- THIS ARTICLE
-
Abstract
- Full Text (PDF)
- Alert me when this article is cited
- Alert me if a correction is posted
- SERVICES
- Email this article to a friend
- Similar articles in this journal
- Similar articles in PubMed
- Alert me to new issues of the journal
- Download to citation manager
- Reprints & Permissions
- CITING ARTICLES
- Citing Articles via HighWire
- Citing Articles via Google Scholar
- GOOGLE SCHOLAR
- Articles by Tanenbaum, S. B.
- Articles by Cagan, R. L.
- Search for Related Content
- PUBMED
- PubMed Citation
- Articles by Tanenbaum, S. B.
- Articles by Cagan, R. L.
A Screen for Dominant Modifiers of the irreC-rst Cell Death Phenotype in the Developing Drosophila Retina
Sara B. Tanenbaum1,a, Sharon M. Gorski1,2,a, Jamie C. Rusconia, and Ross L. Caganaa Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Saint Louis, Missouri 63110
Corresponding author: Ross L. Cagan, Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Campus Box 8103, 660 S. Euclid Ave., St. Louis, MO 63110., cagan{at}molecool.wustl.edu (E-mail)
Communicating editor: K. ANDERSON
 | ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Programmed cell death (PCD) in the Drosophila retina requires activity of the irregular chiasmC-roughest (irreC-rst) gene. Loss-of-function mutations in irreC-rst block PCD during retinal development and lead to a rough eye phenotype in the adult. To identify genes that interact with irreC-rst and may be involved in PCD, we conducted a genetic screen for dominant enhancers and suppressors of the adult rough eye phenotype. We screened 150,000 mutagenized flies and recovered 170 dominant modifiers that localized primarily to the second and third chromosomes. At least two allelic groups correspond to previously identified death regulators, Delta and dRas1. Examination of retinae from homozygous viable mutants indicated two major phenotypic classes. One class exhibited pleiotropic defects while the other class exhibited defects specific to the cell population that normally undergoes PCD.
PROGRAMMED cell death (PCD) is an essential process that occurs during the normal development of both invertebrates and vertebrates. Often referred to as a developmentally regulated cell suicide program, PCD most often culminates in a morphologically defined form of cell death termed apoptosis (
The major mediators of apoptotic death, the caspases, have been identified in a variety of organisms (
Cell death in Drosophila involves a number of conserved components including caspases (
The Drosophila retina is especially useful for investigating the signaling pathways involved in cell survival and cell death due to its simplicity and the powerful tools available for its study. The morphology of death has been well characterized and mutations that affect the death decision result in an adult rough eye phenotype that is readily scored (
750 unit eyes, or ommatidia. Each ommatidium is composed of 14 cells: 8 photoreceptor neurons, 4 (nonneuronal) cone cells, and 2 optically insulating primary pigment cells (1°s). Between ommatidia is an interweaving hexagonal lattice of secondary/tertiary pigment cells (2°/3°s) and mechanosensory bristles. The structure of the interommatidial lattice is the result of the final cell fate decision in the retina, cell death vs. the 2°/3° fate. Cells are organized in part by selective PCD, involving the death of 1500–2000 excess interommatidial cells (Fig 1A and Fig B;
|
One of the first genes demonstrated to be involved in Drosophila retinal PCD was irreC-rst. Loss-of-function mutations in irreC-rst result in a block in retinal PCD during both the larval and pupal stages, leading to an excess of interommatidial cells and a corresponding rough eye phenotype in the adult (
To provide potential new entry points for investigating mechanisms of PCD in the developing retina, we conducted a genetic screen for modifiers of the cell death phenotype conferred by a partial loss-of-function allele of irreC-rst. By using a mutation in a gene encoding a transmembrane protein, our aim was to identify upstream components of the cell death process. This is an important point since it is the upstream, or proximal, portions of cell death signaling pathways that are the least understood. We chose to employ a dominant enhancer/suppressor screening strategy that is based on the rationale that in a sensitized background the removal of a single copy of a gene can result in the dominant modification of a target phenotype. A particular advantage of this type of genetic screen is that it can identify homozygous lethal mutations in the F1 generation. Successful adaptations of the strategy are numerous (
| MATERIALS AND METHODS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Mutagenesis and screening:
Fly cultures and crosses were performed at 22.5° unless stated otherwise. Male irreC-rst[3] flies were starved for a day and then fed overnight a 1% sucrose solution containing 3.3 mM N-ethyl-N-nitrosourea (ENU). In each bottle, 50 male flies were mated with 25 y irreC-rst[3]; dp[ov]; p[p] virgin females (Fig 2A). Each line containing irreC-rst[3] used in the screen was derived from a single pair mating to reduce genetic background variability. F1 progeny were scored for enhancement or suppression of the irreC-rst[3] rough eye phenotype using a dissecting microscope. Because ENU results in mosaicism in the F1 (i.e., some clones of cells contain the mutation while others do not), we scored both eyes of every F1 for an effect. Approximately 150,000 F1 progeny were scored. Each round of mutagenesis/mating was designated by a letter, in alphabetical order, and each mutant F1 was named with its corresponding letter followed by a unique number.
