InteractiveFly: GeneBrief

PvuII-PstI homology 13: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - PvuII-PstI homology 13

Synonyms - Munster (Mu), Hazy

Cytological map position - 21C--D

Function - transcription factor

Keywords - eye imaginal disc, lamina, medulla, terminal differentiation

Symbol - Pph13

FlyBase ID: FBgn0023489

Genetic map position -

Classification - homeodomain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Bernardo-Garcia, F.J., Fritsch, C. and Sprecher, S.G. (2016). The transcription factor glass links eye field specification with photoreceptor differentiation in Drosophila. Development [Epub ahead of print]. PubMed ID: 26952983
Summary:
Eye development requires an evolutionarily conserved group of transcription factors, termed "retinal determination network" (RDN). However, little is known about the molecular mechanism by which the RDN instructs cells to differentiate into photoreceptors. This study shows that photoreceptor cell identity in Drosophila is critically regulated by the transcription factor Glass, which is primarily expressed in photoreceptors and whose role in this process was previously unknown. Glass is both required and sufficient for the expression of phototransduction proteins. Data demonstrate that the RDN member Sine oculis directly activates glass expression, and that Glass activates the expression of the transcription factors Hazy and Otd. Hazy was identified as a direct target of Glass. Induced expression of Hazy in the retina partially rescues the glass mutant phenotype. Together, these results provide a transcriptional link between eye field specification and photoreceptor differentiation in Drosophila, placing Glass at a central position in this developmental process.

Liang, X., Mahato, S., Hemmerich, C. and Zelhof, A. C. (2016). Two temporal functions of Glass: Ommatidium patterning and photoreceptor differentiation. Dev Biol [Epub ahead of print]. PubMed ID: 27105580
Summary:
The downstream mechanisms that maintain identity and regulate differentiation of retinal cells remain poorly understood. This study report that the transcription factor Glass has a dual role in establishing a functional Drosophila eye. Persistent defects in ommatidium patterning combined with cell death correlate with the overall disruption of eye morphology in glass mutants. In addition, Glass exhibits a separable role in regulating photoreceptor differentiation. In particular, the apparent loss of glass mutant photoreceptors is not only due to cell death but also a failure of the surviving photoreceptors to complete differentiation. Moreover, the late reintroduction of Glass in these developmentally stalled photoreceptors is capable of restoring differentiation in the absence of correct ommatidium patterning. Mechanistically, Glass is necessary for the expression of many genes implicated in differentiation, i.e. rhabdomere morphogenesis, phototransduction, and synaptogenesis. Specifically, Glass was shown to directly regulate the expression of Pph13, which encodes a transcription factor necessary for opsin expression and rhabdomere morphogenesis. Finally, Glass choreographing of photoreceptor differentiation is conserved between Drosophila and Tribolium, two holometabolous insects. Altogether, this work identifies a fundamental regulatory mechanism to generate the full complement of cells required for a functional rhabdomeric visual system and provides a critical framework to investigate the basis of differentiation and maintenance of photoreceptor identity.
Bernardo-Garcia, F. J., Humberg, T. H., Fritsch, C. and Sprecher, S. G. (2016). Successive requirement of Glass and Hazy for photoreceptor specification and maintenance in Drosophila. Fly (Austin): [Epub ahead of print]. PubMed ID: 27723419
Summary:
Development of the insect compound eye requires a highly controlled interplay between transcription factors. However, the genetic mechanisms that link early eye field specification to photoreceptor terminal differentiation and fate maintenance remain largely unknown. This study deciphered the function of two transcription factors, Glass and Hazy, which play a central role during photoreceptor development. The regulatory interactions between Glass and Hazy suggest that they function together in a coherent feed-forward loop in all types of Drosophila photoreceptors. While the glass mutant eye lacks the expression of virtually all photoreceptor genes, young hazy mutants correctly express most phototransduction genes. Interestingly, the expression of these genes is drastically reduced in old hazy mutants. This age-dependent loss of the phototransduction cascade correlates with a loss of phototaxis in old hazy mutant flies. It is concluded that Glass can either directly or indirectly initiate the expression of most phototransduction proteins in a Hazy-independent manner, and that Hazy is mainly required for the maintenance of functional photoreceptors in adult flies.
BIOLOGICAL OVERVIEW

