Effects of Mutation or Deletion

Table of contents

Notch and eye differentation

Interactions are described between the Notch locus of Drosophila, and two other loci, scabrous and vestigial, which respectively affect the eyes and wings, were examined. The Notch locus is responsible for mediating decisions of cell fate throughout development in many different tissues. Mutations and duplications of vestigial and scabrous alter the severity of phenotypes associated with Notch mutations and duplications in a manner that is essentially tissue- and allele-specific. These interactions indicate that the products of vestigial and scabrous act in conjunction with Notch to stimulate the differentiation of specific cell types (Rabinow, 1990).

The Drosophila retina represents a particularly accessible tissue to address issues of local cell-cell signaling. Correct pattern is achieved in the Drosophila retina in part through the temporal and spatial control of programmed cell death (PCD). The mature retina is composed of an organized array of some 750 unit eyes (ommatidia), each containing eight photoreceptor neurons, four cone cells, two primary pigment cells (1's), and a hexagonal lattice composed of secondary/tertiary pigment cells (2'/3's) and sensory bristle organules. With the possible exception of the cells of the bristle organule, cell fates in the retina are not determined through lineage-based restriction but instead rely on local signals passed between cells. These signals result in progressive recruitment of undifferentiated cells by their previously differentiated neighbors. Creation of the interommatidial lattice of 2'/3's is the result of the final cell fate decision in the retina: some cells are recruited as 2'/3's, while any remaining excess cells are removed by PCD. Two different cell types have been proposed to be the major regulators of cell death in the retina: 1's and cells of the bristle organule. 1's were implicated as potential regulators of PCD by experiments examining Notch loss-of-function alleles: reduction of Notch function led to loss of both 1's and PCD, leading to the suggestion that 1's direct PCD. Alternatively, bristles have been proposed as regulators of PCD in the retina due to clustering of apoptotic cells (detected by acridine orange staining) around bristle organules. More recent experiments indicate that cell death can occur in the absence of bristles, although their presence may still influence PCD. Evidence is provided that the cone cells and 1's provide a signal that promotes survival of cells in the interommatidial lattice. Further evidence is provided that this signal represents part of a balance between signals of the Ras and Notch pathways, which appear to act in opposition to regulate the number of interommatidial cells permitted to remain (Miller, 1998).

The first cell types to emerge in the developing retina are the photoreceptor neurons and (non-neuronal) cone cells, which arise within the retinal neuroepithelium of the mature larva. The larva then undergoes pupation as the retina evaginates (disc eversion) and is repositioned to lie distally against the pupa's cuticle. Soon after disc eversion, the 1's emerge to enwrap the cone cells (22-24 hours APF). They establish direct contact with the remaining undifferentiated cells which lie between ommatidia, and which are referred to as interommatidial precursor cells or IPCs. Finally, a hexagonal lattice is formed between ommatidia as IPCs are directed into one of two fates: 2'/3' or PCD. The result is a precise hexagonal array of ommatidia, each surrounded by nine 2'/3's and three bristles. Each cell type in the developing retina can be recognized by the position of its nucleus. Typically, nuclei are first found in the basal part of the neuroepithelium and rise apically as a cell begins its differentiation. During early pupal stages, cone cell nuclei are arranged as an apical 'cloverleaf' at the center of each ommatidium and several microns above the photoreceptor nuclei; two 1' nuclei form an apical ring around the cone cells; and the IPC nuclei are found basally between ommatidia (these nuclei are slow to rise apically except bristle nuclei, which are found at an intermediate level early in their differentiation). This stereotyped arrangement permits identification and ablation of each cell type. Experimentally induced ablation alters the arrangement and subsequent identity of cells in the retina, in order to understand the underlying mechanism of cell fate determination. Once the ablation is performed, pupae are permitted to develop for an additional 24 hours to allow for establishment of all cell types; retinae are then removed and stained with cobalt sulfide to highlight each cell type at the surface. In each experiment, the non-ablated partner is used as an internal control. The effects of ablation are limited to the target cell, with little apparent collateral damage to neighboring cells as assessed by their normal subsequent development (Miller, 1998).

Disc eversion is complete by 18 hours APF at 25'C; the first indication of 1' differentiation is the apical migration of its nucleus at 22-24 hours APF. In initial studies, laser ablation of a 1' at this stage results in its rapid replacement. 1' nuclei ablated after 24 hours APF are not replaced. With regard to establishment of the 1' fate, these results indicate: (1) several cells have the potential to differentiate as 1's; (2) this decision remains reversible for several hours; and (3) during this period, established 1's provide a signal inhibiting the 1' fate in their neighbors. Loss of pupal Notch activity blocks both 1' differentiation and PCD, leading to the suggestion that 1's promote PCD. However, it is not clear whether loss of 1's leads necessarily to a block in PCD or whether Notch affects each process independently. Cell ablation provides a more direct assessment of the role of 1's on PCD. To assess whether 1's promote PCD they were ablated 24-27 hours APF, a stage after the point in their development when replacement can occur. Removal of a single 1' results in misplaced and, surprisingly, missing 2'/3's in the neighboring interommatidial lattice when observed 24 hours later. Typically, removal of one 1' results in loss of one or two 2'/3's, suggesting that 2'/3's require input from at least two 1's to survive. Later ablation of 1's (28-30 hours APF) has minimal effects on the number of cells within the interommatidial lattice, defining a developmental window in which 1's can influence the fate of neighboring IPCs. This result would seem to run counter to the hypothesis that 1's direct PCD. However, the rearrangement of the remaining 2'/3's often make it difficult to establish with certainty that the observed lattice defects are due to ectopic loss of IPCs. Ideally, 1's should be removed from a larger region and at an earlier stage (Miller, 1998).

One potential strategy for early removal of 1's is to determine which cell type is required to direct the 1' fate. Morphology and genetic manipulations have indicated that cone cells, which directly abut developing 1's, are the most likely source of such an inductive cue. Ablation of the four cone cells from a single ommatidium at 18-22 hours APF - before the emergence of 1's - prevents recruitment of any 1's to that ommatidium. Sparing just one of the cone cells, however, is sufficient to induce 1-2 1's. No difference is found in the ability of any of the four cone cells to induce a 1'. Ablation of photoreceptor neurons has no effect on 1' differentiation. Thus, the presence of cone cells is necessary for the recruitment of 1's. Importantly, these results indicate that removal of cone cells would provide a reliable and early means of removing 1's. Loss of cone cells and 1's results in ectopic PCD. In order to more clearly examine the effects of signals from 1's on PCD, all four cone cells were removed from four neighboring ommatidia within a 'square'. Within this square are contained 48±3 IPCs (the precursor cells that would normally differentiate as 2'/3's or die. Ablation of the sixteen resident cone cells between 18-22 hours APF leaves most of these IPCs without either the neighboring cone cells or 1's. Normally, approx. 25% of these cells would undergo PCD, leaving the 37 surviving cells to form the interweaving hexagonal lattice of 2'/3's. Again, loss of 1's leads to an increase in PCD. This massive loss of 2'/3's is followed by a rearrangement which brings the apices of the surrounding, unablated ommatidia together to approximate a normal arrangement of ommatidia. The few remaining 2'/3's are associated with these newly displaced 1's. In general, the only indication of the previous apical presence of the four cone cell-ablated ommatidia is a tuft of sensory bristles (normally separated by several 2'/3's) and a 'jog' in the normally straight rows of ommatidia. The hidden photoreceptor core of each ablated ommatidium remains beneath the surface. This result is consistent with the model that 1's do not promote cell death within the interommatidial lattice of IPCs but instead repress it (Miller, 1998).