|
Linkage analysis of modifiers:
F1 mutant progeny were backcrossed to y irreC-rst[3]; dp[ov]; p[p] flies to test for germline transmission of the mutation and to establish chromosomal linkage as outlined in Fig 2B. The F1 mutant males were backcrossed to y irreC-rst[3]; dp[ov]; p[p] virgin females and F2 males and females were rescored for the modified phenotype. If the modified phenotype and the pink[p] phenotype were observed in the same flies, then the mutation mapped to the second chromosome. If the modified phenotype and the dumpy[ov] phenotype were observed in the same flies, then the mutation mapped to the third chromosome. The unmarked fourth chromosome was not considered in these analyses. The F1 mutant virgin females were backcrossed to y irreC-rst[3]; dp[ov]; p[p] males and modified F2 males that were non-dumpy[ov], non-pink[p] were selected. These males were subsequently analyzed like F1 mutant males to establish chromosomal linkage. If in the subsequent F3 generation only modified females were observed, then the mutation was X-linked and viable. If no modified males were observed in the F2 generation (from an F1 mutant female), but modified females were observed, then the mutation was X-linked and lethal.
Balancing modifiers:
Once chromosomal linkage was determined, the dominant modifiers were balanced (Fig 2B). For second chromosome mutations, modified males were mated to y irreC-rst[3]; +/CyO virgin females and non-dumpy[ov], Curly siblings were used to establish a balanced stock. For third chromosome mutations, modified males were mated to y irreC-rst[3]; p[p]/TM3, Ser, p[p] virgin females and non-pink[p], Serrate siblings were used to establish a balanced stock. For X-linked lethal mutations, modified y irreC-rst[3] virgin females were mated to FM7 males. Individual y irreC-rst[3] (*)/FM7 virgins were then selected and mated to FM7 males (an asterisk indicates the mutagenized chromosome). Balanced stocks were maintained from lines producing only FM7 males (Fig 2B).
Genetic interaction tests:
To help classify mutations, a tester strain was placed in combination with the mutations, and possible genetic interactions were scored by viewing under a dissecting microscope. Male flies of the genotype y irreC-rst[3]; */CyO or y irreC-rst[3]; */TM3 were mated to virgin females from the tester strains and the effects of the mutant chromosome vs. the balancer chromosome were compared in the F1 males (an asterisk indicates the mutagenized chromosome). Tester strains included irreC-rst[UB883], irreC-rst[CT] (provided by K. Fischbach), GMR-rpr (1x), GMR-hid (provided by H. Steller), In(1)w[m4] (Bloomington Stock Center), and 39C-34 (provided by L. Wallrath). Line 39C-34 is w- and homozygous for an hsp-70 white transgene located on the fourth chromosome; it is sensitive to known suppressors of position effect variegation (PEV; L. WALLRATH, personal communication;
Complementation tests and mapping:
Complementation tests were based initially on lethality and conducted among modifiers of the same type on the same chromosome. Subsequent complementation tests were conducted among both lethal and nonlethal modifiers of the same type that mapped to the same chromosomal region. These latter tests were based on eye phenotype, performed at 29°, and scored in the absence of irreC-rst[3]. All modifiers, with the exception of 10 lines, were meiotically mapped based on their genetic interaction with irreC-rst[3]. The 10 lines were not meiotically mapped because they were especially weak stocks; they were included, however, in complementation analyses. Second chromosome modifier males, y irreC-rst[3]; */CyO, were mated to al dp b pr c px sp virgin females (an asterisk indicates the mutagenized chromosome). The F1 y irreC-rst[3]/+; */al dp b pr c px sp virgin females were then backcrossed to al dp b pr c px sp males and F2 y irreC-rst[3]; *[al dp b pr c px sp]/al dp b pr c px sp males were scored.
A similar scheme for mapping third chromosome mutations was performed using the st ry sr e mapping strain. Approximately 200 F2 recombinant males were scored for each mutant line. Additional F2 recombinants (50–100) were scored for some third chromosome mutants using the mapping strains ss e tx or mwh jv. Since y and irreC-rst[3] are closely linked and we scored a large number of F2 males, we assumed that effects due to recombination events between y and irreC-rst[3] in F1 virgin females were negligible. Once approximate map positions were determined, further mapping was performed by testing mutants for complementation with deficiencies and known genes in the relevant regions.
One group of special note is the lethal lines in which, based on meiotic and deficiency mapping, the mutation responsible for the eye phenotype is apparently distinct from the mutation causing lethality. This group includes: B37, B183, B246, B273, C14, C109, C137, C329, C451, C463, C383, C478, C532, D49, D17, D151, D187, D207, D231, E156, E218, E252, E304, G30, G67, H74, H94, I19, I42, I47, J88, K45, and K46.
