To determine where the Lyra/sens gene is expressed, in situ hybridization experiments were carried out. Sens mRNA is first expressed in stage 10 embryos in small clusters of 2-4 ectodermal cells. One of the cells in each cluster is the presumptive anterior or posterior sensory organ precursor. This expression quickly refines to ectodermal cells that will give rise to SOP A and P cells. The message then rapidly accumulates in many SOPIs and IIs during stage 11. The mRNA is most abundant in the SOPI and barely detectable or absent in the progeny of the SOPII. During germ band retraction, the remaining SENS mRNA disappears, and by stage 13 the mRNA is only detected in the salivary glands. These observations suggest that sens is most actively transcribed in the SOPI and that other cells of the PNS inherit the message or transcribe the gene at significantly lower levels than SOPIs (Nolo, 2000).
Staining of embryos with anti-Sens antibodies shows a similar expression pattern to the mRNA. With exception of salivary glands and a few ectodermal cells, the protein is confined to proneural fields of some SOPs, nuclei of SOPs, and differentiating cells of the PNS. Protein expression is initiated during stage 10, is maximal during stages 11 and 12, and begins to vanish in differentiated embryos. Double-labeling experiments with P element enhancer detector A37 show that almost all SOPs that express lacZ in A37 embryos express Sens. In contrast to the mRNA, the protein is present in differentiating cells of the PNS. All EMS-induced mutations express the protein, but the levels are either severely reduced or the protein fails to localize to the nucleus in mutants. In summary, Sens is a nuclear protein whose expression is almost exclusively restricted to precursors and early differentiating cells of the PNS (Nolo, 2000).
To establish if Lyra/Sens is expressed in imaginal tissues, discs were stained with the anti-Sens antibody. Sens is expressed in eye-antennal discs in the R8 photoreceptors, two small clusters of cells in the lateral portion of the disc, and the chordotonal organs of Johnston organs. This pattern is similar to that reported for the A101 enhancer detector (a SOP marker). In leg discs, Sens is expressed in the precursors of the femoral chordotonal organ, as well as in other external sensory SOPs. In wing discs, Sens expression is very dynamic. Sens is first expressed in some ectodermal cells surrounding the cells that will become fated as SOPs. The expression levels in these ectodermal cells are much lower than in the SOPs, and the protein is not confined to the nuclei. This is most easily illustrated for SOPs of the bristles of wing margins but is also observed in other areas. Individual cells within these clusters, the presumptive SOPs, start to accumulate higher levels of Sens protein. Double labeling for anti-ßgalactosidase and anti-Sens in wing discs that carry the A101 SOP marker reveals that the onset of Sens expression precedes that of ßgalactosidase. Both ßgalactosidase and Sens are often coexpressed, except in the SOPs of the bristles of the posterior wing margin where Sens is expressed prior to A101 lacZ. It is likely that no proneural gene is expressed in the noninnervated bristles of the posterior wing margin, although they are similar to other es organs (Nolo, 2000).
Two mutations, sensM256 and sensI235 (Salzberg, 1997) fail to complement Lyra1, a deletion [Df(3L)Ly1, 70A2-3;A5-6]. Although Lyra1 embryos are homozygous lethal, a single copy of the Lyra1 mutation causes a dominant loss of the anterior and posterior wing margin. An EMS screen over the Lyra1 deficiency permits isolation of other alleles of sens: E1, E2, E53, E54, E58, and E69 (H. Irick, personal communication to Flybase, cited in Nolo, 2000). Most alleles cause similar phenotypes, but much of the phenotypic analysis is carried out with allele E2 (Nolo, 2000).
Since none of the sens alleles causes a dominant wing phenotype, it is conceivable the phenotype associated with Lyra1 may map elsewhere. However, the following data support the notion that Lyra mutations correspond to gain-of-function mutations of sens. (1) The dominant phenotype associated with Lyra1 could not be recombined onto a sens mutant chromosome, indicating that both mutations map at the same site. (2) An X ray-induced revertant of LyraSX67, a Lyra allele that complements all sens mutations, is homozygous lethal and fails to complement all sens alleles. (3) Lyra mutations have breakpoints in or near the sens gene. (4) Lyra mutations cause ectopic expression of sens precisely in those portions of wing imaginal discs that normally give rise to tissues that are lost in Lyra mutants. (5) Additional copies of Lyra+ in Lyra mutants do not alter the wing phenotype (Abbott, 1990). These and other data provide strong evidence that Lyra mutations cause their phenotype by ectopically expressing sens (Nolo, 2000).
To determine the phenotypic consequences of the loss of Sens protein function in imaginal discs, a clonal analysis was performed with the sensE2 mutation using gamma irradiation and the Minute technique, as well as the FRT/FLP technique. The phenotypes of sensE2/sensE2 and sensE2/sens1228/4 embryos are similar, suggesting that sensE2 is a severe loss of function or null allele (sensE2 contains a stop codon in the middle of the coding sequence). In addition, sensE2/sensE2 embryos and flies can be fully rescued by the transgene, showing that this chromosome does not carry other obvious viable or lethal mutations. Mutant clones lack bristles, sockets, and microchaetae. This loss of bristles and sockets is confined to the mutant cells, indicating a cell-autonomous requirement for sens (Nolo, 2000).
In imaginal discs, sensE2 clones contain low levels of mutant cytoplasmic Sens protein several hours prior to puparium formation. However, they contain undetectable levels of Sens protein at and after puparium formation. Mutant sensE2 clones are marked by the absence of ßgalactosidase driven by the arm-lacZ transgene. After puparium formation, mutant clones in imaginal discs fail to express the Scute protein. However, mutant clones in less mature wing discs do contain Scute protein in proneural clusters, showing that Sens is not required for Scute expression in ectodermal cells. In addition, some newborn SOPs in mutant clones contain higher levels of Scute than surrounding cells of the proneural field. However, these are consistently lower levels of Scute than in neighboring SOPIs. The levels of Scute in mutant SOPIs remain low and are not maintained. No Scute expression is observed at puparium formation. It is concluded that Sens protein is required to further enhance and maintain expression of Scute in SOPs (Nolo, 2000).
Thus, clonal analysis shows that loss of sens during imaginal disc development leads to loss of bristles, socket cells, and neurons. Unlike in embryos, this loss is paired with a loss of all markers tested in SOPs or their daughters (Asense, Scute, Cut, mAb22C10, and Prospero). This phenotype is more severe than in embryos, suggesting a more prominent requirement for Sens in larval SOPs than in embryos. The phenotype associated with loss of sens in wing imaginal disc is similar to the phenotype associated with the loss of scute and achaete. In addition, clonal analysis indicates that sens is required in cells in which it is expressed. It is therefore conclude that sens is a cell autonomous factor required for an early event in PNS differentiation. Unlike the proneural genes, the requirement for sens is not restricted to a type or subpopulation of sensory organs; rather, it is required for most PNS organs and cells (Nolo, 2000).
The specification of external sensory organs of anterior wing margin has been shown to be dependent not only on Scute and Achaete, but also on Asense. Therefore, SOPs of anterior wing margin were stained with anti-Asense. Asense is not expressed in mutant SOPs, showing that Asense expression is also dependent on Sens. No Cut expression was observed in the SOPIIs, although expression of Cut in other cells of the margin is unaffected. Similarly, expression of the antigen recognized by mAb22C10 in early differentiating cells of mutant clones is absent. In summary, these data suggest that SOPIs may at least in part be specified in sens mutant clones because some express higher levels of Scute than the other cells of the proneural cluster. However, loss of Sens activity then causes a failure to further upregulate and maintain proneural gene expression leading to cell loss in the adult imago (Nolo, 2000).
Like Lyra, many mutations that affect wing morphogenesis have mutant phenotypes in which there are missing sectors of the wing margins. These include vestigial, Notch, Delta, cut, apterous, and others, which have been shown to play important roles during fruitfly development. It is known that many of these so-called 'wing scalloping' mutations, including cut and vestigial, are caused by excessive cell death in the prospective wing margins of late larvae following a period of apparently normal development. In Lyra mutants, although there is a significant reduction (10%-20%) of the number of cells in the adult wing, no evidence of apoptotic or necrotic cell death was found by transmission electron microscopy, acridine orange, or trypan blue staining in third instar and pupal discs. These and other data have led to the suggestion that Lyra may affect more fundamental parameters of cell growth and specification. The Lyra mutant has therefore been of interest to those investigating wing margin development (Nolo, 2001).
