In Drosophila, cell-fate determination of all neuroectoderm-derived glial cells depends on the transcription factor Glial cells missing (Gcm), which serves as a binary switch between the neuronal and glial cell fates. Because the expression of Gcm is restricted to the early phase of glial development, other factors must be responsible for the terminal differentiation of glial cells. Expression of three transcription factors, Reversed polarity (Repo), Tramtrack p69 (Ttk69) and PointedP1 (PntP1), is induced by Gcm in glial cells. Repo is a paired-like homeodomain protein, expressed exclusively in glial cells, and is required for the migration and differentiation of embryonic glial cells. To understand how Repo functions in glial terminal differentiation, the mechanism of gene regulation by Repo was analyzed. Repo is shown to act as a transcriptional activator through the CAATTA motif in glial cells, and three genes are defined whose expression in vivo depends on Repo function. In different types of glial cells, Repo can act alone, or cooperate with either Ttk69 or PntP1 to regulate different target genes. Coordination of target gene expression by these three transcription factors may contribute to the diversity of glial cell types. In addition to promoting glial differentiation, it was found that Repo is also necessary to suppress neuronal development, cooperating with Ttk69. It is proposed that Repo plays a key role in both glial development and diversification (Yuasa, 2003).

Although ectopic Repo induces the appearance of many non-glial lacZ-expressing cells in the dorsal epidermis, cells within the CNS do not respond to ectopic Repo. In fact, even in the wild-type background, not all Repo-positive glia in the CNS expressed the ftz HDS reporter. This suggests that the mechanism by which Repo regulates transcription may be different in the CNS from the one for peripheral glia. One possible scenario is that the functions of Repo in the CNS require cooperation with one or more other factors, and that these interactions preclude Repo from acting through the CAATTA motif. Ttk69 and PntP1 are good candidates for such co-factors, because ttk and pointed are both required for the development of CNS glial cells. Although repo, ttk and pointed are expressed in overlapping subsets of CNS glial cells, their expression is mutually independent; Repo continues to be expressed in the ttk or pointed mutant background, and lacZ expression levels in enhancer-trap lines of ttk or pointed are unaffected in repo mutant embryos. Moreover, ectopic expression of Repo in the entire neuroectoderm does not increase the expression of pointed P1 mRNA or Ttk69, nor does ectopic expression of either Ttk69 or PntP1 affect Repo expression. All three genes are most probably regulated independently, downstream of the glial determinant Gcm (Yuasa, 2003).

Although glial specification by Gcm is well established, how the characteristics of individual glial cells are determined is poorly understood. Gcm expression is confined to the early stage of glial development, suggesting that Gcm itself does not participate in the terminal differentiation of glia. Moreover, Gcm also directs blood cell development; Gcm is expressed in macrophage precursors and ectopic expression of Gcm in crystal cell precursors causes the transformation of crystal cells to macrophages. These results clearly show that the expression of Gcm does not always lead to the determination and terminal differentiation of glia. In glial cells, Gcm induces the expression of three transcription factors, Repo, Ttk69, and PntP1, and the loss of these proteins causes abnormal glial development, although Gcm expression remains normal. Although gcm can direct repo expression in various contexts, repo is not expressed endogenously in blood cells, but is confined to Gcm-positive glial cells, lasting even after gcm expression has ceased. In repo mutant embryos, the migration, survival and terminal differentiation of glial cells are abnormal. This study shows that Repo activates gene expression in glia, and also demonstrates that Repo mediates the suppression of neuronal differentiation. These results suggest that Repo is the major factor that is necessary for glial development (Yuasa, 2003).

Effects of Mutation or Deletion

The CNS midline of Drosophila should not be considered as an isolated autonomous entity but as an organizing center for the rest of the CNS. Cells located at the midline of the developing central nervous system perform a number of conserved functions during the establishment of the lateral CNS (the rest of the CNS as distinguished from the midline). The midline cells of the Drosophila CNS are required for correct pattern formation in the ventral ectoderm (which gives rise to the rest of the CNS) and for induction of specific mesodermal cells. The midline cells are also required for the correct development of lateral CNS cells. Embryos that lack midline cells through genetic ablation show a 15% reduction in the number of cortical CNS cells. A similar thinning of the ventral nerve cord can be observed following mechanical ablation of the midline cells. A number of specific neuronal and glial cell markers have been identifed that are reduced in CNS midline-less embryos, as for example in single-minded embryos, in early heat-shocked Notch(ts1) embryos or in embryos where the midline cells have been mechanically ablated. Genetic data suggest that both neuronal and glial midline cell lineages are required for differentiation of lateral CNS cells. One marker, the rR226 enhancer trap insertion, reveals a reduction in the number of marker positive cells in midline ablated embryos. In embryos lacking pointed transcript P2, the number of rR226-positive cells decreases. It is thus concluded that the CNS midline plays an important role in the differentiation or maintenance of the lateral CNS cortex (Menne, 1997).

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called ‘head midline’) that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobes, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ‘cyclops’ like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

A specific P-element insertion into pointed has a ß-gal pattern which strongly resembles the pattern of glia, including glia of the stomatogastric nervous system. In these pointed P-element insertion mutants the SNS is often considerably reduced in size and defasciculated. This situation is reminiscent of the phenotype seen in the CNS of pointed mutants. pnt is expressed in both glia and support cells of the VUM cluter of neurons and the midline glial cells, and both groups of cells are involved in the formation of the commisural pathways in the developing CNS. Another P-element insertion into pointed has a considerably different ß-gal pattern from the first. The embryonic ß-gal in the second is seen in the SNS anlage and all three invaginations of the SNS as well as in commisural glia. Outside of the SNS it is found in the CNS, PNS, visceral mesoderm, and malpighian tubules. The differential ß-gal expression in the two lines may be due to the influence of different enhancers in the different P-elements. However, that both lines show expression in the glia is consistent with the SNS glial pattern of pointed. The phenotype of the two insertions are similar. There is a dramatic reduction in the invaginations and a failure of vesicle formation (Forjanic, 1997).

Loss of pointed function leads to poorly differentiated glial cells and a marked decrease in neuronal cells known to interact directly with pointed P1-expressing longitudinal glial cells (Klaes, 1994)

pnt mutants display a pointed head skeleton and deletion of the medial portion of all denticle belts (Jurgens, 1984). There are no pointed minus photoreceptors, and pointed minus cells never express Elav, an early marker of neuronal differentiation. This suggests that pointed is required for cell fate determination (O'Neill, 1994). Interaction of pointed with ras pathway genes indicates that pointed is activated by the ras pathway (O'Neill, 1994).

To assess the individual functions of the two different Pointed protein forms, new pointed alleles were generated affecting either the P1 or the P2 transcript, termed P1 and P2 alleles, respectively. Genetic analysis reveals partial heteroallelic complementation between certain pointed P1 and P2 alleles. Surviving trans-heterozygous flies have rough eyes, abnormal wings and halters, suggesting a requirement for Pointed function during their imaginal disc development. Further genetic analysis demonstrates that expression of a given pointed P2 allele depends on trans-acting transcriptional regulatory sequences (Scholz, 1993).