Construction of non-irreC-rst[3] lines:
Males from second chromosome modifier lines, y irreC-rst[3]; */CyO were mated to y; SLM, Bl, y+/Sp virgin females (an asterisk indicates the mutagenized chromosome), F1y; */SLM, Bl y+ males were selected and backcrossed to y; SLM, Bl, y+/Sp virgin females. F2 y; */SLM, Bl, y+ siblings were mated to construct a balanced line. Homozygous */* larvae were detected as yellow animals. For third chromosome modifiers, y irreC-rst[3]; */TM3, Ser males were mated to TM3, Sb/TM6b, Tb virgin females. F1 +; */TM6b, Tb males were backcrossed to parental balancer females and F2 */TM6b siblings were used to construct a balanced stock. Homozygous mutant larvae and pupae were detected as non-Tubby animals.
Immunohistochemistry:
Post-death stage pupal retinae were analyzed at 42 hr after puparium formation (APF) at 25°, 34 hr APF at 29°, or 50 hr APF at 22.5°. Immunostaining for IrreC-rst and Armadillo was performed with mAb 24A5.1 to IrreC-rst (1:50; gift from K. Fischbach;
| RESULTS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Identification of a weak allele of irreC-rst:
To identify dominant modifiers of irreC-rst, we required an allele of irreC-rst that results in an intermediate "sensitized" eye phenotype. The severity of several irreC-rst mutations (irreC-rst[UB883], irreC-rst[IR34], irreC-rst[3], irreC-rst [CT]) was analyzed by examining the exterior roughness of the adult eye and cellular morphology of the pupal eye. The irreC-rst[3] mutation best fits the phenotypic criteria: it gives rise to an adult eye that is intermediate in roughness compared to wild type and irreC-rst[CT], an allele that appears to have a strong hypomorphic eye phenotype (
The irreC-rst[3] mutation is due to a chromosomal rearrangement that places the irreC-rst locus next to heterochromatin, resulting in PEV of gene expression (
The genetic screen:
The protocol used to isolate dominant enhancers and suppressors of irreC-rst[3] is outlined in Fig 2A. Approximately 150,000 ENU mutagenized F1 flies were screened for modifications of the irreC-rst[3] rough eye phenotype. In particular, we looked for effects on the alignment of ommatidial rows, which requires normal lattice formation and PCD. F1 dominant modifiers were isolated and backcrossed to parental strains to test for germline transmission and to determine chromosomal linkage (Fig 2B; MATERIALS AND METHODS). Because ENU results in mosaicism, many mutagenized F1 progeny carried a mutation in their eye primordia that was not found in the germline primordia. We found that 15% of mutations recovered in the F1 were transmitted to the F2 generation. Of these, we were able to construct 170 stable balanced lines (Fig 2B; MATERIALS AND METHODS). These represent 84 dominant enhancers and 86 dominant suppressors of irreC-rst[3].
Genetic tests identify nonspecific modifiers:
Due to the nature of the irreC-rst[3] mutation and our method of screening eye roughness, we expected that only a subset of the modifiers would be involved specifically in irreC-rst function and retinal PCD. Examples of types of genes in which we expected to recover mutations include the following: (i) modifiers of PEV, (ii) general transcription and translation factors, (iii) genes with dominant rough eye phenotypes, (iv) genes involved in processes in the eye other than cell death/cell survival, and (v) genes involved specifically in the regulation of death. Several genetic tests and phenotypic assays, described below, were used to help distinguish these classes.
To identify irreC-rst[3] modifiers that acted through regulation of PEV, we tested each mutant line for an effect on two different tester lines sensitive to known modifiers of PEV, 39C-34 and In(1)w[m4] (see MATERIALS AND METHODS). Of our 170 modifiers of irreC-rst[3], we identified 49 that behaved as general suppressors of PEV and no modifiers that behaved as general enhancers of PEV (Table 1). Notably, other loci that exhibit PEV are sensitive to suppressors but not enhancers of PEV (
|
Fly lines with dominant rough eye phenotypes were identified by removing irreC-rst[3] from each modifier line. Five second chromosome "enhancers" exhibited a dominant rough eye phenotype (Table 1). Complementation tests indicated that all five mutations were alleles of Star (
Remaining modifiers:
The remaining 116 dominant modifiers of irreC-rst[3] are enumerated in Table 2 according to modifier activity and chromosomal location. Two mutations, both enhancers, are located on the X chromosome. A total of 24 suppressors are located on the second chromosome, and 13 suppressors are located on the third chromosome. A total of 5 and 72 enhancers mapped to the second and third chromosomes, respectively. Examples of an enhancer and suppressor phenotype in combination with irreC-rst[3] are shown in Fig 1J and Fig K.
|
Complementation tests and mapping:
To determine allelism among the modifiers, complementation tests and genetic mapping were undertaken. Initially, complementation tests were based on lethality and conducted between homozygous lethal modifiers of the same type on a given chromosome. These tests yielded four lethal complementation groups containing multiple alleles, all third chromosome enhancers (Table 3, underlined). These represent only 10 of the 64 homozygous lethal lines. The remaining lines represent either single alleles of essential genes or they contain a second site lethal mutation distinct from the modifying mutation. The latter is most likely the case for at least 33 of the homozygous lethal lines: in these lines, deficiencies failed to complement the eye phenotypes (see below and MATERIALS AND METHODS) but did complement the lethality, indicating that the two are distinct.
