The Interactive Fly

Genes involved in tissue and organ development

The Eye-Antenna Disc and Eye Morphogenesis

  • Genes expressed in eye morphogenesis
  • Genes expressed in antennal morphogenesis
  • Bolwig's Organ, the Hofbauer-Buchner Eyelet and Ocelli
  • Structure of the eye
  • Development of the eye

    Eye-antenna disc
  • Specification of the eye versus the antenna
  • 5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila
  • Transformation of eye to antenna by misexpression of a single gene
  • Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

    Eye development
  • A quantitative analysis of growth control in the Drosophila eye disc
  • Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis
  • Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference
  • Stochastic spineless expression creates the retinal mosaic for colour vision
  • Binary cell fate decisions and fate transformation in the Drosophila larval eye
  • The spatiotemporal order of signaling events unveils the logic of development signaling
  • dachshund potentiates Hedgehog signaling during Drosophila retinogenesis
  • Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye
  • Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway
  • Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye
  • Single-base pair differences in a shared motif determine differential Rhodopsin expression
  • Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila
  • An eye on trafficking genes: Identification of four eye color mutations in Drosophila
  • A model of the spatio-temporal dynamics of Drosophila eye disc development
  • Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye
  • The cuticular nature of corneal lenses in Drosophila melanogaster

    Photoreceptor development
  • Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis
  • The actomyosin machinery is required for Drosophila retinal lumen formation
  • Does Unc-GFP uncover ciliary structures in the rhabdomeric eye of Drosophila?
  • Ire1 supports normal ER differentiation in developing Drosophila photoreceptors
  • Past1 modulates Drosophila eye development

    Vision
  • Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila
  • Color discrimination with broadband photoreceptors
  • Genetic dissection reveals two separate retinal substrates for polarization vision in Drosophila
  • Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace
  • Loss of Na/K-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration
  • Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors
  • Anatomical reconstruction and functional imaging reveal an ordered array of skylight polarization detectors in Drosophila
  • Fly photoreceptors encode phase congruency


    Structure of the eye

    The adult eye consists of an array of approximately 800 hexagonal ommatidia, or facets. There is also an array of sensory hairs projecting from the surface.

    Each ommatidium contains eight photoreceptor cells, each associated with a rhabomere. A rhabomere is a rod-like element containing photoreceptor elements (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). Photoreceptor cells one through six of each ommatidium are placed radially around cells 7 and 8, forming an irregular trapezoid. Rhadomeres of cells seven and eight occupy a central position, cell seven above cell eight. Each ommatidium is surrounded by two primary pigment cells and these are surrounded by six secondary pigment cells, shared by adjacent ommatidia. Thus each ommatidium contains a total of 22 cells, making the total number of cells in each eye over 16,000 (Ready, 1976). From each mature cluster a bundle of eight axons runs posteriorly into a pre-optic stalk. For information on inductive relationships between the projecting axons and the innervated medula of the brain optic lobe see the brain site.

    In the differentiation of the eye, R8 is the first cell population to have its fate determined. Subsequently and successively comes the differentiation of three cell pairs: R2/5, R3/4 and R1/6. Following this is the differentiation of R7 and the surrounding cone cells.

    Development of the eye

    The eye is derived from the eye-antennal disc. The disc itself arises from approximately 20 cells of the optic primordium in the embryonic blastoderm. The disc is formed by invagination at stage 12, to produce a flattened sac of epithelium. By the third instar larva the disc contains about 2000 cells. During the middle of the third instar phase, a dorsal ventral furrow forms, advancing from posterior to anterior. The furrow, whose progression requires hedgehog function, is the site where commitment to photoreceptor fate is initiated. hh activates the expression of decapentaplegic and the proneural gene atonal in the furrow. ato expression is refined to the future R8 photoreceptor and is required for the development of this cell. The area anterior to the furrow is rich in synchronously dividing cells, but lacks a pattern. The furrow itself is caused by a shortening of cells at its center. The area posterior to the furrow shows preclusters of cells, each with a recognizable core of five cells, corresponding to cells 2, 3, 4, 5 and 8 of the photoreceptor. These photoreceptors and accessory cells are recruited to each ommatidial cluster (composed of developing photoreceptors) by waves of expression of the ligand spitz (spi) for the EGF receptor. A diagram is presented describing the progression of the morphogenetic furrow across the eye disc.

    In addition to the dorso-ventral furrow described above, there is an axis of dorso-ventral symmetry. Associated with the advancing boundary of cluster formation, the area ahead of this boundary is indented along the anterior posterior axis. This groove can be seen in the early third instar disc. It is this anterior-posterior groove that structures the DV symmetry of the eye (Ready, 1976). A diagram is presented describing the specification of the eye disc primordium and the establishment of dorsal/ventral asymmetry.

    Cells in the Drosophila eye are determined by inductive signalling. A model of eye development has been built that explains how simple intercellular signals could specify the diverse cell types that constitute the ommatidium. This model arises from the observation that the Drosophila homologue of the EGF receptor (EGF-R) is used reiteratively to trigger the differentiation of each of the cell types -- successive rounds of EGF-R activation recruit first the photoreceptors, then cone and finally pigment cells. It seems that a cell's identity is not determined by the specific signal that induces it, but is instead a function of the state of the cell that receives the signal. EGF-R signalling is activated by the ligand, Spitz, and inhibited by the secreted protein, Argos. Spitz is initially produced by the central cells in the ommatidium and diffuses over a small distance. Argos has a longer range, allowing it to block more distal cells from being activated by low levels of Spitz; This interplay between a short-range activator and a long-range inhibitor is termed 'remote inhibition'. Since inductive signalling is common in many organisms and its components have been conserved, it is possible that the logic of signalling may also be conserved (Freeman, 1997).

    Specification of the eye versus the antenna

    The Drosophila compound eye is specified by the concerted action of seven nuclear factors: Twin of eyeless (Toy), Eyeless (Ey), Eyes absent (Eya), Sine oculis (So), Dachshund (Dac), Eye gone (Eyg), and Optix (Opt). These factors have been called 'master control' proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, several of these eye specification genes are not coexpressed in the same embryonic cells -- or even in the presumptive eye. Surprisingly, the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes; specification occurs much later than previously thought -- not during embryonic development but in the second larval stage (Kumar, 2001).

    The Egfr and Notch pathways function in the specification or determination of the eye. An ey-GAL4 driver was used to express target proteins; this element drives expression first in the eye and antenna anlagen in the embryo (by stage 11) and then in regions ahead of the furrow in just the eye imaginal disc. Egfr function was removed in this domain by expressing a dominant negative form of the receptor. Both the eye and antenna were deleted from eclosed adults indicating that both structures require Egfr signaling for their specification, determination, or survival. Under these conditions, the larval discs do not form, making analysis of later developmental phenotypes impossible. The same phenotype was obtained with dominant negative Ras indicating that this activity is Ras dependent. Wild-type and activated forms of several components of the Ras pathway were expressed in the eye anlagen using the same driver and it was found that hyperactivation of many elements leads to the homeotic transformation of the eye into a morphologically complete antenna. Homeotic transformation of the eye to antenna can also be induced by the Egfr ligand Spitz but not by two other known activators. The membrane bound version of Spitz does not induce homeotic transformations, suggesting a requirement for paracrine signaling. Wild-type and constitutively active forms of the Egfr and two other Drosophila RTKs (Breathless [Btl] and Heartless [Htl]) were expressed but only Egfr is able to induce the transformation. Expression of the constitutively active version of Egfr gives a significantly stronger phenotype than the wild-type version of Egfr, suggesting that the level of Egfr signaling is important for maintaining the balance between eye and antennal identities. The downstream elements of the pathway that can induce this transformation include Ras, Raf, and PntP1, while neither MEK, MAPK, nor PntP2 induced this effect in this assay. Aop, Tramtrack (Ttk), and BarH1/H2, each of which mediates negative feedback inhibition of Egfr signaling, delete the eye. The failure of Mek, Mapk, and PntP2 to induce this transformation reflects the existence of actual branch points in the pathway. However, it is also possible that the quantitative levels of expression of these three elements are not limiting for this signal at this time and place; indeed, their phosphorylation states may be more relevant (Kumar, 2001).

    Notch and Egfr have been shown to often antagonize each other during cell fate decisions in the fly eye. Notch function was removed with a dominant negative form and results similar to the effects of Egfr signal hyperactivation were obtained. Consistent with this, when an activated form of Notch was expressed, the size of the eye was reduced and there were severe dysmorphies. Expression of dominant negative transgenes of the ligands Delta (Dl) or Serrate (Ser) also results in the eye to antenna transformation. Elevated expression levels of both Su(H) and many of the proteins of the E(spl) complex (m4, m7, m8, m8DN, malpha, mß, mgamma, and mdelta) were also expressed but no effect on either eye or antenna disc development was observed. However, homeotic eye to antenna transformations occurred when Mastermind (Mam) was expressed using a dominant negative construct. Mam is a member of the neurogenic gene group that encodes a nuclear protein of unknown function. These results suggest a Su(H) and E(spl)C independent pathway for eye and antenna disc development that involves Mam (Kumar, 2001).

    The Wg and Hh pathways have been shown to regulate the size and shape of the eye. Overexpression of a transgene containing the full-length wild-type Cubitus interruptus (Ci) protein, a downstream component of Hh signaling, can mediate eye to antenna homeotic transformations. It is uncertain if these data show a positive or negative Hedgehog signal; Ci may be cleaved into the repressor form, thereby mimicking the effects on eye development seen in loss of Hedgehog signaling. Overexpression of Wg results in the near complete deletion of the eye. Downstream of Wg lie two transcription factors: Sloppy Paired 1 and 2 (Slp1, Slp2). The effect of Wg on eye specification appears to be mediated through Slp2 but not Slp1. Taken together, these observations suggest that along with Egfr and Notch, both Wg and Hh signaling function during eye and antenna disc specification (Kumar, 2001).

    Do Egfr and Notch Act upstream of the eye specification genes? A molecular epistasy study was undertaken, examining the expression of some of the eye and antennal specification genes in the transforming conditions during the third larval stage (before cell types differentiate). In eye specification gene mutants (such as ey), ommatidial development is blocked, but the eye disc remains in a reduced form. Conditions that produce eye to antenna transformations, whether through hyperactivation of Egfr or downregulation of Notch signaling, show a complete replacement of the eye disc with an antenna disc. Distal-less (Dll) and Spalt-Major are normally expressed within subdomains of the antenna disc and are required for antenna development. Dll and SalM are expressed in the correct locations in the transformed antenna disc suggesting that both endogenous and transformed antenna are also both morphologically and molecularly equivalent (Kumar, 2001).

    The transcription of five of the seven known eye specification genes (toy, ey, eya, so, and eyg) was examined. In transforming conditions, transcription levels of all five of the seven genes are below the levels of detection. This is consistent with both Egfr and Notch signaling acting genetically upstream to both the eye and antennal specification genes. The downregulation of ey suggests that the ey-GAL4 driver may also be downregulated via an autoregulatory mechanism. That the transformation occurs despite this may reflect a phenocritical period for the eye-antenna transformation; once the transformation has occurred the system is refractory to the loss of Egfr signaling (Kumar, 2001).

    When and where are the eye and antenna specified? The seven known eye specification genes are thought to act in a genetic and biochemical complex; by pairwise tests, their products have been shown to either directly regulate each other's transcription or to interact at the protein level, or both. From the few published reports of the early expression patterns of eye specification genes and from fate mapping experiments, it has been suggested that eye versus antennal fate specification occurs during the latter stages of embryogenesis. These concepts lead to a straightforward hypothesis: at some point in the developing embryo, the seven eye specification genes' products are coexpressed in the presumptive eye and act to specify its fate. A similar event (with the action of different genes) also specifies the antenna (Kumar, 2001).

    If this hypothesis is true, then three predictions should hold: (1) At some time during embryonic development, there should be two domains of expression of the eye specification genes that correspond to the future eyes, and anterior to these, should be two domains of antenna specification gene expression marking and acting to direct antenna fate. These gene products should be specific to the future structures they mark, and should not be found elsewhere. (2) The eye specification genes should be coexpressed in the same cells. This is known to be true of toy, ey, and eyg. (3) The phenocritical period for the eye to antenna transforming function of Egfr and Notch pathway signaling should be coincident with, or earlier than, the time at which the eye and antenna specification genes are first specifically coexpressed. All three of these predictions were tested (Kumar, 2001).

    To test the first prediction above, embryos were collected (at 1 hr intervals from 1 to 16 hr after egg deposition, AED) and analyzed for expression of the canonical eye specification gene ey (Pax6) and the antenna specification protein Dll. Dll is first detected at 7 hr in the leg imaginal disc primordia and in several segments in the embryonic head. ey transcription in the eye imaginal disc is first detectable at 11 hr while Dll is seen in an adjacent region as well as other sites. In latter stages of embryogenesis, the eye imaginal disc invaginates and assumes a more dorsal-medial position within the embryonic head, just above the developing embryonic brain. Regions of ey expression are observed that correspond to this. Furthermore, this ey expression corresponds to domains of Escargot expression (Esg), a general imaginal disc marker. However, Dll expression is more anterior and it is not clear if these sites correspond to the presumptive antennae. It thus appears that ey is expressed in both the presumptive eye and antenna by 13 hr and remains there through the last embryonic time point observed, and that Dll is not expressed in the future antenna at any embryonic time. It is also quite clear that ey is expressed in many sites in the embryo that will never form eye (such as the segmental grooves). In short, the position of the presumptive eye or antenna during embryonic development cannot be distinguish based on the specific expression of their respective 'master control' genes—neither ey nor Dll expression are sufficient to specify the eye or the antenna; therefore, prediction 1 (above) does not hold true (Kumar, 2001).

    Are the eye specification genes coexpressed during embryonic development? The expression pattern of Eya and Dac proteins and so transcription were examined at 1 hr time points (from 1 to 16 hr AED) and it was found that none of these three eye specification genes is coexpressed with ey within the presumptive eye. The fact that these genes are not expressed within the same cells during embryonic development precludes any possibility that their products act in a multiprotein complex critical for eye specification in the embryo and, thus, prediction 2 (above) does not hold true either. However, eye specification might occur later in development (Kumar, 2001).

    The eye specification genes are first coexpressed in the second larval stage. In second stage larva, the eye specification gene products are completely segregated into the eye portion of eye-antennal disc, but the antennal marker Dll is evenly expressed in both the eye and antennal segments. Interestingly, the expression patterns of the eye specification genes are still not completely overlapping. For instance, toy appears to be expressed throughout the entire eye field while both eya and dac are expressed just in the posterior portions of the eye disc. In the third larval stage, the eye specification genes remain within the eye portion and Dll is now segregated to just the antennal segments (Kumar, 2001).

    The phenocritical period for eye to antenna transformation is in the second larval stage. Use was made of the cold sensitivity of the GAL4 protein to determine the phenocritical period. GAL4 is a yeast protein and is fully functional at 25°C but is less active at 18°C. Flies of the ey-GAL4/UAS-SerDN genotype were raised at 18°C, shifted to 25°C for a consecutive series of 24 hr periods, and then returned to 18°C until late third instar imaginal discs could be examined. The use of the dominant negative Ser construct in this experiment effectively eliminates Notch pathway function in the developing eye. Indistinguishable transformations were observed in other experiments with constructs that either hyperactivate Egfr pathway signaling or inactivate Notch. The developing eye-antennal complex is completely normal if kept continuously at 18°C (negative control) while constant exposure to 25°C temperatures resulted in the eye to antenna transformation (positive control). These controls confirm that the cold sensitivity of GAL4 protein activity is sufficient to control the transformation (Kumar, 2001).