Through years of research, considerable insight has been garnered into the regulatory mechanisms of photoreceptor cell specification and the mechanisms of phototransduction. However, the events that occur between the specification of a photoreceptor neuron and the final organization and function of the phototransduction signaling cascade are poorly defined. PvuII-PstI homology 13 (Pph13), also known as Munster, is a homeodomain transcription factor expressed only in photoreceptor cells. Pph13 expression correlates with the differentiation and not specification of photoreceptor cells. In agreement with its expression profile, Pph13 is found to be required for both rhabdomere morphogenesis and for the proper detection of light. In addition, Pph13 exerts its effect by the regulation of photoreceptor specific gene expression (Zelhof, 2003).

Upon specification, photoreceptor neurons immediately send axonal projections into the optic lobe of the Drosophila brain. The outer photoreceptor cells project into the lamina whereas the inner photoreceptor cells (R7, R8) send axonal projections deeper into the optic lobe and terminate in the medulla. Furthermore, the differentiation of the photoreceptor neurons is not complete until 4 days later, at the end of metamorphosis. One unique feature of vertebrate and Drosophila photoreceptor neurons is the creation of a specialized light-sensing organelle on the apical cell surface. In Drosophila, the rhabdomere is the photoreceptor light-sensing organelle and is the functional equivalent of the outer segment of vertebrate rod and cone cells. Each rhabdomere consists of 60,000 tightly-packed microvilli, each 50 nm in diameter and 1-2 µm in length. This results in a tremendous increase in surface area to house the tens of millions of rhodopsin molecules and associated signaling molecules that are responsible for the detection of light (Zelhof, 2003).

A key aspect of eye structure is the organization of the phototransduction machinery into the rhabdomere. As the rhabdomere develops, the signaling molecules required for the detection and translation of light into a receptor potential are expressed and localized to the rhabdomere. In Drosophila, the activation of rhodopsin leads to the activation of Phospholipase C (PLC) via a coupled heterotrimeric G protein. PLC catalyzes the breakdown of phosphatidyl 4,5-bisphosphate [PtdIns(4,5)P2] into the two intracellular messengers inositol triphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). This reaction leads to the opening of light sensitive cation-selective channels (TRP, TRPL and TRPgamma) and the generation of a depolarizing receptor potential (Zelhof, 2003).

To identify genes required for photoreceptor cell differentiation, a screen was performed for the presence or absence of the deep pseudopupil (DPP) in Drosophila adult eyes. The presence of the DPP is an indication of the overall integrity of the photoreceptor cells and their associated rhabdomeres. Such screens have been effective in isolating mutations that affect eye structure and development. To limit the search for those mutants that affect aspects of differentiation and not specification, mutants were excluded that had incorrect external morphology, particularly rough or irregular shaped eyes. Consequently, 6,000 viable second chromosome EMS mutated lines, generated from 38,000 F3 lines were screened for the absence of a DPP and 33 mutant stocks were isolated that represent 18 complementation groups (Zelhof, 2003).

Since import of the signaling components into the developing rhabdomere occurs late in photoreceptor differentiation, it was reasoned that flies that lacked a DPP and could not correctly respond to light would be the best candidates for mutants defective in photoreceptor terminal differentiation. Using electroretinogram assays (ERGs) that measure the capacity of photoreceptor cells to convert light into a receptor potential, the collection of 33 mutants was screened for those that had defects in light perception. The results indicated that among the group that had irregular ERGs, one had a severe deficiency in the detection of light. This mutant responds reproducibly only to long durations of high intensity light. The characteristic on/off transients of wild-type responses are undetectable and by 10 days post eclosion, the mutants appear to lose all responses to light (Zelhof, 2003).