|
Modifying mutations were meiotically mapped to a chromosomal region based on their phenotypic interaction with irreC-rst[3]; lethality was not used as a basis for mapping to ensure that we mapped the modifier mutation and not any unrelated second site lethal mutations present in the stock. Based on the resultant map positions, we then conducted complementation tests between mutants of the same type that mapped to the same chromosomal region. The modifier lines in which irreC-rst[3] was removed (see above) were used for these tests: trans-heterozygotes were analyzed for a rough eye phenotype. These tests were carried out at 29° since we observed that a greater number of the homozygous viable mutants yielded a detectable phenotype at this temperature compared to our standard 22.5°. Similar complementation tests with deficiency lines were used to localize the mutations more precisely and to assist in construction of complementation groups. The complementation groups and their members are shown in Table 3. We identified 3 groups of suppressors and 1 group of enhancers on the second chromosome, and at least 2 groups of suppressors and 10 groups of enhancers on the third chromosome. (Mutations in the 97B-97E region demonstrated complex complementation, suggesting the presence of complex loci or synthetic interactions.) A total of 43 mutations have not yet been placed into complementation groups and are listed also in Table 3 by modifying activity and map position. Some of the modifiers are mapped to a chromosome or chromosome arm only; these modifiers were difficult to map due to weak stocks or subtle phenotypes.
Additional genetic tests:
To characterize further the second and third chromosome modifiers, we tested their ability to alter the eye phenotype of other alleles of irreC-rst. Suppressors were examined for an effect on irreC-rst[CT], a mutation that results in a truncation of the IrreC-rst protein (
Finally, one line (H42) was identified as a suppressor of GMR-hid. No genetic modifiers of GMR-rpr were identified.
Description of phenotypic classes:
To distinguish between mutants with early eye defects and those with abnormalities related to pupal stage cell death/cell survival, we conducted phenotypic analyses at the cellular level. Our analyses presented here consider the homozygous viable lines; further clarification regarding the nature of the modifying mutation in the homozygous lethal lines, i.e., determining whether the modifying activity and lethality are coincident, is required prior to their characterization (see MATERIALS AND METHODS). A total of 52 homozygous viable lines with the irreC-rst[3] mutation removed were analyzed. To assess cellular organization at the surface, 34-hr pupal retinae (at 29°, equivalent to 42 hr at 25°) were visualized with anti-Armadillo antibody.
A number of mutants (11) did not have a detectable pupal eye phenotype (Table 4). These may represent genes that do not have independent eye phenotypes or they may be extremely weak alleles that result in a viable eye phenotype only in trans with stronger alleles. The remaining mutants fall into two major classes (Table 4).
|
Class 1. Pleiotropic defects: A total of 27 mutations affected the early development of the retina, as assessed by abnormal cone cell clusters in 34-hr (29°) pupal retinae (defects in cone cells typically reflect earlier defects in the underlying photoreceptor neurons). Some of these mutant lines exhibited a reduced number of cone cells accompanied by an increase or decrease in interommatidial cells (Fig 3). Other mutant lines displayed an increase in the number of cone cells; these cone cell defects were often, but not always, accompanied by an increase in the number of interommatidial cells (Fig 3). The majority of mutant lines that exhibited pleiotropic defects had variable numbers of cone cells accompanied by lattice defects and/or other general defects including variable numbers of 1°s and misarranged bristles (Fig 3).
|
Class 2. Lattice-specific defects:
Our screen also identified several mutant lines (11 as homozygotes and 1 as a heteroallelic combination) with defects that appear to be limited to the 2°/3° pigment cells of the interommatidial lattice (Fig 4). These mutations result in a small number of excess 2°/3°s but do not appear to disrupt early retinal development. The majority of extra cells were found adjacent to bristles. This is not surprising because regions next to bristles normally contain the greatest number of extra cells prior to PCD (
|
Two of the well-defined complementation groups contained member lines that were homozygous viable and thus allowed a comparison of phenotypes within complementation groups. We found that the homozygous mutant phenotypes within these two complementation groups [mapped to Df(2L)M24F-B and Df(3R)dr-rv1; Table 3] were consistent with respect to the two major phenotypic classes. Members of a single complementation group demonstrated either pleiotropic or lattice-specific defects, but not both. In addition, both pleiotropic and lattice-specific complementation groups included a homozygous viable allele that appeared wild type, supporting the notion that at least some members of the wild-type class represent weak alleles that result in a detectable eye phenotype only in trans with stronger alleles.
| DISCUSSION |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
In this article we present the results of a large-scale enhancer/suppressor screen for modifiers of irreC-rst[3], a mutation that blocks PCD in the Drosophila retina. In addition to the intermediate nature and specificity of its phenotype, the irreC-rst[3] mutation was particularly advantageous for studying PCD because of the transmembrane, i.e., upstream, position of the IrreC-rst gene product. It is the upstream or proximal portions of cell death pathways that are perhaps the least well understood. We have identified mutations in a number of genes that represent candidates for the regulation or execution of the death process.