    Temperature shifts during the embryonic and first larval stages failed to induce any effects. The eye-antennal discs are completely normal. This is consistent with the expression data suggesting that the eye is not specified during embryogenesis. A temperature shift during the first half of the second larval stage results in a reduced eye field, but no transformation to antenna. Interestingly, this phenotype is similar to that seen in ey mutant homozygotes. The eye to antenna transformation is fully induced in all cases when the temperature shift occurs during the latter half of the second larval stage. The transformed antenna expresses Dll in an identical pattern as seen in the endogenous antenna. Subsequent temperature shifts during the earliest phase of the third larval stage do not result in a complete transformation. Interestingly, loss of Notch during the next day of the third larval stage results in defects in the regulation of the morphogenetic furrow. These results clearly indicate that the phenocritical period is chiefly within the latter half of the second larval stage. This phenocritical period does not predate the expression of any of the eye specification genes, but it is coincident with their first coexpression and, thus, prediction 3 (above) holds true (Kumar, 2001).

    Is the presumptive eye actually transformed into a second antenna under these conditions, or does the eye degenerate and get replaced by regrowth from elsewhere? The former interpretation (transformation) is favored because in hundreds of transformed L2 disc complexes dissected, a degenerating eye disc, or a small (presumably regrowing) antennal disc was never observed. In all cases, the transformed antenna is equal in size to the normal one. Indeed, both are somewhat larger than normal. This might suggest that a fixed number of cells that normally distribute preferentially to the eye are now equally allocated to both antennae (Kumar, 2001).

    The notch pathway signals differentially in the eye and antenna primordia in the second larval stage. Loss of Notch activity during the second larval stage results in the transformation of the eye into an antenna. Thus, it is predicted that Notch signaling should be elevated in the presumptive eye versus the antenna at the critical time. Cells that are actively receiving a Notch signal upregulate Notch protein expression. Thus elevated Notch antigen expression can be used as a reporter of elevated Notch signaling. Notch and ey expression were examined in imaginal discs from first, second, and third stage larvae. Both Notch and ey are expressed throughout the entire eye-antennal disc anlagen during the first larval stage. By the second larval stage, Notch is differentially upregulated within the presumptive eye. Interestingly, Notch appears especially active along the eye margins and midline, where it is thought to regulate retinal polarity. In contrast, ey appears to be exclusively within the eye field. In the third larval stage, Notch expression is upregulated in the morphogenetic furrow, where it acts to control ommatidial spacing while ey remains upregulated ahead of the furrow (Kumar, 2001).

    Therefore, Egfr signaling promotes an antennal fate while Notch signaling promotes an eye fate. This role for Notch is consistent with the observation that removal of Notch signaling can partially inhibit compound eye development. Furthermore, several of the eye and antennal specification genes (ey, toy, eya, so, eyg, salM, and Dll) are downstream of the Egfr and Notch inputs. Wg and Hh pathway signaling affect this specification. The eye specification genes form a regulatory network and the direct control of any one of these genes may affect the others. Thus, which (if any) of the known eye specification genes is a direct target of Notch or Egfr signals may require direct biochemical assays (Kumar, 2001).

    While activating Egfr or blocking Notch signals transforms the eye cleanly into an antenna, the reciprocal transformation is not complete, suggesting that there may be additional positive regulators of eye fate. The reciprocal transformation experiment could not be conducted (i.e., antenna to eye switch via hyperactivation of Notch or downregulation of Egfr signaling solely within the antennal anlagen). Unlike the ey-GAL4 driver, there is not an equivalent known driver that is expressed solely with the antennal anlagen. All known antennal-determining genes are also expressed in other imaginal discs. For instance, the Dll-GAL4 driver is expressed in several places within the embryonic head and leg imaginal disc. Expression of Egfr or Notch constructs with this driver results only in embryonic lethality. It may be that the antenna can be changed to an eye via alterations of Egfr or Notch signaling provided that the appropriate tools for their missexpression are available (Kumar, 2001).

    Why do homozygous mutants for eye specification genes not transform the eye into an antenna? While it may be that some alleles are not nulls (e.g., ey1), a more interesting possibility is that there may be functional redundancy in some cases -- particularly that of ey and toy. Thus, only when both genetic functions are eliminated will a true null condition exist. Just such a situation confused the phenotypic analysis of two other twin homeodomain proteins, engrailed and invected. Unfortunately, mutations of the toy gene do not yet exist (Kumar, 2001).

    How do the eye specification genes function? Published genetic epistasy and biochemical interaction data suggest that the seven known eye specification genes' products interact at the transcriptional and protein levels to direct cells toward eye fate. This requires that they are expressed in the same cells. Furthermore, it has been suggested that many, if not all, of these genes are 'master regulators' of eye fate -- that is, they are both necessary and sufficient for eye specification. Many very compelling experiments have been described showing the induction of ectopic eyes through the ectopic expression of these genes alone or in synergistic combinations. It is suggested that these genes come under separate regulation by different patterning signals in early development and that there are overlapping domains. Only when all of the domains coincide (during the second larval stage) do eye specification genes specify the eye. This seems to be the simplest explanation since the eye specification genes form a very tight genetic, biochemical, and transcriptional regulatory network suggesting that they are together required for eye specification. It may be that the final coexpression of the eye specification genes' products (and the exclusion of the antennal specification factors) is the last step required to allow the morphogenetic furrow to initiate in response to the next local expression of hh and for the final specification of retinal cell types and pattern (Kumar, 2001).

    5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

    Although the eye-antenna disc primordium primordium (EADP) is thought to be derived from the visual primordium, which is defined by so expression, ey and so expressions do not overlap. Consistently, loss of so does not affect ey and toy expressions and the formation of the EADP. Other genes in the retinal determination network, namely, dachshund (dac) and eyes absent (eya), are not expressed in the EADP. These observations strongly suggest that the EADP formation is independent of the retinal determination network genes so, dac and eya. Its origin and the course of formation need further clarification. 5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments (Huang, 2017).

    Transformation of eye to antenna by misexpression of a single gene

    In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance understanding of the mechanisms that restrict stem cell fate. This study shows that Dorsal interacting protein 3 (Dip3), a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and this observation has been taken advantage of to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, these results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae (Duong, 2008).

    Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor (Bhaskar, 2002).

    Antagonism between the N and EGFR signaling pathways influences developmental fate in the eye-antennal disc leading to a loss of eye tissue and the appearance of extra antennae. Although this phenotype was originally suspected to represent eye-to-antennal transformation, subsequent analysis suggests that it most likely represents antennal duplication. Specifically, the absence of the N signal leads to a failure in eye disc proliferation resulting in compensatory over-proliferation of the antennal disc and its subdivision into multiple antennae. Consistent with the idea that the extra antennae result from under-proliferation of the eye field, it was found that the phenotype was largely suppressed by over-expression of CycE to drive the cell cycle (Duong, 2008).

    In this study, it was found that inhibition of eye disc growth leads to antennal duplication. But in addition, it was shown that the same treatment that leads to antennal duplication can also direct the transformation of eyes to antennae. These two phenotypes are anatomically distinct. This anatomical distinction is evident in adults: antennae resulting from antennal duplication are found anterior to the antennal foramen, while the antennae resulting from eye-to-antenna transformation are found posterior to the antennal foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication discs exhibit multiple circular dac expression domains within a single sac of epithelium (the antennal disc), while eye-to-antennal transformation discs exhibit two or more circular dac expression domains spread over both the eye and antennal discs. The two types of discs display distinct molecular signatures as well: the antennal duplication discs exhibit duplicated Dll expression domains, while the eye discs undergoing transformation to antennae lack Dll expression (Duong, 2008).

    Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal transformation is provided by the observation of eyes that are only partially transformed to antennae since is very difficult to reconcile these partial transformations with the idea of antennal duplication. In some cases, proximal antennal segments tipped with eye tissue are observed. In accord with this phenotype, some third instar larval eye discs display a central domain of Elav-positive differentiating photoreceptors surrounded by a circular dac domain (Duong, 2008).

    These arguments support the idea that antennal duplication and eye-to-antennal transformation are mechanistically distinct phenomena, and the remainder of the discussion assumes this to be the case. However, the possibility that these two phenotypes are two manifestations of a single mechanism cannot be excluded. For example, the discs exhibiting duplicated Dll domains may represent complete transformations, while the discs lacking duplicated Dll domains, but containing Elav may represent partial transformations (Duong, 2008).

    The data show that discs undergoing antennal duplication as a result of Dip3 expression are comprised of a severely diminished eye region and an enlarged antennal region. As shown by BrdU labeling experiments, these antennal duplication discs most likely result from suppression by Dip3 of cell proliferation in the eye field leading to overproliferation of the antennal disc. This conclusion is supported by the ability of factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression defect (Duong, 2008).

    Many experimental manipulations that reduce the size of the eye disc (e.g., surgical excision, induction of cell death, or suppression of cell proliferation) lead to enlargement and duplication of the antennal primordium. How might reduction of the eye field lead to antennal field over-growth? One possibility is that the eye field produces a growth inhibitory signal. Alternatively, the eye field and the antennal field may compete with each other for limited nutrients or growth factors. In support of this latter possibility, recent studies of the role of dMyc in wing development have demonstrated growth competition between groups of imaginal disc cells (Duong, 2008).

    While the results imply that antennal disc overgrowth is required for antennal duplication, overgrowth is thought not to be sufficient for duplication. This conclusion derives from experiments in which an antennal disc specific driver is used to direct over-expression of CycE or Nact. This resulted in antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal duplication was not observed. Thus, in addition to antennal overgrowth, antennal duplication also appears to require reduction or elimination of the eye disc. Regulatory signals from the eye disc may act to prevent antennal duplication (Duong, 2008).

    The eye and antenna discs differ in several respects: (1) Specific expression of the organ-specification genes. The eye disc expresses the retinal determination gene network (RDGN) genes, including eyeless (ey), twin of eyeless (toy), eyes absent (eya), sine oculis (so), and dachshund (dac), while the antennal disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern from that seen in the antennal disc. In the second instar eye disc, hth is expressed throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell proliferation. The hth expression domain later retracts to only the anterior-most region of the eye disc. This pattern is different from the circular expression pattern observed in the antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is required to specify the proximodistal axis in the leg and antenna. In the early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg and dpp expression domain are separated, so that there is probably no intersection between high levels of Wg and Dpp signaling. (3) Whereas the partial overlap of Dll and hth expression domains in the antennal disc is important for proximodistal axis specification, there is no Dll expression in the eye disc. Dll expression in the center of the antennal and leg discs is induced by the combination of high levels of Dpp and Wg signaling. Because there is no overlap of Dpp and Wg signaling in the eye disc, Dll is not induced (Duong, 2008).

    Therefore, efficient transformation of the eye disc into an antennal disc requires at least three things: (1) repression of the eye fate pathway; (2) activation the antennal fate pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in the antenna and leg disc that induces proximodistal axis formation. Any one of these three conditions by itself is not sufficient. (1) Loss of the RDGN genes leads only to the loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra antenna are formed. The induction of Dll is not ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to the expression of Dll and the transformation to the antennal fate. (2) Simply expressing the antennal determining genes Dll or hth in the eye disc does not change the eye fate into antennal fate. It was found that uniform expression of Dll in the eye disc (ey>Dll) resulted in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll and C68a>Dll induced extra antenna but not within the eye field. Therefore, although Dll and hth are important determinants for antennal identity, it is their specific spatial expression patterns that determine antennal development. (3) Creating the intersection of Wg and Dpp signaling does not change the eye into antenna. Such manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc. Therefore, the specific genes induced by Dpp and Wg signaling may depend on disc-specific factors. In the eye disc, turning on Wg signaling in the dpp expressing morphogenetic furrow only blocked furrow progression (Duong, 2008).

    In this study, it was found that the ectopic expression of a single gene, Dip3, can cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1) Overexpression of Dip3 repressed (non-cell-autonomously) ey and dac. The repression of ey may be due to the induction of ct. The ability of Dip3 to simultaneously repress multiple retinal determination genes is completely consistent with the many known cross-regulatory interactions between these genes. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P border and induced ectopic dpp/wg expression (Duong, 2008).

    Interference with cell cycle progression appears to be a common link between the two phenotypes described in this study. In the case of antennal duplication, interference with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows the required intersection between Dpp and Wg signaling (Duong, 2008).

    The observation that Dip3 misexpression can transform the eye field, but not other tissues, to an antennal fate suggests a close evolutionary relationship between the eye and the antenna. Previous studies have emphasized the homology between antennae and legs. The findings presented here that misexpression of a single transcription factor, namely Dip3, can transform eyes to antennae provides support for the notion that the eye and antenna may also, in some sense, be homologous to one another. Previous evidence in support of this idea comes from the observation that similar spatial arrangements of Wg and Dpp signaling along with a temporal cue provided by the ecdysone signal are required for the formation of the eye and the mechanosensory auditory organ. Small mechanosensory sensilla, such as Johnston's organ and the chordotonal organs (stretch receptors) are thought to represent the earliest evolving sense organs. Perhaps the eye resulted from a duplication and specialization of such a sensillum (Duong, 2008).

    Notch-dependent epithelial fold determines boundary formation between developmental fields in the Drosophila antenna

    Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).

    This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).

    Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).

    Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).

    This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).

    A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).

    Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).

    Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).

    It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).

    N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).

    Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).

    The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).

    A quantitative analysis of growth control in the Drosophila eye disc

    The size and shape of organs is species-specific, and even in species in which organ size is strongly influenced by environmental cues, such as nutrition or temperature, it follows defined rules. Therefore, mechanisms must exist to ensure a tight control of organ size within a given species, while being flexible enough to allow for the evolution of different organ sizes in different species. This study combined computational modelling and quantitative measurements to analyse growth control in the Drosophila eye disc. It was found that the area growth rate declines inversely proportional to the increasing total eye disc area. Two growth laws were identified and found to be consistent with the growth data that would explain the extraordinary robustness and evolutionary plasticity of the growth process and thus of the final adult eye size. The study discusses how each of these laws constrains the set of candidate biological mechanisms for growth control in the Drosophila eye disc (Vollmer, 2016).

    Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis

    Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).

    In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).

    This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).

    Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes Sde, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).

    This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).

    Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference

    Genome control is operated by transcription factors (TFs) controlling their target genes by binding to promoters and enhancers. Conceptually, the interactions between TFs, their binding sites, and their functional targets are represented by gene regulatory networks (GRNs). Deciphering in vivo GRNs underlying organ development in an unbiased genome-wide setting involves identifying both functional TF-gene interactions and physical TF-DNA interactions. To reverse engineer the GRNs of eye development in Drosophila, this study performed RNA-seq across 72 genetic perturbations and sorted cell types and inferred a coexpression network. Next, direct TF-DNA interactions were derived using computational motif inference, ultimately connecting 241 TFs to 5,632 direct target genes through 24,926 enhancers. Using this network, network motifs, cis-regulatory codes, and regulators of eye development were found. The predicted target regions of Grainyhead were validated by ChIP-seq and this factor was identified as a general cofactor in the eye network, being bound to thousands of nucleosome-free regions (Potier, 2014).

    The development of the Drosophila eye is a classical model system to study neuronal differentiation and patterning. The TFs that represent the core of the retinal determination network are Eyeless (Ey), Twin of Eyeless (Toy), Dachsund (Dac), Sine Oculis (So), and Eyes Absent (Eya). Although many regulatory interactions are known between these TFs, as they intensively cross-regulate each other, knowledge about interactions with downstream target genes and of other TFs involved in the eye-antennal gene regulatory network (GRN) is sparse. This study aimed at combining classical reverse genetics-starting from a mutant allele and analyze its (molecular) phenotype-with genomics. Doing so, attempts were made to unveil genetic regulatory interactions in an unbiased way, and many regulators of the eye and antennal developmental programs were identified; most of these did not require or use any mutation or direct perturbation (Potier, 2014).

    The mapping approach began by systematically perturbing the developmental system. Attempts were made to include multiple perturbations into one data matrix to obtain a broad spectrum of expression profile changes. These perturbations included TF mutants, TF overexpression, TF knockdown, and cell sorting (Potier, 2014).