While weak or no response to light can be the result of numerous factors (e.g. missing phototransduction molecules or subsequent retinal degeneration), the absence of a DPP in newly eclosed flies suggests that the rhabdomeres did not form. To examine whether the rhabdomeres are present, an EM-ultrastructural analysis was performed on the adult mutant photoreceptor cells. This examination revealed that all photoreceptor cells and associated rhabdomeres were present. However, the rhabdomeres were severely malformed. They were significantly smaller in size and often the microvilli within each rhabdomere were misaligned. In addition, the rhabdomeres did not consistently extend the entire thickness of the retina. Given the malformed rhabdomeres and the inability of this mutant to detect light, this mutant was tentatively name hazy; subsequently hazy was found to represent a null allele of the photoreceptor specific homeodomain gene Pph13 (Zelhof, 2003).

Pph13 is expressed only in photoreceptor cells, the larval cells of Bolwig's organ and the adult R1-R8 photoreceptor cells, and no defect has been detected in specification of these cells. Pph13 does not have the ability to activate transcription outside the context of a photoreceptor cell nor does ectopic expression of Pph13 affect the fate of any of the photoreceptor or associated accessory cells. The only phenotypes observed are in photoreceptor cell morphogenesis and function, suggesting Pph13 function is restricted to photoreceptor cell terminal differentiation (Zelhof, 2003).

Pph13hazy mutants have two striking defects: the ability of the photoreceptor cell to detect light and the biogenesis of the light sensing organelle, the rhabdomere. Are the two phenotypes connected? The possibility that the malformed rhabdomeres are contributing to the inability of these mutants to detect light or vice versa cannot be eliminated. However, the severity of the rhabdomere defect cannot be solely responsible. For example, mutants have been isolated that result in malformed rhabdomeres equal to those seen in Pph13hazy but have a normal ERG. In addition, the loss of Chaoptin and NinaC both result in a considerable loss of rhabdomeric size and rhodopsin levels but they have a better response to light then Pph13hazy mutants (Zelhof, 2003).

In addition, Pph13 is required for the transcription of phototransduction proteins. Clearly, trpl, trpgamma and Gß are not expressed in mutant photoreceptor cells, and the absence of Pph13 affects the full expression of several other signaling components. This is clearly observed with Arr1 expression. (1) The data demonstrate that in the inner (R7/R8) wild-type photoreceptor cells have a considerable higher expression of Arr1 when compared with the outer photoreceptor cells (R1-R6). (2) The loss of Pph13 does not eliminate expression of Arr1 in photoreceptor cells. Arr1 expression can be seen in mutant R7/R8 photoreceptor cells and the lack of signal in the outer photoreceptors is not due to the absence of Arr1 expression but rather the fact that these cells start out with lower levels of Arr1. Taken together, while all of the detected protein aberrations can explain the severe loss of light sensitivity, the results do not eliminate the possibility of a yet unidentified molecule required for proper phototransduction. Selective rescue and identification of any other missing components will be needed to explain the complete molecular mechanisms responsible for the decrease in light sensitivity (Zelhof, 2003).

The molecular mechanisms for rhabdomere biogenesis are for the most part unknown. Nonetheless, the data do provide a few insights into rhabdomere biogenesis. Pph13 is required for the generation or execution of a late acting signal necessary for the elaboration and growth of the microvilli into a rhabdomere. Immunofluorescent and EM analyses demonstrate that the defects observed in Pph13hazy mutants are the result of a developmental flaw and not of retinal degeneration. The disorganized rhabdomeres do not show any of the characteristic signs of degeneration and more significantly a clear halt is detected in rhabdomere development by 72 hours APF. In addition, by all measurements, the early events (36 to 60 hours APF) of rhabdomere biogenesis occur normally (Zelhof, 2003).

The data also indicate the failure of growth is not due to the improper localization or delivery of proteins to the rhabdomere. For example, Chaoptin, which is required for the cross-linking of microvilli still localizes to the developing rhabdomere before and after the rhabdomere has stalled in development. In addition, the proteins composing the phototransduction machinery, especially rhodopsin, which has a role in phototransduction and in maintaining the structural integrity of the rhabdomere, are imported and stabilized within the malformed rhabdomeres. The characteristic expansion of the endoplasmic reticulum associated with defects in rhabdomeric protein cell trafficking was not detected (Zelhof, 2003).