The Lyra1 mutation is associated with an X-ray-induced deletion uncovering cytological bands 70A2-3;A5-6. It is a dominant mutation that causes a regular and predictable pattern of loss of the anterior and posterior wing margins along with a small amount of nearby wing surface tissue. The presence of a duplication of the chromosomal region carrying the wild-type Lyra locus in a Lyra1 background does not suppress the Lyra phenotype (Abbott, 1990). Hence, the dominant phenotype is not due to haploinsufficiency. This suggests that Lyra is a gain-of-function mutation and is likely to be a neomorphic allele characterized by spatial and/or temporal misregulation of expression of a gene product. In addition, the results of clonal analysis with wild-type clones in Lyra/+ flies indicate a non-cell-autonomous function (Abbott, 1986), suggesting that the Lyra mutation may affect processes requiring cross-talk among cells, such as specification of positional information or lateral inhibition (Nolo, 2001).
Properties of the Lyra1 phenotype have been studied extensively by Abbott (1986, 1990). Five key observations and conclusions follow: (1) excessive cell death in the putative wing margins of third instar and early pupal wing discs does not account for margin loss in Lyra adult wings; (2) although Lyra mutants do not form anterior and posterior wing margins, there is a normal dorsal/ventral compartment boundary as well as a zone of nonproliferating cells (ZNC) along the 'de facto' Lyra wing margin; (3) a monoclonal antibody, which binds the unidentified E1C antigen expressed in the larval wing margin precursor, allows for the demonstration that an effect of Lyra on anterior and posterior wing margins is apparent early in the third larval instar; (4) margin rescue experiments using clonal analysis show that wild-type Lyra is not required for bristle development per se; (5) further analysis of shape and position of clones indicates that the wing margin, defined as a set of several rows of cells along either side of the dorsal/ventral boundary, plays an important role in wing morphogenesis. This observation presaged the current paradigm that interactions among a number of gene products expressed in the margin region, often acting across the compartment boundary, serve to organize wing development (Nolo, 2001).
Salzberg (1994, 1997) has shown that the senseless gene affects the development of the PNS and report three alleles: two ethyl methanesulfonate (EMS)-induced alleles (sensM256 and sensI235) and one induced by P-element dysgenesis (sens1228/4). The lethality associated with these alleles has been mapped by meiotic recombination to 3-40.5, the same location to which Lyra1 maps (Nolo, 2000). senseless mutations M256 and I235 failed to complement the lethality of Lyra1, which is associated with deletion 70A2-3;70A5-6. The Lyra1 deletion uncovers three essential complementation groups: l(3)70Aa, l(3)70Ab, and l(3)70Ac. Unfortunately, these mutants no longer exist. However, Holly Irick and Peter Cherbas carried out an EMS mutagenesis to identify lethal mutations uncovered by Df(3L)BK10 (71C3;71E5). Because the Df(3L)BK10 chromosome is marked with Lyra1 it is suspected that some of the Irick and Cherbas lethals are in the Lyra1 deficiency. Twenty-seven lethal mutants that failed to complement the Lyra1 Df(3L)BK10 chromosome were tested and 8 lethal mutations were isolated that fail to complement the Lyra1 deficiency (Nolo, 2001).
Complementation tests for the lethal phenotype show that six of these mutations are alleles of senseless, referred to as complementation group l(3)70Ad in FlyBase. This complementation group is presumably allelic to one of the lost l(3)70A complementation groups. The sens alleles were designated E1, E2, E53, E54, E58, E64, E69, and E87. (Irick and Cherbas, personal communication to FlyBase cited in Nolo, 2001). All but E64 and E87 are still available (Nolo, 2001).
The Lyra1 deficiency in trans to other senseless alleles causes lethality but these mutant embryos do not display a severe loss of neurons as typically seen in homozygous senseless mutations (Nolo, 2000). Indeed, the following observations suggest that the Lyra1 mutation is not a loss-of-function allele of senseless. (1)Lyra1/sens mutant embryos display either no loss of PNS neurons or a very subtle loss, indicating that the deficiency associated with Lyra1 does not result in the lack of the senseless gene product; (2) none of the senseless alleles cause a loss of wing margin phenotype in heterozygous flies (sens/+), indicating that haploinsufficiency of sens does not cause the Lyra phenotype; (3) a second, independently generated dominant allele of Lyra, LyraSX67, interacts additively with Lyra1 to produce a more severe margin loss, but complements all senseless alleles. Since Lyra mutations are dominant and their phenotype is not caused by haploinsufficiency of senseless, they are presumed to be either antimorphic (dominant negative) or neomorphic (gain of function) in nature. An antimorphic nature is most unlikely since duplications of the chromosomal region do not ameliorate the phenotype associated with Lyra1 (Abbott, 1990). Furthermore, the Lyra1/sens mutants do not display obvious defects in the PNS, as would be expected if Lyra was a dominant-negative allele of senseless. It is therefore concluded that Lyra mutations are neomorphic mutations (Nolo, 2001 and references therein).
The following data support the neomorphic nature of the Lyra mutations, that is, that they are gain-of-function, regulatory mutations of senseless: (1) the dominant phenotype associated with Lyra1 could not be recombined onto a senseless mutant chromosome, indicating that both mutations map at the same site and that the Lyra phenotype may be breakpoint dependent; (2) molecular analyses show that the distal breakpoint of Df(3L)Ly1 affects a genomic fragment that contains the 3' end, including the 3' untranslated region, of the sens gene; (3) an X-ray-induced revertant of LyraSX67, LyraSX67R12, is homozygous lethal and fails to complement all the senseless alleles, showing that LyraSX67 is associated with senseless; (4) both Lyra mutations cause ectopic expression of Senseless in wing imaginal discs (see below). These observations strongly indicate that the Lyra alleles are neomorphic/gain-of-function mutations of senseless (Nolo, 2001).
To demonstrate that ectopic expression of Senseless can mimic the Lyra phenotype, flies were constructed that carried different UAS-senseless transgenes under the control of GAL4 drivers that express GAL4 rather specifically in the wing disc. Most and possibly all GAL4 drivers that cause widespread expression of GAL4 are lethal in the presence of UAS-senseless. Ectopic expression of Senseless in the wing disc using the C1003-GAL4 driver causes a phenotype that is similar to that observed in Lyra mutants: the wing margins are severely affected whereas the rest of the wing is unaffected. Expression of Senseless in a domain that corresponds to the wing margin using the C96-GAL4 driver also causes a loss of wing margin. In this case the loss is not as severe as that induced by the more ubiquitous driver. These observations clearly show that ectopic expression of Senseless is sufficient to cause loss of wing margin tissue. Conversely, they indicate that other areas of the wing disc do not respond to ectopic Senseless expression with tissue loss. Instead, as reported previously (Nolo, 2000), it is consistently observed that ectopic Senseless causes scattered supernumerary bristles on nonmargin surfaces of adult wings. Moreover, ectopic expression of Senseless in wing discs, using a dpp-GAL4 driver, results in large clusters of extra bristles on the notum as well as parts of the wing surface. In leg discs, this driver causes a severe loss of all distal parts of the leg. It is concluded that ectopic expression of Senseless causes very different phenotypes: loss of tissue in some areas of imaginal discs and extra sensory organs in others (Nolo, 2001).
Four markers were tested to determine the effects of Lyra mutations on the expression of key genes that have been shown to play important roles in wing development. The vestigial gene can be viewed as the wing selector gene because its lack of expression causes wing loss and its ectopic expression causes extra wing tissue. Vestigial expression at the dorso-ventral boundary is essential to wing margin development. Furthermore, vestigial is a marker for wing identity and has an important function in wing growth (Nolo, 2001 and references therein).
The effect of both Lyra mutants on the expression pattern of lacZ driven by the vestigial boundary enhancer vgBE was tested. lacZ expression of the boundary enhancer is almost entirely lost in the anterior and posterior portion of the wing pouch of Lyra1 but is restored in the revertant, which has the same pattern of expression as the wild-type adult wing. The pattern is similar in LyraSX67, except that slightly more prospective margin is missing, in agreement with the more severe margin loss in the adult wing. Lyra has no effect on the vestigial quadrant enhancer, vgQE, which controls later Vestigial expression and growth of the nonmargin portion of the wing pouch. In this case Vestigial is expressed throughout the wing blade but not in the prospective margin. Immunocytochemical staining with the anti-Vestigial antibody shows a different pattern of expression in Lyra wing discs in the anterior and posterior area of the wing pouch when compared to wild type. It is not known what underlies this altered pattern, but it may be due to loss of Vestigial expression at the dorso-ventral boundary. Since loss-of-function clones of vestigial (vg-/vg-) do not proliferate in the wing, the loss of wing margin tissue in Lyra mutants could be caused by a partial loss of Vestigial expression at the anterior and posterior wing boundary. This in turn may cause loss of cell proliferation during pupal wing development (Nolo, 2001).