Both vertebrate Ets-1 and Ets-2 Ets domains are highly homologous to the Ets domain of Pointed. In addition, the N-terminal region of Drosophila Pointed P2 and vertebrate Ets proteins share another homologous domain, the so-called RII/pointed box, which appears to mediate the ras-dependent phosphorylation/stimulation of Pointed. The vertebrate Ets proteins are functionally homologous to Pointed. Pointed P2 effectively binds to vertebrate Ets-binding sites in vitro, and stimulates Ets responsive sequences when transiently expressed in vertebrate cells. Conversely, when vertebrate ets transgenes are expressed during fly development, they are capable of rescuing the pointed mutant phenotype in both midline glia and photoreceptor development. As ectopically expressed Pointed P1 can also rescue the Pointed P2 deficiency in photoreceptor development, it appears that the ability of ets products to phenocopy each other in vivo does not require the conserved RII/pointed box, but rather, primarily relies on the presence of the highly conserved ETS domain (Albagli, 1996).

The spitz-group mutants (spitz, rhomboid, and pointed) are embryonic lethal and have similar cuticle phenotypes; they are shorter than wild type and have deletions of ventral cuticle. vein mutant are shorter and the Keilin's organs and ventral black dots are closer together than in wild-type. Ventral cuticle is deleted between Keilin's organs. The deletions occur in a similar region in spitz-group mutants; spitz and rhomboid have a larger portion of ventral cuticle deleted than vein mutants, but pointed embryos have similar deletions. In vein mutants sensory hairs surrounding the pit structure of Keilin's organs are missing. Unlike the spi-group genes, vein is not critical for embryonic survival and head skeleton and sense organs are normal. Most vein mutants die either as embryos or as larvae, but a small number do pupariate. Individuals that survive to pupariate secrete a pupal case with pattern abnormalities (Schnepp, 1996).

Mutations in the genes spitz (spi), Star (S), single-minded (sim), pointed, rhomboid (rho) (all zygotic), and sichel (sic) (maternal), collectively termed the spitz group, cause similar pattern alterations in ventral ectodermal derivatives of the Drosophila embryo. The cuticle structures lacking in mutant embryos normally derive from longitudinal strips of the ventro-lateral blastoderm. Defects are found in the median part of the central nervous system. In addition, the nerve cells expressing the even-skipped protein appear abnormally arranged. These results suggest that groups of cells from the same region, including both epidermal and neural precursor cells, require spitz-group gene activity for normal development. The members of the spitz group differ from one another: sim affects a more median strip of the ventral ectoderm than the other zygotic genes and pnt causes separation rather than deletion of pattern elements. As shown by pole cell transplantations sim, rho, and pnt appear to be exclusively zygotically expressed (Mayer, 1988).

The identification of pnt, Ras1, and rolled mutations as enhancers of an E2F overexpression phenotype suggests that there might be cross-talk between the signaling pathways downstream of receptor tyrosine kinases and the E2F pathway. The basis for this interaction is not clear and may be complex because both loss-of-function mutations (in Ras1 and pnt) and a gain-of-function mutation (in the rolled MAP kinase) enhance the E2F-dependent phenotype (Staehling-Hampton, 1999).

Signaling by Egfr, the Drosophila epidermal growth factor receptor tyrosine kinase (RTK), is essential for proper migration and survival of midline glial cells (MGCs) in the embryonic central nervous system (CNS). A gene called split ends (spen) was isolated in a screen designed to identify new components of the RTK/Ras pathway. Drosophila Spen and its orthologs are characterized by a distinct set of RNA recognition motifs (RRMs) and a SPOC domain, a highly conserved carboxy-terminal domain of unknown function. To investigate spen function in the context of RTK signaling, the consequences of spen loss-of-function mutations on embryonic CNS development were examined. spen is required for normal migration and survival of MGCs; embryos lacking spen have CNS defects strikingly reminiscent of those seen in mutants of several known components of the Egfr signaling pathway. In addition, spen interacts synergistically with the RTK effector pointed. Using MGC-targeted expression, it was found that increased Ras signaling rescues the lethality associated with expression of a dominant-negative spen transgene. Therefore, spen encodes a positively acting component of the Egfr/Ras signaling pathway (Chen, 2000).

To examine the consequences of complete loss of spen function, spen germline clones were generated using the ovoD-FLP/FRT system. Embryos lacking both maternal and zygotic spen will be referred to as 'spen mutants'. These mutants exhibit moderate defects in CNS morphology, as visualized by anti-Elav antibody, a pan-neuronal marker. In stage 16 spen mutant embryos, the space separating the two longitudinal halves of the CNS is reduced compared with the wild type and, in some segments, the two sides are completely collapsed across the midline. Because of this collapse, the midline neurons are difficult to detect; however, labeling with the antibody 22C10 reveals that the ventral midline neurons are present and have no obvious defects in their projections. Because such collapsed CNS phenotypes might be indicative of defects in the midline, spen mutant embryos were examined for expression of the MGC-specific marker Slit. Initial determination of the MGCs appeared normal in spen mutants. The first detectable defects occur at late stage 12/early stage 13 when the MGCs normally initiate their migration. In the wild type, the glial cells migrate in a tightly packed configuration along the dorsal surface of the ventral nerve cord, whereas in spen mutants, the glia migrated aberrantly and became spread out in a more diffuse pattern. The results of these analyses differ from a recent report that excessive numbers of MGCs are initially specified in spen mutants. Using the Slit-lacZ nuclear enhancer trap marker to count the MGCs, comparable numbers of MGCs in the wild type and in spen mutants were detected up until stage 13, and a reduction in MGC number in spen mutants beginning at stage 14. Thus, in these spen mutants, which appear to be genetic and protein nulls, normal numbers of MGCs are initially specified, a phenotype consistent with what has been reported for other Egfr pathway mutants (Chen, 2000).

By stage 16, in a wild-type embryo, the Slit-positive MGCs have migrated and elongated to ensheathe the anterior commissure (AC) and posterior commissure (PC) axons, thereby maintaining proper separation and bundling. In similarly staged spen mutant embryos, the MGCs had not properly migrated or wrapped themselves around the commissure bundles. In addition, while apoptosis reduces the number of MGCs in wild type embryos from ~8 per segment at stage 13 down to only ~3 per segment by stage 16-17, in spen mutants this reduction is even more drastic, leaving only 1-2 MGCs per segment. Thus, in spen mutants, although initiation of MGC differentiation appears normal, the later aspects of glial development, including migration, wrapping, and survival or maintenance of the MGC fate, are defective. To confirm that Spen is expressed in the MGCs at stage 13 when its function is required, embryos carrying the MGC-specific enhancer trap line AA142, were double labeled with anti-beta-galactosidase and anti-Spen antibodies. The highest level of Spen expression is seen in the MGCs (Chen, 2000).

Because defects in glial cell development are likely to perturb organization of the CNS, spen mutant embryos were labeled with the antibody BP102, which highlights all axon tracts in the CNS. As predicted, the AC and PC axon bundles are not properly organized or separated and, in some segments, are completely fused. In addition, the two longitudinal connectives appear closer together than normal and are occasionally fully collapsed across the midline. Staining with the anti-Fasciclin II (FasII) antibody, which highlights a distinct set of three axon bundles in each longitudinal branch, further clarifies this phenotype. These longitudinal axon tracts never cross the midline in a wild-type embryo. In contrast, the FasII-positive axons cross and recross the midline in spen mutants, producing a fragmented and disorganized longitudinal axonal array (Chen, 2000).