What is responsible for the flaw in rhabdomere biogenesis? Most notably for a transcription factor believed to be necessary for the activation and not repression of gene transcription, a grossly abnormal accumulation of Rac1 is seen in Pph13hazy mutant photoreceptor cells. However, the presence of Rac1 is in agreement with the fact that the terminal web does form in Pph13hazy mutants. Given that the exact function of Rac1 has not been resolved in rhabdomere biogenesis and that small Rho GTPases have been implicated in mediating signals required for actin reorganization, future experiments will address the function of Rac1 in photoreceptor terminal differentiation and determine how the misregulation of Rac1 accumulation and activity may be contributing to the Pph13hazy rhabdomere phenotypes (Zelhof, 2003).

The molecular cloning of Pph13hazy has identified another homeodomain gene required for photoreceptor morphogenesis. Previous reports have established or implicated eyeless (Pax6), orthodenticle (otd) and Onecut homeodomain genes in eye development. What is the relationship between these various homeodomain transcription factors and how do they coordinate photoreceptor terminal differentiation? Numerous possibilities exist in which each of these transcription factors could control a unique subset of molecular mechanisms required for a functional photoreceptor cell; alternatively, they could act in concert on the same genes to promote differentiation. To eliminate or confirm any one of these possibilities would be premature and further extensive characterization of each of these genes in photoreceptor development is necessary (Zelhof, 2003).

Nevertheless, the preliminary data does allow for some speculation. It is clear that eyeless is required for photoreceptor cell specification and without it a photoreceptor cell or a gene like Pph13 could not function. Besides its early role in photoreceptor cell specification, eyeless is also necessary for rhodopsin expression and superficially, characterization of the late transcriptional targets of Eyeless and Pph13 appears to be different. Pph13 is absolutely required for trpl, trpgamma and Gß expression but is not necessary for rhodopsin expression. This result would suggest that once a cell has committed to a photoreceptor cell fate, both Eyeless and Pph13 have separate and distinct molecular pathways that contribute to photoreceptor differentiation (Zelhof, 2003).

However, comparison of otd and Pph13 mutants suggest a more complex mode of coordination for photoreceptor differentiation. The rhabdomere defects observed otduvi and Pph13hazy mutants are similar. In each case, the defects appear not to be the result of degeneration but a failure in their biogenesis. The rhabdomere terminal web does form in both cases but the overall size and morphology are abnormal. Both Otd and Pph13 are required in the same developmental time window for rhabdomere morphogenesis, but neither is necessary for the expression of the other. Whether Otd and Pph13 represent two parallel pathways directing the expression of the same genes or two distinct pathways with different genetic targets to promote rhabdomere biogenesis will require further investigation. In addition, they do not share a defect in phototransduction. otduvi photoreceptor cells exhibit normal ERGs and no loss of phototransduction proteins downstream of rhodopsin, as seen in Pph13 mutants. Clearly, Pph13 is responsible for two aspects of photoreceptor cell differentiation: phototransduction and rhabdomere morphogenesis (Zelhof, 2003).

Given that the molecular mechanisms orchestrating the differentiation of photoreceptor cells remain largely undefined, the goal of the genetic approach was to isolate genes required for photoreceptor terminal differentiation. Work with Pph13hazy has shed some light on the regulation of this process. However, additional studies that combine the accessibility and genetic amenability of Drosophila eye development, with whole genome expression profiling techniques in both wild-type and Pph13hazy mutant photoreceptor cells, should identify additional transcriptional targets necessary for photoreceptor cells to achieve and maintain a functional state (Zelhof, 2003).


REGULATION

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 Pph13hazy mutants. Western analysis revealed undetectable levels of four phototransduction proteins -- eye Gß, TRPL, TRPgamma and Arr1 in Pph13hazy 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 Pph13hazy 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 Pph13hazy mutants. Nonetheless, the defect observed in Pph13hazy mutants may be a cumulative effect of all the missing proteins identified (Zelhof, 2003).

The majority of the proteins examined are present in Pph13hazy 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 Pph13hazy 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 Pph13hazy 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 Pph13hazy 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).

Binary cell fate decisions and fate transformation in the Drosophila larval eye

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. This study shows that interplay of two transcription factors Senseless (Sens) and homeodomain transcription factor Hazy [PvuII-PstI homology 13, Pph13] control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. These findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner (Mishra, 2013).