In addition to vestigial, wingless has also been shown to play an essential role in wing development. Wingless protein is secreted and is produced in a stripe of three to four cell rows stradling the dorso-ventral boundary. The stripe of Wingless-expressing cells induces neighboring cells to differentiate into the bristles that are present at the wing margin. Removing Wingless in second or early third instars results in the loss of tissue from the wing margin. The role of Wingless with respect to regulation of Vestigial expression at the dorso-ventral boundary is still controversial. However, it is fairly clear that Notch signaling is the primary inducer of vgBE. Hence, Wingless expression in Lyra mutants may provide an independent means to assess the effect of Lyra mutations on wing development. Wingless expression is severely reduced in the anterior and posterior domain of the wing pouch of Lyra mutants. With the exception of the central domain of the dorso-ventral boundary, where Wingless expression is apparently normal (as is the Lyra wing margin), Lyra's expression is confined to a narrow domain in which levels of Wingless protein are reduced severely. Since Wingless is an important secreted factor for wing margin development, this reduction in expression in Lyra mutants may act in an additive fashion with the loss or severe reduction of Vestigial expression (Nolo, 2001).
Given the similarities between the loss of wing margin tissue in some cut and Lyra mutants, Cut expression was examined in Lyra mutants. Cut is expressed in a row that is two to five cells wide at the dorso-ventral boundary. This expression is largely overlapping with that of Wingless and the vestigial boundary enhancer but occurs in the mid-third instar, much later than either Wingless or vgBE. Loss of Cut expression on both sides of the wing boundary results in extensive notching of the margin. cut has been shown to be a direct target of Notch, but not of wingless. In addition, while the initiation of Wingless expression is not dependent on cut, maintenance of Wingless expression is dependent on cut. Cut expression is shown to be essentially abolished in Lyra mutants in the anterior and posterior region of the wing pouch (Nolo, 2001).
Since Notch signaling plays a prominent role in the regulation of the expression of vgBE, wingless, and cut, attempts were made to determine if Notch signaling is affected. Lyra wing discs were stained with anti-Delta antibodies since anti-Notch antibody immunohistochemical staining of wing discs failed because of high background levels. There is an obvious reduction in the expression of Delta in the anterior and posterior wing pouch along the presumptive wing margin. Hence, one of the key known activators of Notch signaling at the dorso-ventral boundary is altered and reduced in its expression pattern. In summary, four known markers that have previously been shown to be required for the development of the wing margin and the rows of bristles along the margin are not expressed properly in Lyra mutants. In addition, the domains of expression that are affected in these mutants correspond to the domains that are affected in Lyra mutant discs and adult wings and are contained within the domains in which Senseless is expressed ectopically. These data suggest that ectopic expression of Senseless in Lyra mutants may be able to downregulate the expression of several genes that play a pivotal role in wing margin development, possibly by downregulating Notch signaling (Nolo, 2001).
To further investigate the ability of Senseless to downregulate the expression of specific genes, the effect of ectopic expression of Senseless on Wingless and Cut expression was examined. Senseless was ectopically expressed using the C96-GAL4 wing margin driver; this was followed by staining with anti-Senseless. Senseless overexpression causes a dramatic downregulation of Wingless and Cut protein levels, although in both cases clusters of immunoreactive cells along the wing margin remain. Similarly, when using the dpp-GAL4 driver to ectopically express Senseless along the anterior-posterior wing boundary, a precise disruption in the continuity of Wingless and Cut expression was found where the dpp stripe is normally expressed. This downregulation correlates with a loss of the distal tip of the wing. Note also that ectopic Senseless expression causes ectopic Cut expression in some cells of the wing pouch that normally do not express Cut, as expected from previous observations (Nolo, 2000). In summary, these data demonstrate that ectopic expression of Senseless in the wing margin is a potent repressor of expression of key players previously shown to function in wing margin development (Nolo, 2001).
The loss of wing margin in Lyra mutants can be viewed as the sum of two components. The first component is an effect on margin determination in the developing wing disc. Indeed, these data are in agreement with numerous observations showing that loss of Notch signaling causes loss of expression in the wing margin of the patterning genes wingless and vestigial. The second component corresponds to an effect on cell proliferation. Indeed, loss and gain of Notch signaling experiments have been shown to cause a severe decrease and increase in cell proliferation, respectively. The data suggest that loss of Delta causes a loss of Notch signal and a loss of cell proliferation in the wing margin. The reduction in cell proliferation in Lyra wing development begins shortly after pupariation and continues during the first half of pupal development (Abbott, 1990). This is the time window in normal development when differentiation of bristles and trichomes takes place as well. To further examine how ectopic Senseless affects wing margin specification and differentiation during Lyra wing development, the expression patterns of Scute and string were studied (Nolo, 2001).
scute is a proneural gene belonging to the achaete/scute complex and a basic Helix Loop Helix (bHLH) transcription factor required for determination of SOPs in the anterior wing margin. A downregulation of Scute expression in the anterior pouch of the wing disc is observed. Indeed, in LyraSx67 wings, there are few SOPs expressing Scute at the anterior wing margin. This is in sharp contrast to ectopic expression of senseless in other epithelial cells of the wing disc where it causes induction of Scute expression (Nolo, 2000 and Nolo, 2001).
Wingless is required for differentiation of bristles late in margin development. Indeed, high levels of Wingless are known to be required for the proper expression of the proneural SOP determinants acheate and scute. In addition, Cut expression in third instar discs has been shown to be dependent on Wingless expression, while Cut is also required for the maintenance of Wingless expression. Since Cut is essential for all wing margin bristles, both innervated and noninnervated, it is proposed that the combined reduction in Wingless and Cut expression in Lyra mutants may cause a secondary reduction in proneural gene expression in the wing margin, as revealed by Scute staining. This reduction in expression should lead to a loss of numerous bristles in the anterior and posterior wing margin. However, these observations do not provide a rationale for the loss of wing blade cells adjacent to the margin, which are also observed in Lyra mutants (Nolo, 2001).
The failure to form SOPs in the wing discs of Lyra mutants predicts that the set of two cell divisions required for differentiation of margin bristles in the early pupa will not take place. The reason for the loss of the surrounding unspecialized margin cells in the adult wings of Lyra is not as obvious, but one hypothesis is that these cells also fail to proliferate. To test this, the mRNA expression pattern of String was examined. String mRNA is normally expressed in the central cells of both the anterior and posterior wing margin during the later third instar larval stage even though margin cells are arrested at this time and cell proliferation does not begin until early pupariation. in situ experiments with string confirmed this expression pattern in wild-type discs. But in Lyra third instar wing discs, the mitosis-inducing phosphatase String (Cdc25) is severely downregulated in the anterior and posterior area of the prospective wing margin, as indicated by in situ hybridization. This is consistent with an overall lack of proliferation in the anterior and posterior margin region. However, it is also possible that the non-bristle-forming cells are present in the wing margin, but that they lose their capacity to flatten and secrete margin elements (trichomes), which serve as the visible hallmark of each cell. This could be caused by their lack of exposure to the sequence of proteins required for determination of the wing margin (Nolo, 2001).
vestigial can be viewed as a 'wing selector' gene, a view that is supported by the observation that its ectopic expression can rescue loss of Wingless. Loss of Vestigial in the wing disc also causes a failure of wing cells to proliferate. It has been proposed that the vgBE is induced by Notch signaling when and where Wingless is active at the developing wing margin. The main function of Wingless is to enforce gene expression in the wing disc rather than to initiate it. Hence, the combined loss of vestigial expression at the boundary and the strong reduction in Wingless expression at the wing margin may affect cell proliferation and cell identity not only in the wing margin, but also in a few cell rows adjacent to the anterior and posterior wing margin. This model is in agreement with the observation that no alterations in the expression pattern of the quadrant enhancer of vestigial are found in Lyra mutants and that Lyra wing discs exhibit a dramatic reduction in string expression in the cells along the dorso-ventral boundary. Since string has been shown to induce mitosis, and since Lyra mutants exhibit no cell death and a loss of cells in pupal development, a causal relationship between these observations is proposed. At the root of the Lyra phenotype may be the observation that the Delta signal is impaired, which should lead to a decrease in Notch signaling. This decrease may explain the loss of Wingless, Vestigial, and Cut expression, which have all previously been shown to depend on Notch signaling. This defect in Lyra mutants is proposed to underlie the effect on margin determination in the developing wing disc and the reduction in cell proliferation in early pupae (Nolo, 2001).