Experiments indicate that Notch signaling promotes programmed cell death. To provide further evidence for signaling within the interommatidial lattice and to assess the nature of these signals, the role of two signaling pathways were examined in the ablation paradigm. Previous work has implicated the Notch signaling pathway in all cell fate decisions in the developing retina, including patterning of the 2'/3' lattice. Notch encodes a large transmembrane signaling receptor which is expressed exclusively in IPCs during the stage of PCD. Alleles that reduce Notch activity in the young pupa both prevent 1' differentiation and block PCD, which has led to the suggestion that 1's promote death in neighboring IPCs. However, cells in the position of 1's are observed to demonstrate some differentiation. In addition, it appears that 1's actually promote the 2'/3' fate at the expense of PCD. Either the Notch-mediated block in PCD is due to partial differentiation of 1's (which can then block PCD), or Notch is in fact required to promote PCD and its role in 1' differentiation is separable. To determine whether Notch has a direct role in PCD, its function was reduced in the absence of 1's. The N fa-g2 mutation is one of a series of alleles that remove Notch activity specifically in pigment cells, resulting in loss of both 1' differentiation and PCD. Sixteen cone cells were ablated prior to 22 hours APF in four ommatidia as described above to create a zone that lacks cone cells and 1's. The number of 2'/3's which subsequently differentiated was then compared between the zone receiving ablation and unablated N fa-g2 controls. Ablated N fa-g2 retinae retain 51±3 2'/3's, a number similar to those retained in unablated controls. The ability of Notch mutations to block PCD in the absence of 1's, and Notch's exclusive expression within the IPCs during the 2'/3' versus PCD decision, indicates that it acts directly within the interommatidial lattice to promote PCD (Miller, 1998).

Ras signaling promotes the 2'/3' fate at the expense of PCD. One pathway which can act in opposition to Notch is the Ras signal transduction pathway Ras is required for a variety of cell fate decisions in the developing retina. To test the role of Ras signaling during PCD, flies were used in which an inducible heat shock promoter was fused to the activated Ras form Dras1 Val12. A 1-hour pulse of Dras1 Val12 throughout the retina beginning at 26 hours APF rescues IPCs from PCD. Early removal of cone cells and 1's in four neighboring ommatidia has no effect on this rescue. This result indicates that Ras signaling acts to prevent PCD and/or promote the 2'/3' fate. With regard to PCD, therefore, Ras acts in opposition to Notch signaling (Miller, 1998).

This Ras-mediated rescue of cells is similar to, and epistatic to, the rescue provided by cone cells and 1's. Are the two signals linked? The Ras pathway has been demonstrated to be activated by a variety of extracellular stimuli, including signaling through receptor tyrosine kinases (RTKs). In the developing retina, the Egf receptor ortholog is an RTK that regulates a variety of cell fate determination steps including 2'/3' determination. Consistent with the results described above for activation of Dras1, loss of Egfr activity leads to a loss of 2'/3's, presumably due to an excess of PCD. To determine whether Egfr signaling is sufficient to block PCD, flies containing an activated form of Egfr (l-DER) fused to an inducible heat shock promoter received a 1-hour heat shock. Expression of l-DER throughout the young pupal retina results in a block in PCD. The loss of PCD is not complete, perhaps due to the relatively weak activation of Egfr provided by the l-DER protein. Egfr is a receptor that acts autonomously: Egfr expression in IPCs is anticipated in the cells where it is active during the stage of PCD. Consistent with this view, Egfr is found to be expressed primarily in the IPCs. These results suggest the possibility that IPCs receive a signal from their neighbors that activates their own Egfr signaling and represses PCD. The ablation results suggest this signal is derived from the 1's and perhaps the cone cells. Interestingly, the TGFalpha ortholog, Spitz, is expressed at high levels in the cone cells and bristles and can be detected at lower levels in the 1's. Spitz is a diffusible ligand of Egfr and may represent the 'life' signal provided by the ommatidium. Together, these observations suggest a model in which patterning requires local Spitz/Egfr signaling by (at least two) 1's to rescue neighboring IPCs from a Notch-imposed apoptotic fate. One important test of this model will require the removal of spitz function specifically in cones cells and 1's (Miller, 1998).

It has been suggested that lateral specification of cell fate by Notch signaling depends on feedback on Notch (N) and Delta (Dl) transcription to establish reciprocal distributions of the receptor and its ligand at the protein level. In Drosophila neurogenesis the predicted reciprocal protein distributions have not been observed. Either the current model of lateral specification or the description of N and/or Dl protein distributions must be incomplete. R8 photoreceptor specification in the developing eye has been reexamined to resolve this question with regard to this example of lateral specification. N and Dl protein levels were assessed in the cell as a whole and at the cell surface, where these proteins were mostly found at the intercellular adherens junctions. Protein levels do not correspond to Notch signaling in wild type. Dl protein first appears on cell surfaces just ahead of the furrow and becomes elevated in column 0 on the posterior cells of the so-called rosettes, including the future photoreceptors and the posterior core cell. The R8 cell surface exposes much greater amounts of Dl than its neighbors. However, Dl transcription and protein levels do correlate with altered N signaling in mutant genotypes. These findings suggest the difference relates to the speed of lateral specification in vivo. The time required for N signaling to inhibit atonal expression, an indication of the time required for the cell to respond to N signaling, is at most 90 min, but changes in the Dl protein distribution in mutant genotypes arise more slowly. N expression is little regulated by N signaling, but protein encoded by the N^ts1 allele is temperature-sensitive for appearance at the cell surface. Some aspects of the pattern of Dl protein appears to be due to endocytosis. It is concluded that feedback of N signaling on Dl transcription does occur but is too slow to account for the pattern of R8 specification. Studies of the ommatidia mosaic for a Notch duplication, or for the N^ts1 allele at semi-restrictive temperatures, have found that cells beginning with less N activity are not necessarily predisposed to be selected for R8 differentiation. These results are inconsistent with the notion that small fluctuations in N signaling levels between equivalent cells are assessed and as a consequence initiate the process of R8 selection. The data argue that other signals may be responsible for the pattern of R8 cell fate allocation by N. Other genes are known where mutations affect the R8 pattern and may constitute a second signal. These include that coding for the EGF receptor, where certain alleles can block R8 specification. Additional genes affecting the R8 pattern include the one coding for the secreted protein Scabrous and another coding for the homeodomain protein Rough. It is also possible that lingering effects of any prior N signaling in the same cells might render them unequal to a competition to become R8 (Baker, 1998).