In the larval eye, determination of primary or secondary precursors to acquire either Rh5-PR or Rh6-PR identity depends on the transcription factors Sal, Svp and Otd. Primary as well as secondary precursors have the developmental potential to express Rh5 or Rh6. During differentiation, a pulsed expression of Sens acts as a trigger to initiate a distinct developmental program: Sens acts genetically in a feedforward loop to inhibit the Rh6-PR cell-fate determinant Svp and to promote the Rh5-PR cell-fate determinant Sal. Similarly, in the adult retina, differentiation of 'inner' PRs R7 and R8 requires sens and sal. Sal is necessary for Sens expression in R8-PRs and misexpression of Sal is sufficient to induce Sens expression in the 'outer' PRs R1-R6 (Mishra, 2013).

Svp is exclusively expressed in R3/R4 and R1/R6 pairs of the outer PRs in early retina development. Initially, Sal is expressed in the R3/R4 PRs in order to promote Svp expression. Later, Svp represses Sal in R3/R4 PRs in order to prevent the transformation of R3/R4 into R7. Similarly in larval PRs Svp is repressing Sal in secondary precursors (Mishra, 2013).

Intriguingly, in R8 development in the adult retina Sens also provides two temporally separable functions: First, during R8 specification, lack of Sens in precursors results in a transformation of the cell into R2/R5 fate; second, during differentiation, Sens counteracts Pros to inhibit R7 cell fate and promotes R8 cell fate. Thus, Sens is an early genetic switch in R8-PRs and larval Rh5-PRs that represses an alternate cell fate (Mishra, 2013).

The lack of Sens results in a larval eye composed of only Rh6-PRs. Thus, the default state for both primary and secondary precursors is to differentiate into Rh6-expressing PRs. Rh6 is also the default state in adult R8 PRs: In the absence of R7 PRs (e.g. sevenless mutants) that send a signal to a subset of underlying R8 PRs, the majority of R8 PRs express Rh6. Thus, the genetic pathway initiated by the Sens pulse ensures that primary precursors choose a distinct developmental pathway by repressing the Rh6 ground state. The mechanisms that initiate and control this pulse of Sens remain to be discovered (Mishra, 2013).

In larval PRs as well as in the formation of sensory organ precursors (SOP) in the wing, Sens functions as a binary switch between two alternative cell fates. In the larval eye, this switch occurs when Sens is expressed in one cell type and not in the other. However, during wing disc development the cell fate choice in SOP formation is controlled by the levels, and not the presence or absence of Sens expression: high levels of Sens act synergistically with proneural genes to promote a neuronal fate, while in neighboring cells, low levels of Sens repress proneural gene expression, thereby promoting a non-SOP fate. Thus, Sens uses distinct molecular mechanisms to act as a switch between Rh5 versus Rh6-PR cell fate and SOP versus non-SOP cell fate (Mishra, 2013).

Transcription factors regulate developmental programs in a context- dependent fashion. An example is Sens, which has distinct functions in BO and eyelet development. First, during embryonic development, Sens acts as a key cell fate determinant by regulating transcription factors controlling PR-subtype specification. Second, during metamorphosis Sens inhibits ecdysone-induced apoptotic cell death. Third, in the adult eyelet Sens promotes Rh6 expression. Interestingly, the pro-survival function of Sens appears to be a conserved feature of Sens in other tissues and also in other animal species. In the salivary gland of Drosophila, Sens acts also as a survival factor of the salivary gland cells under the control of the bHLH transcription factor Sage. pag-3, a C.elegans homolog of Sens is involved in touch neuron gene expression and coordinated movement (Jia, 1996; Jia, 1997). Pag-3 was shown to act as a cell-survival factor in the ventral nerve cord and involved in the neuroblast cell fate and may affect neuronal differentiation of certain interneurons and motorneurons. In mice, Gfi1 is expressed in many neuronal precursors and differentiating neurons during embryonic development and is required for proper differentiation and maintenance of inner ear hair cells. Gfi1 mutant mice lose all cochlear hair cells through apoptosis, suggesting that its loss causes programmed cell death (Wallis, 2003). Taken together, these findings support that Sens and its orthologs function in cell fate determination and cell differentiation both in nervous system formation, but also play an essential role in the suppression of apoptosis (Mishra, 2013).

