph13 have a role in photoreceptor cell fate specification? To address this possibility, several features associated with changes in photoreceptor cell fate were examined. (1) The expression and distribution of photoreceptor cell specific rhodopsins were checked. No misexpression or absence of the unique rhodopsins of the outer and inner photoreceptor cells was detected. In addition, there are no changes in the stereotypical position of the rhabdomeres as present in the late specification mutant spalt. (2) Nor are there any pathfinding or target choice mistakes of the photoreceptor axons. (3) Unlike transcription factors involved in the early specification of photoreceptor cells [e.g., eyeless, eyes absent and sine oculis], misexpression of Pph13 in other tissues does not lead to the appearance of ectopic eyes (Zelhof, 2003).

Dessain, S. and McGinnis, W. (1993). Drosophila homeobox genes. Adv. Dev. Biochem. 2: 1-55

Fortini M. E. and Rubin G. M. (1990). Analysis of cis-acting requirements of the Rh3 and Rh4 genes reveals a bipartite organization to rhodopsin promoters in Drosophila melanogaster. Genes Dev. 4: 444-463. PubMed Citation: 2140105

Goriely, A., Mollereau, B., Coffinier, C. and Desplan, C. (1999). Munster, a novel paired-class homeobox gene specifically expressed in the Drosophila larval eye. Mech. Dev. 88: 107-110. 10525194

Mishra, M., et al. (2010). Pph13 and orthodenticle define a dual regulatory pathway for photoreceptor cell morphogenesis and function. Development 137(17): 2895-904. PubMed Citation: 20667913

Papatsenko D., Nazina A. and Desplan C. (2001). A conserved regulatory element present in all Drosophila rhodopsin genes mediates Pax6 functions and participates in the fine-tuning of cell-specific expression. Mech. Dev. 101: 143-153. PubMed Citation: 11231067

Tahayato A., et al. (2003). Otd/Crx, a dual regulator for the specification of ommatidia subtypes in the Drosophila retina. Dev. Cell 5: 391-402. PubMed Citation: 12967559

Zelhof, A. C., Koundakjian, E., Scully, A. L., Hardy, R. W. and Pounds, L. (2003). Mutation of the photoreceptor specific homeodomain gene Pph13 results in defects in phototransduction and rhabdomere morphogenesis. Development 130: 4383-4392. 12900454

date revised: 15 December 2010

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