Planar polarity is seen in epidermally derived structures throughout the animal kingdom. In the Drosophila eye, planar polarity is reflected in the mirror-symmetric arrangement of ommatidia (eye units) across the dorsoventral midline or equator; ommatidia on the dorsal and ventral sides of the equator exhibit opposite chirality. Photoreceptors R3 and R4 are essential in the establishment of the polarity of ommatidia. The R3 cell is thought to receive the polarizing signal, eminating from the equator, through the receptor Frizzled (Fz), before or at higher levels than the R4 cell, generating a difference between neighbouring R3 and R4 cells. Both loss-of-function and overexpression of Fz in the R3/R4 pair result in polarity defects and loss of mirror-image symmetry. Notch and Delta (Dl) are identified as dominant enhancers of the phenotypes produced by overexpression of fz and dishevelled (dsh); dsh encodes a signaling component downstream of Fz, and it is shown that Dl-mediated activation of Notch is required for establishment of ommatidial polarity. Whereas fz signaling is required to specify R3, Notch signaling induces the R4 fate. These data indicate that Dl is a transcriptional target of Fz/Dsh signaling in R3, and Dl activates Notch in the neighboring R4 precursor. This two-tiered mechanism explains how small differences in the level and/or timing of Fz activation reliably generate a binary cell-fate decision, leading to specification of R3 and R4 and ommatidial chirality. How Notch signaling induces the R4 fate remains unclear, as it usually represses photoreceptor development at this stage. However, the precursor cells are already committed to form the R3/R4 pair by transcription factors (such as Seven-up) that are required for both R3/R4-cell fate and polarity generation (Fanto, 1999).

Eye development in Drosophila involves the Notch signaling pathway at several consecutive steps. At first, Notch signaling is required for stable expression of the proneural gene atonal (ato), thereby maintaining the neural potential of the cells. Subsequently, in a process of lateral inhibition, Notch signaling is necessary to confine neural commitment to individual photoreceptor founder cells. Later on, the successive addition of cells to maturing ommatidia is under Notch control. In contrast to previous assumptions, the recessive Notch allele split (N^spl) specifically involves loss of the early proneural Notch activity in the eye, which is in agreement with bristle defects as well. As a result, fewer cells gain neural potential and fewer ommatidia are founded. N^spl alleles are characterized by a smaller number of ommatidia, which usually contain less than the normal set of photoreceptors. Enhancement of this phenotype by the dominant mutation Enhancer of split [E(spl)^D] happens within the remaining proneural cells (in which Ato expression has been abolished). In line with genetic data, this process occurs primarily at the protein level due to altered protein-protein interactions between the aberrant E(spl)^D and proneural proteins. Indeeed, in a yeast two-hybrid assay, the mutant M8*, representing the E(spl)^D alteration, binds significantly more strongly to proneural proteins, especially Achaete and Atonal. The mutant M8* protein does not interfere with the establishment of high Ato levels within intermediate-group cells. In contrast, m8* transcripts accumulate to a much higher level due to increase of mRNA stability caused by the deletion. Heterodimerization of M8* with other E(spl) bHLH proteins is indistinguishable from that of the wild-type M8 protein. Therefore the N^spl mutation reduces the inductive, proneural activity of N, which is normally required to stabilize expression of the proneural gene atonal. As a consequence fewer intermediate groups arise: these groups serve as reservoirs for future R8 cells. E(spl)^D potentiates the deficits of N^spl because in the compromized background the already lowered Ato levels in most presumptive R8 cells now drop below the threshold required to maintain neuronal fate. N^spl is the first Notch mutation known to specifically affect Notch inductive processes during eye development (Nagel, 1999).

The receptor protein Notch plays a conserved role in restricting neural-fate specification during lateral inhibition. Lateral inhibition requires the Notch intracellular domain to coactivate Su(H)-mediated transcription of the Enhancer-of-split Complex. During Drosophila eye development, Notch plays an additional role in promoting neural fate independent of Su(H) and E(spl)-C, and this finding suggests an alternative mechanism of Notch signal transduction. Genetic mosaics were used to analyze the proneural enhancement pathway. Proneural enhancement involves upregulation of proneural gene expression in single cells that will become neurons. In Drosophila eye development, Notch (N) is required for proneural enhancement in addition to lateral inhibition. The molecular mechanism of proneural enhancement has not been determined. As in lateral inhibition, the metalloprotease Kuzbanian, the EGF repeat 12 region of the Notch extracellular domain, Presenilin, and the Notch intracellular domain are required. By contrast, proneural enhancement becomes constitutive in the absence of Su(H), and this leads to premature differentiation and upregulation of the Atonal and Senseless proteins. Ectopic Notch signaling by Delta expression ahead of the morphogenetic furrow also causes premature differentiation. It is concluded that proneural enhancement and lateral inhibition use similar ligand binding and receptor processing but differ in the nuclear role of Su(H). Prior to Notch signaling, Su(H) represses neural development directly, not indirectly through E(spl)-C. During proneural enhancement, the Notch intracellular domain overcomes the repression of neural differentiation. Later, lateral inhibition restores the repression of neural development by a different mechanism, requiring E(spl)-C transcription. Thus, Notch restricts neurogenesis temporally to a narrow time interval between two modes of repression (Li, 2001).

In the developing eye, lateral inhibition restricts the proneural gene atonal (ato) to individual R8 photoreceptor cells, which found each ommatidium. Earlier, ato must first have reached levels of activity sufficient to sustain expression by autoregulation, in conjunction with its bHLH heterodimer partner encoded by daughterless (da) and with a zinc-finger protein encoded by senseless (sens). Such 'proneural enhancement' depends on N and Dl but not on Su(H) or E(spl)-C. Clones of cells mutant for the E(spl)-C or for Su(H) lead to neural hyperplasia because they lack lateral inhibition, but clones of cells mutant for N or Dl show reduced neural differentiation because they lack proneural enhancement. These divergent phenotypes show that proneural enhancement occurs by a mechanism distinct from that of lateral inhibition (Li, 2001).

Mosaic analysis with Notch pathway mutations have been used to elucidate the mechanism of proneural enhancement. Requirements similar to those of canonical N signaling for processed forms of Dl, Notch EGF repeats 10-12, and proteolytic processing of the N intracellular domain have been found. Proneural enhancement is independent of any Su(H)-mediated gene activation but is mimicked by the complete absence of Su(H) protein, and this indicates that proneural enhancement depends on the disruption of Su(H)-mediated gene repression (Li, 2001).

The phenotypes of other mutations can be compared to the E(spl) or N phenotypes. A neurogenic mutant phenotype indicates a role in lateral inhibition, not in proneural enhancement. A hyponeural phenotype indicates a requirement in proneural enhancement (Li, 2001).

The neurogenic phenotype of the metalloprotease kuz suggests that processed Dl might be important for lateral inhibition and that unprocessed, transmembrane Dl may not be sufficient. It is unknown what form of Dl is required for proneural signaling. Clones mutant for kuz show neural hyperplasia. The distribution of R8 cells labeled by Boss antibody is intermediate between the distributions of clones null for E(spl) and for N. This indicates either partial loss of lateral inhibition or a weak proneural phenotype that still permits some neurogenesis to occur. Ato expression was examined to distinguish these possibilities. In kuz clones, Ato protein appears at the same time as it does in neighboring wild-type regions, but it remains at a low level. Posterior to the furrow, small clusters of R8 cells express Ato at a higher level, but many fewer cells do so than in E(spl) clones. This shows that proneural enhancement is affected in kuz mutant clones, but to a lesser degree than in N null clones, so that more cells go on to take the R8 cell fate. An intermediate phenotype associated with small clusters of R8 cells results in combination with the kuz lateral-inhibition defect. This is consistent with a role for processed Dl in proneural enhancement as well as in lateral inhibition, although it is important to note that kuz might have roles besides Dl processing. Such roles might include other aspects of N function (Li, 2001).