Hazy plays distinct roles in larval PRs and during metamorphosis. First, Hazy is essential during embryogenesis for proper PR differentiation. This early function of Hazy is essential for PRs to differentiate properly during embryogenesis, to express Rhodopsins and to subsequently maintain Rhodopsin expression during larval stages. This function of Hazy is similar to its role in rhabdomere formation in adult PRs and subsequent promotion of Rh6 expression, although it is not required for Rh5 in the adult retina. It is likely that Hazy exerts this function by binding to the RCSI site of the rhodopsin promoters, as has been suggested for the adult retin. Second, during metamorphosis Hazy is required in Rh6-PRs to repress sens, thus allowing these cells to undergo apoptosis. This highlights the reuse of a small number of TFs for distinct functions in the same cell type at distinct time points of PR development. How these temporally distinct developmental programs are controlled on a molecular level remains unresolved. It seems likely that the competence of the cell to respond to a specific transcription factor changes during development (Mishra, 2013).

rh5 and rh6 are expressed in different PRs at different developmental stages: rh5 is expressed in the larval eye and in the adult retina, whereas rh6 is expressed in the larval eye, the adult eyelet and the adult retina. However, the gene regulatory networks controlling rhodopsin expression are distinct in these organs. In the adult retina, a bi-stable feedback loop of the growth regulator melted and the tumor suppressor warts acts to specify Rh5 versus Rh6 cell fate, respectively, while in the larva, Sens, Sal, Svp and Otd control Rh5 versus Rh6 identity whereas Hazy has been shown to maintain Rhodopsin expression. A third genetic program acts downstream of EcR during metamorphosis in Rh5-PRs to switch to Rh6, which requires Sens (Mishra, 2013).

An intriguing question is how the developmental pathways to specify Rh5- or Rh6-cell fates converge on the regulatory sequences of these two genes. It seems likely that parts of the regulatory machinery acting on the rh5 and rh6 promoters are shared between the larval eye, adult retina and eyelet, especially as short minimal promoters are functional in all three different contexts. Future experiments will show how the activity of the identified trans-acting factors is integrated on these promoters to yield context-specific outcomes (Mishra, 2013).


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 Pph13hazy mutant embryos or photoreceptor cells (Zelhof, 2003).


EFFECTS OF MUTATION

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 Pph13hazy 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 Pph13hazy 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 Pph13hazy 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 Pph13hazy 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 Pph13hazy mutants, the appearance of normal Rac1 expression and localization was expected in Pph13hazy mutants. The data indicate that the temporal expression of Rac1 is normal in Pph13hazy 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 Pph13hazy photoreceptor cells. Interestingly, this misregulation of Rac1 activity or accumulation may be contributing to the rhabdomere defects observed in Pph13hazy 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).


REFERENCES

Search PubMed for articles about Drosophila PvuII-PstI homology 13

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

Jia, Y., Xie, G. and Aamodt, E. (1996). pag-3, a Caenorhabditis elegans gene involved in touch neuron gene expression and coordinated movement. Genetics 142: 141-147. PubMed ID: 8770591

Jia, Y., Xie, G., McDermott, J. B. and Aamodt, E. (1997). The C. elegans gene pag-3 is homologous to the zinc finger proto-oncogene gfi-1. Development 124: 2063-2073. PubMed ID: 9169852

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

Mishra, A. K., Tsachaki, M., Rister, J., Ng, J., Celik, A. and Sprecher, S. G. (2013). Binary cell fate decisions and fate transformation in the Drosophila larval eye. PLoS Genet 9: e1004027. PubMed ID: 24385925

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

Wallis, D., Hamblen, M., Zhou, Y., Venken, K. J., Schumacher, A., Grimes, H. L., Zoghbi, H. Y., Orkin, S. H. and Bellen, H. J. (2003). The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival. Development 130: 221-232. PubMed ID: 12441305

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


Biological Overview

date revised: 23 July 2014

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