Our analyses indicated that the variegated nature of the irreC-rst[3] phenotype was actually quite useful for isolating modifying mutations: ectopic cells are concentrated in patches in irreC-rst retinae making them more likely to cause visible alterations in the ommatidial rows of the adult eye. We found that examining mutant adult eyes in a non-irreC-rst background was a less reliable method for detecting mutations in PCD. Mutations that resulted in a small number of ectopic but evenly dispersed interommatidial cells, while detectable in pupal retinae, were sometimes difficult or impossible to detect in the adult.
Surprisingly, both enhancer mutations and suppressor mutations, when placed in a background wild type for irreC-rst, blocked PCD. None were seen to reduce unambiguously the number of interommatidial cells; however, the arrangement of interommatidial cells was extremely aberrant in some lines and assessing levels of PCD was difficult. One might have anticipated that enhancers would resemble mutations in irreC-rst, having extra interommatidial cells, but that suppressors would direct ectopic cell death. Our finding may be explained by the observation that ectopic overexpression of irreC-rst also blocks PCD (
Finally, none of our identified dominant suppressors of irreC-rst[3] also suppressed irreC-rst[CT]. This result is consistent with the view that irreC-rst[CT] represents close to a "null" for irreC-rst activity in the retina. The extremely weak nature of the irreC-rst[UB883] allele also made it a poor identifier of enhancers and it was unusable for identifying suppressors. It appears, then, that irreC-rst[3] was the best choice for the screen.
Secondary screens:
Several secondary screens were conducted to identify those mutants that warranted future study. First, we anticipated identifying modifiers of PEV. Several suppressors of irreC-rst[3] that also suppressed other PEV phenotypes were identified. Most of these mutants behaved similarly with the two PEV tester lines; however, a small number of the mutants only suppressed the variegation phenotype of one of the tester lines, suggesting that still more tester lines might identify additional PEV suppressors. We did not identify any enhancers of PEV with our tester lines. In addition, only one of four known enhancers of PEV that were tested directly enhanced irreC-rst[3], suggesting that the number of enhancers of PEV that we may have obtained in the screen is small.
Interaction tests between our mutants and GMR-rpr or GMR-hid yielded only a single (GMR-hid) suppressor. The failure of most irreC-rst[3] modifiers to affect GMR-rpr or GMR-hid is not surprising: direct tests (i) between irreC-rst[3] and GMR-rpr or GMR-hid and (ii) between irreC-rst[3] and a deficiency (Df(3L)H99) that removes reaper, hid, and grim also failed to demonstrate any interaction (data not shown). Lack of a genetic interaction suggests that these genes may act in separate pathways to regulate the death process.
Enhancer and suppressor complementation groups:
On the basis of complementation testing, we isolated at least 4 second chromosome and 12 third chromosome complementation groups. Most of these irreC-rst[3] modifier groups consist of multiple members that display a rough eye in trans to other members of their group. We cannot exclude the possibility that some apparently allelic combinations were instead due to synthetic interactions between alleles of different genes, although synthetic interactions of this sort are rare and are often allele specific.
Surprisingly, we identified only two interacting mutations on the X chromosome, both enhancers. In part this is due to the fact that we mutagenized males: X chromosome mutations could be isolated only in F1 females, effectively halving the screen size for the X chromosome. In addition, an unexpected skewing in the distribution of mutations was realized by the observation that only a small number of the mutants isolated on the second and third chromosomes came from F1 females, and all were enhancers. The starting irreC-rst[3] stock had a weaker, less visible eye phenotype in females. This observation suggests that suppression was difficult to detect in females and that females are less sensitive to enhancement of the irreC-rst[3] phenotype. Together, these observations indicate that the true effective size of the screen for mutants on the X chromosome was far smaller than for the second and third chromosomes.
dRas1:
We identified one line in the genetic screen, J108, which presented a weak dominant rough eye and is an allele of dRas1. Because the eye phenotype of irreC-rst[3]; dRas1[J108]/+ was significantly more severe than either the irreC-rst[3] hemizygous phenotype or the dRas1[J108] dominant phenotype alone, we believe that this allele of dRas1 represents a true enhancer of irreC-rst. Supporting this contention, two loss-of-function alleles of dRas1 [alleles l(3)06677 and 
C406] acted as dominant suppressors of the irreC-rst[3] phenotype (C. BRACHMANN, personal communication). This further demonstrates that dRas1 may act near irreC-rst in a pathway regulating PCD. The unusual dominant phenotype of dRas1[J108], in addition to its failure to complement dRas1 loss-of-function alleles, indicates that the mutation may represent an antimorphic or neomorphic form of the gene. Identifying the molecular lesion will provide a better understanding of the nature of this dRas1 allele.