In Drosophila, the genes rhomboid, Star, pointed and spitz, all positively acting components of the Egfr pathway, share a characteristic CNS phenotype similar to that of spen mutants. Specifically, whereas the proper number of MGCs are initially specified, they later migrate abnormally and eventually degenerate and die. The phenotypic similarities between spen and the Egfr pathway genes, as well as the isolation of spen as an enhancer of an activated yan allele, are consistent with the hypothesis that spen may be a positively acting factor in the Egfr/Ras signaling pathway. To explore this possibility, whether spen and the RTK pathway effector pointed interact synergistically in the midline was investigated. The expectation is that a reduction in activity of a proven positive effector of the Egfr pathway, such as pnt, should dominantly enhance the spen phenotype. Embryos lacking maternal spen can be partially rescued by zygotic spen expression from a paternally inherited wild-type allele (this genotype is referred to as spen/+). Stage 15-17 spen/+ embryos appear phenotypically wild type, with only ~4% of the embryos exhibiting CNS defects. Embryos heterozygous for a pnt loss-of-function mutation (pnt/+) have no apparent dominant defects. Reducing the pnt dosage in the spen/+ background increases the frequency of axonal defects to ~25%. The predominant phenotype is reduced separation between the two longitudinal axon pathways and a single inappropriate crossing of the midline by one of the FasII-positive axon tracts. This dose-sensitive interaction between pnt and spen strongly supports a role for spen as either a positively acting component of the Egfr pathway or as a component of a parallel pathway synergizing with Egfr during MGC development (Chen, 2000).

To investigate further the connection between spen and Egfr/Ras signaling in the MGCs, a putative dominant-negative spen transgene was generated that truncates the carboxy-terminal ~1500 amino acids, including the highly conserved SPOC domain. When transfected into S2 cultured cells, this construct (SpenDeltaC) is expressed at high levels and localizes to the nucleus just as is found for the endogenous wild-type Spen protein. Ubiquitous expression of SpenDeltaC is unable to rescue the lethality or phenotypes associated with spen mutants, implying an essential function for the conserved carboxy-terminal SPOC domain. To determine whether SpenDeltaC might behave as a dominant-negative mutation, the Slit-Gal4 driver was used to induce high levels of expression specifically in the MGCs. MGC-specific expression of SpenDeltaC results in completely penetrant lethality. In contrast, and consistent with the lack of primary neuronal defects associated with spen mutants, pan-neural expression of SpenDeltaC using the Elav-Gal4 driver does not compromise the viability or patterning of the fly. To test the hypothesis that the Slit-Gal4/SpenDeltaC lethality might be due to compromised RTK/Ras pathway signaling, a determination was made of whether increasing the level of Egfr/Ras pathway signaling, specifically in the MGCs, could compensate for the reduction in spen function associated with expression of the dominant-negative SpenDeltaC transgene. Whereas Slit-Gal4-driven expression of either an activated RasV12 or the SpenDeltaC transgene results in lethality, flies expressing both RasV12 and SpenDeltaC in the MGCs are viable and appear normally patterned. The mutual suppression is extremely penetrant, since over 50% of the expected class of flies was recovered. Similar, but less penetrant, rescue was obtained when SpenDeltaC and a secreted form of the Egfr ligand Spitz were coexpressed in the MGCs. Together, these results strongly suggest that spen functions autonomously in the MGCs, acting either downstream of or in parallel to Ras as a positive effector or regulator of RTK signaling (Chen, 2000).

Although the molecular mechanisms underlying Spen function in the RTK/Ras pathway remain to be elucidated, given its membership of the RRM family, one possibility is that Spen might directly regulate the processing and/or stability of specific transcripts to generate functionally distinct protein isoforms in response to, or required for, Ras signaling events. Post-transcriptional regulation of gene expression allows quick responses to external or developmental signals, and RRM family members have been shown to mediate many different cellular processes including mRNA splicing, stabilization, localization and transport. Two attractive potential targets of such activity in the CNS are the Ras pathway effector pointed and the zinc finger transcription factor tramtrack. Both genes produce alternatively spliced transcripts and are required in the MGCs. The synergistic interactions detected between spen and pointed make pointed a particularly appealing candidate. A third possibility, given that spen was isolated as an enhancer of an activated yan allele, is that Spen might function to destabilize yan transcripts in response to RTK-initiated signals. In this model, spen would contribute a second level of post-translational regulation that would reinforce the transient mitogen-activated protein (MAP) kinase signal that downregulates Yan protein, thereby stabilizing release from the Yan-mediated block to differentiation. In all these scenarios, spen could either function in parallel to the Ras/MAP kinase cascade, or could itself be directly regulated or activated by the pathway (Chen, 2000).

The spitz class genes, pointed (pnt), rhomboid (rho), single-minded (sim), spitz (spi) and Star (S), as well as the Drosophila Epidermal growth factor receptor (Egfr) signaling genes, argos (aos), Egfr, orthodenticle (otd) and vein (vn), are required for the proper establishment of ventral neuroectodermal cell fate. The roles of the CNS midline cells, spitz class and Egfr signaling genes in cell fate determination of the ventral neuroectoderm were determined by analyzing the spatial and temporal expression patterns of each individual gene in spitz class and Egfr signaling mutants. This analysis shows that the expression of all the spitz class and Egfr signaling genes is affected by the sim gene, which indicates that sim acts upstream of all the spitz class and Egfr signaling genes. Overexpression of sim in midline cells fails to induce the ectodermal fate in the spi and Egfr mutants. In contrast, overexpression of spi and Draf causes ectopic expression of the neuroectodermal markers in the sim mutant. Ectopic expression of sim in the en-positive cells induces the expression of downstream genes such as otd, pnt, rho, and vn, which clearly demonstrates that the sim gene activates the Egfr signaling pathway and that CNS midline cells, specified by sim, provide sufficient positional information for the establishment of ventral neuroectodermal fate. These results reveal that the CNS midline cells are one of the key regulators for the proper patterning of the ventral neuroectoderm by controlling Egfr activity through the regulation of the expression of spitz class genes and Egfr signaling genes (Chang, 2001).

Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitz group genes, yet its underlying cause is unknown. This phenotype has been studied and a new function has been discovered for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes, Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning (Urban, 2004).

Pointed is an Ets domain-containing transcription factor that is responsible for transducing most known instances of EGFR signalling. Although it was previously clear that pointed mutant embryos rarely display denticle belt fusions (Mayer, 1988), analysis of a more recent null allele that removes both P1 and P2 transcripts demonstrates that even complete loss of pointed leads only to a very low frequency of denticle belt fusions. This is also consistent with the milder effects of pointed clones in the developing eye, and in particular the late onset of their apoptosis. These observations raise the possibility that EGFR-mediated survival signalling in general occurs primarily at a non-transcriptional level. Consistent with this model, EGFR signalling has been shown to reduce Hid protein stability, thus directly inhibiting apoptosis (Urban, 2004).

pannier and pointedP2 act sequentially to regulate heart development

The Drosophila heart consists of two major cell types: cardioblasts, which form the contractile tube of the heart; and pericardial cells, which flank the cardioblasts and are thought to filter and detoxify the blood or hemolymph of the fly. This study presents the completion of the entire cell lineage of all heart cells. Notably, a previously unappreciated distinction has been detected between the lineages of heart cells located in the posterior seven segments relative to those located more anteriorly. Using a genetic screen, the ETS-transcription factor pointed has been detected as a key regulator of cardioblast and pericardial cell fates in the posterior seven segments of the heart. In this domain, pointed promotes pericardial cell development and opposes cardioblast development. This function of pointed is carried out primarily if not exclusively by the pointedP2 isoform and, that in this context, pointedP2 may act independently of Ras/MAPK pathway activity. The GATA transcription factor pannier acts early in dorsal mesoderm development to promote the development of the cardiac mesoderm and thus all heart cells. Finally, it is demonstrated that pannier acts upstream of pointed in a developmental pathway in which pannier promotes cardiac mesoderm formation, and pointed acts subsequently in this domain to distinguish between cardioblast and pericardial cell fates (Alvarez, 2003).