EGF repeats 10-12 bind Dl and are important during lateral inhibition because a glutamic acid-to-valine substitution in EGF repeat 12 in the N^M1 mutant is embryonic lethal and neurogenic. Clones of N^M1 mutant cells in the eye affect proneural enhancement and lateral inhibition, as does kuz, and this finding indicates that Dl interacts with the EGF repeat 12 region of N for proneural enhancement as well as for lateral inhibition (Li, 2001).

Clones mutant for the psn mutation were examined to test whether the novel proneural pathway requires proteolytic processing of N. Clones of psn exhibit an intermediate phenotype. Small patches of R8 cells differentiate, as in N^M1 or kuz clones but unlike in E(spl) clones. Ato expression initiates normally but never elevates to the same levels seen in the wild type. Lateral inhibition is deficient in psn clones as judged by the loss of E(spl) expression [E(spl) mDelta], so the intermediate psn phenotype indicates an effect on proneural enhancement in addition (Li, 2001).

In lateral inhibitory signaling, the processed intracellular domain enters the nucleus. Clones mutant for the N^CO mutation were examined to test whether proneural enhancement is also mediated by the released intracellular domain or, alternatively, by other parts of the processed protein. In place of Gln-1865, N^CO encodes a termination codon that truncates the N intracellular domain close to the transmembrane domain. Eye clones of N^CO almost completely lack R8 cells or other neurons. Ato expression is greatly reduced, and only rare R8 cells form posterior to the furrow. Expression of the Senseless protein, a marker for Ato activity, is also greatly reduced, and this finding confirms the failure to establish high levels of Ato expression and function. These results show that the N intracellular domain is required for proneural enhancement. Similar results were obtained with N^60g11, which truncates the intracellular domain carboxy-terminal to the ankyrin/CDC repeats (Li, 2001).

It is noteworthy that the N^CO phenotype is 'stronger' than clones of the N protein null, in which occasional patches of neurogenesis are seen. If this is attributed to the dominant-negative effect of the protein encoded by N^CO, then residual neurogenesis in N null clones must reflect residual N protein, perhaps persisting from before the mitotic recombination event (Li, 2001).

The N intracellular domain converts nuclear Su(H) protein from a transcriptional repressor into a transcriptional activator during lateral inhibition. What is its role in proneural enhancement? It has been concluded that proneural enhancement does not require Su(H) based on the neurogenic phenotype of Su(H) mutant clones. However, the original Su(H) mutants seem not to have eliminated the Su(H) repressor function. Recently, deletion alleles of the Su(H) gene have been recovered that eliminate all Su(H) function (Li, 2001).

Clones homozygous for the Su(H)^Delta47 allele are neurogenic, as described previously for other alleles. In addition, however, Su(H)^Delta47 mutant cells differentiate prematurely. Ato expression begin earlier in Su(H)^Delta47 clones than in neighboring tissue, and it soon reaches high levels. The senseless gene is expressed in response to ato activity. Senseless is also expressed prematurely in Su(H)^Delta47 clones. Daughterless protein is ubiquitous but upregulated in ato-expressing cells of the furrow. It was hard to see premature elevation of Daughterless in Su(H)^Delta47 clones, and this must be subtle if it occurs (Li, 2001).

Premature differentiation in Su(H)^Delta47 clones might be explained if Su(H) normally antagonizes proneural enhancement. Then, in the total absence of Su(H) protein, Ato would enhance prematurely and initiate eye differentiation. Accelerated differentiation would in turn accelerate the progress of the morphogenetic furrow, induce Atonal expression more anteriorly, and begin the cycle again. To investigate the effect of N signaling on this Su(H) function, Dl was misexpressed ahead of the morphogenetic furrow. A transposon insertion in the hairy gene provided GAL4 protein expression. Ato expression is expanded anteriorly throughout the domain of h expression in h^GAL4; UAS-Dl eye discs. The sca gene, which is expressed in response to ato activity, is also expressed more anteriorly in response to ectopic Dl. Neural differentiation begins normally in the most posterior part of h^GAL4;UAS-Dl eye discs but becomes progressively disorganized more anteriorly as differentiation accelerates (Li, 2001).

The similiarity between activating N signaling ahead of the morphogenetic furrow and deleting Su(H) indicates that N signaling overcomes repression mediated by Su(H). If Su(H) antagonizes proneural enhancement by activating gene transcription, activating N ahead of the furrow should have released the N intracellular domain, elevated gene transcription, and antagonized morphogenetic furrow progression and differentiation, opposite that of what was observed (Li, 2001).

Different forms or complexes of N intracellular domain might be required to antagonize Su(H)-mediated repression during proneural enhancement from those that coactivate Su(H)-mediated gene transcription. The possible role of bib, mam, and neur in proneural enhancement has not been assessed. The bib gene encodes a transmembrane protein required for lateral inhibition in embryonic neurogenesis. Ommatidia that are mutant for bib contain occasional extra photoreceptor cells, and some ommatidia have multiple R8 cells. Ato expression begins and progresses normally, but posterior to the morphogenetic furrow small clusters of two or three cells, instead of single cells as in the wild type, often retain Ato expression. Sections through the adult retinas often reveal ommatidia with extra photoreceptor cell rhabdomeres, both of the R8/R7 small rhabdomere class and of the larger R1-R6 outer photoreceptor class. Since bib affects lateral inhibition only slightly, it is possible that an equally subtle requirement for bib in proneural enhancement might be undetected in these experiments (Li, 2001).

These findings suggest a model for proneural enhancement. The release of N intracellular domain in response to Dl derepresses genes that are repressed by Su(H). The relevant targets do not require Su(H)-mediated transcriptional activation, so deletion of Su(H) mimics N signaling. The mechanism contrasts with lateral inhibition. N signaling provides N intracellular domain as a coactivator for Su(H), which is essential for the transcription of E(spl)-C. Lateral inhibition cannot proceed in the absence of Su(H) because blocking repression by Su(H) is not sufficient for E(spl)-C transcription (Li, 2001).

The ato gene could be a direct target of proneural enhancement. ato regulatory sequences have been examined for activity control regions, but possible repression sites have not been assessed. Another candidate is daughterless, which encodes a bHLH heterodimer partner of Ato that is required for Ato function in eye development. A third candidate is senseless, a zinc finger protein that enhances and maintains proneural gene expression. Expression of ato and sens is prematurely elevated in the absence of Su(H), which is consistent with regulation by Su(H)-R. However, each might depend on Su(H)-R only indirectly because elevated expression of ato or sens requires the function of all three genes (Li, 2001).

Why does proneural enhancement precede lateral inhibition if both depend on Su(H) and nuclear N intracellular domain? (1) Multiple lines of evidence indicate that proneural enhancement requires less N activation than does lateral inhibition. These include the greater sensitivity of lateral inhibition to the N^ts mutation, nonautonomous rescue of proneural enhancement by Dl over distances for which lateral inhibition go unrescued, and neurogenesis in N mutant clones due to undetectably low levels of N protein (which is eliminated by dominant-negative protein from the N^CO allele). Therefore proneural enhancement is expected to occur sooner in response to N signaling. (2) The evolving transcriptional regulation of ato changes sensitivity to lateral inhibition over time. Even recombinant N intracellular domain expression does not prevent initial ato expression ahead of the furrow, but ato is exquisitely sensitive later when its expression depends on autoregulation (Li, 2001).