Prior studies demonstrated a role for the dEGFR/dRas1 pathway in the regulation of PCD within the Drosophila retina. Laser ablation studies in the Drosophila retina revealed that the cone cells and 1°s provide a survival signal to the neighboring interommatidial lattice, perhaps through localized activation of the dEGFR/dRas1 pathway (
Delta:
We isolated four alleles of Delta as dominant enhancers of irreC-rst[3]. Although we can detect very subtle phenotypic defects in some of these Delta lines, the observed dramatic enhancement was significantly more than additive; in addition, we detect strong modifier activity with other Delta alleles that do not exhibit any dominant eye phenotype on their own. Again, this is intriguing as Delta and its receptor Notch represent another pathway implicated previously in PCD (
Phenotypes:
Our isolated homozygous viable mutant lines corresponded to two major phenotypic classes. The first class demonstrated pleiotropic phenotypic effects while mutants in the second class appeared to affect specification of interommatidial cells exclusively.
Mutant lines with pleiotropic effects exhibited an aberrant number of cone cells. Alteration in the number of cone cells is often an indicator of earlier defects: for example, abnormal photoreceptor differentiation can lead to subsequent abnormal cone cell recruitment (photoreceptor differentiation has not been assessed in these lines). Many mutant lines contained a variable number of cone cells within each ommatidium, often five and sometimes three; these were in addition to ectopic 2°/3° cells. Cone cells provide a signal that rescues 2°/3° precursors from death (
Perhaps of greatest interest are the lines exhibiting defects specific to the interommatidial lattice. Ommatidia from these lines often contained an additional cell, the cone-contact cell, positioned between their two 1°s. Cone-contact cells have been observed also in retinae overexpressing the caspase inhibitor p35 (
The collection of mutants created in this work represent modifiers of the irreC-rst[3] retinal phenotype. Previous studies have indicated a role for irreC-rst in cell adhesion and in the rearrangement of interommatidial cells that occurs prior to and during the pupal PCD stage (
| FOOTNOTES |
|---|
1 These authors contributed equally to this work. 
2 Present address: Genome Sequence Centre, British Columbia Cancer Research Centre, Vancouver, BC V5Z 4E6, Canada.
| ACKNOWLEDGMENTS |
|---|
We thank I. Duncan for important advice on design of the genetic screen and M. Marra for helpful comments and discussions. We also thank H. Steller, L. Wallrath, I. Duncan, and the Bloomington and Umeå Stock Centers for fly strains, and K. Fischbach for fly strains and mAb 24A5.1. This work was supported by grant R01 EY11495 from the National Institutes of Health (NIH); J.R. received support from NIH grant EY07057.
Manuscript received November 18, 1999; Accepted for publication May 9, 2000.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
ALNEMRI, E. S., D. J. LIVINGSTON, D. W. NICHOLSON, G. SALVESEN, and N. A. THORNBERRY et al., 1996 Human ICE/CED-3 protease nomenclature. Cell 87:171[Medline].
BERGMANN, A., J. AGAPITE, K. MCCALL, and H. STELLER, 1998 The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell 95:331-341[Medline].
BOSCHERT, U., R. G. RAMOS, S. TIX, G. M. TECHNAU, and K. F. FISCHBACH, 1990 Genetic and developmental analysis of irreC, a genetic function required for optic chiasm formation in Drosophila. J. Neurogenet. 6:153-171. (Erratum. J. Neurogenet. 6: 263).[Medline].
CAGAN, R. L. and D. F. READY, 1989a Notch is required for successive cell decisions in the developing Drosophila retina. Genes Dev. 3:1099-1112
CAGAN, R. L. and D. F. READY, 1989b The emergence of order in the Drosophila pupal retina. Dev. Biol. 136:346-362[Medline].
CARRERA, P., S. ABRELL, B. KERBER, U. WALLDORF, and A. PRIESS et al., 1998 A modifier screen in the eye reveals control genes for Kruppel activity in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 95:10779-10784
CARTHEW, R. W., T. P. NEUFELD, and G. M. RUBIN, 1994 Identification of genes that interact with the sina gene in Drosophila eye development. Proc. Natl. Acad. Sci. USA 91:11689-11693
CHEN, P., A. RODRIGUEZ, R. ERSKINE, T. THACH, and J. M. ABRAMS, 1998 Dredd, a novel effector of the apoptosis activators reaper, grim, and hid in Drosophila. Dev. Biol. 201:202-216[Medline].
CHINNAIYAN, A., K. O'ROURKE, B. R. LANE, and V. M. DIXIT, 1997 Interaction of ced-4 with ced-3 and ced-9: a molecular framework for cell death. Science 275:1122-1126
DEMEREC, M. and H. SLIZYNSKA, 1937 Mottled white 258-18 of Drosophila melanogaster.. Genetics 22:641-649
DEVERAUX, Q. L., R. TAKAHASHI, G. S. SALVESEN, and J. C. REED, 1997 X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388:300-304[Medline].