Pioneering work in C. elegans established the importance of elucidating cell lineages to obtain a thorough understanding of animal development. Prior work established the cell lineage of 10 out of the 16 heart cells that arise in each hemisegment of the posterior seven heart segments. To define the lineage of the remaining heart cells, the FLP/FRT lineage tracing system was used to determine the lineal relationship of the two Eve-positive pericardial cells and four Tin-positive pericardial cells that arise in each hemisegment. This system creates random clones marked by tau-lacZ reporter gene activity. Briefly, clones were induced during stage 8 just as the pan-mesodermal divisions are being completed. This allowed clones to be induced in mesodermal cells prior to the emergence of heart precursors. To identify the lineage of Eve-positive pericardial cells, embryos were double labeled for ß-galactosidase to mark clones, and with Eve to identify Eve-positive pericardial cells. To identify the lineage of Tin-positive pericardial cells, embryos were double labeled for ß-galactosidase to mark clones, and Tin to identify Tin-positive pericardial cells. In addition to the Tin-positive pericardial cells, Tin labels cardioblasts and Eve-positive pericardial cells. However, based on position and morphology one can unambiguously distinguish Tin-positive pericardial cells from Tin-positive cardioblasts and Eve-positive pericardial cells (Alvarez, 2003).

Eighteen clones were identified that contained at least one Eve-positive pericardial cell. Eleven of these clones (61.1%) consisted solely of two Eve-positive pericardial cells, six clones consisted of two Eve-positive pericardial cells and one or two nearby heart or other mesodermal cells, and one clone consisted of a single Eve-positive pericardial cell. Thus, when one Eve-positive pericardial cell is observed within a clone of two or more cells a second Eve-positive pericardial cell always exists within this clone. These data demonstrate that the two Eve-positive pericardial cells within a hemisegment are siblings and arise from an Eve-positive pericardial cell precursor (Alvarez, 2003).

Tin-positive pericardial cell clones fall into two classes: those that contained two Tin-positive pericardial cells, and those that contained one Tin-positive pericardial cell and one cardioblast. These two classes of clones arise in mutually exclusive regions of the heart. Clones that contain two Tin-positive pericardial cells arise in the posterior seven segments of the heart (this region is referred to as the posterior heart domain), whereas clones that contain one Tin-positive pericardial cell and one cardioblast arise anterior to this domain (this region is referred to as the anterior heart domain). The point of demarcation between these clonal types coincides precisely with the location of the first pair of Svp-positive cardioblasts. These data demonstrate that heart cells exhibit distinct cell lineages as a function of position along the anteroposterior axis (Alvarez, 2003).

A total of 24 Tin-positive pericardial cell clones were identified in the posterior heart domain. Fifteen of these clones consisted solely of two Tin-positive pericardial cells, eight clones consisted of two Tin-positive pericardial cells and two nearby mesodermal cells, and one clone consisted of a single Tin-positive pericardial cell. Thus, when one Tin-positive pericardial cell is observed within a clone of two or more cells, a second Tin-positive pericardial cell always exists within this clone. These data indicate that the four Tin-positive pericardial cells found in each hemisegment of the posterior domain arise from two Tin-positive pericardial cell precursors. The inability to identify any clones that contain four Tin-positive pericardial cells indicates that adjacent Tin-positive pericardial cell precursors are unlikely to share a common lineage (Alvarez, 2003).

Eighteen Tin-positive pericardial cell clones were identified in the anterior heart domain. All 18 clones consisted of one Tin-positive pericardial cell and one cardioblast. These data indicate that within this region Tin-positive pericardial cells arise from bi-potent heart precursors, each of which produces one Tin-positive pericardial cell and one cardioblast. These data also demonstrate that cardioblasts and Tin-positive pericardial cells in the anterior heart domain develop via a different cell lineage than cardioblasts and Tin-positive pericardial cells that develop in the posterior domain (Alvarez, 2003).

The analysis of ten additional cardioblast clones in the anterior heart domain support a distinct cell lineage for anterior versus posterior cardioblasts. Nine clones consisted of one cardioblast and one non-Tin-expressing pericardial cell, whereas a single clone consisted of two cardioblasts. Thus, most, if not all, anterior domain cardioblasts share a sibling relationship with a pericardial cell. In addition, all anterior domain cardioblasts exhibit cell lineages distinct from posterior domain cardioblasts. Together with the lineage data on Tin-positive pericardial cells, these results support the idea that cardioblasts and Tin-positive pericardial cells in the anterior heart domain carry out distinct functions from those found in the posterior heart domain (Alvarez, 2003).

Interestingly, the lineage of the twelve cardioblasts in the anterior heart domain appears fixed with respect to whether they share a sibling relationship with a Tin-positive or Tin-negative pericardial cell. These cardioblasts were numbered 1-12 from anterior to posterior with cardioblast 12 being immediately anterior to the first Svp-positive cardioblast. Four clones were identified that contained cardioblast 12 and in each clone this cardioblast shared a sibling relationship with a Tin-negative pericardial cell. By contrast, cardioblasts 10 and 11 each share a sibling relationship with a Tin-positive pericardial cell. No multiple clones were obtained for all twelve cardioblasts; nonetheless, these data suggest a fixed relationship between the position of a cardioblast and whether its sibling pericardial cell expresses Tin. It is speculated that the differences in gene expression between different pairs of sibling cardioblasts and pericardial cells in the anterior domain may reflect functional differences between such pairs of heart cells (Alvarez, 2003).

pnt was identified as an inhibitor of cardioblast development in a screen for mutations that affect cardioblast and/or pericardial cell development. To identify genes that regulate heart development ~2000 third chromosomal lethal P element lines were screened for defects in the expression of Mef2 a protein expressed in all cardioblasts and Eve. Two P element mutations were uncovered that cause an approximate twofold increase in cardioblasts. One of these P elements [l(3)S012309] maps to cytological position 94F1-3 and was known to be an allele of pnt. To verify that lesions in pnt result in the formation of ectopic cardioblasts, the phenotype of five additional pnt alleles was assayed. Although the severity of the phenotype varies for each pnt allele, all alleles display a significant increase in cardioblast number relative to wild-type embryos. With respect to the excess cardioblast phenotype, these alleles can be grouped into an allelic series. The presence of excess cardioblasts in embryos homozygous mutant for each pnt allele indicates that pnt normally functions in heart development to repress cardioblast development (Alvarez, 2003).

The published heart phenotype of the GATA transcription factor pnr is opposite that of pnt. In pnr mutant embryos, too many pericardial cells and too few cardioblasts are thought to develop. As a first step towards examining the potential regulatory interactions between pnr and pnt, a detailed analysis was carried out of heart development in pnr mutant embryos. pnrVX6, a null allele that contains a small deletion that removes all but the N-terminal nine amino acids of pnr, was used as well as pnr1, a molecularly uncharacterized allele. In contrast to a prior study, a loss of both cardioblasts and pericardial cells was found in pnr embryos. The dorsal mesodermal phenotypes were quantified for Eve-positive pericardial cells as well as for all pericardial cells using the pan-pericardial marker Zfh1. In wild-type embryos an average of 22.7 Eve-positive pericardial cells and 61.1 Zfh1-positive pericardial cells were observed per embryo side. pnrVX6 embryos exhibit the most severe effect with an average of 9.4 and 16.9 Eve- and Zfh1-positive pericardial cells, respectively. pnrVX6/pnr1 embryos exhibit an intermediate phenotype with an average of 16.4 Eve-positive pericardial cells and 27.4 Zfh1-positive pericardial cells, while pnr1 embryos exhibit the mildest phenotype with an average of 21.2 and 37.4 Eve- and Zfh1- positive pericardial cells, respectively. A severe loss of cardioblasts and Odd-positive pericardial cells were observed in these backgrounds although these phenotypes were not quantified. The loss of cardioblasts and Odd-positive pericardial cells is most severe in pnrVX6 embryos and least severe in pnr1 embryos where short stretches of cardioblasts are still visible. These results indicate that pnr normally functions to promote the development of all heart cells. The results demonstrate that the earliest manifestation of cardiac mesoderm development is defective in pnr embryos, suggesting that the general lack of heart cells in pnr embryos arises indirectly via a defect in the specification of the cardiac mesoderm. Double mutant studies support the model that pnr acts upstream of pnt in a developmental pathway\ (Alvarez, 2003).