    Eye-antennal discs were dissected at the stage where in the WT discs about half of the eye disc contains pluripotent cells that are dividing asynchronously, while the other half contains differentiating PR neurons, in consecutive stages of differentiation. Simultaneously, the antennal disc contains neuronal precursors that are undergoing specification. The expression changes induced by the perturbations often result from a shift in proportion of cell types. This is trivial for the cell-sorting experiments; for example, the GMR>GFP-positive cells show, as expected, a very strong enrichment of genes related to PR differentiation. TF mutants and TF perturbations can also result in cell type shifts; for example, overexpression of Atonal yields more R8 PRs, and the glass mutant results in fewer differentiated PRs. Other TF perturbations cause changes in gene expression downstream of the TF without changing the cell type composition, such as Retained, which disturbs axonal projection. The key technique that was applied, however, was not to compare each TF perturbation with WT discs to identify differentially expressed genes. Rather, linear and nonlinear correlations of gene expression profiles were used across the entire vector of 72 gene expression measurements. This TF-gene coexpression network contains both direct and indirect edges, and although this network is informative, a second layer of predicted TF-DNA interactions was added, thus making this a direct GRN. To increase the sensitivity, a very large collection was used of TF motifs, also including position weight matrices derived for yeast and vertebrate TFs and including computationally derived motifs (e.g., highly conserved words). Using motif-motif similarity measures and TF-TF orthology relationships, each motif was linked to a candidate binding factor. This yielded a large network with 335 TFs and their predicted direct targets. The only functional network of comparable size and comparable directedness to this in vivo network is the TH17 GRN that was derived in vitro in a recent study (Yosef, 2013). That study used a microarray time course of naive CD4+T cells differentiating into TH17. From these gene expression data, they derived TF-gene interactions by clustering and filtered those with TF-DNA interactions obtained by ChIP-seq data, TF perturbations, and cis-regulatory sequence analysis (Potier, 2014).

    The predicted direct and functional eye-antennal GRN includes many previously reported interactions, such as known target genes for Eyeless and Sine Oculis. Target genes in the network were also found for late factors (e.g., Glass, Onecut) and very late factors (e.g., Pph13). The fact that information was captured at different time points during development is because several cell populations were sorted that are loosely correlated with the temporal axis of development, consisting of undifferentiated pluripotent cells anterior to the furrow, all PR cells undergoing differentiation posterior to the MF, R8 PR cells, and late populations of chp-positive cells. However, the temporal information encoded in the network is limited to these broad domains, and a more detailed reconstruction of the time axis would require higher resolution cell sorting or microdissection experiments. Although the perturbed TFs were mainly chosen for their development of the retina, master regulators of antennal development, such as aristaless were also identified (Potier, 2014).

    Interestingly, general factors like Grainyhead were found that were ubiquitously expressed. Grh was found as one of the TFs with the largest number of target enhancers and its binding correlates with open chromatin. Previous studies have shown that Grainyhead may interact with Polycomb and Trithorax proteins to regulate (both activate and repress) target gene expression. It is speculated that this observed correlation can be explained by the fact that Grh is present ubiquitously in the eye disc, thus yielding many sequence fragments from bound and nucleosome-free enhancers by FAIRE-seq (Potier, 2014).

    It is well known that network motifs such as FFLs play an important role in biological networks. One such network motif was examined in more detail, namely the TF pair Glass-Lozenge, and their common targets. These TFs constitute a double-feedback loop (Glass regulates Lozenge, Lozenge regulates Glass, and they together regulate 36 targets). For this network motif, it was found that Glass and Lozenge motifs co-occur at the same enhancer, where they furthermore overlap; this may indicate competition for binding between Glass and Lozenge. Given that Lozenge, an important regulator of cone cell differentiation, could be a repressor and Glass, an important regulator of PR differentiation, could be an activator, such a competition at the CRM level could indeed be a plausible mechanism for their regulatory action (Potier, 2014).

    Another interesting feature that can be derived from a GRN is the proportion of autoregulatory TFs (108 autoregulatory TFs in the eye network) and the proportion of activating versus repressive TFs. A recent large-scale study in yeast found a small majority of yeast TFs to have a repressive role. In that study, each individual TF was perturbed, thereby providing information on positive versus negative edges from the TF to its direct predicted targets, whereby TF-DNA information was used from ChIP-chip data. Since the eye GRN was started from a coexpression TF-gene network, the correlations between TFs and their candidate targets were revisited, and 151 TFs were found that have their motif enriched in the positively correlated target genes, but not in the negatively correlated targets, and 127 TFs showing the opposite; 62 TFs show enrichment in both. This finding agrees, to some extent, with the results in yeast concerning the high amounts of gene-specific repressors. On the other hand, the eye network suggests relative more TFs with a dual activator/repressor function, while the yeast study found only a few such cases (Potier, 2014).

    In conclusion, starting from an expression matrix derived from large-scale perturbations and combining TF-gene coexpression with TF-DNA interactions based on motif inference enabled drawing an extensive eye-antennal GRN. All predicted regulatory interactions, target genes, and candidate regulatory regions are stored in a Neo4J database and can be queried from a laboratory website. The database can also be accessed directly from Cytoscape using the CyNeo4j plugin or can be queried programmatically using the Neo4j query language Cypher. Although many known regulators and cis-regulatory elements were uncovered and several other ones were revealed, a large part of the predicted network, including how the dynamics of the developmental program are encoded in the cis-regulatory regions and in the topology of the network, remains to be explored (Potier, 2014).

    The actomyosin machinery is required for Drosophila retinal lumen formation

    Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, Actin5C was identifed as a key molecule. The results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter (see Model for Drosophila retinal lumen formation). Together, these results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen (Nie, 2014; Pubmed

    Does Unc-GFP uncover ciliary structures in the rhabdomeric eye of Drosophila?

    The Uncoordinated (Unc) gene product, a potential ortholog of orofaciodigital syndrome 1 (Ofd1), is involved in the assembly of the ciliary axoneme in Drosophila and it is, therefore, constrained to cell types that have ciliary structures, namely type 1 sensory neurons and male germ cells. This study shows that evenly spaced Unc-GFP spots are present in the eye imaginal discs of third instar larvae. These spots are restricted to the R8 photoreceptor cell of each ommatidium in association with mother centrioles. This finding is unexpected since the Drosophila eye is of rhabdomeric type and should lack ciliary structures (Gottardo, 2016).

    Ire1 supports normal ER differentiation in developing Drosophila photoreceptors

    The endoplasmic reticulum (ER) serves virtually all aspects of cell physiology and, by pathways incompletely understood, is dynamically remodeled to meet changing cell needs. Inositol-requiring enzyme 1 (Ire1), a conserved core of the Unfolded Protein Response (UPR), participates in ER remodeling and is particularly required during the differentiation of cells devoted to intense secretory activity, "professional" secretory cells. This study characterize Ire1's role in ER differentiation in developing Drosophila compound eye photoreceptors (R cells). As part of normal development, R cells take a turn as professional secretory cells with a massive secretory effort that builds the photosensitive membrane organelle, the rhabdomere. Rough ER sheets proliferate as rhabdomere biogenesis culminates and Ire1 is required for normal ER differentiation. Ire1 is active early in R cell development and is required in anticipation of peak biosynthesis. Without Ire1, rough ER sheets are strongly reduced and the extensive cortical ER network at the rhabdomere base, the subrhabdomere cisterna (SRC), fails. Instead, ER proliferates in persistent, ribosome-poor tubular tangles. A phase of Ire1 activity early in R cell development thus shapes dynamic ER (Xu, 2016).

    From its initial specification in the morphogenetic furrow, to its adult service as a photosensor, a developing Drosophila R cell plays many roles, calling upon a sequence of conserved, core cell functions that target the developmental task at hand. In the last quarter of pupal life that task is the enormous expansion of the photosensory membrane via secretory delivery of membrane rich in rhodopsin and allied elements of phototransduction. This study shows a corresponding expansion of stacked rER meets this secretory challenge; at this developmental stage, R cells are typical of cells devoted to intensive secretion, 'professional' secretory cells, generally. As seen with other professional secretory cells, this study found that R cells require Ire1 for normal ER expansion and differentiation. When normal cells proliferate rER stacks, Ire1 mutant ER proliferates in a dense, chaotic tangle of reticulon-rich tubules. The mechanisms by which Ire1 supports normal R cell ER differentiation remain unknown and in view of its multiple and far-reaching effectors are likely to be multiple. Shown in this study recapitulated in R cells, a common theme in Ire1 regulation of ER differentiation is a requirement in anticipation of secretory activity, suggesting Ire1 builds in secretory capacity as part of normal organelle programming. It is unlikely Ire1 acts alone and temporal overlap of Ire1 activity, shown in this study, and also by (Coelho, 2013), with that of PERK, a second conserved UPR pathway suggests they may cooperate in ER programming. The profuse tangle of reticulon-rich tubules that arises during peak secretory effort, resembles the pathology seen in Ire1 mutant yeast upon ER stress: in response to unfolded protein stress, normal yeast proliferate ER sheets but, although normal in the absence of stress, stressed Ire1 mutant yeast expand ER in dense, reticulon-rich tangles. Together, results suggest the possibility that Ire1 contributes essential shaping activity to a program of ER expansion and, in its absence, an unbalanced drive expands dysmorphic ER (Xu, 2016).

    Shown in this study, and previously (Coelho, 2013), Ire1 loss compromises Rh1 production and secretory delivery needed to build the rhabdomere. Prior work has shown that Ire1's contribution to these tasks is Xbp1-independent, which is in accord with the current study, and now has been extended to ER shaping; R cell ER is normal in severe Xbp1 hypomorphs. Previously, it has been shown that Ire1 contributes to Rh1 delivery via degradation of fatty acid transporter, fatp, mRNA: Ire1 loss elevates fatp mRNA and its reduction using RNAi rescues Rh1 delivery (Coelho, 2013). As elevated levels of phosphatidic acid (PA) have been shown to disrupt R cell apical membrane transport, it is proposed that increased fatp elevates PA and thus degrades Rh1 delivery (Coelho, 2013). Abnormal, expanded rER is seen in R cells with increased PA, but appears distinct from the tangled tubular ER seen in this study. Tubulated ER seen in this study also differs from the dilated ER lumens commonly noted when misfolded secretory protein products over-accumulate, e.g., in Akita mice that accumulate misfolded proinsulin, and distinct from the strongly amplified, well-formed rER seen in ninaA mutants caused by lumenal Rh1 accumulation. Failure to assemble expanded rER sheets with accumulation of irregular, ribosome-poor ER tubules in Ire1 mutant R cells is reasonably the ultrastructural substrate for the severe reduction of Rh1 levels and growth deficit generally; without a definitive measure of reduced Rh1 synthesis, it is possible that reduced Rh1 levels are attributable to enhanced ER-Associated Degradation (ERAD) in an out of control ER. Loss of a normal SRC in Ire1 mutants plausibly contributes to the failure to deliver secretory traffic to the growing rhabdomere. Collapse of normal ER morphology is thus catastrophic for multiple cell activities (Xu, 2016).

    The mechanism by which lower temperature rescues ER differentiation in Ire1 mutant cells remains to be determined, but may be connected to numerous observations showing basal, Ire1-independent, ER capacity supports many aspects of normal cell development and physiology. Indeed, despite profound ER disruption during peak rhabdomere growth, mutant R cells are viable and execute a wide range of normal cell physiologies, including normal cell polarity, cell fate specification and the complex choreography that assembles ommatidia. Similarly, Ire1 null nematodes are viable and morphologically normal, but die when challenged with mutations in other UPR branches or tunicamycin. Mouse hepatocytes lacking Ire1 α show reduced rER content but are otherwise phenotypically normal; however upon ER stress they fail to maintain lipid homeostasis. The gut of mice lacking Ire1beta is phenotypically normal, but sensitized to experimental colitis. In the absence of stress, ER morphology is normal in yeast ire1 mutants. It is speculated that when pupal development is slowed more than, twofold, 9-10 days at 19°C, versus 4 days at 25°C, basal ER capacity meets secretory demand (Xu, 2016).

    The distribution of peripheral ER between sheet and tubule domains has been likened to a "tug-of-war", with activity promoting ER sheets poised against tubule promoting activity, particularly that of membrane curvature-inducing reticulons. The near disappearance of rER sheets and concomitant emergence of dense tubular tangles in Ire1 mutants suggests a runaway win for tubule promotion, potentially due to a failure of sheet-forming activities, an abnormal regulation of tubule formation, or a combination of both. Ribosomes promote ER sheets and it is possible the loss ribosomes in mutant R cells destabilizes rER. Alternately, abnormal, dense Rtnl1 accumulations seen in mutant R cells may reflect a cannibalization of ER sheets by misregulated Rtnl1 with a consequent reduction in Rh1 synthesis; misregulated Rtnl1 could also account for the loss of the SRC cortical ER network. The dynamic, stereotyped differentiation of ER morphology underlying Drosophila R cell differentiation presents a genetically accessible system to investigate how developmental programs shape ER (Xu, 2016).

    Past1 modulates Drosophila eye development

    Endocytosis is a multi-step process involving a large number of proteins, both general factors, such as clathrin and adaptor protein complexes, and unique proteins, which modulate specialized endocytic processes, like the EHD proteins. EHDs are a family of Eps15 Homology Domain containing proteins that consists of four mammalian homologs, one C. elegans, one Drosophila melanogaster and two plants orthologs. These membrane-associated proteins are involved in different steps of endocytic trafficking pathways. The Drosophila EHD ortholog, PAST1, has been shown to associate predominantly with the plasma membrane. Mutations in Past1 result in defects in endocytosis, male sterility, temperature sensitivity and premature death of the flies. Also, Past1 genetically interacts with Notch. The present study investigated the role of PAST1 in the developing fly eye. In mutant flies lacking PAST1, abnormal differentiation of photoreceptors R1, R6 and R7 was evident, with partial penetrance. Likewise, five cone cells were present instead of four. Expression of transgenic PAST1 resulted in a dominant negative effect, with a phenotype similar to that of the deletion mutant, and appearance of additional inter-ommatidial pigment cells. These results strongly suggest a role for PAST1 in differentiation of photoreceptors R1/R6/R7 and cone cells of the fly ommatidia (Dorot, 2017).

    Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis

    Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Tau, a microtubule associated protein, was analyzed in this study to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. Tau was found to have a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation (Nam, 2016).

    Stochastic spineless expression creates the retinal mosaic for colour vision

    Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called 'pale' and 'yellow', defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. The Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless (Wernet, 2006).

    The ability to discriminate between colours has evolved independently in vertebrates and invertebrates. However, despite the obvious differences in eye development and design, both flies and humans have developed retinal mosaics where classes of photoreceptor cells (PRs) with different spectral sensitivity are randomly distributed. The compound eye of Drosophila consists of ~800 optical units (ommatidia), each containing eight PRs in addition to accessory cells. In each ommatidium, the six 'outer PRs' (R1-R6) function like the vertebrate rod cells, as they are required for motion detection in dim light. These cells express the broad-spectrum rhodopsin, Rh1. The 'inner PRs' (R7 and R8) may be viewed as the equivalent of the colour-sensitive vertebrate cone cells, which express a range of different rhodopsin molecules (Wernet, 2006).

    The general rule of sensory receptor exclusion also applies to Drosophila ommatidia, where only one rhodopsin gene is expressed by a given PR. The expression of inner PR rhodopsins can be used to distinguish three ommatidial subtypes. Two of the subtypes are distributed randomly throughout the retina: ~30% of ommatidia express ultraviolet-sensitive Rh3 in R7 cells and blue-sensitive Rh5 in R8 cells, and therefore are specialized in the detection of short wavelengths ('pale' ommatidia). The remaining ~70% express another ultraviolet-sensitive opsin (Rh4) in R7 and green-sensitive Rh6 in R8, making them more responsive to longer wavelengths ('yellow' ommatidia). The coupled expression of Rh3/Rh5 or Rh4/Rh6 within the same ommatidium results from communication between R7 and R8. In the dorsal rim area (DRA), a third type of ommatidia exists in which both R7 and R8 express ultraviolet-sensitive Rh3. These ommatidia are used to detect the e-vector of polarized sunlight for orientation. Spatially localized polarized light detectors and stochastically distributed colour-sensitive ommatidia therefore reflect two fundamentally different specification strategies that shape the retinal mosaic of Drosophila (Wernet, 2006).