The main result of this study is that neural development in the Drosophila eye depends on two functions of the N intracellular domain in response to ligand binding: (1) N relieves Su(H)-mediated repression to enhance ato expression and function and to permit neurogenesis (proneural enhancement); (2) later, another pathway requires N to coactivate Su(H)-dependent E(spl) transcription (lateral inhibition). No genes or regions of N have yet been found to be required to affect one function but not the other. By means of these two functions stimulated by the same ligand, N signaling coordinates the upregulation of ato in proneural cells and represses ato in cells not specified as neural precursor cells, and N restricts neural patterning to a narrow time interval between two distinct modes of repression (Li, 2001).

Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signaling. Previous work has shown that the Notch signaling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors -- a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signaling pathways involved. Evidence suggests that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae (Baonza, 2001).

Loss of Notch signaling leads to a loss of neural differentiation. Cells within clones of a null allele of Notch fail to upregulate Atonal expression from its initial low, uniform level. This implies that Notch signaling is required for the initiation of neural development but not for the first low level expression of Atonal. To examine in detail the role of Notch signaling in promoting neural differentiation, clones of cells expressing the Notch ligand Delta were made and their ability to induce neural differentiation was examined. In the wing disc, similar ectopic expression of Delta in clones induces the activation of Notch signaling within the clone as well as non-autonomously in cells surrounding it (Baonza, 2001).

Clones were generated using the Gal4/UAS system combined with the Flip-out technique and third instar larval eye discs were labelled with different markers to assess neural development. The phenotype of Delta-expressing clones depends on their position with respect to the morphogenetic furrow. Clones in the anterior part of the disc have no effect unless they are within 12-15 cell diameters of the furrow. Within this zone close to the furrow, Delta induces the ectopic expression of Atonal, both autonomously within the clone and non-autonomously, in cells surrounding the clone. In some of these clones there are also cells ectopically expressing the neural antigen Elav. This indicates that once Atonal expression is activated, the full neural program is initiated. Thus, the primary proneural function of Notch signaling is the activation of Atonal (Baonza, 2001).

Consistent with the neural-promoting properties of Delta, clones that span the furrow from posterior to anterior cause the anterior displacement of Atonal and Elav expression. This displacement implies that the furrow accelerates as it moves through the clone. In the region of these clones that lies posterior to the furrow, the domain of Atonal expression is expanded and the Atonal-expressing cells are disorganized and more numerous. In this region repression of neural differentiation, visualized with the expression of Elav, is also observed. This later phenotype reflects the function of Notch signaling pathway in preventing neural differentiation posterior to the morphogenetic furrow (Baonza, 2001).

Similar clones were also produced expressing the alternative Notch ligand, Serrate, and unlike Delta-expressing cells, these clones cause no neural induction ahead of the furrow. Conversely, when posterior to the furrow, Ser-expressing clones behave like those expressing Delta and prevent neural differentiation. This implies that anterior to the furrow, the two Notch ligands are not equivalent in their ability to activate the receptor. The reason for this has not been explored, but it is noted that the Notch glycosyltransferase Fringe, which makes Notch resistant to Serrate, is strongly expressed anterior to the furrow. The inability of Serrate to induce proneural Notch signaling is consistent with previous reports, which show that loss of Serrate caused no effects on eye development (Baonza, 2001).

These results imply that there is a zone of about 12-15 cell diameters ahead of the morphogenetic furrow, where the activation of Notch signaling by Delta, but not by Serrate, is sufficient to trigger neural fate (Baonza, 2001).

The progression of the morphogenetic furrow correlates with the modulated expression of the negative regulators of Atonal expression, Emc and Hairy. Hairy is expressed in a broad stripe anterior to the furrow and rapidly switched off in the furrow. Emc protein is present in all cells but the highest levels are present in a dorsoventral stripe of cells anterior to the domain of Hairy expression, whereas the lowest levels are observed in the furrow. Thus, the increase of Atonal expression in the proneural groups within the furrow is associated with the downregulation of both Emc and Hairy. Whether this downregulation of Emc and Hairy is mediated by Notch was tested by analyzing the expression of Emc and Hairy when Notch signaling is blocked and when it is ectopically activated (Baonza, 2001).

In mitotic clones of the Notch null allele N^54/9, the expression of Hairy is displaced posteriorly extending behind the morphogenetic furrow. The consequent ectopic expression of Hairy within the furrow is accompanied by a reduction in Atonal expression: Atonal levels remain at the low level normally observed anterior to the furrow. Similar results were obtained with Delta clones. Reciprocally, when Notch signaling is ectopically activated in clones of Delta-expressing cells, Hairy is downregulated, both within the clone and in the cells immediately surrounding it. In these clones Emc is also downregulated within the clone, although for reasons that are not understood, Emc levels are unusually high in the wild-type cells that border the clone. The downregulation of Emc and Hairy caused by the ectopic expression of Delta correlates with increased expression of Atonal ahead of the furrow. It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001).

The most well characterized role of Notch signaling in R8 photoreceptor determination is mediating the process of lateral inhibition, which refines Atonal expression from a small group of cells to a single cell. However, an earlier and opposite role for Notch, this time promoting neural determination, has also been recognized, although how this 'proneural' function integrates with other pathways necessary for neural differentiation has been unclear. In this work, it has been shown that in normal eye development the proneural function of Notch signaling depends on prior Dpp signaling. Emc and Hairy, two negative regulators of Atonal expression, mediate the proneural function of Notch signaling in the eye. Thus, a model is proposed that links the upregulation of Atonal in the proneural groups with the downregulation of Hairy and Emc through the activation of Delta/Notch signaling (Baonza, 2001).

Thus a model is proposed specifically to integrate proneural Notch signaling into the concept of a progression of cell states, from undetermined to pre-proneural to proneural. Hh in the cells posterior to the morphogenetic furrow activates the expression of Dpp in the furrow. The data support the idea that as Dpp acts at a longer range than Hh, this relays a signal to a zone extending about 15 cells anterior to the furrow, priming these cells for differentiation. This makes cells competent to receive a later signal that upregulates Atonal expression, thereby initiating overt neural differentiation. This second signal is also dependent on Hh, but operates only much closer to the furrow: the evidence implies that it consists of Delta activating Notch signaling. The initial 'pre-proneural' state is molecularly defined by the accumulation of the repressors of atonal transcription Hairy and Emc, as well as by the positive regulator of Atonal, the HLH transcription factor Daughterless. Therefore, although Atonal and Daughterless are both expressed in this pre-proneural zone, neural differentiation is not initiated, as Hairy and Emc ensure that Atonal activity remains below a threshold. The Hh-dependent activation of Delta/Notch signaling triggers the transition from this pre-proneural state to the proneural state by downregulating both Hairy and Emc. This negative regulation of the Atonal repressors is sufficient to allow the accumulation of active Atonal in the proneural groups to a level where R8 determination is initiated (Baonza, 2001).

Notch can only trigger Atonal upregulation in a zone extending 12-15 cells anterior to the furrow, and this zone is defined as the cells that receive the diffusible factor Dpp, whose source is in the furrow. Dpp acts to define a ‘pre-proneural’ state that prepares cells for the imminent initiation of neural determination. This pre-proneural state is defined as the zone of cells that initiate Hairy and Atonal expression in response to Dpp signaling. A functional definition to this state can be added: all these cells are primed for neural differentiation because all can respond to Notch activation by upregulating Atonal levels (Baonza, 2001).