This paper indicates that pnr and pnt act sequentially to regulate heart development. pnr acts early in mesoderm development to enable the cardiac mesoderm to form. Subsequent to this event, pnt acts within the cardiac mesoderm to regulate the ability of cells to choose between the pericardial or cardioblast fate. In this context, pnt inhibits the development of the Svp-class of cardioblasts and appears to function independently of Ras/MAP kinase pathway activity (Alvarez, 2003).

The effect of pnt on heart development is restricted to the posterior seven heart segments where Svp cardioblasts normally develop. Interestingly, the lineage studies identify a clear difference in the cell lineage of cardioblasts that develop in the posterior seven heart segments versus those that develop more anteriorly. These results identify a genetic and developmental distinction between these two regions of the heart. In addition, they suggest that cells in different regions of the heart carry out different functions and that these functions are probably under homeotic gene control. Future work that addresses the physiological role of these cells in heart function and the control of their development by homeotic genes should provide a more comprehensive understanding of heart development (Alvarez, 2003).

The data suggest that PntP2 may regulate cardioblast and pericardial cell development independently of Ras/MAP kinase activity. Given that every other developmental function of pnt has been traced back to receptor tyrosine kinase/Ras signaling activity, the apparent Ras independent activity of PntP2 is puzzling. Since PntP2 is expressed broadly throughout the mesoderm, a number of models can explain the apparent Ras-independent activity of PntP2 in the heart. For example, PntP2 may not require MAP-kinase-mediated phosphorylation to carry out a subset of its function. Consistent with this, phosphorylation of PntP2 does not appear to affect its DNA-binding ability. Thus, in the absence of MAP-kinase stimulation, PntP2 is still probably able to bind target promoters alone or in complexes with other proteins. Such an activity of PntP2 could on its own regulate target gene expression by blocking the ability of other transcriptional effectors to bind to and activate target gene transcription, or through an obligate association with other proteins required to activate (or to repress) target genes (Alvarez, 2003).

A second model is that PntP2 requires MAP kinase activation but that this activity is carried out by one of the other MAP kinase pathways in Drosophila: the JNK pathway or the p38 pathway. Preliminary phenotypic analyses indicate that heart development is normal in embryos mutant for basket, the Drosophila JNK-kinase. Analysis of p38 kinase activity is presently limited because of the absence of suitable genetic backgrounds. A third possibility is that a novel Ras-dependent pathway does in fact activate PntP2 during heart development. This model is consistent with the recent identification of a novel receptor tyrosine kinase expressed in the developing visceral mesoderm. Experiments that failed to identify a pnt-like excess cardioblast phenotype upon mesodermal overexpression of a dominant-negative form of Ras argue against this model. However, Ras is maternally loaded and it is extremely difficult to eliminate all Ras activity in this manner. Thus, even though Ras-like mesodermal phenotypes were observed in these experiments, a role for Ras in regulating cardioblast number may have been missed because of differential sensitivity of different developmental pathways to partial Ras inactivation. Future work that (1) addresses the ability of MAP-kinase insensitive forms of PntP2 to regulate heart development, and (2) identifies PntP2 target genes in the heart and elucidates how PntP2 regulates such genes should help clarify the molecular basis through which PntP2 governs heart development (Alvarez, 2003).

Phenotypic analysis of pnr conflicts with a prior study that showed an increase in pericardial cells in pnr mutants. This study used Eve to identify a subset of pericardial cells in pnr1 embryos. The difference in results are attributed to use of the pnrVX6 null allele, the ability to distinguish unambiguously Eve-positive pericardial cells from Eve-positive somatic muscle progenitors, and to specific defects in dorsal closure exhibited by pnr embryos that result in the local aggregation of cells in the dorsal region of the embryo. Genetic results identify pnr1 as a hypomorphic allele and Eve-positive pericardial cell formation is found to be almost wild type in this background. In these experiments, Eve-positive pericardial cells were unambiguously identified via their co-expression of Zfh1 and it was possible to quantify precisely Eve-positive pericardial cell number in pnr1 embryos. This is important as one can observe local increases in Eve-positive mesodermal cells in pnr embryos. However, such apparent increases arise from the local aggregation of dorsal mesodermal cells in pnr1 embryos caused by defects in dorsal closure and not by an overall increase in Eve-positive mesodermal cells (Alvarez, 2003).

The genetic identification of pnr1 as a hypomorphic allele is intriguing given that molecular and expression analyses indicate the pnr1 lesion results from a premature stop codon in the middle of the first zinc finger and that the Pnr1 protein localizes predominantly to the cytoplasm. This lesion is expected to abrogate the DNA-binding ability of the Pnr protein. However, genetic experiments indicate that the Pnr1 protein retains residual activity at least with respect to heart development. These results raise the possibility that Pnr may be able to carry out some of its functions independently of DNA binding. Future work that focuses on a detailed structure function analysis of the Pnr protein should clarify whether Pnr can act independently of its DNA-binding ability in some developmental contexts (Alvarez, 2003).

The pnt allelic series indicates that pntDelta88 exhibits a milder excess cardioblast phenotype than pntS012309, pnt2, and pntRR112. This result is surprising as pntDelta88 deletes the exons pntP2 shares with pntP1 and as a result pntDelta88 is assumed to be an amorphic allele of the pnt locus. Using antisense RNA probes specific for the unique exons of pntP2, an essentially wild-type pattern of pntP2 transcription is observed in pntDelta88 mutant embryos. These data raise the possibility that the N-terminal regions of pntP2 may also retain partial activity. Studies along the lines of those suggested for Pnr should also help elucidate whether truncated forms of PntP2 retain residual activity (Alvarez, 2003).

Significant similarity exists between the embryology and molecular regulation of early heart development in Drosophila and vertebrates. In this context, the identification of a role for pnt (a member of the evolutionarily conserved ETS transcription factor family) in Drosophila heart development raises the possibility that ETS family proteins regulate vertebrate heart development. Consistent with this, ETS1 and ETS2, the two most closely related vertebrate ETS proteins to pnt, are expressed in the developing vertebrate heart -- functional studies indicate these genes regulate the expression of specific genes in the heart. However, knockout studies have not yet revealed a clear role for ETS1 or ETS2 in the morphological development or differentiation of the vertebrate heart. The existence of multiple vertebrate ETS-family members highly homologous to pnt, as well as a total of 25 ETS family members in humans suggests the possibility of functional redundancy in ETS protein function during vertebrate and mammalian heart development. Thus, a full understanding of ETS protein function during heart development awaits construction and analysis of animals multiply mutant for different ETS family members (Alvarez, 2003).

Pointed and tracheal development

Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Genetic mosaic analysis has been used in Drosophila to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them (Ghabrial, 2006).