    The current model for specifying colour-sensitive ommatidia combines stochastic and instructive steps. First, a subset of R7 (pale R7, pR7) stochastically chooses Rh3 expression over the 'R7 default', Rh4. Second, these cells then impose the p fate (Rh5) onto R8 (pale R8, pR8) of the same ommatidium (Wernet, 2006).

    This study reports the identification of spineless (ss) as a key regulatory gene for establishing the retinal mosaic. ss encodes the Drosophila homologue of the human arylhydrocarbon ('dioxin') receptor, a member of the bHLH-PAS (basic helix-loop-helix- Period-Arnt-Single-minded) family of transcription factors. At mid-pupation, ss is stochastically expressed in a majority of R7 that seem to correspond to the y subtype. ss is both necessary and sufficient to specify the yellow R7 (yR7) fate and subsequently the entire y ommatidia; pR7 cells are thus specified by default, and stochastic expression of ss represents the key regulatory event defining the retinal mosaic required for fly colour vision (Wernet, 2006).

    homothorax (hth) has been identified as the key regulatory gene necessary and sufficient for the specification of DRA ommatidia. ss and hth cause similar homeotic phenotypes: that is, complete (hth) or partial (ss, 'aristapedia') transformation of antennae into legs. Therefore, a potential role of ss in ommatidial subtype specification was tested by generating whole-mutant eyes, as well as mitotic clones, lacking ss function using the null allele ssD115.7 and the ey-FLP/FRT technique. Owing to ey-FLP expression in the antennal imaginal disc, ss mutant flies showed a strong aristapedia phenotyp, but lacked any obvious morphological eye phenotype. However, expression of rhodopsin genes was severely affected. In wild-type eyes, Rh3 is found in ~30% of R7 cells, as well as in both R7 and R8 of DRA-ommatidia, whereas the remaining ~70% of R7 contain Rh4. In ss mutant eyes, Rh4 was completely absent, whereas Rh3 was expanded into all R7 cells. The total number of ommatidia was not reduced, indicating that R7 cells were mis-specified into pR7, rather than yR7 being specifically eliminated. ss mutant mitotic clones were morphologically wild type; however, Rh3 was always present in mutant R7 cells (marked by the absence of ß-galactosidase (ß-gal) expression), whereas Rh4 was always lost (Wernet, 2006).

    To test whether the R7 ss phenotype was cell autonomous, individual mutant R7 cells were generated using the MARCM technique. All mutant R7 cells [marked by the presence of green fluorescent protein (GFP) expression] contained Rh3 and never Rh4, demonstrating that ss is required cell autonomously in R7 to induce Rh4 expression. DRA ommatidia were correctly specified in ss mutant eyes, since Rh3 was expressed normally in both DRA R7 and R8 cells. Therefore, ss is necessary for the establishment of the yR7 subtype without affecting PR fate specification (Wernet, 2006).

    The ommatidial subtypes are first specified in R7, which then instruct R8. Therefore, ss mutant eyes should exhibit a rhodopsin phenotype in R8. In wild types, ~30% of R8 cells contain Rh5, and the remaining ~70% contain Rh6. In ss mutant eyes, the large majority (up to 95%) of R8 contained Rh5, with some R8 still containing Rh6. However, most of these remaining yR8 cells were located in the dorsal third of the eye. In this part of the retina, instruction of pale R8 (pR8) by pR7 is less efficient, resulting in ommatidia with odd-coupled (Rh3/Rh6) rhodopsin expression. In ss mutants, the frequency of such ommatidia was significantly increased in the dorsal region. To test whether the R8 opsin phenotype of ss mutants resulted from the inability of some mutant R7 cells to properly instruct R8, rather than from ss being directly required in R8, sevenless; spineless (sev; ss) double-mutant eyes were generated (Wernet, 2006).

    These eyes, which lacked R7 cells, always exhibited the sev single-mutant phenotype, with virtually all R8 cells containing Rh6. This indicates that ss is required in R7 for the formation of the yR7 subtype, and consequently for the formation of yR8, without being directly required in R8 PRs (Wernet, 2006).

    Whether ss was also sufficient to induce the y ommatidial subtype was tested. Overexpression of ss in all developing PRs using a strong LGMR (long glass multiple reporter)-Gal4 driver and UAS-ss (LGMR.ss flies) resulted in a rough eye phenotype, as well as a dramatic rhodopsin phenotype: Rh4 was activated in all PRs throughout the eye (R1-R6 as well as R7 and R8), as revealed by ectopic expression of an Rh4-GFP reporter in many PRs per ommatidium compared with wild type. To avoid the strong phenotype in the eye, ss was misexpressed using the weaker, variegated GMR driver, sGMR (short GMR)-Gal431 (sGMR.ss flies). This led to strong ectopic induction of Rh4 in many PRs without severely affecting retinal morphology. This ectopic induction of Rh4 was also observed in sev mutants, and was thus independent of R7. Rh3 was still detected in some R7 in sGMR.ss flies, presumably due to the lack of variegated Gal4 expression in these cells, whereas Rh4 was expanded to some outer PRs. However, co-localization of Rh3 and Rh4 was never observed, confirming that gain of Rh4 in R7 cells always leads to the exclusion of Rh3. In contrast, gain of Rh4 in outer PRs did not lead to the exclusion of Rh1; frequent coexpression of Rh1 and Rh4 was observed (Wernet, 2006).

    Using an Rh4-lacZ reporter construct in LGMR.ss flies, it was found that ß-gal-positive PR axons projected to both lamina and medulla, confirming the expansion of Rh4 into outer PRs. However, Rh4-expressing outer PRs were not transformed into genuine R7 cells, since they maintained their lamina projections. Notably, DRA inner PRs were the only cells not expressing Rh4, suggesting that the DRA fate, specified by the gene hth, antagonizes ss function. Expression of Rh3 and Rh5 was completely lost (including in the DRA, where no rhodopsin was detected), while Rh6 expression was found in most R8 cells. This resulted in R8 coexpressing Rh4 and Rh6, demonstrating that the 'one sensory receptor per cell' rule can be broken in Drosophila PRs, as has been shown in other insects. Therefore, ectopic induction of the yR7 fate by ss specifically excludes the formation of pR7 cells. As a consequence, R8 cells expressing Rh5 are not induced, with most R8 expressing Rh6. Rh6 was never found in outer PRs, supporting the hypothesis that ss is required only in R7 for the choice between Rh3 and Rh4, and not directly in R8 for the Rh6 choice. In LGMR.ss flies, the specification of outer versus inner PRs (markers spalt and seven up) or of R7 versus R8 (prospero and senseless) was normal. Thus, ss acts by segregating ommatidial subtypes downstream of early PR specification events (Wernet, 2006).

    Colour PR cell fate determination seems to be a late event in PR differentiation. To test whether ss can transform the R7 fate at late stages of development, the PanR7-Gal4 driver (which is also expressed in DRA R8 cells) was used. Late mis-expression of ss induced the y fate (Rh4) in all R7 cells, whereas Rh3 was absent. Opsin expression in the DRA was also altered, with Homothorax-positive cells (both R7 and R8) now expressing Rh4. Hence, it is possible to reprogramme the R7 fate at later stages of differentiation, as PanR7-Gal4 becomes activated at the time of rhodopsin expression. Surprisingly, expression of R8 rhodopsins outside the DRA was not affected, since the distribution of Rh5 and Rh6 resembled the wild type. As a result, many ommatidia manifested the very unusual coupling of Rh4 in R7 and Rh5 in R8. Therefore, although ss is able to reprogramme all R7 late in development, R8 cannot revert their fate once they have been instructed to become pR8, and they maintain Rh5. Two antagonistic genes expressed in either of the two R8 subtypes have been identified that act together as a molecular consolidation system responsible for this inertia of R8. To confirm that late expression of ss exclusively in R7 is sufficient to transform R7, ss was mis-expressed in ssD115.7 mutants using PanR7-Gal4. This was sufficient to induce Rh4 and to repress Rh3. R8 were again not reprogrammed and exhibited the ss mutant phenotype, with many R8 cells expressing Rh5 (Wernet, 2006).

    All of the results presented above strongly indicated that ss must be expressed in the y subtype of R7 at some point during pupal development. Since several attempts to generate an anti-Spineless antibody had failed, in situ hybridization was used to detect ss messenger RNA in the retina at mid-pupation. At ~50% pupation, ss mRNA was detected in four neuronal cells per ommatidial cluster, one PR and three bristle cells. The PR was also labelled by anti-Prospero, confirming its identity as R7. Although the expression levels of ss in bristle cells seemed uniformly high, levels of ss expression varied considerably among R7 cells, ranging from very faint to very strong in 60%-80% of R7. A 1.6 kilobase 'eye enhancer' fragment (sseye) was also identified within the ss promoter that drives PR-specific expression. After crossing ss eye-Gal4 to UAS-ß-gal::NLS (nuclear localization sequence) reporters, PR-specific ss expression was first detected at mid-pupation-that is, approximately one day before rhodopsins are expressed, and before any visible molecular or morphological distinction between ommatidial subtypes. A single PR per ommatidium, which was identified as R7 through co-staining with Prospero, expressed ss. Thus, the ss eye enhancer recapitulates endogenous ss expression in PRs. ss expression was detected in 60%-80% of R7, correlating well with the distribution of Rh4 in adult retina. Like Rh4-expressing ommatidia, ß-gal-positive ommatidia were more abundant in the dorsal half of the eye, and no ß-gal expression was detected in the DRA (marked by Homothorax), where Rh4 is also never expressed. ss eye-Gal4 expression was detectable for only ~2 h at midpupation (Wernet, 2006).

    Although it was not possible to directly co-stain for ss and Rh4 (which starts to be expressed one day later during pupation), it seems that at mid-pupation a short pulse of ss is deployed in a large subset of R7, which will become yR7 (Wernet, 2006).

    Whether a short pulse of ectopic ss expression was able to modify the entire retinal mosaic was tested using a heat shock-Gal4 driver (hs-Gal4) to temporally control ss expression (hs.ss flies). A 30-min heat shock at ~50% pupation indeed resulted in an increase of Rh4 expression with a concomitant reduction of Rh3 in adults. The phenotype varied extensively, from only R7 cells expressing Rh4 (~25% of the flies analysed had Rh4 in most R7), to almost every PR expressing Rh4. In contrast, a 30-min pulse of ss in one-day-old adult flies had no effect. Heat shocks during larval or early pupal stages were lethal. Thus, PRs are extremely sensitive to a short pulse of ss during mid-pupation, at the time when endogenous ss is normally expressed. To further study the mechanism of the stochastic choice between p and y ommatidia, the retinal mosaic was examined in different mutant backgrounds. Flies heterozygous for ssD115.7 had fewer Rh4-expressing R7 cells. Since the ssD115.7 allele affects only the ss coding sequence, heterozygous flies have two functional promoters, only one of which produces a functional protein, suggesting that the non-productive promoter might sequester limiting factor(s) that regulate(s) the expression levels of ss. If this hypothesis is correct, addition of extra copies of the ss promoter should have a similar effect. Indeed, the addition of two functional copies of the ss eye enhancer (ss eye-Gal4) in an otherwise wild-type background also caused a significant reduction of the yR7 subtype. Therefore, the level of Spineless expression is important for the induction of the yR7 fate, which is less efficient in cells where the amount of Spineless is reduced (Wernet, 2006).

    Retinal patterning in Drosophila reveals an original mechanism for how PR mosaics can be generated: stochastic expression of a single transcription factor (Spineless) acts as a binary switch that transforms the seemingly homogeneous compound eye into a mosaic, distinguishing p and y subtypes. However, subtype specification and rhodopsin expression can be separated, since ss expression in yR7 has ceased well before the time of rhodopsin expression. Additional factors are therefore required downstream of ss to ensure expression of adult p- and y-specific markers such as rhodopsins and additional screening pigments. A revised two-step model is proposed for the stochastic specification of p and y ommatidia. First, R7 are stochastically divided into two subtypes by the induction of ss in yR7. ss-positive R7 express Rh4, whereas the remaining R7 choose the pR7 fate and express Rh3 by default. Second, only those R7 cells that did not express ss (pR7) retain the ability to induce the pR8 fate (Rh5), whereas yR8 express Rh6 by default. The 'default states' of R7 (Rh3) and R8 (Rh6) therefore belong to opposite subtypes. Expression of R8 rhodopsin genes is maintained by a bistable regulatory loop containing the genes warts and melted. Notably, the localized specification of polarization-sensitive DRA ommatidia by hth antagonizes the stochastic choice executed by ss, placing these two genes into a new regulatory relationship during retinal patterning. Therefore, the role of the transcription factor Spineless is to generate the retinal mosaic required for fly colour vision by distinguishing yR7 from pR7 cell fates, and preventing R7 from instructing the underlying R8 cells. Mosaic expression of sensory receptors has been described in detail for the olfactory system of both vertebrates and insects, and random PR mosaics have been described for humans and amphibians, as well as insects. Two transcription factors have been shown to regulate the specification of blue versus red/green cone cell fates in mammals. Upon mutation of either - the human nuclear receptor NR2E3 (also known as PNR) or the rodent thyroid hormone ß2 receptor - the number of blue cones is dramatically increased at the expense of green cones, leading to 'enhanced S-cone syndrome'. It should be noted that this retinal phenotype bears important similarity to the altered ommatidial mosaic in Drosophila ss mutants, where long wavelength-sensitive y ommatidia are lost at the expense of the short wavelength-sensitive p type (Wernet, 2006).

    The stochastic cell fate choice occurs at the level of the ss promoter: the very short pulse of ss expression at mid-pupation is not only controlled temporally, but its levels are also critical, and only ~70% of R7 receive enough Spineless to commit to the yR7 fate. Elucidating the mechanism that controls ss expression will shed some light into the fascinating process of stochastic gene expression, and the identification of its downstream targets will provide insights into consolidation and maintenance of cell fates (Wernet, 2006).

    Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila

    The visual systems of most species contain photoreceptors with distinct spectral sensitivities that allow animals to distinguish lights by their spectral composition. In Drosophila, photoreceptors R1-R6 have the same spectral sensitivity throughout the eye and are responsible for motion detection. In contrast, photoreceptors R7 and R8 exhibit heterogeneity and are important for color vision (see Normalized spectral quantum sensitivity of the different rhodopsins in the photoreceptor subtypes R7p, R7y, R8p, R8y, and R1-R6). This study investigated how photoreceptor types contribute to the attractiveness of light by blocking the function of certain subsets and by measuring differential phototaxis between spectrally different lights. In a 'UV vs. blue' choice, flies with only R1-R6, as well as flies with only R7/R8 photoreceptors, preferred blue, suggesting a nonadditive interaction between the two major subsystems. Flies defective for UV-sensitive R7 function preferred blue, whereas flies defective for either type of R8 (blue- or green-sensitive) preferred UV. In a 'blue vs. green' choice, flies defective for R8 (blue) preferred green, whereas those defective for R8 (green) preferred blue. Involvement of all photoreceptors [R1-R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis from motion detection that is mediated exclusively by R1-R6 (Yamaguchi, 2010).

    Phototaxis consists of at least three behavioral components: (1) detection of an object or a light source in visual space; (2) the attractiveness or aversiveness of the light stimulus at this location; and (3) a motor output (goal-directed walking and turning). Genetic dissection of the visual input to phototaxis relies on the assumption that phototaxis stays intact even if some of these inputs are deleted, i.e., that the mutants can still detect the locations of the two stimuli and move toward or away from them. Although in mutants affecting the R1-R6 subsystem, walking speed, turning, and orientation toward stationary objects are affected, these behaviors are sufficiently intact to support phototaxis. In the experiments reported in this study, light intensities were chosen such that all mutants had at least one photoreceptor type that could detect the light sources and guide walking and turning behavior. This allowed measuring of the second component, the differential attractiveness of the two stimuli, which determined the choice between the two monochromatic lights depending upon the photoreceptor types available in the respective fly strains. Flies could judge the attractiveness of a light source on the basis of its spectral composition and/or spectrally weighted brightness. In the following paragraphs the contributions of the four types of photoreceptors R1-R6, R7, R8p, and R8y to the attractiveness function are discussed (Yamaguchi, 2010).