Apoptosis in developing Drosophila embryos is rare and confined to specific groups of cells. How do salivary glands of Drosophila embryos avoid apoptosis? senseless (sens), a Zn-finger transcription factor, is expressed in the salivary primordium and later in the differentiated salivary glands. The regulation of sens expression in the salivary placodes is more complex than observed in the embryonic PNS. sens expression is initiated in the salivary placodes by fork head (fkh), a winged helix transcription factor. The expression of sens is maintained in the salivary glands by fkh and by daughterless (da), a bHLH family member. Salivary gland-expressed bHLH (Sage), a salivary-specific bHLH protein, has been identified as a new heterodimeric partner for Da protein in the salivary glands. In addition, the data suggest that sage RNAi embryos have a phenotype similar to sens and that sage is necessary to maintain expression of sens in the embryonic salivary glands. Furthermore, in the salivary glands, sens acts as an anti-apoptotic protein by repressing reaper and possibly hid (Chandrasekaran, 2003).
In situ hybridization shows that sens mRNA is first expressed in the dorsal cells of the salivary placodes at stage 11 of embryogenesis. As the embryo undergoes germ band retraction, sens mRNA expression expands to include all the cells of the salivary placodes, but is excluded from the salivary duct precursors.A similar expression pattern is observed for Sens protein, though the protein is not expressed at high levels in the ventral part of the salivary placodes. Though sens mRNA and protein disappear from the embryonic PNS by stage 13, both continue to be expressed in the embryonic and larval salivary glands (Chandrasekaran, 2003).
Embryos mutant for sensE2 have small salivary glands, about half to a third the size of normal salivary glands. In addition, the salivary glands of stage 16 sensE2 embryos are smaller than those in stage 13 embryos, suggesting that the loss of cells may be progressive. Similar phenotypes were obtained for two other alleles, sensE58 and sensI235, as well as for transheterozygotes of sensE2 and a deficiency for sens, suggesting that sensE2 behaves as an amorph in these studies. The phenotype seen in the sensE2 mutant salivary glands can be rescued by overexpressing sens in the embryo, indicating that the observed phenotype is due to the lack of sens function in the salivary primordium (Chandrasekaran, 2003).
Because the salivary glands are the only non neural tissue in the embryo to express sens, it was of interest to see how different the regulation of sens transcription is in this tissue. In the PNS, Da forms heterodimeric complexes with proneural bHLH proteins. These complexes are necessary for both the initiation and maintenance of sens expression in the sensory organ precursors. The proneural genes achaete, scute, lethal of scute, asense and atonal are mainly expressed in the proneural clusters and are absent from the salivary placodes. By contrast, da expression is ubiquitous in the early embryo and is upregulated in the salivary glands of older embryos, suggesting that da might be involved in regulating the expression of sens in the salivary placodes. If so, da mutants would have a salivary phenotype similar to sens mutants. In confirmation of this hypothesis, salivary glands in da mutants were smaller than in wild-type embryos. In situ hybridization showed that the levels of sens mRNA (and protein) are dramatically reduced in the salivary glands of da mutants, suggesting that Da regulates sens in both the PNS and salivary gland. However, unlike the PNS, salivary gland sens expression initiates in the absence of da (Chandrasekaran, 2003).
Although known Da partners are not expressed during salivary development, a genome-wide survey for genes encoding bHLH proteins identified sage, a gene whose expression is salivary gland-specific in the embryo. The expression of sage in the salivary placodes is first observed at stage 10, the stage at which the first Scr targets begin their salivary expression. sage continues to be expressed in the salivary glands throughout embryogenesis and into larval development. Scr-mutant embryos lack salivary glands and do not express sage. Double stranded RNA interference was used to test whether sage is required for salivary gland development. Forty percent of the embryos injected with sage dsRNA, showed small salivary glands, compared with 10% for the injection buffer control. Sens levels are reduced by sage dsRNA injection. These observations indicate that sage is required for regulation of sens in the salivary glands. Sens expression does initiate in the absence of sage, as it does in da mutant embryos (Chandrasekaran, 2003).
It has been suggested that class II bHLH proteins, the class that includes Sage, can heterodimerize with Da. To test whether Sage indeed forms a complex with Da, a GST pulldown assay was used with 35SDa protein and GST-Sage. Da protein binds to GST-Sage. In addition, Da does not bind to a truncated Sage that lacks the C-terminal bHLH domain. These observations show that Da can partner with Sage in vitro and suggest that Sage and Da form a complex in vivo to regulate the expression of sens in the salivary glands (Chandrasekaran, 2003).
In the sensory organ precursors of the PNS, sens is necessary to maintain the expression of the proneural genes. Similarly, sage RNA is decreased in sens mutants, suggesting a positive feedback loop between sens and sage. However, expression of da appears to be unaffected in sens mutants (Chandrasekaran, 2003).
Although da and sage are necessary for maintaining sens expression, initiation of sens in the salivary placodes did not depend on either of these genes. Since sens expression in the salivary placodes initiates at stage 11, later than primary Scr target genes, it was thought sens might be indirectly activated by Scr through one of these primary targets. As expected, sens expression was found to be absent in Scr mutant embryos. sens expression is unchanged in embryos mutant for several Scr-regulated early transcription factors such as huckebein, trachealess and eyegone. However, fkh mutant embryos show a complete absence of sens expression in the salivary placodes and never express sens at the later stages. The expression of sens in the PNS is unaffected in these mutants. da and sage RNAs were unchanged at stages 10 and 11 in fkh mutants, indicating that the lack of sens is not due to the effects on sage or da expression. There was a slight reduction in sage RNA at stage 12, which may be due to the positive feedback loop between sens and sage in the salivary placodes. Thus, sens expression in the salivary placodes is initiated by fkh and is maintained at high levels throughout embryogenesis by da and sage (Chandrasekaran, 2003).
Thus, the regulation of sens in the salivary glands is more complicated than in the proneural tissues. sens expression in the salivary glands can be divided into two parts: initiation and maintenance. sens is initiated in the salivary placodes in response to fkh, one of the initial set of salivary genes that are directly activated by Scr at the beginning of stage 10 (4.3 hours AEL). sens expression begins about an hour later and may be directly regulated by fkh. There are FKH binding sites present at the 3' end of sens and a fragment carrying these sites is sufficient to recapitulate the expression in the salivary glands (Chandrasekaran, 2003).
Since sens is a fkh target and because both sens and fkh embryos show extensive salivary apoptosis, it was thought that apoptosis in fkh mutants might be caused by lack of sens. Because rescuing cell death in fkh mutants does not rescue normal morphogenesis, it was suggested that sens normally protects salivary cells from cell death, and other fkh target genes direct the cell movements and shape changes needed to form the salivary gland. However, the apoptosis of the salivary placodes in fkh mutants could not be rescued by ubiquitous expression of sens. There are two explanations for this result. The first possibility is that sens was not overexpressed at high enough levels to overcome cell death. However, this is likely not to be the case because the same arm-GAL4:UAS-sens combination was used to rescue the sens phenotype. Furthermore, arm-Gal4:UAS-P35 rescues cell death in sens mutants. Thus, the second possibility is favored, that loss of fkh leads to multiple proapoptotic changes, only one of which is the failure to activate sens (Chandrasekaran, 2003).
Although Fkh can initiate expression of sens in the salivary placodes, both Da and Sage are required for high level sens expression at later stages. Da is also known to control the expression of sens in the PNS. There, it partners with the proteins of the Achaete-Scute Complex or with Atonal to regulate sens expression. For sens regulation in the salivary primordium, a new Da partner, Sage, which belongs to the bHLH proteins of the Mesp family, has been identified. These results are the first to demonstrate the ability of Mesp family members to heterodimerize with Da. It is shown, using RNAi, that absence of sage leads to a decrease in the size of the glands and a reduction in levels of Sens. In turn, Sens appears to positively regulate the levels of sage mRNA in the salivary glands. The existence of this positive feedback loop leads to the question of which protein, Sage or Sens, is the true antagonist of apoptosis in the salivary glands. The presence of sage mRNA in sens mutants sheds some light on this issue. In sens mutants, high levels of Rpr-11-lacZ are induced at stage 12, in the salivary placodes. At this stage, sens mutant embryos still express sage and da mRNA in the placodes at normal levels. Reduction in sage mRNA is not observed until stages 13-14, by which time the salivary glands of sens mutants are already reduced in size. These results indicate that sens, not sage, is necessary to maintain the survival of the salivary gland cells (Chandrasekaran, 2003).
A similar circuit controls the regulation of expression of Gfi1 (Wallis, 2003), the vertebrate ortholog of sens, in the inner ear cells of mice. The bHLH protein Math1, termed Atoh1, a homolog of atonal, is necessary to maintain Gfi1 mRNA, but not for its initiation in the inner ear cells. It would be interesting to examine if fkh family members are involved in this case to initiate the Gfi1 expression. However, the feedback regulation of sens onto sage or proneural genes is not observed between Gfi1 and Math1 (Chandrasekaran, 2003).
Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).
To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4221) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).
To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4221-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).
In contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).
Proper dendritic routing is important for primary dendrites of ddaD and ddaE to grow in parallel toward the dorsal midline without crossing each other and for secondary branches of ddaD and ddaE to avoid the space between ddaD and ddaE. Therefore, there must be mechanisms that promote this stereotyped arborization pattern, including signals that promote anterior arborization of ddaD and posterior arborization of ddaE, as well as signals that antagonize posterior arborization of ddaD and anterior arborization of ddaE. Indeed, RNAi of 10 TFs disrupted the dendritic routing patterns of ddaD and ddaE, resulting in aberrantly oriented primary dendrites. RNAi of cg1244, bap55 (brahma associated protein of 55kD), cg9104, cg4328, and cg7417 resulted in inappropriate anterior arborization of ddaE as well as inappropriate posterior arborization of ddaD. Anterior or even ventral displacement of ddaD concomitant with anterior arborization of ddaE was also observed as well as displacement of ddaE arbors concomitant with misrouting of ddaD. Finally, reducing sens function by RNAi or genetic mutation caused extensive mixing of dendritic arbors from ddaD and ddaE, in addition to dorsal overextension of primary dendrites and an overall reduction in the number of class I neurons (Parrish, 2006).
In addition to the seven genes that function to restrict class I neuron number and control dendrite morphology, three other genes are required to maintain the number of class I neurons. Reduction of their function caused a reduction of class I neurons and defects in dendrite morphogenesis in the remaining neurons. For example, RNAi of the zinc finger TF senseless (sens) reduced the number of class I neurons, consistent with previous findings that sens is required for development of most cells in the PNS. In addition, sens(RNAi) or a sens loss-of-function mutation caused an increase in dendrite outgrowth and mixing of dendrites in segments with both ddaD and ddaE present. Similarly, RNAi of the proneural bHLH TF atonal (ato) reduced the number of class I neurons, consistent with previous findings that chordotonal organs and some md neurons are absent in embryos lacking ato. Consistent with reports that ato functions in neurite arborization in the larval brain, it was also found that ato(RNAi) caused altered arborization patterns of class I dendrites. Thus, it is likely that multiple TFs that regulate neuron number also regulate aspects of post-mitotic neuronal differentiation (Parrish, 2006).
Since group A and B TFs regulate aspects of dendritic growth and branching, potential epistatic relationships among TFs was explored in these phenotypic classes. To do this, RNAi was used to knockdown expression of select TFs in Drosophila embryos carrying a loss-of-function mutation in either the group B/C gene senseless (sens) or the group A gene abrupt (ab). sens mutant class I dendrites overextend dorsally and have reduced lateral branching in addition to routing defects. In sens mutants, RNAi of the group A genes Su(z)12 and ab, which cause increased lateral branching following RNAi in wild-type embryos, led to an increase in lateral branching compared with injected controls. Therefore, Su(z)12 and ab function are still required to limit arborization in sens mutants, and the increased dendritic branching as a result of Su(z)12(RNAi) or ab(RNAi) is epistatic to the increased dorsal extension and reduced lateral branching of sens mutants. In contrast, RNAi of the group A genes cg1244 and cg1841, which caused reduced arborization following RNAi in wild-type embryos, led to a reduction in primary dendrite outgrowth and lateral dendrite branching compared with injected controls. Therefore, at least in the instances described above, loss of group A genes is epistatic to loss of group B genes (Parrish, 2006).
Senseless (Sens) is a conserved transcription factor required for normal development of the Drosophila peripheral nervous system. In the Drosophila retina, sens is necessary and sufficient for differentiation of R8 photoreceptors and interommatidial bristles (IOBs). When Sens is expressed in undifferentiated cells posterior to the morphogenetic furrow, ectopic IOBs are formed. This phenotype was used to identify new members of the sens pathway in a dominant modifier screen. Seven suppressor and three enhancer complementation groups were isolated. Three groups from the screen are the known genes Delta, lilliputian, and moleskin/DIM-7 (msk), while the remaining seven groups represent novel genes with previously undefined functions in neural development. The nuclear import gene msk was identified as a potent suppressor of the ectopic interommatidial bristle phenotype. In addition, msk mutant adult eyes are extremely disrupted with defects in multiple cell types. Reminiscent of the sens mutant phenotype, msk eyes demonstrate reductions in the number of R8 photoreceptors due to an R8 to R2,5 fate switch, providing genetic evidence that Msk is a component of the sens pathway. Interestingly, in msk tissue, the loss of R8 fate occurs earlier than with sens and suggests a previously unidentified stage of R8 development between atonal and sens (Pepple, 2007).
Sens, along with its homologs Gfi-1 and Pag-3, comprises a conserved family of proteins required for normal neural development. In Drosophila, sens is both necessary and sufficient for development of the PNS. In mice, loss of Gfi-1 leads to neurodegeneration of cerebellar Purkinje cells and sensoneural deafness due to loss of inner ear hair cells. Despite the obvious importance of the GPS proteins in normal neural development and their place near the top of the neuronal development cascade, few targets of these proteins in the process of neurogenesis are known. To identify members of this pathway required in neurogenesis, an F1 dominant modifier screen was performed using an ectopic Sens phenotype in Drosophila. Advantage was taken of a dominant, modifiable phenotype generated by ectopic expression of Sens in undifferentiated cells posterior to the morphogenetic furrow. This ectopic Sens led to the recruitment of undifferentiated cells to the bristle fate (Pepple, 2007).
Both known and novel genes have been identified as potential members of the sens pathway by their ability to modify an ectopic Sens phenotype. The Notch signaling pathway is known to regulate Sens function during the resolution of the proneural cluster. This interaction was identified in the screen by the ability of heterozygous loss of Dl to enhance the ectopic Sens phenotype. The nuclear import gene moleskin (msk) was able to strongly suppress the effect of ectopic Sens. msk plays a role in normal eye development and R8 photoreceptor differentiation. Identification of the genes that are represented in the remaining complementation groups will lead to a better understanding of the GPS pathway and normal neural development. It is likely that the remaining complementation groups represent components of the Sens pathway due to their specific effect on lz and not the secondary screens as well as their requirement for normal bristle development in adult thoracic clones. Further characterization of these genes will offer new insight into the highly conserved Sens pathway (Pepple, 2007).
Alleles of msk were found to be suppressors of lz (the expression of UAS-sens in undifferentiated cells by the lozenge-GAL4 driver) with the highest frequency of any complementation group in the EMS screen. Usually such high representation of alleles indicates that the gene has an important role in the phenotype being tested and/or is readily mutagenized. The results presented here suggest a model in which Msk plays a role in the sens pathway. Initial observations of the effect of Msk on the lz phenotype suggested that Msk was needed to maintain high levels of Sens expression. It is possible that in this ectopic situation, Msk contributes to Sens import, but more likely Msk contributes to Sens expression indirectly by importing another component of the pathway that regulates Sens expression. Characterization of the ey-GAL4, UAS-flp (EGUF); msk phenotype strongly suggests that Msk is not the only import factor involved in the Sens pathway during normal development. Clearly, there is functional redundancy with another importin since complete loss of Msk function during early eye development does not remove Sens expression in all R8 cells. In third instar discs, Msk appears to play a role in the maintenance of the R8 cell fate very early in development. Little is known about the early stages of R8 differentiation after specification by Atonal. Previous work on R8 specification and development outlined a hierarchy of events in which Atonal is expressed first and appears to simultaneously activate expression of the downstream targets sens and sca-lacZ. Work on the sens phenotype determined that sca-lacZ expression is still present in sens clones, thereby establishing an epistatic relationship between sca-lacZ expression and sens. The data indicate that there is yet another step in the relationship between Atonal and these two downstream factors. The data suggest that in the msk eye, after specification of the R8 by Atonal but before the onset of sca-lacZ expression, R8 development is disrupted in some clusters, leading to an R2,5 fate switch. This is the first genetic evidence for factors positioned between ato and sca-LacZ/sens (Pepple, 2007).
Nuclear transport is required for the viability of all cells. Interestingly, the loss or decrease in function of some importins can cause specific defects during development. For example, the nuclear exportin Dcas is required for the export of Importin α3 in Drosophila. While null mutants in dcas are not viable, hypomorphs lead to specific cell fate changes in mechanosensory bristles. This phenotype is likely due to extreme sensitivity of Notch signaling to disruption of nuclear transport of one of its pathway members by Importin α3. It is possible that the Msk/Sens interaction was detectable for a similar reason. In the Sens gain-of-function situation, the high level of Sens required to generate ectopic bristles is very sensitive to decreased Msk levels, while during wild-type SOP differentiation, Sens is far less sensitive to Msk levels and exhibits only sporadic effects (Pepple, 2007).