Simultaneous loss of Hairy and Emc activity leads to the precocious differentiation of photoreceptors in a competent region ahead of the morphogenetic furrow, a phenotype that resembles that caused by ectopic expression of Delta. In addition, ectopic Notch signaling downregulates Hairy and Emc ahead of the morphogenetic furrow, causing the accumulation of Atonal at high levels; conversely, loss of function of Notch signaling increased the levels of Hairy. It is concluded that Delta/Notch signaling regulates the expression of these negative regulators in the eye. Consistent with this proposal, Emc is also regulated by Notch in the developing wing disc (Baonza, 2001).

Although Notch signaling negatively regulates both Hairy and Emc, the ectopic expression of Delta does not affect both genes identically. Thus, whereas Hairy is removed both within the clone and in the neighboring cells, Emc is only downregulated autonomously within the clone. This distinction could be an artifact caused by the perdurance of ß-galactosidase. Alternatively, these differences may reflect a different requirement for Notch signaling in the regulation of both genes. Furthermore, the expression pattern of Hairy and Emc is different during the normal progression of the morphogenetic furrow. Hairy is precisely regulated, being expressed only in the cells anterior to the furrow, and is rapidly downregulated in the furrow. This precise regulation is crucial as shown by the ectopic expression of hairy. Emc has a much broader expression pattern in the eye disc, although it shows a similar upregulation followed by downregulation in the zone immediately anterior to the furrow (Baonza, 2001).

It is also worth pointing out that not only does the expression pattern of Emc and Hairy differ, but their exact mechanism of repression is also distinct. Hairy regulates bHLH proteins by a mechanism of direct DNA binding and transcriptional repression. Emc, however, forms complexes with bHLH proteins, preventing their DNA binding. Thus, Emc can antagonize the proneural function of Atonal by two distinct mechanisms: (1) Emc presumably binds to Atonal, rendering it incapable of activating its targets; (2) Emc controls the levels of Atonal. By analogy to its regulation of two other bHLH transcriptional regulators, Achaete and Scute, it is expected that Emc interferes with the autoregulatory upregulation of atonal expression. This positive autoregulation is an essential component of its accumulation in cells within the morphogenetic furrow. In conclusion, the proneural action of Notch signaling increases Atonal activity by two mechanisms: atonal is transcriptionally upregulated, and at the same time a repressive co-factor is removed. These concerted actions lead to the accumulation of active Atonal and thereby the initiation of neural differentiation (Baonza, 2001).

Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody (CM1) raised against a peptide from human caspase 3 was used to investigate how extracellular signals control spatial patterning of cell death. The antibody crossreacts specifically with dying Drosophila cells and labels the activated effector caspase Drice. The initiator caspase Dronc and the proapoptotic gene head involution defective are important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered the EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affects caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appear to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway (Yu, 2002).

A particularly useful feature of the CM1 antiserum is the detection of cells that would otherwise be marked for death but which are protected by baculovirus p35 expression. The morphological protection provided by p35 may permit better investigation of the location and autonomy of death and survival signals. On Western blots the CM1 antibody detects activated Drice but not the Drice zymogen; Drice is the Drosophila sequence most similar to the immunizing peptide. Definitive evidence that CM1-stained cells are apoptotic comes from the dependence of embryonic CM1-staining on the 75C1,2 chromosome region, and from the p35-sensitivity of larval and pupal CM1-stained cells. The morphology of all CM1-stained cells is altered by p35 expression. In the presence of p35, CM1-stained cells become indistinguishable from normal cells by morphological criteria, and are not distinguishable except by CM1 staining. Since baculovirus p35 blocks cell death by inhibiting caspase activity, p35-dependent morphology of CM1-stained cells shows that such morphology depends on caspase activity in the cells, which are therefore apoptotic. These results show that only apoptotic cells are labelled by the CM1 antiserum. Apoptotic cells that are unlabelled by CM1 might also exist, although none have been noticed (Yu, 2002).

Survival in pupal retina is regulated by two further extracellular signals that are not involved in eye imaginal discs. In principle, such signals might act to modulate Egfr signaling, to regulate Hid or DIAP activity in parallel to Egfr, or to activate initiator caspases. Notch (N) is required for caspase activation in the pupal retina. Epistasis experiments show that N is not required for pupal cell death in the absence of Egfr function, and therefore that the normal function of N is to inhibit the Egfr survival signaling pathway in pupae. Such results place N upstream of Egfr and indicate that N acts ultimately through hid and the anti-apoptotic DIAP proteins that prevent caspase activation, rather than through N-mediated caspase activation. Survival in pupal retinas also depends on signals from primary pigment cells and/or cone cells. Such signals must antagonize proapoptotic N activity, since N is epistatic to the primary pigment cell/cone cell signal. The data now imply a pathway in which primary pigment cells and/or cone cells promote survival by inhibiting activation of N, thus preventing N antagonism of Egfr activity in the interommatidial cells (Yu, 2002).

The essential role of Egfr now seems to be downstream of N, whereas the cone cell/primary pigment cell signal must act upstream. Downstream Egfr function raises anew the question of identity of the primary pigment cell/cone cell signal. Primary pigment cells or cone cells do not seem essential for Egfr activation, because N is still required for apoptosis after ablation of these cells. Pupal photoreceptor cells express the Egfr ligand SPI and its processing/presenting factor Rhomboid, and are one possible source of Egfr activation. One model suggests that primary pigment cells and/or cone cells are the source of an unidentified signal or mechanism that prevents N activation (in particular interommatidial cells) so that Egfr survival signaling can continue (Yu, 2002).

Notch (N) activation at the dorsoventral (DV) boundary of the Drosophila eye is required for early eye primordium growth. Despite the apparent DV mirror symmetry, some mutations cause a preferential loss of the ventral domain, suggesting that the growth of individual domains is asymmetrically regulated. The Lobe (L) gene is required non-autonomously for ventral growth but not dorsal growth; it mediates the proliferative effect of midline N signaling in a ventral-specific manner. L encodes a novel protein with a conserved domain. Loss of L suppresses the overproliferation phenotype of constitutive N activation in the ventral, but not in the dorsal eye, and gain of L rescues ventral tissue loss in N mutant background. Furthermore, L is necessary and sufficient for the ventral expression of a N ligand, Serrate (Ser), which affects ventral growth. These data suggest that the control of ventral Ser expression by L represents a molecular mechanism that governs asymmetrical eye growth (Chern, 2002).

It is known that N activation at the DV boundary is vital for eye disc growth. Since L is required specifically for ventral growth, it raises the possibility that L may mediate the proliferative effect of midline N signaling in the ventral eye. The Gal4-UAS system was used to test this hypothesis. Overexpression of a constitutively active N (N^intra) by the dpp-Gal4 driver, which drives expression along the posterior edge of the eye disc, causes gross overgrowth of the eye in both dorsal and ventral domains. Reducing L gene dose strongly suppresses the ventral overgrowth but has much less of an effect on dorsal overgrowth. This ventral-specific suppression of N gain-of-function phenotype suggests that L acts downstream of N (Chern, 2002).