    Blocking or removing any one of the photoreceptor types shifts the preference away from the point of neutrality in at least one of the choice tests. Silencing the R1-R6 cells leaves the flies with a modest blue preference in the UV/B choice, and replacing their broadly sensitive opsin Rh1 by the UV-sensitive opsin Rh3 shifts the preference in the B/G choice to blue. Removing or silencing R7 in the presence of R1-R6 shifts the preference in the UV/B choice to blue and in the B/G choice to green. Inactivating the green-sensitive Rh6 opsin in R8y cells shifts the preference in the B/G choice to blue and in the UV/B choice to UV. Inactivating blue-sensitive Rh5 in the R8p cells causes a green preference in the B/G choice and a UV preference in the UV/B choice (Yamaguchi, 2010).

    Each of the three photoreceptor subsystems (R1-R6, R7, R8) alone can drive phototaxis. Flies with only photoreceptors R1-R6 (sev rh52 rh61 triple mutants) show a blue preference in the UV/B choice, implying that this subsystem not only mediates optomotor responses and orientation but also can mediate the attractiveness function in phototaxis, i.e., that, with only R1-R6 photoreceptors, flies compare the quantum flux of two light sources 180° apart (Yamaguchi, 2010).

    The R8 subsystem alone can also mediate the attractiveness function in phototaxis (sev ninaE17), as had been shown before for the sev rdgB double mutant. The sev ninaE17 flies have a pronounced green preference in the B/G choice test, consistent with the absorption spectrum of Rh6 expressed in all R8 photoreceptors (Yamaguchi, 2010).

    Flies that have only R7 photoreceptors operating (ninaE17, rh52, rh61) show phototaxis. Surprisingly, they have a very strong preference for blue in both choice tests. This could be explained if R7p inhibit R7y photoreceptors. Otherwise, the flies would show a strong UV preference in the UV/B choice. As an alternative explanation, however, it is possible that phototaxis is mediated by the ocelli in flies with only R7 photoreceptors (Yamaguchi, 2010).

    As long as only one of the central photoreceptor subsystems R7 or R8 is inactivated, the results can be explained by a model in which the respective photoreceptors contribute roughly additively to the attractiveness function. In the honeybee, all three subtypes of photoreceptors (UV, blue, and green) also feed into phototaxis. Spectral mixing experiments in phototaxis are consistent with simple summation of quantal fluxes, and the action spectra of phototactic behavior also suggest pooling of all three photoreceptor types. Color information, i.e., comparison between different photoreceptors, does not appear to be used in honeybee phototaxis (Yamaguchi, 2010 and references therein).

    A simple summation model thus can no longer explain the results of all of the genetic manipulations of photoreceptor types in Drosophila presented in this study. When all four photoreceptor types are intact, UV light is more attractive than would be expected from the sum of the UV attractiveness values of the two isolated major subsystems, R1-R6 and R7/R8. In the UV/B choice test that is balanced for wild type, both flies with only the R1-R6 subsystem (sev rh52 rh61) and flies with only the R7/R8 subsystem (rh1 > shits or ninaE17) show a blue preference. It is thus necessary to postulate that an interaction between the two major retinal subsystems R1-R6 and R7/R8 enhances UV attractiveness. The interaction cannot be explained by an attenuation of the attractiveness of blue because no increased blue preference of the two mutants is observed in the B/G choice. The interaction can unambiguously be traced to the R7 cells. In fact, the strength of the contribution of R7 to the attractiveness function in the UV/B choice depends upon which of the other photoreceptor types are functional. No effect of R7 on spectral preference can be detected without a functional R1-R6 subsystem (comparing ninaE17 and sev ninaE17 in both choice tests. A moderate effect is found in the presence of R1-R6 and R8 (comparing wild type and sev; whereas the effect of R7 is large in the absence of the R8 subsystem (comparing rh52 rh61 and sev rh52 rh61 in UV/B choice (Yamaguchi, 2010).

    The most parsimonious explanation of the data is to assume that, in the presence of a functional R8 subsystem, neither the R1-R6 nor the R7 subsystems on their own have a direct input to the attractiveness function. In the absence of R7 (mutant sev), the R1-R6 subsystem seems not to matter for attractiveness although the R8 subsystem is lacking spectral sensitivity in the UV. Likewise, in the absence of a functional R1-R6 subsystem, the R7 subsystem seems not to contribute. Interestingly, as soon as R8y photoreceptors (or R8p and R8y) are inactivated, the other two subsystems exert their influence on the attractiveness function independently of each other. A full model of these interactions would require a more complete investigation (Yamaguchi, 2010).

    R1-R6 rhabdomeres degenerate in ninaE17 mutants, and this degeneration may have secondary effects on the R7/R8 cells. However, there is no significant difference between ninaE17 mutants and flies expressing rh1>shits, in which R1-R6 function is disrupted without affecting rhabdomere structures. Moreover, it was recently reported that R8y are functional in ninaE17 mutants because circadian entrainment to red light, which is still observed in ninaE17, is abolished in ninaE17 rh61 double mutants (Yamaguchi, 2010).

    The data reveal a further deviation from a simple summation of photoreceptor inputs to the attractiveness function. In the meltGOF mutant, green-sensitive R8y cells are transformed into blue-sensitive R8p cells. This should shift their preference in the UV/B choice to blue. Yet, a UV shift is observed. In contrast, in the absence of R7 cells, the transformation of R8y to R8p photoreceptors (comparison between sev and sev meltGOF) increases the blue preference in the UV/B choice as would be expected from spectral sensitivities. This might indicate that R8p cells enhance the input of the UV channel (R1-R6 × R7) to the attractiveness function. These findings, however, should be treated with caution as unknown developmental defects in the sev and meltGOF mutants might account for the phenotypes (Yamaguchi, 2010).

    Using a different phototaxis paradigm, Jacob (1977) proposed a model to explain the nonadditive effects observed in his phototaxis experiments. An inhibition of R1-R6 by R7/R8 was postulated and it was suggested that R7 cells function only when the R1-R6 system is intact. The current results ask for revision of this model. No general inhibition of the R1-R6 receptor subsystem by R7/R8 was found. The data suggest that neither the R1-R6 nor the R7 subsystems have access to the attractiveness function on their own, except in the absence of functional R8y photoreceptors. Moreover, R8p cells in the current tests had an effect only in the presence of functional R8y cells (Yamaguchi, 2010).

    Rh3-expressing R8 cells in the dorsal rim area account for about 10% of all Rh3 expressing cells. These R8 are still present in sev flies, but are switched off in panR7> shits flies. The comparison of these two lines does not reveal an effect of the Rh3-expressing R8 cells in the UV/B choice. Taking these cells into consideration therefore does not change any of the above conclusions (Yamaguchi, 2010).

    This study has shown that all four photoreceptor types [R1-R6, R7(p, y), R8p, and R8y] are involved in phototaxis, in contrast to motion detection, which relies exclusively on R1-R6 photoreceptors. The wild-type attractiveness function cannot be described as the sum of the attractiveness functions of flies lacking one or more functional photoreceptor types. A multiplicative interaction is observed between photoreceptors R1-R6 and R7. In the absence of functional R1-R6 or R7, the attractiveness function is governed by R8. Only in the absence of R8 and one of the other two subsystems does the remaining subsystem govern the attractiveness function. A recent study on the neuronal substrate of spectral preference identified postsynaptic interneurons in the medulla (Gao, 2008) that are good candidates for mediating some of these interactions. Their involvement in differential phototaxis can now be tested (Yamaguchi, 2010).

    The attractiveness function of differential phototaxis is easy to record, robust, and sensitive (for example, Rh5-expressing blue-sensitive cells account for only ~4% of all photoreceptors, yet yield a significant phenotype when they are not functional). Differential phototaxis may lend itself to mutant screens affecting other chromaticity computations in the brain, including color vision, at the circuit level (Yamaguchi, 2010).

    Color discrimination with broadband photoreceptors

    Color vision is commonly assumed to rely on photoreceptors tuned to narrow spectral ranges. In the ommatidium of Drosophila, the four types of so-called inner photoreceptors express different narrow-band opsins. In contrast, the outer photoreceptors have a broadband spectral sensitivity and were thought to exclusively mediate achromatic vision. Using computational models and behavioral experiments, this study demonstrated that the broadband outer photoreceptors contribute to color vision in Drosophila. The model of opponent processing that includes the opsin of the outer photoreceptors scored the best fit to wavelength discrimination data. To experimentally uncover the contribution of individual photoreceptor types, phototransduction of targeted photoreceptor combinations were restored in a blind mutant. Dichromatic flies with only broadband photoreceptors and one additional receptor type can discriminate different colors, indicating the existence of a specific output comparison of the outer and inner photoreceptors. Furthermore, blocking interneurons postsynaptic to the outer photoreceptors specifically impaired color but not intensity discrimination. These findings show that receptors with a complex and broad spectral sensitivity can contribute to color vision and reveal that chromatic and achromatic circuits in the fly share common photoreceptors (Schnaitmann, 2013).

    Combining modeling with genetic manipulations and behavioral experiments, this study identified the photoreceptor types for blue/green discrimination in Drosophila. Functional color discrimination with the opsin pairs Rh1-Rh4 and Rh4-Rh6 indicates that postreceptoral computations underlying color vision may occur within an optic cartridge deriving from a single ommatidium. Neuronal comparison of differential receptor outputs may be through color opponent mechanisms. TM5 cells in the medulla neuropil are a candidate for color opponent cells comparing Rh1 and Rh4 signals, since they integrate the outputs of lamina monopolar cells (especially L3) and R7. Alternatively, the postreceptoral comparisons might take place further downstream in the optic neuropils. Future physiological studies will be necessary to further elucidate this neuronal computation (Schnaitmann, 2013).

    The findings of this study redress the longstanding assumption that solely narrow-band inner photoreceptors mediate color vision. The sensitivity of Rh1 covers a wide spectral range, but it is not uniform. While this spectral sensitivity is not optimal to represent colors, it nevertheless provides information about differences in wavelength composition. This is in line with the rescued color discrimination with the dichromatic opsin pair Rh1-Rh4. Considering the sufficiency of inner photoreceptors for blue/green discrimination, the role of the outer photoreceptors may be to create an additional opponency dimension for enhanced color discrimination in specific wavelength regions (Schnaitmann, 2013).

    The outer photoreceptors have predominant functions in achromatic vision, such as motion detection. Exploitation of the outer photoreceptor pathway for multiple visual functions is advantageous for animals with limited neuronal resources. A recently discovered contribution of Drosophila R7/R8 to motion detection corroborates the current findings of a differential use strategy. Downstream mechanisms for decoding converged color and motion information await future studies (Schnaitmann, 2013).

    Genetic dissection reveals two separate retinal substrates for polarization vision in Drosophila

    Linearly polarized light originates from atmospheric scattering or surface reflections and is perceived by insects, spiders, cephalopods, crustaceans, and some vertebrates. Thus, the neural basis underlying how this fundamental quality of light is detected is of broad interest. Morphologically unique, polarization-sensitive ommatidia exist in the dorsal periphery of many insect retinas, forming the dorsal rim area (DRA). However, much less is known about the retinal substrates of behavioral responses to polarized reflections. Drosophila exhibits polarotactic behavior, spontaneously aligning with the e-vector of linearly polarized light, when stimuli are presented either dorsally or ventrally. By combining behavioral experiments with genetic dissection and ultrastructural analyses, it was shown that distinct photoreceptors mediate the two behaviors: inner photoreceptors R7+R8 of DRA ommatidia are necessary and sufficient for dorsal polarotaxis, whereas ventral responses are mediated by combinations of outer and inner photoreceptors, both of which manifest previously unknown features that render them polarization sensitive. It is concluded that Drosophila uses separate retinal pathways for the detection of linearly polarized light emanating from the sky or from shiny surfaces. This work establishes a behavioral paradigm that will enable genetic dissection of the circuits underlying polarization vision (Warnet, 2012).

    This study defines the retinal substrates for both dorsal and ventral polarization vision in Drosophila. The DRA is necessary and sufficient for dorsal polarotactic responses, a result that strengthens studies in other insects, concluding that this region mediates responses to celestial polarized light. In addition, this work defines the retinal substrate for responses to ventral polarotactic stimuli, as would occur naturally by reflections from shiny surfaces like water or leaves. This work resolves the differences between previous behavioral studies of polarotactic behavior in flies by demonstrating that flies possess separate detectors to respond to distinct wavelengths, and sources, of polarized light (Warnet, 2012).

    A ventral POL region has previously been described in the backswimmer Notonecta, which uses polarized reflections to locate water bodies. In this insect, inner photoreceptors in a small ventral region form orthogonal analyzer pairs with untwisted rhabdomeres much like a DRA. Drosophila uses a different strategy by exploiting the fact that photoreceptors with moderate or weak twist still provide enough PS to serve as polarization analyzers. In this way, other visual senses, such as the detection of motion and spectral cues, should be affected only minimally by the polarization of light Hence, unlike Notonecta with its specialized ventral retina, the generalist Drosophila incorporates ventral POL detectors while preserving other critical visual capacities (Warnet, 2012).

    An interesting feature of this design is that different classes of photoreceptors form ventral POL analyzers depending on stimulus wavelength. Two subtypes are named 'pale' (p) and 'yellow' (y) and are randomly distributed across the retina. R7 cells each express one of two UV opsins rh3 (R7p), or rh4 (R7y), whereas the underlying R8 cells express either rh5 (R8p) or rh6 (R8y). Due to this chromatic heterogeneity, inner photoreceptors are thought to mediate color vision. In the UV range, R7p cells are necessary for normal polarotactic responses; correspondingly, ventral R7 cells are described with moderate to high estimated PS. However, sufficiency experiments also revealed the involvement of outer photoreceptors in polarization vision. Whereas R1-R3 appear to be weakly polarization sensitive, R4, R5, and possibly R6 show pronounced estimated PS due to reduced rhabdomeric twist. These results are consistent with intracellular recordings in Calliphora describing two classes of R1-R6, one of which retains some PS, even in the UV (Warnet, 2012).

    Whereas R4 to R6 with their pronounced PS provide a basis for ventral polarotaxis via the outer photoreceptors, the contribution of R8 is less clear. It was found that R8 rhabdomeres twist strongly and, thus, R8 cells are expected to have low PS. In contrast, behavioral tests demonstrate that R8 can rescue polarotaxis in norpA mutants. Consistent with this, rare cases were found of very short R8 rhabdomeres exhibiting small twist ranges and correspondingly high expected PS. However, we cannot exclude the possibility that the apparent behavioral contributions of R8 could reflect low-level expression of the driver lines in R1-R6 (Warnet, 2012).

    In larger flies, R7y and R8y as well as R1-R6 contain a UV-sensitizing pigment. Because this molecule is not covalently linked to the opsin protein, its function is independent of microvillar orientation, thereby diminishing PS in the UV range. In addition, these cells contain a C40 carotinoid, which both gives them their yellow appearance and induces anomalous dichroism, which further reduces PS. This may explain why R7p, but not R7y, can mediate ventral UV polarotaxis in Drosophila. The data describe an unexpected new role for 'pale' ommatidia outside the DRA (Warnet, 2012).

    Moreover, the behavioral data confirm that in R1-R6 the UV-sensitizing pigment does not completely eliminate PS in the UV, as was previously reported. The contributions of the outer photoreceptors therefore become more pronounced when polarized green light is presented. That cellular contributions to ventral POL vision differ as a function of wavelength is particularly interesting because reflections from leaves contain much less UV (and more green light) than reflections from water. Hence, activation of distinct combinations of photoreceptors might convey specific meanings to the fly (Warnet, 2012).

    The combination of polarization-sensitive outer and inner photoreceptors represents a new analyzer design, differing from those described in the DRA and the ventral retina of Notonecta. In particular, morphological data does not reveal an orthogonal organization of ventral analyzers. However, comparison between these channels might still increase quality and robustness of the signal. Nothing is known about the subtype-specific connectivity of R7p/R8p and their postsynaptic partners, and no electrophysiological data on polarization-opponent interneurons, or 'compass neurons', exist in flies. By establishing Drosophila as a model of polarization vision, these studies will enable genetic screens using quantitative behavioral assays to allow a complete dissection of the neural circuits involved in responding to this fundamental quality of light (Warnet, 2012).