One question still remains: How does the EGUF; msk eye survive at all given the important cargo that Msk is known to transport? The functional redundancy in the Importin family likely provides the cell with enough transport for survival and development in the absence of Msk. However, this idea raises a new question: Why was only Msk identified in the screen and no other importins? A model is proposed in which Msk is the key importin utilized by the cell for high levels of signaling. The lz phenotype requires high levels of signaling to generate ectopic bristles, and this model would explain why an effect with Msk and no other importin was detected. The model does not preclude the ability of other importins to provide transport redundancy for Msk cargos, and in fact evidence is seen for this redundancy in the ability of the EGUF; msk eye to survive and produce some normal ommatidia. Another importin must have the ability to import some level of Sens, pMAPK, and other unidentified factors into the nucleus. Data existst that indirectly support such a model for the role of Msk. In the Atonal intermediate groups within the morphogenetic furrow, Msk must be sequestered away from the nucleus to prevent the very high levels of cytoplasmic pMAPK from entering the nucleus. Although whether other nuclear importins are also sequestered to block pMAPK nuclear entry was not tested, overexpression of Msk in the intermediate groups allows pMAPK to enter the nucleus and affect nuclear signaling. The fact that the cell needs to sequester Msk to prevent high levels of EGFR pathway signaling supports a model in which Msk is important for high levels of signaling (Pepple, 2007).
It has been suggested in other developmental systems that importins are part of a mechanism that regulates the nuclear protein composition of transcription factors and chromatin remodeling factors. In Drosophila, Msk has been shown to import two other developmentally significant cargos, pMAPK and Caudal. In addition to these previously defined roles, the additional data that Msk and nucleocytoplasmic transport play an important role in Sens expression and R8 development. Perhaps more importantly, the fact that abnormalities seen in msk mutant eye discs arise between Atonal and Senseless expression suggests roles for as-yet undiscovered factors and new modes of regulation in this critical pathway (Pepple, 2007).
Specificity of sensory neurons requires restricted expression of one sensory receptor gene and the exclusion of all others within a given cell. In the Drosophila retina, functional identity of photoreceptors depends on light-sensitive Rhodopsins (Rhs). The much simpler larval eye (Bolwig organ; see The Extraretinal Eyelet of Drosophila: Development, Ultrastructure, and Putative Circadian Function) is composed of about 12 photoreceptors, eight of which are green-sensitive (Rh6) and four blue-sensitive (Rh5). The larval eye becomes the adult extraretinal 'eyelet' composed of four green-sensitive (Rh6) photoreceptors. This study shows that, during metamorphosis, all Rh6 photoreceptors die, whereas the Rh5 photoreceptors switch fate by turning off Rh5 and then turning on Rh6 expression. This switch occurs without apparent changes in the programme of transcription factors that specify larval photoreceptor subtypes. It was also shown that the transcription factor Senseless (Sens) mediates the very different cellular behaviours of Rh5 and Rh6 photoreceptors. Sens is restricted to Rh5 photoreceptors and must be excluded from Rh6 photoreceptors to allow them to die at metamorphosis. Finally, Ecdysone receptor (EcR) was shown to function autonomously both for the death of larval Rh6 photoreceptors and for the sensory switch of Rh5 photoreceptors to express Rh6. This fate switch of functioning, terminally differentiated neurons provides a novel, unexpected example of hard-wired sensory plasticity (Sprecher, 2008).
The adult Drosophila eyelet comprises approximately four photoreceptors located between the retina and the optic ganglia. It directly contacts the pacemaker neurons of the adult fly, the lateral neurons. In conjunction with the compound eye and the clock-neuron intrinsic blue-sensitive receptor cryptochrome it helps shift the phase of the molecular clock in response to light. All eyelet photoreceptors express green-sensitive Rh6, and are derived from photoreceptors of the larval eye that mediate light avoidance and entrainment of the molecular clock by innervating the larval lateral neurons (Sprecher, 2008).
Larval photoreceptors develop in a two-step process during embryogenesis. Primary precursors are specified first and develop as the four Rh5-subtype photoreceptors. They signal through Epidermal growth factor receptor (EGFR) to the surrounding tissue to develop as secondary precursors, which develop into the eight Rh6-subtype photoreceptors. Two transcription factors specify larval photoreceptor subtypes. Spalt (Sal) is exclusively expressed in Rh5 photoreceptors, where it is required for Rh5 expression. Seven-up (Svp) is restricted to Rh6 photoreceptors, where it represses sal and promotes Rh6 expression. A third transcription factor, Orthodenticle (Otd), expressed in all larval photoreceptors, acts only in the Rh5 subtype to promote Rh5 expression and to repress Rh6 (Sprecher, 2008 and references therein).
To address the relation between the larval Rh5 and Rh6 photoreceptors and the adult eyelet, they were tracked through metamorphosis. To permanently label them, UAS-Histone2B::YFP, which is stably incorporated in the chromatin, and thus remains detectable in post-mitotic neurons throughout pupation, was used. Surprisingly, all Rh6 photoreceptors degenerate and disappear during early phases of metamorphosis. In contrast, Rh5 photoreceptors can be followed throughout pupation. Expression of Rh5 ceases during early stages of pupation and, at mid-pupation, neither Rh5 nor Rh6 can be detected. About four cells are still present, however, and can be identified by rh5-Gal4/UAS-H2B::YFP or GMR-Gal4/UAS-H2B::YFP. Eyelet photoreceptors only express Rh6, even though H2B::YFP driven by rh5-Gal4 is detectable in those cells. Therefore, the four larval Rh5 photoreceptors must switch rhodopsin expression at metamorphosis to give rise to the four eyelet Rh6 photoreceptors. The remaining eight Rh6 photoreceptors die, their axon becoming fragmented before disappearing. A 'memory experiment' (rh5-Gal4/UAS-Flp;Act-FRT > STOP > FRT-nlacZ) also showed that eyelet Rh6 photoreceptors did express Rh5 earlier (Sprecher, 2008).
The death of Rh6 photoreceptors and transformation of Rh5 photoreceptors was further verified by three independent sets of experiments (Sprecher, 2008).
(1) Rh5 photoreceptors were ablated by expressing pro-apoptotic genes rpr and hid (rh5-Gal4/UAS-rpr,UAS-hid). This results in the absence of larval Rh5 photoreceptors and the complete absence of the eyelet. Conversely, preventing cell death of the Rh6 subtype by expressing the apoptosis inhibitor p35 (rh6-Gal4/UAS-p35) leads to an eyelet that consists of 12 photoreceptors, all expressing Rh6 (Sprecher, 2008).
(2) Larval Rh6 photoreceptors development was blocked by expressing a dominant negative form of EGFR (so-Gal4/UAS-H2B::YFP; UAS-EGFRDN). The eyelet of these animals is not affected and three or four cells express Rh6 normally. This shows that larval Rh6 photoreceptors do not contribute to the eyelet (Sprecher, 2008).
(3) The expression of Sal (Rh5-subtype specific) and Svp (Rh6-subtype specific) was analyzed in the adult eyelet: eyelet photoreceptors still express Sal, but not Svp even though these photoreceptors now express Rh6. Rh5 requires Sal expression in the Bolwig organ, but Otd function is also necessary to activate Rh5 and to repress Rh6. In otd mutants, larval Rh5 photoreceptors marked by Sal express Rh6 and lack Rh5 expression, thus mimicking the switch at metamorphosis. Thus, Rh6 could be expressed in Rh5 photoreceptors if otd function were lost in the eyelet. However, Otd expression does not change during the transition from the Bolwig organ to eyelet although it might be inactive in the eyelet (Sprecher, 2008).
What is the trigger that controls the switch from rh5 to rh6? Ecdysone controls many developmental processes during metamorphosis. EcR is expressed during the third larval instar and pupation in all larval photoreceptors and surrounding tissues. To evaluate EcR activity, a reporter line was used in which lacZ is under the control of multimerized ecdysone response elements (7XEcRE-lacZ). The expression of lacZ is absent until late third instar and prepupation, whereas thereafter all larval photoreceptors (and surrounding tissue) express 7XEcRE-lacZ. EcR expression decreases during late pupation and is no longer detectable by the time Rh6 expression starts in the eyelet (Sprecher, 2008).