The Drosophila tracheal system develops from epithelial sacs of about 80 cells from which primary, secondary and terminal branches sprout without cell division or cell death. Primary branch sprouting is induced by Branchless (Bnl) FGF, a chemoattractant secreted by clusters of cells surrounding each sac, which activates Breathless (Btl) FGF receptor (FGFR), a receptor tyrosine kinase expressed on tracheal cells. Primary branches contain 3-20 cells that organize into a tube as they migrate out from the sac. Bnl also induces the expression of secondary branching genes, such as the transcription factor pointed (pnt), and specifies terminal cells at the ends of outgrowing branches. Terminal cells ramify in the larva in response to Bnl to form fine terminal branches. Other cells at the ends of primary branches become fusion cells that connect with neighbouring branches to form a continuous tracheal network. Terminal and fusion cell fate decisions are also influenced by the Notch, Dpp and Wingless signalling pathways. Dorsal branches, the primary branches that were focused on here in this study, typically consist of five or six cells: two cells near the branch tip, one (DB1) that becomes a terminal cell and another (DB2) that becomes a fusion cell, and three or four cells (DB3-DB6) that form the branch stalk (Ghabrial, 2006).

What does it take to become the leader? The lead cell (DB1) is specified to become a terminal cell by Bnl-Btl signalling. If terminal cell specification is required, then null mutations in the downstream gene pnt, which abolish this function, should have the same effect as btl mutations. Cell clones homozygous for pntDelta88 or two new pnt alleles isolated in the screen (198 and 1318) failed to develop as terminal cells, as expected. However, unlike btl mosaic branches, pnt mosaic branches often lacked a terminal cell. When a terminal cell was missing, there was usually a pnt-/- cell in the stalk position nearest the tip, presumably the DB1 cell that failed to differentiate into a terminal cell. This suggests that pnt-/- cells are able to assume the lead position but fail to differentiate as terminal cells, and that the bias against btl mutant terminal cells is due to the earlier, pnt-independent, function of Btl in primary branch budding and outgrowth. If cells lacking Btl cannot migrate in response to Bnl during budding, they should not be able to move to the lead position necessary to be selected as a terminal cell. Consistent with this, genetic mosaic analysis of stumps (dof/heartbroken), which encodes a Btl adaptor required for cell migration, showed a dearth of terminal cell clones similar to btl (Ghabrial, 2006).

Pointed and Eye Development

The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. faf also interacts with pointed: the extra-photoreceptor phenotype observed in faf mutants is clearly suppressed by pointed mutation; many more ommatidia have six outer photoreceptors in a trapezoidal arrangement characteristic of wildtype ommatidia. yan mutation in combination with faf strongly enhances the faf phenotype. Reducing the D-Jun activity suppresses the faf mutant phenotype. In sevenless;faf double mutants, R7 cells, normally absent in sevenless mutants, form in 60% of the ommatidia. Thus, faf can alleviate the requirement for sev in the R7 precursor. These results indicate that RTK/Ras signaling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf mutant eye discs when the ectopic photoreceptors are induced. It is proposed that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism (Isaksson, 1997).

The activity of the E2F transcription factor is regulated in part by pRB, the protein product of the retinoblastoma tumor suppressor gene. Studies of tumor cells show that the p16ink4a/cdk4/cyclin D/pRB pathway is mutated in most forms of cancer, suggesting that the deregulation of E2F, and hence the cell cycle, is a common event in tumorigenesis. Extragenic mutations that enhance or suppress E2F activity are likely to alter cell-cycle control and may play a role in tumorigenesis. An E2F overexpression phenotype in the Drosophila eye was use to screen for modifiers of E2F activity. Coexpression of dE2F and its heterodimeric partner dDP in the fly eye induces S phases and cell death. Thirty three enhancer mutations of this phenotype were isolated by EMS and X-ray mutagenesis and by screening a deficiency library collection. The majority of these mutations sorted into six complementation groups, five of which have been identified as alleles of brahma (brm), moira (mor), osa, pointed (pnt), and polycephalon (poc). osa, brm, and mor encode proteins with homology to SWI1, SWI2, and SWI3, respectively, suggesting that the activity of a SWI/SNF chromatin-remodeling complex has an important impact on E2F-dependent phenotypes. Mutations in poc also suppress phenotypes caused by p21CIP1 expression, indicating an important role for Polycephalon in cell-cycle control (Staehling-Hampton, 1999).

Receptor tyrosine kinases such as the EGF receptor transduce extracellular signals into multiple cellular responses. In the developing Drosophila eye, Egfr activity triggers cell differentiation. This study focuses on three additional cell autonomous aspects of Egfr function and their coordination with differentiation, namely, withdrawal from the cell cycle, mitosis, and cell survival. Whereas differentiation requires intense signaling, dependent on multiple reinforcing ligands, lesser Egfr activity maintains cell cycle arrest, promotes mitosis, and protects against cell death. Each response requires the same Ras, Raf, MAPK, and Pnt signal transduction pathway. Mitotic and survival responses also involve Pnt-independent branches, perhaps explaining how survival and mitosis can occur independently. These results suggest that, rather than triggering all or none responses, Egfr coordinates partially independent processes as the eye differentiates (Yang, 2003).

In order to test whether differentiation requires more activity of Pnt than maintaining G1 arrest, small deletions were used that specifically mutate one of the two pnt transcripts that encode alternative forms of Pnt protein. Both transcripts encode proteins that bind to the same DNA sequences, but Pnt-P1 is a constitutive activator of transcription, while Pnt-P2 requires MAPK phosphorylation for activity. In clones mutant for Pnt-P1, only 4% of R2-5 differentiation remained, but three times as many cells remained in G1. This indicates that, in the absence of Pnt-P1, Pnt-P2 is able to maintain some cells in G1 but rarely triggers their differentiation. In clones mutant for Pnt-P2, R8 cells were the only cells to differentiate, but other cells remained in G1, in addition. Thus, in the absence of Pnt-P2, Pnt-P1 is able to maintain some cells in G1 but did not trigger their differentiation. These findings indicate that both Pnt-P1 and Pnt-P2 proteins are necessary for normal levels of G1 maintenance and R2-5 differentiation but that Pnt proteins are required more stringently for differentiation than for G1 arrest (Yang, 2003).

These findings demonstrate that Egfr signaling through Ras and Raf to Pnt can trigger different responses at different activity levels. Increased Egfr activity increases the activity of nuclear Pnt proteins to promote differentiation, in addition to G1 arrest. The level of Egfr signaling of each cell depends on multiple activating ligands (Yang, 2003).

Does Pnt have any direct role in mitosis? Although the mitotic index is reduced in pnt mutant clones, reduced ligand secretion due to absent R2-5 cells might account for this. However, wild-type regions barely elevate the mitotic index in adjacent pnt mutant cells, and mitoses is often delayed, even when RasV12 or RasV12S35 is expressed to restore the mitotic index in pnt mutant clones. These observations suggest that both Pnt and another pathway downstream of Raf contribute to mitosis (Yang, 2003).

Ommatidial rotation in the Drosophila eye provides a striking example of the precision with which tissue patterning can be achieved. Ommatidia in the adult eye are aligned at right angles to the equator, with dorsal and ventral ommatidia pointing in opposite directions. This pattern is established during disc development, when clusters rotate through 90°, a process dependent on planar cell polarity and rotation-specific factors such as Nemo and Scabrous. Epidermal growth factor receptor (Egfr) signalling is required for rotation, further adding to the manifold actions of this pathway in eye development. Egfr is distinct from other rotation factors in that the initial process is unaffected, but orientation in the adult is greatly disrupted when signalling is abnormal. It is proposed that Egfr signalling acts in the third instar imaginal disc to 'lock' ommatidia in their final position, and that in its absence, ommatidial orientation becomes disrupted during the remodelling of the larval disc into an adult eye. This lock may be achieved by a change in the adhesive properties of the cells: cadherin-based adhesion is important for ommatidia to remain in their appropriate positions. In addition, there is an error-correction mechanism operating during pupal stages to reposition inappropriately oriented ommatidia. These results suggest that initial patterning events are not sufficient to achieve the precise architecture of the fly eye, and highlight a novel requirement for error-correction, and for an Egfr-dependent protection function to prevent morphological disruption during tissue remodelling (Brown, 2003).