    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).

    The spatiotemporal order of signaling events unveils the logic of development signaling

    Animals from worms and insects to birds and mammals show distinct body plans; however, the embryonic development of diverse body plans with tissues and organs within is controlled by a surprisingly few signaling pathways. It is well recognized that combinatorial use of and dynamic interactions among signaling pathways follow specific logic to control complex and accurate developmental signaling and patterning, but it remains elusive what such logic is, or even, what it looks like. This study developed a computational model for Drosophila eye development with innovated methods to reveal how interactions among multiple pathways control the dynamically generated hexagonal array of R8 cells. Two novel findings were obtained. First, the coupling between the long-range inductive signals produced by the proneural Hedgehog signaling and the short-range restrictive signals produced by the antineural Notch and EGFR signaling is essential for generating accurately spaced R8s. Second, the spatiotemporal orders of key signaling events reveal a robust pattern of lateral inhibition conducted by Atonal-coordinated Notch and EGFR signaling to collectively determine R8 patterning. This pattern, stipulating the orders of signaling and comparable to the protocols of communication, may help decipher the well-appreciated but poorly defined logic of developmental signaling (Zhu, 2016).

    dachshund potentiates Hedgehog signaling during Drosophila retinogenesis

    Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. This study shows that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, dac was demonstrated to enhance Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis (Bras-Pereira, 2016).

    Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye

    Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. This study examined how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. This study investigated here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. Neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response (Kumar, 2015).

    This paper used the Drosophila eye as a model system with which to study how morphogen gradients can be converted into sharply constrained tissue patterns. The action of the Wg morphogen gradient was examined and it was asked how the highest threshold response, the death of the peripheral ommatidia, is orchestrated. Three observations argue that the secondary Wg expressed by the cone cells combines with the primary Wg from the head capsule to generate a sufficient concentration to kill the ommatidia. First, when the Wg pathway is activated in all cone cells (pros->arm* were arm* is an N-terminally truncated form of Armadillo, a constitutive, cell autonomous activator of the Wg transduction pathway) there is an extended zone of apoptosis in the region where the primary Wg source is known to be high. Second, when the secondary Wg (that secreted by the cone cells) is removed the extended band of ommatidial death is lost. Third, when a level of Wg equivalent to that normally found in the peripheral regions is supplied to pros-arm* eyes all ommatidia now die. Thus, this represents a novel gradient read-out mechanism in which the primary morphogen (Wg derived from the head capsule) elicits a secondary morphogen expression (Wg expressed by the cone cells) in the target cells. Thereafter, the two sources unite to generate the high local morphogen concentration needed to direct the appropriate cell behaviors at that position (Kumar, 2015).

    If there is a permissive zone in the periphery (∼3 ommatidial rows) in which the ommatidia will die if cone cell Wg expression occurs, then this raises the question of how the cone cells responses are normally tightly restricted to the peripheral-most row of ommatidia to ensure that only these ommatidia die. The following describes 1he mechanisms likely responsible for this restriction (Kumar, 2015).

    (1) The high threshold of the ommatidial response: It is surmised that the cone cells have a high threshold response to the morphogen, and the initial responses to the primary Wg source (diffusing from the head capsule) is restricted to the outermost ommatidia. However, it can be envisioned that the secondary Wg secreted by the outer cone cells could diffuse and elicit the same output in the next ommatidial row, and an extreme view could see a relay mechanism in which even more internal rows of ommatidia could express Wg in their cone cells (Kumar, 2015).

    (2) The role played by Notum:
    The expression of Notum is similar to Snail family transcription factors in that it is expressed in the cone cells and 2°/3° PCs of the outermost ommatidia, and since Notum functions to inhibit the free diffusion of Wg, it likely acts to prevent Wg diffusion into more interior ommatidia. Indeed previous studies have shown that in notum mutant clones the zone of death expanded out into more interior rows. Thus Notum (and other mechanisms for preventing Wg diffusion) is seen as playing a critical role in restricting the ommatidial death to the outermost row of ommatidia (Kumar, 2015).

    (3) Combining the high threshold response with the restriction of Wg diffusion: Consider the primary Wg diffusing from the head capsule. It enters the outer row of ommatidia and is of sufficient concentration to elicit the appropriate responses (the various expressions in the cone cells and 2°/3° PCs) but not at a level high enough to kill the ommatidia. The cone cells of the outermost row now begin to secrete the secondary Wg, but the concomitant expression of Notum by the cone cells and 2°/3° PCs of these ommatidia provide a barrier to the movement of both the primary and secondary sources of Wg. This restriction of Wg movement not only protects the more internal ommatidia, but ensures that the high levels of morphogen are constrained in the outermost ommatidia to provide the requisite signal for apoptosis (Kumar, 2015).

    In addition to uncovering the synergy between the Wg derived from the head capsule and the cone cells, a number of phenomena relating to the behavior of the various cell types have been detected (Kumar, 2015).

    (1) The early cone cell death: Following the collapse of the cone cells, the ommatidial apoptosis program begins with the death of cone cells themselves, followed ∼two hours later by the other ommatidial cells. This precocious cone cell death may represent a lower apoptosis threshold for these cells, but it is noted that they are sources of Wg secretion and likely experience autocrine and paracrine (between cone cells of the same ommatidium) Wg signaling as they collapse, and as such are more likely to reach the critical Wg activation level before the other cells (Kumar, 2015).

    (2) The cone cell immunity to death: In pros-arm* eyes, in which all cone cell nuclei fall to the photoreceptor layer and express Wg, there is a wide swath of extended death at the periphery in which all cells of the ommatidia die (including the cone cells). But upon prevention of the cone cell nuclear fall by the expression of esg RNAi, the cone cells survive while the photoreceptors in the extended peripheral zone still die. In these ommatidia, levels of Wg needed to drive apoptosis are achieved, but the cone cells appear invulnerable to it. Whether this invulnerability results from the absence of Snail family transcription factors needed to prime the cone cells for the death signal, or whether by remaining in the apical location they somehow avoid the full level of Wg exposure remains unclear (Kumar, 2015).

    (3) The fall of the cone cell nuclei: The maintenance of cone cell cell-bodies in the appropriate apical location is seemingly critical for the ommatidial stability and integrity, as their fall leads to the disruption of corneal lens units and delamination of photoreceptors. This fall appears to be directed by their expression of Snail family transcription factors. In pros-arm* eyes, the expression of esg.RNAi prevents the fall, and correspondingly the ectopic expression of esg in otherwise wild type cone cells engenders their nuclear fall (albeit prematurely). It was asked whether the fall of the cone cell nuclei resulted from a wholesale collapse of the apical junctions of the cone cells, but D/E-cadherin staining showed a normal apical junction pattern many hours after the nuclei had migrated basally. Thus it does not appear that the cone cells nuclei move basally because the cells lose their apical attachments, rather it is inferred that expression of the Snail family transcription factors reprograms some other behaviors of the cone cells. Such a behavior could be a switch in cell-type affinity. If cone cells normally maintain an apical location by adhesive differences with the photoreceptors, and if these adhesive differences are switched, then cone cell plasma membranes will then preferentially move to the photoreceptor layer. Since the nucleus defines the site of maximum cell body profile with corresponding maximum membrane area, then the fall of the nuclei may simply result from the cone cells acquiring an adhesive affinity with the photoreceptors. Other mechanisms can also be envisaged, in which, for example, motor machinery of the cell is used to reposition the cone cell nuclei in the more basal location (Kumar, 2015).

    An appropriate Gal4 driver line is not available to activate gene expression selectively in the 1° PCs, and the mechanism of their death remains unresolved. In GMR.wg eyes, their death was observed coincident with the photoreceptors (following the apoptosis of the cone cells) and it is surmised that it is the high level of Wg derived from head capsule and the cone cells that directs their death. However, there are a number of indications from that offer clues to a more nuanced understanding of their behavior. Initially the nuclei of the 1° PCs flank the clustered photoreceptor nuclei in their more apical region, but when the cone cell nuclei fall, those of the 1° PCs are shunted more basally. This movement deeper into the photoreceptor layer may play a role in their death. A similar argument can be made from the analysis of * eyes in which 1° PCs are lost, but when Snail family transcription factors are removed from this background, the cone cell nuclei do not fall, and the 1° PCs do not die. Hence the 1° PCs behave in a similar manner as the cone cells; if their position is maintained they do not die even though ambient Wg concentrations are sufficient for their death. This may indicate a general principle; that cells need to be in the correct topological position to experience the death signal (Kumar, 2015).

    Furthermore, in * eyes, the cone cell nuclear fall is accompanied by the loss of the 1° PCs even though the cone cells themselves do not die. The removal of Wg expression from the pros-arm* cone cells rescues the 1° PCs indicating that their loss is normally triggered by the cone cell Wg expression, and it is suspected that the low-level apoptosis seen in the main body of pros-arm* eyes may represent the death of the 1° PCs. If this is the case, then this suggests that the 1° PCs have a lower threshold Wg response for their apoptosis than the cone cells and photoreceptors (Kumar, 2015).

    The death of the photoreceptors appears to simply require the additive of effects of the two Wg sources to trigger their death. But another feature has emerged from these studies – the idea that chronic exposure to sub-lethal levels of Wg triggers photoreceptor degeneration. Consider pros-arm*/esgRNAi eyes; here the photoreceptor death occurs only at the widened zone of peripheral apoptosis, but in the main body of the adult eyes ommatidia show degenerate rhabdomere-like tissue in the apical retinas. The presence of rhabdomere-like tissue suggests the differentiation and subsequent degeneration of the photoreceptors leaving them alive but in a runtish condition. Since this phenomenon is Wg dependent (it is absent when wgRNAi is additionally included) it is inferred that the persistent Wg expression from the cone cells chronically signals to the photoreceptors. Indeed, when GMR.wg/GMR.P35 eyes (in which the apoptosis mechanism is suppressed and the photoreceptors are therefore subject to chronic Wg exposure), were examined a similar degenerate phenotype occurred. This observation suggests another function for the removal of the outer-most row of ommatidia: if they were not removed, chronic exposure to high levels of Wg emanating from the head capsule would lead them to deteriorate into a runtish condition (Kumar, 2015).

    A striking feature of the peripheral patterning mechanism is the timing aspect. The peripheral ommatidia are exposed to head capsule-derived Wg from the time of their birth. And yet they only respond to this Wg signal at defined times. The first occurs shortly after pupation when ac/da transcription is repressed and hth expression is induced. This corresponds with the surge in ecdysone expression that occurs in the animals at this time. The second response is the death of ommatidia at 42 h APF and this mechanism is closely tied with the large peak of ecdysone expression that occurs in the second day of pupation. Thus, it is speculated that Wg provides the spatial signal for peripheral patterning, but that the hormone system of the fly provides the temporal cue that determines when the spatial information can be utilized (Kumar, 2015).

    It is concluded that the periphery of the fly eye is an excellent model system with which to study how morphogen gradients are decoded into discrete tissue types, and this study has delved into the mechanism that precisely restricts the spatial positioning of one of those tissue types. An intricate mechanism has been uncovered in which initial threshold responses lead to the local boosting of the morphogen signal while at the same time upregulating mechanisms to prevent the spread of the morphogen. Evidence is also provided to support the idea that appropriate spatial, temporal and topological context is required for the peripheral ommatidia to undergo developmental apoptosis (Kumar, 2015).

    Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway

    The helicases human DDX3 and Drosophila Belle (Bel) are part of a well-defined subfamily of the DEAD-box helicases. Individual subfamily-members perform a myriad of functions in nuclear and cytosolic RNA metabolism. It has also been reported that DDX3X is involved in cell signaling, including IFN-alpha and IFN-beta inducing pathways upon viral infection as well as in Wnt signaling. This study used a collection of EMS-induced bel alleles recovered from a Wingless (Wg) suppressor screen to analyze the role of the Drosophila homolog of DDX3 in Wg/Wnt signaling. These EMS alleles, as well as a P-element induced null allele and RNAi-mediated knock down of bel, all suppressed the phenotype of ectopic Wg signaling in the eye. However, they did not affect the expression of known Wg target genes like senseless, Distalless or wingful/Notum. Ectopic Wg signaling in eye imaginal discs induces apoptosis by increasing grim expression. Mutations in bel revert grim expression to wild-type levels. Together, these results indicate that Bel does not function as a core component in the Drosophila Wg pathway, and that mutations affecting its helicase function suppress the effects of ectopic Wg signaling downstream of the canonical pathway (Jenny, 2016).

    Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye

    Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

    Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye . R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp) , as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens) , as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce , Scm , or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

    The GMR-FLP/MARCM system allowed allowed the removal of Sce and Scm function specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the limited number of binary fate choices that R1/R6/R7 precursors face. In order to adopt the R7 fate, these precursors must choose to: (1) become neurons in response to high RTK activity-they would otherwise become non-neuronal cells; (2) fail to express Svp in response to N activity-they would otherwise become R1/R6s; and (3) fail to express Sens in response to N activity-they would otherwise become R8s. Loss of Sce or Scm from R7s specifically was found to compromises maintenance of the last of these choices. By contrast, no evidence was found that PcG genes maintain either of the other two choices. Sce mutant R7s were examined throughout larval and pupal development and none were found none misexpressed Svp, nor were Sce or Scm mutant R7s that displayed other R1/R6 characteristics found, such as large rhabdomeres positioned at the periphery of the ommatidium or expression of the R1-R6-specific rhodopsin Rh1. While loss of the Abelson kinase was recently shown to cause R neurons to lose expression of the neuronal marker Elav and switch to a non-neuronal pigment cell fate, this study found that Sce and Scm mutant R1/R6s and R7s maintain expression of Elav and the photoreceptor-specific protein Chaoptin, indicating that their commitment to a neuronal fate is also independent of PcG gene function. It is concluded that R7s use Sce and Scm to maintain repression of one but not all alternative binary fate choices (Finley, 2015).

    The Sens-encoding region is bound by Pc in Drosophila embryos and by Sce in Drosophila larvae , suggesting that Sens is directly regulated by these proteins in at least some cell types. However, because of the technical difficulty in isolating sufficient quantities of chromatin specifically from R7 cells, it was not possible to determine whether PcG proteins bind the Sens locus in R7s. It remains possible, therefore, that Sce, Scm, and Pc maintain Sens repression indirectly in R7s-however, the evidence suggests that they do so independently of their canonical role in repressing Hox genes (Finley, 2015).

    Considerable differences were observed in the strengths of the R7 defects caused by loss of Sce, Scm, Pc, or Psc. One possibility is that these proteins do not contribute equally to PRC1's gene-silencing ability. Indeed, the fly genome contains a second Psc-related gene that plays a redundant role with Psc in some cells, possibly accounting for the lack of defect in Psc mutant R7s. Alternatively, the different wild-type PcG proteins may perdure to different degrees within the mutant R7 clones (the cells that divide to generate the mutant R1/R6/R7 precursors contain a wild-type copy of the mutant gene). Attempts were made, but it was not possible to measure the time course of Sce and Scm protein levels in Sce and Scm mutant R7s, respectively, to test their perdurance directly. However, this thought that perdurance is likely, as this study found that Gal80 perdures until early pupal development within GMR-FLP/MARCM-induced R7 clones (Finley, 2015).

    Sce and Scm were found to be required to maintain Sens repression in R7s generated either in the presence or absence of N activity. What might be regulating the deployment of Sce and Scm in these cells? One possibility is that Sce and Scm repress Sens in R1/R6/R7 precursors by default, since these cells never normally express Sens. However, it was found that neither Sce nor Scm is required to maintain the repression of Sens that is established by Svp. Alternatively, Sce and Scm may be deployed to repress Sens as part of a cell's initial commitment to the R7 fate. As mentioned above, wild-type Sce or Scm protein is likely to perdure in newly created homozygous Sce or Scm mutant R7s, respectively, leaving open the possibility that these genes are required not only for the maintenance but also for the establishment of the R7 fate. Previous work showed that the NF-YC subunit of the heterotrimeric transcription factor nuclear factor Y (NF-Y) is also required to maintain Sens repression in R7s. Like the PcG proteins, NF-YC is broadly expressed in all photoreceptor neurons and is not sufficient to cause R7s to adopt R8 fates, indicating that NF-YC is not responsible for the specific role of PcG proteins in R7s. However, the resemblance between the R7 defects caused by loss of Sce, Scm, and NF-YC suggests that NF-Y may participate in PRC1 function. In support of this possibility, loss of the NF-YA subunit from Caenorhabditis elegans also causes defects similar to those caused by loss of the PcG gene sop-2, including derepression of the Hox gene egl-5 (Finley, 2015).