To test the role of ecdysone, a dominant negative form of EcR was expressed specifically in larval Rh5 photoreceptors, while permanently labelling these cells (rh5-Gal4/UAS-H2B::YFP;UAS-EcRDN). This causes no disruption of larval photoreceptor fate, but the eyelet of these animals now consists of four photoreceptors that all express Rh5 instead of Rh6. A comparable phenotype is observed after expression of an RNA interference (RNAi) construct for EcR (rh5-Gal4/UAS-H2B::YFP;UAS-EcRRNAi). Therefore, loss of EcR function prevents larval photoreceptors from switching to Rh6 expression. In both cases, larval Rh6 photoreceptors still degenerate and are not observed in the eyelet (Sprecher, 2008).
The dominant negative form of EcR was also expressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP; UAS-EcRDN). In this case, the Bolwig organ is not affected but the resulting adult eyelet consists of about 12 photoreceptors, all expressing Rh6. This presumably results from Rh6 photoreceptors not undergoing apoptosis whereas larval Rh5 photoreceptors still switch expression to Rh6 in the eyelet. Expression of UAS-EcR-RNAi in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-EcRRNAi) leads to the same results (Sprecher, 2008).
Although EcR could directly control the switch of rhodopsin expression through binding to the promoters of rh5 and rh6, these promoters contain no potential EcR binding sites. Moreover, as no EcR expression is detectable when Rh6 starts to be expressed, this would make it unlikely for EcR to control directly the switch to Rh6. Finally, only allowing expression of the dominant negative form of EcR starting at mid-pupation (GMR-Gal4/Tub-Gal80ts,UAS-EcRDN), after rh5 is switched off, does not prevent activation of Rh6 in the eyelet. Thus EcR most likely acts in an indirect manner in regulating rhodopsins, likely through the activation of transcription factors that bind to rh5 and rh6 promoters (Sprecher, 2008).
The differential response to ecdysone of Rh6 photoreceptors (which die) and of Rh5 photoreceptors (which switch to Rh6) must be due to intrinsic differences between the two subtypes before EcR signalling. Likely candidates are Sal and Svp. However, late misexpression of Svp in Rh5 photoreceptors (rh5-Gal4/UAS-H2B::YFP;UAS-svp) or of Sal in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sal) neither affects rhodopsin expression or cell number in the eyelet nor alter the expression of rhodopsins in the Bolwig organ (which is only affected by very early expression of these transcription factors, through so-Gal4. Thus neither Sal nor Svp are sufficient to alter the response of larval photoreceptors to EcR (Sprecher, 2008).
An additional factor, independent from svp and sal, must therefore allow survival of Rh5 photoreceptors, or promote Rh6 photoreceptor death. It was found that the transcription factor Sens is specifically expressed in larval Rh5 photoreceptors and remains expressed in all cells in the eyelet where it might act to promote cell survival. To test this, sens was misexpressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sens). This results in an eyelet that consists of 12 photoreceptors, all expressing Rh6. Thus, expression of Sens in Rh6 photoreceptors is sufficient to rescue them from death, without affecting Sal and Svp expression and subtype specification of larval photoreceptors (Sprecher, 2008).
Ecdysone hormonal signalling thus acts in two independent ways during the formation of the adult eyelet. First, it induces the degeneration of the Rh6 subtype, thereby assuring the correct number of eyelet photoreceptors. This apoptotic death requires the absence of Sens, whose expression is restricted to Rh5 photoreceptors that survive. Second, ecdysone signalling is also required to trigger the switch of spectral sensitivity of blue-sensitive (Rh5) larval photoreceptors to green-sensitive (Rh6) eyelet photoreceptors (Sprecher, 2008).
Thus terminally differentiated sensory neurons switch specificity by turning off one Rhodopsin and replacing it with another. Although examples of such switches in sensory specificity of terminally differentiated, functional, sensory receptors are extremely rare, this strategy might be more common than currently anticipated. In the Pacific pink salmon and rainbow trout, newly hatched fish express an ultraviolet opsin that changes to a blue opsin as the fish ages. As in flies, this switch might reflect an adaptation of vision to the changing lifestyle. The maturing salmon, born in shallow water, later migrates deeper in the ocean where ultraviolet does not penetrate. The rhodopsin switch in the eyelet may similarly be an adaptation to the deeper location of the eyelet within the head, as light with longer wavelengths (detected by Rh6) penetrates deeper into tissue than light with shorter wavelengths (detected by Rh5) (Sprecher, 2008).
The eyelet functions with retinal photoreceptors and Cryptochrome to entrain the molecular clock in response to light. The larval eye, on the other hand, functions in two distinct processes: for the entrainment of the clock and for the larva to avoid light. Interestingly, the Rh5 subtype appears to support both functions whereas Rh6 photoreceptors only contribute to clock entrainment. Thus, the photoreceptor subtype that supports both functions of the larval eye is the one that is maintained into the adult and becomes the eyelet. Why are Rh6-sensitive photoreceptors not maintained? As these photoreceptors are recruited to the larval eye secondarily, the ancestral Bolwig organ might have had only Rh5 photoreceptors and had to undergo a switch in specificity. Larval Rh5 photoreceptors appear to maintain their overall connectivity to the central pacemaker neurons. However, they are also profoundly restructured and exhibit widely increased connectivity during metamorphosis. This might be due to the increase in number of their target neurons, and the switch of Rh might be part of more extensive plasticity during formation of the eyelet, including increased connectivity and possibly the innervation of novel target neurons (Sprecher, 2008).
The general model that sensory neurons express only a single sensory receptor gene does not hold true for salmon and the fruitfly. Interestingly, reports from several other species, including amphibians, rodents and humans, show co-expression of opsins. In humans, for instance, it has been proposed that cones first express S opsin and later switch to L/M opsin. However, this likely reflects a developmental process rather than a functional adaptation (Sprecher, 2008).
This study identified two major players in the genetic programme for the transformation of the larval eye to the eyelet. (1) EcR acts as a trigger for both rhodopsin switch and apoptosis. Surprisingly, the upstream regulators specifying larval photoreceptor-subtype identity, Sal, Svp and Otd, do not contribute to the genetic programme of sensory plasticity of the rhodopsin switch. Therefore a novel genetic programme is required for regulating rhodopsin expression in the eyelet, which likely depends on downstream effectors of EcR (Sprecher, 2008).
(2) Larval Rh5 and Rh6 photoreceptors respond differently to ecdysone, either switching rhodopsin expression or undergoing apoptosis. This appears to depend on Sens, which is likely to be required for the survival of Rh5 photoreceptors. The role of Sens in inhibiting apoptosis is not unique to this situation: Sens is essential to promote survival of salivary-gland precursors during embryogenesis. The vertebrate homologue of sens, Gfi-1, acts to inhibit apoptosis of T-cell precursors in haematopoiesis and cochlear hair cells of the inner ear. Thus the anti-apoptotic function of Sens/Gfi-1 may be a general property of this molecule (Sprecher, 2008).
Ecdysone acts in remodelling neurons during metamorphosis. In γ-neurons of the mushroom body, a structure involved in learning and memory, ecdysone is required for the pruning of larval processes. Similarly, dendrites of C4da sensory neurons undergo large-scale remodelling that depends on ecdysone signalling. Interestingly, in the moth Manduca, 'lateral neurosecretory cells' express cardio-acceleratory peptide 2, which is switched off in response to ecdysone before expression of the neuropeptide bursicon is initiated in the adult (Sprecher, 2008).
The transformation of larval blue-sensitive photoreceptors to green-sensitive photoreceptors of the eyelet reveals an unexpected example of sensory plasticity by switching rhodopsin gene expression in functional, terminally differentiated sensory neurons (Sprecher, 2008).
In vitro data suggest that the human RbAp46 and RbAp48 genes encode proteins involved in multiple chromatin remodeling complexes and are likely to play important roles in development and tumor suppression. However, to date, understanding of the role of RbAp46/RbAp48 and its homologs in metazoan development and disease has been hampered by a lack of insect and mammalian mutant models, as well as redundancy due to multiple orthologs in most organisms studied. This study reports the first mutations in the single Drosophila RbAp46/RbAp48 homolog Caf1, identified as strong suppressors of a senseless overexpression phenotype. Reduced levels of Caf1 expression result in flies with phenotypes reminiscent of Hox gene misregulation. Additionally, analysis of Caf1 mutant tissue suggests that Caf1 plays important roles in cell survival and segment identity, and loss of Caf1 is associated with a reduction in the Polycomb Repressive Complex 2 (PRC2)-specific histone methylation mark H3K27me3. Taken together, these results suggest suppression of senseless overexpression by mutations in Caf1 is mediated by participation of Caf1 in PRC2-mediated silencing. More importantly, the mutant phenotypes confirm that Caf1-mediated silencing is vital to Drosophila development. These studies underscore the importance of Caf1 and its mammalian homologs in development and disease (Anderson, 2011).