In contrast to N-induced overgrowth, eliminating N signaling by expressing a dominant-negative form of N (N^DN) using the eyeless (ey)-Gal4 driver consistently results in small-eye or no-eye. ey-Gal4 drives Gal4 expression in early eye discs and anterior to the furrow in the third instar discs. Co-expression of L and N^DN partially suppresses this N^DN overexpression phenotype in the ventral domain: ventral eye was selectively restored in close to 20% of ey-Gal4/UAS-N^DN UAS-L animals. The size of the restored ventral eye was either smaller or equal to the reduced dorsal eye, and in no instances was ventral tissue detected without the presence of at least some dorsal tissue. The presence of residual dorsal eye indicates that N^DN overexpression may not completely eliminate endogenous N functions. It also suggests that N activity, even at a low level, is a prerequisite for L to induce ventral proliferation (Chern, 2002).

Given that the requirement of L functions is early and transient, the suppression by L of N^DN phenotype may be specific to undifferentiated cells. This is indeed the case. N^DN was overexpressed using GMR-Gal4 that induces Gal4 expression in all cells posterior to the furrow. GMR-Gal4/UAS-N^DN animals show a rough eye phenotype with a relatively normal eye size, and the eye roughness is not suppressed by overexpression of L in GMR-Gal4/UAS-N^DN UAS-L animals (Chern, 2002).

If L and N act in the same pathway, transheterozygous mutations of these two genes may result in enhanced phenotypes. L^si/+ flies have nicks at the anterior edge of the eye, but the defect is not so severe to result in half-eyes. Loss of one copy of N does not cause visible eye defects. Transheterozygote N^264-47/+; L^si/+ adults, however, had half-eyes in one or both eyes with approximately 50% penetrance. Similar enhancement is observed of L phenotype by mutations in Enhancer of split, a major downstream effector of N signaling. In summary, genetic interactions between L and N support the hypothesis that L mediates the proliferative effect of N signaling specifically in the ventral domain (Chern, 2002).

The domain-specificity of L phenotype indicates that the eye disc is partitioned, and the growth of individual domain is differentially regulated. Loss of the ventral eye in L mutants does not seem to affect DV boundary formation or the associated midline N activation, because disruptions of either of these events would result in abnormal dorsal growth. Additional data suggest that L does not affect the initial DV domain specification: (1) L is functionally downstream of N activation; (2) L mutation does not affect Ser expression at the DV boundary; and (3) domain-specific expression patterns of dpp, fng and wg are not affected in the first instar L mutant eye discs (Chern, 2002).

Consistent with this model, it is proposed that in the seemingly homogenous Ser-expressing, first instar ventral domain, there are actually two distinct groups of Ser-expressing cells: ventral midline cells abutting the dorsal midline cells, and the rest of the ventral cells. Their putative functions are different and their Ser expression is independently regulated. In the ventral midline cells, Ser is involved in setting up the DV boundary, and its expression is regulated by the Ser-N-Dl positive-feedback loop. The midline Ser expression can be further modified by Fng and Hedgehog, both of which can induce Ser expression only near the DV boundary but not elsewhere in the eye field, emphasizing again the distinctiveness of these midline cells (Chern, 2002).

By comparison, in the rest of the ventral domain, Ser is directly involved in controlling local growth. Loss of Ser in the ventral domain causes ventral-specific growth defects similar to the loss of L. Ser expression in the ventral domain may not be sustained by the Ser-N-Dl loop, since ventral Fng inhibits potential Ser-N interaction which is necessary to initiate the positive feedback loop. Instead, ventral Ser expression is regulated by L (Chern, 2002).

The data suggest the eye primordium is partitioned into dorsal, midline and ventral domains with different gene expression and growth properties. It highlights the importance of local cellular context in interpreting signals released from the domain boundaries and shows that the growth of symmetrical domains may be asymmetrically regulated. The model may also be applicable to the development of other imaginal discs as well as other developmental systems (Chern, 2002).

Phospholipid membrane composition affects EGF Receptor and Notch signaling through effects on endocytosis during Drosophila development

The role of phospholipids in the regulation of membrane trafficking and signaling is largely unknown. Phosphatidylcholine (PC) is a main component of the plasma membrane. Mutants in the Drosophila CTP-phosphocholine cytidylyltransferase 1 (Cct1), the rate-limiting enzyme in PC biosynthesis, show an altered phospholipid composition with reduced PC and increased phosphatidylinositol (PI) levels. Phenotypic features of Cct1 indicate that the enzyme is not required for cell survival, but serves a role in endocytic regulation. Cct1^- cells show an increase in endocytosis and enlarged endosomal compartments, whereas lysosomal delivery is unchanged. As a consequence, an increase in endocytic localization of EGF receptor (Egfr) and Notch is observed, and this correlates with a reduction in signaling strength and leads to patterning defects. A further link between PC/PI content, endocytosis, and signaling is supported by genetic interactions of Cct1 with Egfr, Notch, and genes affecting endosomal traffic (Weber, 2003).

Drosophila Cct1 is highly homologous to fly Cct2 and to CCT genes from other species ranging from yeast to human. Cct2, is located immediately downstream of Cct1. CCT catalyzes the formation of CDP-choline during PC biogenesis. Thus, it was surprising to find specific phenotypes associated with Cct1. To confirm that the Cct1 mutations indeed affect PC biogenesis, the membrane composition of wild-type and Cct1 mutant larvae were compared. Strikingly, PC levels were reduced in membrane preparations from CCT1¹⁶⁹¹⁹/Df(3L)emc5 and dCCT1^EP0831/Df(3L)emc5 animals, whereas an increase in PI levels was detected. These data confirm that Cct1 is required for PC biogenesis and demonstrate that in Cct1 mutants the ratio of PC, PI, and PE is changed (Weber, 2003).

To determine the requirements of Cct1 during cellular differentiation and development and because Cct1 null mutants are lethal, Cct1 mutant tissue was generated during eye development. Surprisingly, CCT1⁻ cells initially develop normally with no obvious defects in cellular architectureand give rise to tissue comparable in size to wild-type, indicating that Cct1 is not required for cell growth or survival. Cct1⁻ eye disc clones revealed patterning defects similar to those observed in adult eyes of the hypomorphic P alleles. Mutant ommatidial clusters often showed typical polarity defects. Chirality defects are apparent using the (largely) R4-specific mδ-lacZ reporter, which reflects cell fate selection in the R3/R4 pair and thus ommatidial chirality. Mutant or mosaic clusters often show aberrant mδ-lacZ expression, reflecting defects in Fz/Notch signaling in the R3/R4 pair. The photoreceptor R1/R6-specific marker Bar reveals the general orientation of developing ommatidial clusters and their degree of rotation. In Cct1 clones, many clusters have an aberrant orientation, displaying defects in ommatidial rotation. These data suggest that Cct1 can affect specific processes during eye development, in particular those related to planar cell polarization (PCP; Frizzled or Notch signaling) and ommatidial rotation (Egfr signaling) (Weber, 2003).

Cct1 is additionally required during terminal photoreceptor differentiation. Although Cct1 null mutant photoreceptor cells initially appear normal, they do not form the light-harvesting organelle, the rhabdomere. The rhabdomeres, containing the rhodopsin proteins, are large stacks of membrane microvilli that are formed at the apical membrane domain of each photoreceptor during their terminal differentiation. Transmission electron microscopy (TEM) analysis of Cct1 mutant photoreceptors has revealed that the rhabdomeres failed to properly form (with only remnants of rhabdomeres sometimes to be found. This defect is not light or age dependentas is the case in many phototransduction mutants, indicating that Cct1 is autonomously required during the terminal differentiation of the rhabdomeric membrane stacks. Rhabdomere morphogenesis is thought to be a “burst” of apical membrane synthesis, and it is also affected by blocking dynamin function (via photoreceptor-specific shi^ts expression. Thus, this phenotype can be attributed to membrane trafficking defects (Weber, 2003).