    PcG proteins have been shown to silence many regulators of development in addition to their canonical Hox targets, suggesting that PcG proteins are likely to play broad roles in maintaining cell fate commitments. However, whether PcG proteins are used to maintain specific binary fate choices as cells differentiate is unclear. In fact, the opposite is true during stem cell differentiation, when the repression of terminal differentiation genes by PcG proteins must instead be relieved. This paper has have identified a role for PRC1-associated PcG proteins in maintaining a specific binary fate choice made during adoption of the R7 fate-a choice that does not involve Hox gene regulation or misregulation. The same PRC1-associated proteins are not required to maintain two other binary fate choices that R7s must make. It is concluded that PcG genes are indeed used to maintain some though not all binary fate choices (Finley, 2015).

    Single-base pair differences in a shared motif determine differential Rhodopsin expression

    The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, this study compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11-base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli (Rister, 2015).

    In the visual system, different photoreceptor neurons express specific light-sensing pigments; however, common downstream factors amplify and convert the response to the visual stimulus into a neuronal signal. For instance, each unit eye (ommatidium) of the Drosophila retina contains eight photoreceptors (R1 to R8) that express different light-sensing Rhodopsins (Rhs) that are restricted to specific photoreceptor subsets. Outer photoreceptors R1 to R6 express Rh1. Inner photoreceptors R7 and R8 express either Rh3 in pR7s coupled with Rh5 in pR8s, or Rh4 in yR7s with Rh6 in yR8s. R1 to R8 all share broadly expressed phototransduction factors that amplify and convert the response to the visual stimulus into a neuronal signal (Rister, 2015).

    This study examined the cis-regulatory mechanisms that distinguish restricted from broad expression patterns for Rhodopsins and downstream phototransduction factors, respectively. All Rhs share the conserved Rhodopsin Core Sequence I (RCSI), which resembles the palindromic P3 motif (TAATYNRATTA), an optimal binding site for paired-class homeodomain proteins. Almost all known broadly expressed phototransduction genes contain a P3 motif in their proximal promoter. The presence of a conserved P3/RCSI motif within 100 base pairs (bps) of the Rh transcription start site (TSS) is significantly associated with enrichment in adult eyes. P3/RCSI is required for activation in photoreceptors because its mutation caused either a loss or a strong reduction in expression of 16 broad or restricted reporters, with the exception of Arr1. Moreover, expression of 10 out of 15 reporters was lost in mutants for the photoreceptor-specific transcription factor Pph13, a paired-class homeodomain protein that binds P3 and the Rh6 RCSI in vitro (Rister, 2015).

    Because each Rh promoter has a highly conserved RCSI variant, the sufficiency of P3 and RCSI were tested to determine the significance of the specific differences between perfectly palindromic (P3) and imperfect motifs (RCSI). Four copies of the P3 motif (including four neighboring bps for spacing; the contribution of these additional bps was only tested for Rh4) from the broadly expressed ninaC, rdgA, or trpl drove broad expression in all photoreceptors, consistent with previous results. In sharp contrast, multimerized RCSI motifs drove expression in subsets of photoreceptors. The RCSI of Rh3 and Rh6 contains a K50 motif, a binding site for K50 homeodomain proteins such as the Dve repressor or the Otd activator. Expression of [Rh3 RCSI]4 and [Rh6 RCSI]4 was biased to inner photoreceptors: [Rh3 RCSI]4 mediated restricted expression in R8 and R7, with a strong bias toward the pR7 subset, where Rh3 is normally expressed. This pattern is complementary to the expression of Dve, which is indeed responsible for the restricted expression as [Rh3 RCSI]4 drove a broad, P3-like pattern in dve mutants. [Rh6 RCSI]4 drove restricted expression in R8s and R7s; expression in R1 to R6 was very weak in comparison to P3 motifs, which was due to dve-dependent repression (Rister, 2015).

    [Rh1 RCSI]4 drove variable expression in R1 to R6, where Rh1 is expressed. This outer photoreceptor-specific pattern is complementary to the inner photoreceptor expression of [Rh3 RCSI]4. Rh4 has the same RCSI as Rh1. However, adding the synergistic 3' RCSII motif led to expression in yR7s, where Rh4 is expressed. Although [Rh5 RCSI]4 was not sufficient for reporter expression, adding three K50 motifs to a single Rh5 RCSI ([K50]3 + [Rh5 RCSI]1) led to expression in R8 and pR7 photoreceptors (Rister, 2015).

    In summary, the RCSI motifs of specific Rhs differ from palindromic P3 motifs in broadly expressed genes: They drive expression that is biased toward the endogenous Rh expression patterns. It is shown below that full subtype specificity and activation often requires the repetition of motifs that are present in the RCSI (Rister, 2015).

    As specific RCSI motifs directed restricted expression in different photoreceptor subsets, albeit with incomplete subtype specificity and with some variability in expression levels, it was asked whether the single-bp differences are required for subtype specificity in a wild-type promoter context and which other motifs are required for full restriction. The K50 (Otd/Dve) motifs (TAATCC) were mutated to Q50 (Pph13) motifs (TAATTG/A) to disrupt repression while preserving RCSI-mediated activation. Mutating the Rh3 RCSI resulted in an expansion to yR7s, where Dve is present at low levels. Mutating the Rh6 RCSI caused derepression in R1 to R6 and the ocelli. Rh3 and Rh6 have K50/Dve repressor motifs repeated upstream, and mutation of individual motifs also caused derepression in yR7s and R1 to R6 and the ocelli, respectively. Taken together, single-bp changes create K50 motifs in the Rh3 and Rh6 RCSI, which are required for subtype-specific expression together with their upstream repeats (Rister, 2015).

    The importance of the disrupted P3 palindrome-i.e., the imperfect 3' homeodomain binding motif in the RCSIs of Rh1 toRh5 was examined. Creating a palindromic motif in theRh3 RCSI (TAATCCAATTC->TAATCCAATTA) caused derepression in yR7s that depended on Pph13. Therefore, derepression appears to be due to increased activation through the newly created Q50/Pph13 site. The same ATTC→ATTA mutation in the Rh5 RCSI led to partial derepression in yR8s. This single-bp change created abinding site for the activator Otd (AGATTA), and indeed derepression in yR8s was lost in otd mutants, as was activation in pR8s (Rister, 2015).

    The 3' ATTC motif in the RCSI of Rh3 and Rh5 is repeated upstream. Mutating the upstream repeat without creating a Q50/Pph13 site (ATTC->CAAA) also caused derepression in yR7s (Rh3) or yR8s (Rh5). Mutating both ATTCs of Rh5 enhanced derepression into almost all yR8s. Therefore, this study has identified repressor motifs in the RCSIs of four Rhs (K50/Dve motifs in Rh3/Rh6 and ATTC motifs in Rh3/Rh5). These motifs are repeated upstream within less than 100 bps and are required for full subtype specificity (Rister, 2015).

    A single-bp ATTT->ATTA mutation in the Rh4 RCSI caused derepression in R1 to R6, pR7s, and the ocelli. The correct pattern was restored by crossing the mutant Rh4 reporter in a Pph13 mutant background, indicating that the A -> T change prevents Pph13 from overcoming repression in the 'wrong' photoreceptor subsets, as was the case for Rh3 and Rh6. The same mutation in the Rh1 RCSI caused no detectable derepression. Replacing two bps in the RCSI of the ocelli-specific Rh2 to obtain a Q50/Pph13 site led to derepression in R1 to R6 photoreceptors that depended on Pph13 (Rister, 2015).

    In vivo data revealed that a cell-fate decision requires single-bp differences in RCSI motifs. They complement previous findings in cell culture that subtle sequence differences in a glucocorticoid receptor or nuclear factor κB (NF-κB) binding site can specify the mode of transcriptional regulatio and that small differences in binding-site sequences can lead to distinct Hox specificities in vivo and in vitro. (1) Single bps in RCSI prevent binding of dimers of broadly expressed activators such as Pph13, tipping the balance of activator/repressor binding. This weakened activation allows repressors to prevent activation in other photoreceptor subtypes. (2) They generate specific combinations of overlapping activator and repressor motifs, often repeated upstream to provide robust expression and full subtype specificity. Creating overlap of activator and repressor motifs is an efficient way of blocking a key activator site in the 'wrong' cell types that express a repressor, especially because the RCSI motifs are very close to the transcription start site and repression there could block other activators. The precise tuning of RCSI motifs within their respective promoter context leads to incompatibility in other Rh promoters, as revealed by RCSI swap experiments: Replacing a given RCSI with another one resulted in two main outcomes: loss of expression or derepression in specific subsets of photoreceptors (Rister, 2015).

    The RCSI/P3 motif resembles 'terminal selector' motifs that allow the coordinated expression of effector genes that define a particular neuron type. Yet, RCSI motifs exhibit additional layers of regulation that are integrated in a single regulatory element, as their sequence is modified for subtype specificity. Mutating a cis-regulatory motif in many cases appears to be the shortest evolutionary path toward a novel phenotype. Although it was found that it is possible in some cases to eliminate ectopic expression by removing the broadly expressed activator Pph13, this simultaneously causes a loss of expression of several broad phototransduction genes, defects in photoreceptor morphology, and a severe loss of light sensitivity (Rister, 2015).

    It is proposed that the modification of a P3-type motif into different RCSI-type motifs allowed partitioning Rh expression to different subtypes of photoreceptors. This opened the possibility to discriminate wavelengths and likely conveyed a selective advantage. In this model, P3 motifs represent a positive regulatory element shared by ancestral genes that were expressed in all photoreceptors. This regulation is conserved, as the promoter of the long-wavelength Rh, as well as Gβ76C that are both expressed in all photoreceptors in the beetle Tribolium, contain a palindromic P3-type motif and depend on Pph13 (Rister, 2015).

    Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila

    Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, this study examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, this study proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, alpha5, beta5 and beta6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In all, these findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development (Velentzas, 2013).

    A population of G2-arrested cells are selected as sensory organ precursors for the interommatidial bristles of the Drosophila eye

    Cell cycle progression and differentiation are highly coordinated during the development of multicellular organisms. The mechanisms by which these processes are coordinated and how their coordination contributes to normal development are not fully understood. This study determined the developmental fate of a population of precursor cells in the developing Drosophila melanogaster retina that arrest in G2 phase of the cell cycle and investigated whether cell cycle phase-specific arrest influences the fate of these cells. Retinal precursor cells that arrest in G2 during larval development were shown to be selected as sensory organ precursors (SOPs) during pupal development and undergo two cell divisions to generate the four-cell interommatidial mechanosensory bristles. While G2 arrest is not required for bristle development, preventing G2 arrest results in incorrect bristle positioning in the adult eye. It is concluded that G2-arrested cells provide a positional cue during development to ensure proper spacing of bristles in the eye. The results suggest that the control of cell cycle progression refines cell fate decisions and that the relationship between these two processes is not necessarily deterministic (Meserve, 2017).

    Loss of Na/K-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration

    The activity of Na+/K+-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na+/K+-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na+/K+-ATPase deficiency to in vivo neurodegeneration has been lacking. This study used Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na+/K+ ATPase. Loss of ATPα, an α subunit of Na+/K+-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K+. Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na+/K+-ATPase β subunit (see Nervana 1 and Nervana 2), partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na+/K+-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na+/K+ ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na+/K+ ATPase deficiency-mediated neurodegeneration (Luan, 2014).

    The Na+/K+-ATPase transports Na+ and K+ against their concentration gradients across the cell membrane to maintain a low Na+ and high K+ concentration within the cells. These ion gradients determine the resting membrane potential and form the basis of the excitability of neurons. The Na+ gradient also provides the driving force for various secondary active transporters that import glucose, amino acids, and other nutrients into the cell. Additionally, the ion concentrations maintained by Na+/K+-ATPase are important for regulating cellular volume and preventing cells such as neurons from swelling and lysing (Luan, 2014).

    Considering the importance of Na+/K+-ATPase in basic cellular functions, it is not surprising that either dysregulation or deficiency of neuronal Na+/K+-ATPase was observed in many neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD) and rapid-onset dystonia Parkinsonism (RDP). Thus, disrupting normal Na+/K+-ATPase activity in neurons has been proposed to contribute to the pathogenesis of neurodegeneration. Nevertheless, the link between the disrupting neuronal Na+/K+-ATPase activity and neuronal dysfunction/degeneration has yet to be clarified (Luan, 2014).

    Na+/K+-ATPase is composed of at least two subunits: a large catalytic α subunit and a regulatory, single-transmembrane-domain β subunit. Mammals have three α-subunit and two β-subunit genes and may express six structurally distinct Na+/K+-ATPase isoforms. In the brain, although the α3 and β2 subunits are expressed predominantly in neurons, the α2 and β1 subunits are found primarily in glia, and the α1 subunit is ubiquitously expressed. The Na+/K+-ATPase in glia is required to maintain a low K+ level in the neuronal environment and thus has a large impact on neuronal function and survival. Na+/K+-ATPase inhibitors like ouabain act on all Na+/K+-ATPase isoforms and cannot differentiate between the cell-autonomous effects of the Na+/K+-ATPase in neurons from those derived from the neighboring glia. Thus, genetic approaches are needed to modulate the Na+/K+-ATPase level in neurons to investigate the function of neuronal Na+/K+-ATPase. Genetic studies on the impact of neuronal Na+/K+-ATPase deficiency in the past decade, which were mostly based on characterization of heterozygous mutant mice of the α3 subunit, have identified defects in the function of central brain neurons but have not provided direct evidence of neurodegeneration (Luan, 2014).

    The Drosophila visual system expresses only one type of α subunit, ATPα, and three β subunits, Nrv1–3. This study used Drosophila photoreceptors as a genetic model to study the cell-autonomous functions of neuronal Na+/K+-ATPase. Although ATPα mutants in Drosophila exhibit extensive neurodegeneration, these mutants were not used because the degeneration is due to the loss of Na+/K+-ATPase not only in neurons but also in neighboring non-neuronal cells. Instead, using a UAS/Gal4-mediated RNAi approach, knocked down ATPα and Nrv1-3 were generated specifically in photoreceptors, and the impact of this knockdown was assessed on visual signaling and photoreceptor integrity in the fly (Luan, 2014).

    Na+/K+-ATPase activity establishes and maintains the characteristic transmembrane gradients of Na+ and K+, which underlie essentially all vertebrate and invertebrate cellular physiology. Although the importance of Na+/K+-ATPase to the function and survival of both neurons and non-excitable cells has been demonstrated by decades of pharmacological studies, the involvement of neuronal Na+/K+ ATPase defects in neurodegeneration has yet to be demonstrated in vivo. This cell-specific RNAi study reveals for the first time that Na+/K+-ATPase is essential for normal neuronal function of Drosophila photoreceptors. More importantly, this work provides in vivo evidence that links neuronal Na+/K+-ATPase deficiency to age-dependent neurodegeneration (Luan, 2014).