DORN, R., V. KRAUSS, G. REUTER, and H. SAUMWEBER, 1993 The enhancer of position-effect variegation of Drosophila, E(var)3-93D, codes for a chromatin protein containing a conserved domain common to several transcriptional regulators. Proc. Natl. Acad. Sci. USA 90:11376-11380
DORSTYN, L., P. A. COLUSSI, L. M. QUINN, H. RICHARDSON, and S. KUMAR, 1999 DRONC, an ecdysone-inducible Drosophila caspase. Proc. Natl. Acad. Sci. USA 96:4307-4312
FERNANDES-ALNEMRI, T., G. LITWACK, and E. S. ALNEMRI, 1994 CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J. Biol. Chem. 269:30761-30764
FRASER, A. G., N. J. MCCARTHY, and G. I. EVAN, 1997 drICE is an essential caspase required for apoptotic activity in Drosophila cells. EMBO J. 16:6192-6199[Medline].
FREEMAN, M., 1994 Misexpression of the Drosophila argos gene, a secreted regulator of cell determination. Development 120:2297-2304[Abstract].
FREEMAN, M., 1996 Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell 87:651-660[Medline].
GRAHAM, A., P. FRANCIS-WEST, P. BRICKELL, and A. LUMSDEN, 1994 The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 372:684-686[Medline].
GRAHAM, A., G. KOENTGES, and A. LUMSDEN, 1996 Neural crest apoptosis and the establishment of craniofacial pattern: an honorable death. Mol. Cell. Neurosci. 8:76-83[Medline].
GRUNEBERG, H., 1935 A new inversion of the X-chromosome in Drosophila melanogaster.. J. Genet. 31:163-184.
HAMBURGER, V. and R. LEVI-MONTALCINI, 1949 Proliferation, differentiation, and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J. Exp. Zool. 111:457-502[Medline].
HAWKINS, C. J., S. L. WANG, and B. A. HAY, 1999 A cloning method to identify caspases and their regulators in yeast: identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1. Proc. Natl. Acad. Sci. USA 96:2885-2890
HAY, B. A., T. WOLFF, and G. M. RUBIN, 1994 Expression of baculovirus p35 prevents cell death in Drosophila. Development 120:2121-2129[Abstract].
HAY, B. A., D. A. WASSARMAN, and G. M. RUBIN, 1995 Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83:1253-1262[Medline].
JACOBSON, M. D., M. WEIL, and M. C. RAFF, 1997 Programmed cell death in animal development. Cell 88:347-354[Medline].
KARIM, F. D., H. C. CHANG, M. THERRIEN, D. A. WASSARMAN, and T. LAVERTY et al., 1996 A screen for genes that function downstream of Ras1 during Drosophila eye development. Genetics 143:315-329[Abstract].
KAUFMANN, B. P., 1942 Reversion from roughest to wild type in Drosophila melanogaster.. Genetics 27:537-549
KERR, J. F., A. H. WYLLIE, and A. R. CURRIE, 1972 Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26:239-257[Medline].
KOLODKIN, A. L., A. T. PICKUP, D. M. LIN, C. S. GOODMAN, and U. BANERJEE, 1994 Characterization of Star and its interactions with sevenless and EGF receptor during photoreceptor cell development in Drosophila. Development 120:1731-1745[Abstract].
KURADA, P. and K. WHITE, 1998 Ras promotes cell survival in Drosophila by downregulating Hid expression. Cell 95:319-329[Medline].
LI, P., D. NIJHAWAN, I. BUDIHARDJO, S. M. SRINIVASULA, and M. AHMAD et al., 1997 Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479-489[Medline].
MAGNUSSON, C. and D. L. VAUX, 1999 Signalling by CD95 and TNF receptors: not only life and death. Immunol. Cell Biol. 77:41-46[Medline].
MILLER, D. T. and R. L. CAGAN, 1998 Local induction of patterning and programmed cell death in the developing Drosophila retina. Development 125:2327-2335[Abstract].
MORI, C., N. NAKAMURA, S. KIMURA, H. IRIE, and T. TAKIGAWA et al., 1995 Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation. Anat. Rec. 242:103-110[Medline].
NAGATA, S., 1997 Apoptosis by death factor. Cell 88:355-365[Medline].
NEUFELD, T. P., A. H. TANG, and G. M. RUBIN, 1998 A genetic screen to identify components of the sina signaling pathway in Drosophila eye development. Genetics 148:277-286
OPPENHEIM, R. W., 1991 Cell death during development of the nervous system. Annu. Rev. Neurosci. 14:453-501[Medline].
PARKS, A. L., F. R. TURNER, and M. A. MUSKAVITCH, 1995 Relationships between complex Delta expression and the specification of retinal cell fates during Drosophila eye development. Mech. Dev. 50:210-216.
RAMOS, R. G., G. L. IGLOI, B. LICHTE, U. BAUMANN, and D. MAIER et al., 1993 The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein. Genes Dev. 7:2533-2547
REITER, C., T. SCHIMANSKY, Z. NIE, and K. F. FISCHBACH, 1996 Reorganization of membrane contacts prior to apoptosis in the Drosophila retina: the role of the IrreC-rst protein. Development 122:1931-1940[Abstract].