The Egfr ligand Keren was misexpressed in developing photoreceptors and cone cells under the control of sev-Gal4. Surprisingly, this caused a disruption in the orientation of ommatidia relative to WT, a phenotype not previously associated with excess Egfr signalling. In the WT adult eye, all ommatidia are oriented at 90° relative to the equator. By contrast, when Keren is misexpressed, many ommatidia are abnormally oriented, with some ommatidia having rotated more than 90° and some less than 90°. In general, excess Egfr signalling leads to over-recruitment of cells in the eye, but photoreceptor recruitment is not affected when Keren is expressed at these levels. However, analysis of the pupal retina shows that Keren misexpression causes over-recruitment of cone cells, consistent with it acting through the Egfr. Previous work has shown that recruitment of cone cells is more sensitive than photoreceptors to Egfr overactivation; these results support this view, and also suggest that rotation is more sensitive than photoreceptor recruitment to perturbation of Egfr signalling (Brown, 2003).

Further examination of the adult phenotype indicates that it is rotation specifically that is disrupted on overexpressing Keren; the chirality (i.e. the correct specification of R3 and R4) of the ommatidia remains unaffected. This distinguishes the UAS-Keren phenotype from disruption of PCP components, which can cause both rotational and chiral defects (Brown, 2003).

All known cases of Egfr signalling in Drosophila are transmitted through the canonical Ras/Raf/MAPK pathway, and through a transcriptional output. The transcription factor Pointed is involved in most circumstances: PointedP2 is directly phosphorylated and activated by MAPK, and upregulates the expression of PointedP1; both factors mediate the transcription of downstream genes. In the case of rotation, which is envisaged as being a specialized case of cell motility or tissue remodelling, it seemed possible that Egfr signalling might influence the cytoskeleton directly, rather than exerting its effects by transcriptional control. Therefore whether a pointed hypomorph shows rotational defects was tested. Although many ommatidia show under-recruitment of photoreceptors, rotational defects are frequent in those ommatidia that are correctly specified, indicating that this function of the Egfr pathway relies on Pointed-mediated transcription (Brown, 2003).

Combinatorial signaling by the Frizzled/PCP and Egfr pathways during planar cell polarity establishment in the Drosophila eye

Frizzled (Fz)/PCP signaling regulates planar, vectorial orientation of cells or groups of cells within whole tissues. Although Fz/PCP signaling has been analyzed in several contexts, little is known about nuclear events acting downstream of Fz/PCP signaling in the R3/R4 cell fate decision in the Drosophila eye or in other contexts. This study demonstrates a specific requirement for Egfr-signaling and the transcription factors Fos (AP-1), Yan and Pnt in PCP dependent R3/R4 specification. Loss and gain-of-function assays suggest that the transcription factors integrate input from Fz/PCP and Egfr-signaling and that the ETS factors Pnt and Yan cooperate with Fos (and Jun) in the PCP-specific R3/R4 determination. The data indicate that Fos (either downstream of Fz/PCP signaling or parallel to it) and Yan are required in R3 to specify its fate (Fos) or inhibit R4 fate (Yan) and that Egfr-signaling is required in R4 via Pnt for its fate specification. Taken together with previous work establishing a Notch-dependent Su(H) function in R4, it is concluded that Fos, Yan, Pnt, and Su(H) integrate Egfr, Fz, and Notch signaling input in R3 or R4 to establish cell fate and ommatidial polarity (Weber, 2008).

Previous studies established that Fz is required cell-autonomously for R3 fate induction. The current analyses of kay/fos LOF alleles indicate that Fos is also required cell-autonomously in R3 for its fate determination. When overexpressed, Fos also acts like Fz in R3/R4 photoreceptors at the time of PCP establishment, with the cell of the pair that has higher Fos levels adopting the R3 fate. Based on its requirement in R3 and genetic interactions, Fos could act as a nuclear effector of Fz/PCP signaling. This is supported by the observation that it is able to suppress sev-dsh induced PCP defects; the genetic data can however not rule out that Fos could act in parallel to Fz/Dsh-PCP signaling). The subtle differences observed between fz and kay/fos LOF requirements (in fz R3/R4 mosaics the wild-type cell adopts the R3 fate often causing chirality inversions, while in kay/fos mosaic pairs with a mutant R3 the pair often adopts symmetrical R4/R4 appearance) is likely due either to the hypomorphic nature of the kay/fos alleles that had to be used in the analysis or potential redundancy with jun (Weber, 2008).

In addition to the positive Fos signaling input, R3 specification also requires the repressor function of Yan, with Yan inhibiting R4 fate in the R3 precursor. This is evident by the cellular requirement of Yan and highlighted by the increased defects in a kay/fos and yan double mutant scenario, where both aspects are partially impaired causing frequent R3/R4 fate decision defects. The dominant enhancement of kay2 by yan LOF suggests that keeping the R4 fate off in R3 precursors is as important as inducing the R3 fate (Weber, 2008).

Previous work has demonstrated that Fz/PCP signaling leads to Dl and neur upregulation in R3, activating Notch signaling in the neighboring R4 precursor. This study shows that Egfr-signaling is also specifically required for R4 fate determination. The ETS factors Yan and Pnt are nuclear effectors of Egfr-signaling in many contexts including photoreceptor induction, and the data indicate that they act also in R3/R4 determination. Egfr-signaling leads to an inactivation of Yan and an activation of Pnt through their phosphorylation by the Rl/Erk MAPK. As Yan represses the R4 fate it needs to get inactivated in the R4 precursor by Egfr-signaling and conversely Pnt is activated in R4. Together with the Notch-Su(H) activity this leads to R4 fate induction. Thus, for R3 determination Fz/PCP signaling and its nuclear effectors Fos (and Jun) are sufficient, along with Yan mediated repression of the R4 fate in R3 precursors. R4 fate determination, on the other hand, requires the joint activity of two pathways, Notch and Egfr-signaling and their nuclear effectors. A similar Egfr-Notch cooperation is observed in R7 induction and in cone cells (Weber, 2008).

These data support a complex interaction scenario between Fz/PCP, Notch, and Egfr-signaling in R3/R4 fate determination. Whereas the Notch-Su(H) activation in R4 depends on Fz/PCP signaling in the R3 precursor, the Fz/PCP and Egfr-signaling pathways require a fine balance. This is reflected by their genetic interactions, both at the level of the receptors fz and Egfr and their nuclear effectors Fos/Jun and the ETS factors Pnt and Yan, suggesting a cooperative involvement between the Fz/PCP and Egfr pathways (Weber, 2008).

The nuclear Egfr-signaling response is very likely mediated by Pnt in R4. Although this could not be addressed in pnt LOF clones due to the non-autonomous defects, which are caused by feedback loop requirements in which Pnt participates. The sufficiency experiments fully support a cell-autonomous requirement of Pnt in R4 to specify R4 fate, consistent with the Egfr requirement (Weber, 2008).