    Genetic studies on neuronal Na+/K+-ATPase function in the past decade, which were primarily based on the characterization of mice heterozygous for a mutation of the α3 subunit, have focused on central brain neurons; however, the importance of functional Na+/K+-ATPase in sensory neurons remains largely unknown. This study shows that knockdown of ATPα in Drosophila photoreceptor neurons abolishes their response to light, resulting in complete blindness in the fly. These results confirm the importance of Na+/K+-ATPase in animal sensory functions. The light response of Drosophila photoreceptors is mediated by cation influx (mostly Na+) through light-stimulated TRP channels. Photoreceptors in ATPα knockdown flies were unresponsive to light for two reasons. First, without a sufficient K+ gradient across the cell membrane, the cell has no negative resting membrane potential and, thus, no electrical driving force for cation influx through TRP channels. Second, in the absence of Na+/K+-ATPase, photoreceptors may have already accumulated a high intracellular level of Na+ in the dark, which prevents extracellular Na+ flow into the cell through TRP channels during light stimulation. However, loss of the light response may not be attributed to morphological defects in the photoreceptor. First, temporally controlled knockdown of ATPα in the adult stage excludes the involvement of obvious developmental problems. Second, the light response of photoreceptors was abolished in 1-day-old ATPα knockdown flies despite the overall normal shape of the rhabdomeres. Based on these findings, it is concluded that loss of the light response is independent of degeneration in ATPα knockdown photoreceptors (Luan, 2014).

    Drosophila photoreceptors have been used as a genetic model for retinal degeneration studies. Photoreceptor degeneration in many mutants is caused by defects in the regulation of the visual transduction cascade and is light-dependent. In ATPα knockdown photoreceptors, however, the visual cascade may not mediate or regulate degeneration because light deprivation does not change the severity of neurodegeneration in 10-day-old flies. Instead, the progressive degeneration in ATPα knockdown photoreceptors has demonstrated characteristics of apoptotic/necrosis hybrid cell death that are reminiscent of those observed in Na+/K+-ATPase-inhibited mammalian cells. When Na+/K+-ATPase is inhibited by ouabain, mammalian cells undergo hybrid cell death, which has been attributed to a loss of intracellular K+ ions. In vitro studies suggest that the depletion of intracellular K+ may induce apoptosis or act as a necessary cofactor to promote apoptosis. It is estimated that the intracellular levels of K+ in ATPα knockdown photoreceptors could be as low as 10 mM, which is comparable to the 50%-80% decrease in K+ observed in ouabain-treated mammalian cells. Thus, the low levels of intracellular K+ could have contributed to the degeneration of ATPα knockdown photoreceptors. Additionally, Ca2 + overload may also have a role in photoreceptor degeneration. The depolarization of the membrane potential in ATPα knockdown photoreceptors may activate voltage-gated Ca2 + channels and a reversed operation of the Na+-Ca2 + exchanger. Both activities will increase the intracellular Ca2 + concentration and could promote necrotic cell death. Finally, accumulation of Na+ inside ATPα knockdown photoreceptors impairs the driving force of nutrient import through the secondary membrane transporters, which may also play a role in cell degeneration. Hybrid cell death, an intermediate form of cell death falling along an apoptosis-necrosis continuum, can also be found in the neurodegeneration caused by excitotoxicity and ischemia. Therefore, further studies on the role Na+/K+-ATPase in hybrid cell death will elucidate the mechanism of the cell death bearing both apoptotic and necrotic features in different neuropathological conditions (Luan, 2014).

    Until now, most in vivo studies on Na+/K+-ATPase-related neurodegeneration have relied on either pharmacological agents (Bignami, 1966 and Lees, 1994) or Drosophila ATPα mutants. Those studies have suggested that both dysregulation and deficiency of Na+/K+-ATPase lead to extensive neurodegeneration. However, the degeneration observed in those studies could be partially derived from defects in non-neuronal tissues and cells in the brain. For example, Na+/K+-ATPase in the blood-brain-barrier participates in the maintenance of water and ion homeostasis in the central nervous system (CNS), which is critical for neuronal function and survival. In the Drosophila auditory organ (Johnston's organ), Roy found that knocking down ATPα in scolopale cells, principal support cells that enclose neuronal dendrites, results in neuronal dysfunction and complete deafness. Additionally, a defect of Na+/K+-ATPase in astrocytes could be responsible for neonatal seizures and spongiform encephalopathy. In common neurodegenerative disorders such as AD, PD and RPD, however, Na+/K+-ATPase is only reduced in specific subgroups of neurons. To better mimic the neuropathological conditions of neurodegenerative diseases to study this degeneration mechanism, it is necessary to specifically downregulate Na+/K+-ATPase in particular neurons to avoid perturbations in other cells (Luan, 2014).

    Because homozygous mutations in the mouse α3 subunit of Na+/K+-ATPase cause neonatal lethality, genetic studies on neuronal isoforms of Na+/K+-ATPase have so far been primarily based on the characterization of heterozygous α3 mutants. Those mouse studies, however, have not revealed direct evidence of neurodegeneration in the brain most likely due to the relatively moderate reduction of Na+/K+-ATPase activity in the heterozygous mutants. This in vivo model using Drosophila photoreceptors, which mimics the neuropathological conditions of those neurodegenerative disorders, could be a valuable tool for further investigating the mechanism of neuronal Na+/K+-ATPase deficiency-mediated neurodegeneration. In addition to the genetic tools for gene modulation, Drosophila utilizes simple assays such as ERG, phototaxis and optomotor responses, and the optical neutralization assay to evaluate both the function and morphology of photoreceptor neurons. The loss of Na+/K+-ATPase in photoreceptors does not change the environmental K+ levels, allowing the study the cell-autonomous effects of neuronal Na+/K+-ATPase deficiency. Taking advantage of this simple and convenient neuronal model may allow identification of the key players in Na+/K+-ATPase deficiency-mediated neurodegeneration, which will thereby guide in the design of new therapeutic strategies for neurodegenerative disorders (Luan, 2014).

    This study has provided evidence that either dysregulation or deficiency of neuronal Na+/K+-ATPase causes abnormal depolarization of neurons by disrupting the intracellular ion balance instead of extracellular ion homeostasis, which leads to neuronal dysfunction and behavioral abnormality. Furthermore, disrupted neuronal Na+/K+-ATPase activity triggers progressive neurodegeneration. Therefore, this study suggests that early intervention against dysregulation or deficiency of neuronal Na+/K+-ATPase may alleviate the progression of neurodegenerative disorders (Luan, 2014).

    Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors

    Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to beta-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, CG9317, an SLC22 transporter family protein, was identified and named CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals (Xu, 2015).

    Anatomical reconstruction and functional imaging reveal an ordered array of skylight polarization detectors in Drosophila

    Drosophila photoreceptors R7 and R8 in the dorsal rim area (DRA) of the compound eye are specialized to detect the electric vector (e-vector) of linearly polarized light. These photoreceptors are arranged in stacked pairs with identical fields of view and spectral sensitivities, but mutually orthogonal microvillar orientations. This anatomical arrangement in larger flies suggests that the DRA constitutes a detector for skylight polarization, in which different e-vectors maximally excite different positions in the array. To test the hypothesis, responses to polarized light of varying e-vector angles in the terminals of R7/8 cells using genetically encoded calcium indicators were measured. Data confirm a progression of preferred e-vector angles from anterior to posterior in the DRA, and a strict orthogonality between the e-vector preferences of paired R7/8 cells. Decreased activity in photoreceptors was observed in response to flashes of light polarized orthogonally to their preferred e-vector angle, suggesting reciprocal inhibition between photoreceptors in the same medullar column, which may serve to increase polarization contrast. Together, these results indicate that the polarization-vision system relies on a spatial map of preferred e-vector angles at the earliest stage of sensory processing (Weir, 2016).

    Fly photoreceptors encode phase congruency

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although it is now known that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. This study reverse-engineered the neural code of Drosophila photoreceptors and shows for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. To mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli (Friederich, 2016).

    An eye on trafficking genes: Identification of four eye color mutations in Drosophila

    Genes that code for proteins involved in organelle biogenesis and intracellular trafficking produce products that are critical in normal cell function. Conserved orthologs of these are present in most or all eukaryotes including Drosophila melanogaster. Some of these genes were originally identified as eye color mutants with decreases in both types of pigments found in the fly eye. Using these criteria, this study molecularly mapped and evaluated the genome sequences of four eye color mutations: chocolate, maroon, mahogany, and red Malpighian tubules. Mapping was performed using deletion analysis and complementation tests. chocolate is an allele of the VhaAC39-1 gene, which is an ortholog of the Vacuolar H+ ATPase AC39 subunit 1. maroon corresponds to the Vps16A gene and its product is part of the HOPS complex, which participates in transport and in organelle fusion. red Malpighian tubule is the CG12207 gene, which encodes a protein of unknown function that includes a LysM domain. mahogany is the CG13646 gene, which is predicted to be an amino acid transporter. The strategy of identifying eye color genes based on perturbations in quantities of both types of eye color pigments has proven useful in identifying proteins involved in trafficking and biogenesis of lysosome related organelles. Mutants of these genes can provide valuable in vivo models to understand these processes (Grant, 2016).

    A model of the spatio-temporal dynamics of Drosophila eye disc development

    Patterning and growth are linked during early development and have to be tightly controlled to result in a functional tissue or organ. During the development of the Drosophila eye, this linkage is particularly clear: the growth of the eye primordium mainly results from proliferating cells ahead of the morphogenetic furrow (MF), a moving signaling wave that sweeps across the tissue from the posterior to the anterior side, that induces proliferating cells anterior to it to differentiate and become cell cycle quiescent in its wake. Therefore, final eye disc size depends on the proliferation rate of undifferentiated cells and on the speed with which the MF sweeps across the eye disc. A spatio-temporal model of the growing eye disc was developed in this study based on the regulatory interactions controlled by the signals Decapentaplegic (Dpp), Hedgehog (Hh) and the transcription factor Homothorax (Hth) and how the signaling patterns affect the movement of the MF and impact on eye disc growth was explored. Published and new quantitative data were used to parameterize the model. In particular, two crucial parameter values, the degradation rate of Hth and the diffusion coefficient of Hh, were measured. The model is able to reproduce the linear movement of the MF and the termination of growth of the primordium. It was further shown that the model can explain several mutant phenotypes, but fails to reproduce the previously observed scaling of the Dpp gradient in the anterior compartment (Fried, 2016).

    Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye

    During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting (or opposing) proliferation in progenitors is essential to understand normal development and disease. This study used the Drosophila eye as a model where the transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. Hth+Tsh-induced tissue overgrowth was shown to require the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation (Neto, 2016).

    Abnormal maintenance of transcription factors that promote an undifferentiated, proliferative state is often an initiating event in tumors. However, abnormal growth is dependent on specific non-autonomous signals provided by the microenvironment. This study used an experimental system that results in continuous growth to identify these signals and the mechanism of action. In this system, the GAL4-driven maintenance during eye development of hth and tsh, two transcription factors normally transiently co-expressed in eye progenitors, cause cells to increase their avidity for Dpp. This, in turn, leads to a hyper-activation of the pathway, which is necessary to maintain the proliferative/undifferentiated phenotype. The increased avidity for Dpp was shown to be mediated, at least partly, through increased expression of the proteoglycans components encoded by dally and dlp, functionally modified by slf (Neto, 2016).

    Progenitor cells, forced to maintain Hth and Tsh (hth+tsh progenitor-like cells) trap Dpp produced at local sources, which then causes an increased in intracellular signaling. The mechanism responsible of this trapping seems to be the increase of extracellular matrix (ECM) components. First, a cell-autonomous increase was found in dally transcription and Dlp membrane levels, the two glypican moieties of heparane sulphate proteoglycans. Second, the RNAi-mediated attenuation of sfl function, a gene encoding an enzyme required for the biosynthesis of these proteoglycans, is required for the overgrowth/eye-suppression phenotype induced by hth+tsh maintenance. A third line of support comes from examination of the effects of hth+tsh or hth+tsh+slf RNAi on the pMad profiles. Considering that the Dpp production remains unaltered, hth+tsh tissue shows an increase in both pMad signal amplitude and range, which is consistent with the increase in proteoglycans simultaneously augmenting Dpp diffusion and stability. On the contrary, reducing proteoglycan biosynthesis in hth+tsh+slf RNAi cells results in the retraction of the pMad signaling range back towards control values, which again is expected if Dpp's diffusion depends on proteoglycans (Neto, 2016).

    By forcing the expression of hth and tsh in eye precursors, these cells are exposed to signaling levels higher than they would normally encounter. This is because during normal eye development Dpp, produced at the furrow, represses first hth and then, closer to the furrow, also tsh, so that the cells approaching the furrow and receiving the highest Dpp levels no longer co-express hth and tsh. The loss of hth marks the transition between proliferation/undifferentiation and cell quiescence/commitment. This transition coincides with a transient proliferative wave (the so-called 'first mitotic wave') that precedes entry into G1. This transition zone corresponds to a region where low, but not null, levels of Hth and pMad signals overlap. If the interaction between hth+tsh and the Dpp pathway described in this study were to hold also in the zone of hth/Dpp signal overlap during normal eye development (remember that hth-positive cells co-express normally tsh too), one prediction would be that the mitotic wave would be lost if either hth or dpp-signaling were removed. Indeed this has been shown to be the case: RNAi-mediated attenuation of hth or abrogation of Dpp signaling result in the loss of the first mitotic wave. However, it is not thought that the mechanisms driving Dpp-mediated proliferation of optix> hth+tsh cells are necessarily the same as those operating normally in hth+tsh-expressing progenitors during eye development, because of the following experiment. Discs were generated expressing in their dorsal domain an RNAi targeting Hth's partner, the Pbx gene extradenticle (exd). In the absence of Exd, Hth is degraded. Therefore, a depletion of Exd causes an effective loss of Hth. Knowing that in optix>hth+tsh the stability and diffusion of Dpp were increased, the prediction would be that the loss of hth (in exd-depleted cells) should cause a decrease in both the stability and diffusion of Dpp. However, when the dorsal ('exd-') with the ventral ('exd+') pMad profiles of D>exdRNAi discs was quantified, it was found that both the stability and diffusion of Dpp increased by the loss of hth. This result suggests that during normal eye development hth (perhaps together with tsh) influences Dpp signaling, but the mechanisms described in this study as triggered by forced hth+tsh expression are likely different (Neto, 2016).

    The upregulation of dally and dlp by hth+tsh is likely the consequence of the transcriptional activity of Hth+Tsh in partnership with the YAP/TAZ homologue, Yki, as previous work showed that loss of the protocadherin genes fat (ft) and dachsous (ds) , which causes the activation of Yki, results in an upregulation of dally and dlp in the wing primordium. In fact, previous studies have found, in imaginal tissues, binding of Yki and Hth to nearby sites on the dlp locus, suggesting that some of this regulation might be direct. All these data make Yki a necessary component of the molecular machinery responsible for the increased avidity of hth+tsh cells for Dpp. However, in the eye primordium, the overexpression of Yki induces a different phenotype than hth+tsh. More importantly, in the eye primordium, yki+ clones do not cause the autonomous upregulation of pMad signal that hth+tsh clones do. Therefore, a specific stoichiometry among Hth, Tsh and Yki is likely necessary to induce the Dpp signaling-dependent properties of hth+tsh cells, at least in the developing eye. Alternatively, Hth and Tsh may activate Yki-independent targets that would be required for the full expression of the phenotype. Recently, another study has found that Yki and the Dpp pathway synergize in stimulating tissue overgrowth, both in eye and wing primordia, through the physical association between Yki and Mad. The current results suggest that hth+tsh progenitor-like cells establish a positive feedback, in which the growth promoting activity of the Hth:Tsh:Yki complex would be enhanced by increasing levels of pMad activated by Dpp. This feedback would be region-specific, as it depends on sources of Dpp that are localized within the eye primordium. Further work is needed to investigate the molecular mechanisms behind this feedback. Finally, it has been shown recently that tissue growth promoted by the PI3K/PTEN and TSC/TOR nutrient-sensing pathways also requires Dally, which, in turn, increases the avidity of the growing tissue for Dpp. Therefore, increasing the avidity for Dpp by augmenting proteoglycan levels may be a common strategy of tissues to sustain their growth (Neto, 2016).

    The cuticular nature of corneal lenses in Drosophila melanogaster

    The dioptric visual system relies on precisely focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, this study used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, drosocrystallin and retinin, and two newly identified proteins, Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, in situ hybridization was conducted at 50% pupal development, a key age for lens secretion. The results confirm previous reports that drosocrystallin and retinin are expressed in the two primary corneagenous cells-cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes (Stahl, 2017).


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    Genes involved in organ development

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