RODRIGUEZ, A., H. OLIVER, H. ZOU, P. CHEN, and X. WANG et al., 1999 Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway. Nature Cell Biol. 1:272-279[Medline].
SAWAMOTO, K., A. TAGUCHI, Y. HIROTA, C. YAMADA, and M. H. JIN et al., 1998 Argos induces programmed cell death in the developing Drosophila eye by inhibition of the ras pathway. Cell Death Differ. 5:262-270[Medline].
SCHNEIDER, T., C. REITER, E. EULE, B. BADER, and B. LICHTE et al., 1995 Neural recognition in the optic lope of Drosophila: restricted expression of the IrreC-rst protein is required for normal axonal projections of columnar visual neurons. Neuron 15:259-271[Medline].
SIMON, M. A., D. BOWTELL, G. S. DODSON, T. R. LAVERTY, and G. M. RUBIN, 1991 Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67:701-716[Medline].
SINCLAIR, D. A., V. K. LLOYD, and T. A. GRIGLIATTI, 1989 Characterization of mutations that enhance position-effect variegation in Drosophila melanogaster. Mol. Gen. Genet. 216:328-333[Medline].
SO, K. F., G. CAMPBELL, and A. R. LIEBERMAN, 1990 Development of the mammalian retinogeniculate pathway: target finding, transient synapses and binocular segregation. J. Exp. Biol. 153:85-104
SONG, Z. W., K. MCCALL, and H. STELLER, 1997 DCP-1, a Drosophila cell death protease essential for development. Science 275:536-540
VAUX, D. L. and S. J. KORSMEYER, 1999 Cell death in development. Cell 96:245-254[Medline].
VERHEYEN, E. M., K. J. PURCELL, M. E. FORTINI, and S. ARTAVANIS-TSAKONIS, 1996 Analysis of dominant enhancers and suppressors of activated Notch in Drosophila. Genetics 144:1127-1141[Abstract].
VUCIC, D., S. SESHAGIRI, and L. K. MILLER, 1997 Characterization of reaper- and FADD-induced apoptosis in a lepidopteran cell line. Mol. Cell. Biol. 17:667-676[Abstract].
VUCIC, D., W. J. KAISER, and L. K. MILLER, 1998 Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol. Cell. Biol. 18:3300-3309
WALLRATH, L. L. and S. C. R. ELGIN, 1995 Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 9:1263-1277
WANG, S. L., C. J. HAWKINS, S. J. YOO, H. A. J. MULLER, and B. A. HAY, 1999 The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98:453-463[Medline].
WEIL, M., M. D. JACOBSON, and M. C. RAFF, 1997 Is programmed cell death required for neural tube closure? Curr. Biol. 7:281-284[Medline].
WOLFF, T. and D. F. READY, 1991 Cell death in normal and rough eye mutants of Drosophila. Development 113:825-839[Abstract].
YUAN, J. and H. R. HORVITZ, 1992 The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death. Development 116:309-320[Medline].
YUAN, J., S. SHAHAM, S. LEDOUX, H. M. ELLIS, and H. R. HORVITZ, 1993 The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1b-converting enzyme. Cell 75:641-652[Medline].
ZOU, H., W. J. HENZEL, X. S. LIU, A. LUTSCHG, and X. D. WANG, 1997 Apaf-1, a human protein homologous to C. elegans ced-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90:405-413[Medline].
This article has been cited by other articles:
 |
|
 |
|
  J. B. Cordero, D. E. Larson, C. R. Craig, R. Hays, and R. Cagan Dynamic Decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina Development, May 15, 2007; 134(10): 1861 - 1871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Grzeschik and E. Knust IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin Development, May 1, 2005; 132(9): 2035 - 2045. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Kankel, D. M. Duncan, and I. Duncan A Screen for Genes That Interact With the Drosophila Pair-Rule Segmentation Gene fushi tarazu Genetics, September 1, 2004; 168(1): 161 - 180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Hsiung and K. Moses Retinal development in Drosophila: specifying the first neuron Hum. Mol. Genet., May 15, 2002; 11(10): 1207 - 1214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gorski and M. Marra Programmed cell death takes flight: genetic and genomic approaches to gene discovery in Drosophila Physiol Genomics, May 10, 2002; 9(2): 59 - 69. [Abstract] [Full Text] [PDF]
|
|
- THIS ARTICLE
-
Abstract
- Full Text (PDF)
- Alert me when this article is cited
- Alert me if a correction is posted
- SERVICES
- Email this article to a friend
- Similar articles in this journal
- Similar articles in PubMed
- Alert me to new issues of the journal
- Download to citation manager
- Reprints & Permissions
- CITING ARTICLES
- Citing Articles via HighWire
- Citing Articles via Google Scholar
- GOOGLE SCHOLAR
- Articles by Tanenbaum, S. B.
- Articles by Cagan, R. L.
- Search for Related Content
- PUBMED
- PubMed Citation
- Articles by Tanenbaum, S. B.
- Articles by Cagan, R. L.