Several lines of evidence suggest that the participation of Caf1 in PcG complexes may account for many of the phenotypes observed in flies with altered expression of Caf1. First, Caf1 loss-of-function clones in the eye have phenotypes ranging from slight disorganization and bristle defects to almost complete loss of homozygous tissue in adults, and incomplete rescue of Caf1 results in adult eyes that are small and disorganized. Clones of many PcG genes have similar phenotypes in the eye. Loss of E(z) or Pc causes mild defects in differentiation in the third instar disc, but clones fail to survive in adults. An analogous situation occurs in Caf1short clones, where expression of Elav, which marks differentiating neurons, is present in Caf1 clones at third instar but Caf1short tissue is largely missing in the adult. Derepression of Hox genes could account for these phenotypes, as ectopic expression of many Hox genes in the eye field causes small disorganized eyes in adults (Anderson, 2011).
Second, flies with incomplete rescue of Caf1 display a range of homeotic phenotypes, notably transformation of arista to leg. Similar homeotic transformations, including antenna-to-leg transformations, are a hallmark of mutations in PcG genes. Also a genetic interaction was observe between Caf1 and the PRC1 gene Pc, since mutations in Caf1 are able to dominantly suppress the homeotic transformation of second or third leg to first leg in Pc15/+ males (Anderson, 2011).
Third, the disrupted patterning of Caf1short mutant heads may also result from PcG dysfunction. It is possible that these patterning defects are non-cell autonomous and may be an indirect result of widespread apoptosis in the eye disc. Normally, when an imaginal disc is injured, remaining cells proliferate and assume correct identities, leading to a perfectly patterned adult structure. This type of regeneration requires that some determined cells must change their fates and involves substantial chromatin remodeling. Under specific circumstances, the disc can regenerate with incorrect patterning, leading to duplication, deletion or transformation of structures, a phenomenon referred to as transdetermination. Levels of many PcG transcripts are increased in transdetermining imaginal discs, and heterozygous mutations in PcG genes can enhance transdetermination in regenerating imaginal discs. Therefore, one interpretation of the patterning defects in Caf1short mutant discs is that under the stress of widespread apoptosis, the remaining heterozygous tissue is haploinsufficient for the chromatin remodeling activity required to properly regenerate and pattern the injured disc. Consistent with this interpretation, no extra or missing appendages are observed in flies with Caf1long clones, which show less active Caspase 3 staining at third instar (Anderson, 2011).
Finally, in Caf1 mutant tissue, a reduction is observed in levels of the H3K27me3 mark, which is associated with inactive chromatin and PRC2 activity. These data are consistent with a disruption of PRC2 function as a result of loss of Caf1 and represent the first in vivo evidence that Caf1 is an essential member of this chromatin remodeling complex in an animal model (Anderson, 2011).
One obvious question arises from the current study: why were multiple Caf1 alleles identified in a screen for modifiers of the sens overexpression phenotype? Moreover, it is surprising that mutations in Caf1 were not identified in previous Drosophila modifier screens involving PcG or Rb pathway members. It is proposed that the link between sens and Caf1 is due to the role of Caf1 in PcG-mediated silencing (Anderson, 2011).
Recent evidence suggests that Sens and Hox proteins can compete for binding at overlapping sites at an enhancer of the rhomboid (rho) locus (Li-Kroeger, 2008). When the Hox protein Abdominal-A (Abd-A) binds, transcription of rho is activated, whereas binding by Sens leads to repression of rho. In the embryo, this mechanism acts as a molecular switch to allow differentiation of either chordotonal organs (under control of Sens) or hepatocyte-like cells called oenocytes (by the action of Abd-A). It is proposed that a similar mechanism underlies the suppression of the Sens overexpression phenotype. It is hypothesized that one or more targets of Sens in the eye contain similar overlapping sites that can be bound by either Sens or a Hox protein. During normal development, these loci are bound by neither Sens nor Hox in undifferentiated cells posterior to the furrow, as no Hox genes are known to be widely expressed in the eye field. In the absence of both types of factors, these loci are transcriptionally active, and are necessary to ultimately attain the proper fates of the cells in which they are expressed. When Sens is overexpressed, as in ls, Sens binds to its recognition site in the downstream loci, repressing transcription. Repression of these genes initiates a cascade leading to a change in cell fate; for example, some of the cells that would normally become secondary or tertiary pigment cells now become bristle precursors, giving rise to the extra bristles of ls. However, when one copy of Caf1 is lost, a slight derepression of the Hox genes occurs due to loss of PcG activity. Hox proteins are now able to compete with Sens for the overlapping binding sites, tipping the balance towards activation of downstream genes and attainment of normal cell fate - effectively suppressing ls. The ability of ectopic expression of pb and Antp in the eye to suppress ls is consistent with this hypothesis. Suppression of ls by Hox proteins is particularly significant given that ectopic expression of Antp alone in the eye field leads to a small and disorganized eye. As Sens activity is exquisitely sensitive to Hox proteins, especially in the eye, the screen for modifiers of a sens overexpression phenotype was therefore ideal for identifying mutations in Caf1 (Anderson, 2011).
Previous studies have explored pro-apoptotic roles of Hox proteins and anti-apoptotic roles of Sens. It is therefore possible that one effect of Hox gene derepression in ls eyes suppressed by Caf1 may be restoration of an apoptotic fate in cells that would otherwise form bristle precursors due to ectopic Sens. Abd-A expression in the abdomen during normal third instar larval development leads to apoptosis of proliferating neuroblasts of the central nervous system, and ectopic expression of other Hox genes can also cause neuroblast apoptosis. Accordingly, survival of neuroblasts is dependent on PcG activity to repress Hox gene expression. Furthermore, expression of Sens is necessary in the Drosophila embryonic salivary gland to prevent apoptosis. Thus, one possible mechanism for suppression of ls by Caf1 mutations is that in the ls eye, Sens may promote the ectopic bristle fate partly by repressing apoptotic genes in cells normally fated to die, whereas in the ls eye suppressed by mutations in Caf1, ectopic Hox proteins may promote apoptosis and prevent bristle formation. Increased Ubx expression was not detected by antibody staining; however, a very small increase in one or more Hox proteins may be all that is necessary to change the transcriptional state of downstream loci and prevent the ectopic bristles and other defects in the highly sensitized ls eye - especially in the eye field, where no Hox genes are known to be highly expressed. Furthermore, the fact that multiple Hox proteins can recognize the same DNA binding site offers the possibility that the competitive effect of each Hox protein type on genes with overlapping Sens/Hox binding sites would be additive. Therefore, although loss of one copy of Caf1 may only cause a small derepression of any one Hox gene, mild derepression of many Hox genes collectively can lead to strong repression of the ls phenotype (Anderson, 2011).
Biochemical evidence suggests that Caf1 is a member of multiple complexes that effect gene regulation through chromatin remodeling, suggesting that it is a vital component of the cell's arsenal of chromatin modifying factors. Although the results suggest that disruption of PRC2 function may be the most important consequence of Caf1 gain- or loss-of-function, many phenotypes that were observed in Caf1 mutant tissue are also reminiscent of mutations in members of other complexes previously shown to contain Caf1. It is not surprising that all three alleles of Caf1 in the current study are homozygous lethal, and that Caf1short cells have poor viability, considering that Caf1 has been found in the NURF and CAF-1 complexes, which have fundamental roles in nucleosome assembly and spacing. The apoptosis observed in eyes with Caf1short clones is also consistent with a role for Caf1 in the dREAM (Drosophila Rbf, E2F2, and Myb-interacting proteins) complex. Members of the E2f family of transcription factors can complex with Dp proteins and bind short recognition sites to activate transcription. When Rb binds the E2f-Dp complex, transcription is repressed. Like Caf1 homozygotes, homozygous rbf1 null flies die in early larval development. Fully rbf1-deficient embryos display increased apoptosis, a phenotype reminiscent of the increased active Caspase-3 staining seen anterior to the morphogenetic furrow in eye discs with Caf1short clones (Anderson, 2011).
The mammalian homologs of sens, Growth Factor Independence 1 (Gfi1) and Gfi1b are essential to the development of multiple cell types and have been implicated as oncogenes. Therefore, the possibility that Caf1 links Sens with the activity of PcG complexes through parallel, competing pathways has implications for both Drosophila development and the activity of Gfi1 family members in human development and disease, and warrants additional study beyond the scope of the present work. The results underscore the importance of Caf1 to diverse processes, including cell survival and tissue identity, and highlight the participation of Caf1 in multiple chromatin remodeling complexes. Further studies are needed to fully assess the importance of Caf1 in Drosophila development, as well as its developmental role in other chromatin remodeling complexes (Anderson, 2011).
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