The phenotypic features of Cct1 during eye development suggest a requirement in one or several signaling pathways. Thus, genetic interactions were tested between Cct1 and components of most of the common signal transduction cascades. Strikingly, Cct1 interacts with Egfr signaling. Cct1 alleles dominantly suppress the gain-of-function (GOF) Ellipse allele of Egfr (Egfr^ElpB1). The Egfr^ElpB1 phenotype is apparent in reduced eye size and loss of photoreceptors and ectopic veins in the wing. All aspects of the Egfr^ElpB1 phenotype arer suppressed by Cct1 alleles. The degree and quality of suppression was comparable to that of loss-of-function (LOF) alleles of components of the Egfr/Ras pathway, suggesting a positive Cct1 requirement in Egfr signaling. This observation was confirmed by the inverse interaction. The homozygous CCT1¹⁶⁹¹⁹ eye phenotype is dominantly enhanced by Egfr. The interactions between Cct1 and Egfr are consistent with the Cct1 eye phenotypes, as Egfr is required for photoreceptor induction, survival, and ommatidial rotation (Weber, 2003).

The ommatidial chirality defects in Cct1 mutants suggest a role in PCP establishment, which is governed by an interplay of Fz-Notch signaling. This is supported by the observation that Cct1 alleles suppress the PCP GOF phenotypes of sev-Fz and sev-Dsh, which display PCP defects with chirality inversions and symmetrical clusters. Cct1 dominantly suppress these aspects in both backgrounds, with many more ommatidia displaying a chiral arrangement. Since PCP GOF eye phenotypes are modified by components of Fz or Notch pathways, this was addressed further and an interaction between Cct1 and Notch signaling in general was tested (Weber, 2003).

Notch is haploinsufficient, showing a dominant LOF phenotype in strong alleles in the notching of the wing margin. Genetic modifications of this phenotype have been generally used to dissect the requirements of other genes in the context of Notch signaling. In the N null allele, N^55e11/+, the wing notching phenotype is enhanced by removing one copy of Cct1, both with respect to the extent and frequency of the notch. In addition, although N^55e11/+ flies have no dominant eye phenotype in a wild-type background, the simultaneous removal of one copy of Cct1 and aos gives rise to rough eyes with many chirality defects and a partial loss of the ventral eye. Such triple-heterozygous eyes resemble the stronger CCT1¹⁶⁹¹⁹/Df(emc5) phenotype. Thus, although neither N nor Egfr signaling show dominant LOF eye phenotypes, the simultaneous removal of one copy of Cct1 results in a haploinsufficiency of this genotype. This not only supports a requirement for CCT1 in N signaling, but also suggests a link between N and Egfr signaling, possibly mediated through Cct1. Moreover, the homozygous CCT1¹⁶⁹¹⁹ phenotype is strongly enhanced by a Notch⁻/+ background, with severe wing notching and dramatically reduced eyes in rare escapers. These observations support the notion that Cct1 is required for Notch signaling in several developmental contexts (Weber, 2003).

Surprisingly, the CCT enzymes are not required for cellular survival, as even clones of the double null allele (CCT²⁹⁹) show the same phenotypic features as the CCT1¹⁷⁹ null, indicating that a cell can function with reduced PC and increased PI levels. This suggests that the alternate pathway of PE methylation can synthesize enough PC or that cells can function in many aspects with low PC levels. However, a decrease in PC (and an increase in PI) levels affects regulatory aspects of membrane dynamics and endocytosis. In addition, Cct1 is the major gene responsible for the observed defects, since CCT1¹⁶⁹¹⁹ and CCT1¹⁷⁹ show the same phenotypes as the double null CCT²⁹⁹ allele (Weber, 2003).

The primary Cct1 defect is an increase in the rate of endocytosis. In particular, more clathrin-coated pits (CCPs) and vesicles are observed, leading also to a size increase of the endosomal compartment. However, membrane traffic progression through the endosomal compartment to the lysosome is not affected. This is in contrast to dynamin mutants (shi), where CCPs fail to pinch off and thus endocytosis is blocked (Weber, 2003).

An explanation for the increase of endocytosis in Cct1 mutants might lie in the fact that decreased PC and increased PI levels affect the dynamics of the assembly and/or removal of the clathrin coat. PIP₂ (derived from PI) has been suggested to serve as a 'docking site' for clathrin coat-promoting proteins, such as Epsin. It has been proposed that several factors, including dynamin, amphyphysin, and endophilin, can also initiate vesicle budding. An increase in PI and change in the PC/PI ratio is also interesting in the context of the proposal that Sec14p could monitor the PC/PI ratio and influence endocytosis. An alternative cause for the phenotypes observed might be the fact that the plasma membrane is normally asymmetric, with a difference in phospholipid composition between the outer and inner plasma membrane layers. Mammalian plasma membranes have higher PC content in the outer layer as compared to the inside face, suggesting that PC levels in the outer membrane layer serve a specific function (Weber, 2003).

Cell surface receptor internalization and subsequent sorting to multivesicular bodies and lysosomal delivery are important mechanisms of signaling modulation. The phospholipid composition of the plasma membrane affects this process. The enlarged endosomal compartment is likely a consequence of the increase in endocytosis. Genetic and histological analyses suggest that Egfr and N are sensitive to Cct1 function and possibly PC/PI levels. The Garland cell analysis and genetic interactions indicate that Cct1⁻ cells have an increased rate of endocytosis, suggesting that plasma membrane (PM) proteins spend a shorter time at the plasma membrane with a reduced chance to interact with their ligands. As membrane trafficking to the lysosome is functioning in Cct1⁻ cells, the overall levels of PM receptors might be reduced. Although the N and Egfr pathways are affected the most, it is likely that other receptors are also affected. Not all RTKs are equally sensitive to Cct1 activity, however; for example, Sevenless (Sev) does not interact with Cct1. Furthermore, internalization of the Sev/Boss (Sev-ligand) complex is affected by hk mutants but is not manifest in an R7 loss phenotype. This suggests that signaling levels require tight regulation for some RTKs, such as Egfr, with multiple distinct responses at different signaling levels, as compared to others such as Sev, with a simple on/off situation (Weber, 2003).

Both Egfr and N signaling levels are reduced in Cct1 mutants, as deduced from the genetic interactions. The peak signaling levels for both pathways are affected in Cct1, whereas other aspects of their readout are less affected, supporting the notion that Cct1 function is important for signaling level output in certain contexts only. Consistently for both pathways, regulated ligand and receptor processing and trafficking are critical for correct signaling levels. Moreover, since Cct1 expression can be regulated by signaling input, a given cell population could regulate aspects of endocytosis through its membrane PC content (Weber, 2003).

It has recently been shown that a regulatory crosstalk between Egfr and Notch signaling is mediated through the transcriptional upregulation of a hypothetical factor by Egfr/Ras, leading to endocytosis and downregulation of Notch/LIN-12 in C. elegans. Strikingly, a heterozygous condition for both signaling pathways (that has no phenotypic consequence by itself) shows dramatic defects in a Cct1⁻/+ background, also suggesting crosstalk between Egfr and Notch signaling. Thus, some aspects of the crosstalk between these and other pathways might be generally regulated or mediated through membrane phospholipid contents (Weber, 2003).

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