In summary, the behavior of the nuclear effectors of the respective signaling pathways involved in R3/R4 specification reflects the combinatorial nature of the signaling pathway input into the R3 and R4 fates (Weber, 2008).

Although in the embryo Fos and Jun need to act as heterodimeric partners in a non-redundant manner, in imaginal discs the scenario is more complicated. Whereas jun mutant clones display only mild phenotypes and do not affect proliferation/survival, strong kay/fos LOF alleles show severe defects, suggesting that kay/fos is the main AP-1 component acting in imaginal discs. This is supported by recent studies on the role of Fos in cell cycle regulation and proliferation (Hyun, 2006). Nevertheless, the double mutant combination of kay and jun revealed a requirement of both as no kay/fos, jun double mutant cells are recovered, suggesting a partially redundant function of kay/fos and jun in imaginal discs (Weber, 2008).

The specific role of the possible distinct heterodimers between the different Fos isoforms and Jun, or the different Fos isoforms themselves, could be very complex. This complexity is also evident in the fact that overexpression of a dominant-negative Fos protein form or a single wild-type isoform (transcript RA, according to Flybase) causes similar phenotypic defects (e.g. in the eye or in thorax closure). Future experiments will have to address which of the Fos isoforms is required in which context and if and how they interact with Jun (Weber, 2008).

Regulation of neurogenesis and epidermal growth factor receptor signaling by the Insulin receptor/Target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. Growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. The control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. This study shows that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors (McNeill, 2008).

Tight coordination of growth and differentiation is essential for normal development. InR/TOR signaling controls the timing of neuronal differentiation in the eye and leg in Drosophila. This study demonstrates that the InR/TOR pathway regulates neuronal differentiation in an S6K-dependent, but 4EBP/eIF4E-independent manner. It has previously been impossible to determine whether InR/TOR signaling was acting downstream or in parallel to the EGFR/MAPK pathway. Using argos and rho as reporters this study shows that the InR/TOR pathway is able to regulate EGFR/MAPK signaling downstream of MAPK. Moreover, pntP2 expression is up- and downregulated by activation or inhibition of InR/TOR signaling, respectively, and InR/TOR and EGFR pathways interact through pntP2. Taken together these data suggest that temporal control of differentiation by the InR/TOR pathway is achieved by modulation of EGFR pathway transcriptional targets in differentiating PRs (McNeill, 2008).

TOR is part of two multimeric complexes (TORC1 and TORC2) and is a core component of the InR pathway. TORC1 activity is regulated by nutrient and energy levels providing a conduit for hormonal and catabolic cellular inputs. Growth is regulated by two downstream targets of TORC1: S6K and 4EBP. The current data demonstrate that upstream of TORC1, differentiation and growth are regulated by the same factors. Downstream of TORC1, differentiation and growth differ significantly in that loss of s6k, but not eIF4E (or overexpression of 4EBP) affects differentiation. eIF4E regulates 7-methyl-guanosine cap-dependent translation and is the rate-limiting factor in translation initiation. The finding that eIF4E does not affect differentiation suggests that the temporal control of differentiation is not based on a translation initiation-dependent mechanism. Strikingly, loss of s6k blocks the precocious differentiation induced by loss of tsc2. Given the relatively weak effects of loss of s6k this may seem surprising. However, the degree of suppression is similar to the effect of loss of s6k on the overgrowth phenotype caused by loss of tsc2, namely, tsc2, s6k double-mutant cells are the same size as wild-type cells. Although loss of eIF4E has no affect on differentiation it may act redundantly with another factor, such as s6k. Testing this hypothesis though is technically challenging since the Drosophila genome contains eight different eIF4E isoforms. It will be interesting in future to test whether any of these isoforms regulate differentiation or alternatively whether eIF4E and s6k act redundantly. Although further work is required to determine the precise relationship between S6K and the InR/TOR pathway, the data point to a critical role of S6K in coordinating neuronal differentiation and growth (McNeill, 2008).

As in other neuronal systems, differentiation of PRs in the Drosophila eye occurs in a stereotyped manner. The advantage of the Drosophila retina as an experimental system is that the PRs differentiate spatiotemporally. Using this feature, as well as a series of cell-type-specific antibodies, this study has demonstrated that InR/TOR signaling is selective in the cell-types that it affects. The differentiation of PRs 2/5, 3/4, and 8 are unaffected by perturbations in InR/TOR signaling, whereas PRs 1, 6, and 7 and cone cells are dependent on this pathway for temporal control of differentiation. Interestingly the affected cells all differentiate after the second mitotic wave. However, regulators of the cell cycle do not affect the temporal control of differentiation. Why then are PRs 1, 6, and 7 and cone cells specifically affected? In cells with increased InR/TOR signaling, the expression of argos, rho, and pntP2 is precocious and increased throughout the clone, suggesting that the upregulation of EGFR signaling occurs in all cells. However, decreasing EGFR activity using a hypomorphic pntP2 allele specifically affects the differentiation of PRs 1, 6, and 7 and cone cells. Interestingly, pntP2 expression in differentiated cells is also restricted to PRs 1, 6, and 7 and cone cells. These observations suggest that differentiation of PRs 1, 6, and 7 and cone cells is critically dependent on EGFR levels signaling through pntP2. Therefore, although activation of InR/TOR signaling causes upregulation of EGFR transcriptional targets in all cells as they differentiate, the phenotypic effect is seen only in PRs 1, 6, and 7 and cone cells since these cells are highly sensitive to EGFR activity signaling through pntP2. This possibility is supported by the fact that precocious differentiation caused by overexpression of Dp110 can be suppressed by the simultaneous reduction of pntP2 levels. The complete suppression of the Dp110 differentiation phenotype by simultaneous reduction of pntP2 strongly suggests that pntP2 acts downstream of Dp110 and InR/TOR signaling in a pathway that regulates the temporal control of differentiation. It has been suggested that later differentiating PRs require higher levels of EGFR activity than their earlier differentiating neighbors. In particular, the activation of PR 7 requires both EGFR and Sevenless RTKs. In the case of InR/TOR pathway activation it may be that, through its regulation of EGFR downstream targets, the 'second burst' of RTK activity is enhanced causing PRs 1, 6, and 7 and cone cells to differentiate precociously. There may also be other as yet unidentified factors through which the InR/TOR pathway controls the expression of Aos and rho in PRs 2-5 and 8 (McNeill, 2008).

Activation of insulin and insulin-like growth factor receptors in mammalian systems is well known to elicit a response via the Ras/MAPK pathway. However, loss of the InR in the Drosophila eye does not result in a loss of PRs, a hallmark of the Ras pathway, nor does mutation of the putative Drk binding site in chico affect the function of the Drosophila IRS. In accordance with these data no change is seen in dpERK staining when the InR/TOR pathway is activated in the eye disc. Rather than a direct activation of Ras signaling by the InR, the data suggest that in the developing eye crosstalk between these pathways occurs at the level of regulation of the expression of EGFR transcriptional outputs. The most proximal component of the EGFR pathway that is regulated by InR/TOR signaling is pntP2. However, the data suggest that temporal control of PR differentiation requires concerted regulation of EGFR transcriptional outputs, since overexpression of pntP2 alone is not sufficient to cause precocious differentiation, whereas overexpression of activated EGFR is sufficient. Interestingly, microarray analyses of Drosophila and human cells have shown that the InR/TOR pathway regulates the expression of hundreds of genes. The mechanism by which this transcriptional control is exerted has yet to be elucidated. It will be interesting in future to determine the extent of transcriptional crosstalk between InR/TOR and EGFR pathways in developing neurons (McNeill, 2008).

pointed: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | References

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