1 Division of Genetics, Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden
2 Institute for Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Present address: Institute of Veterinary Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
*Author for correspondence (e-mail: noll{at}molbio.unizh.ch)
Accepted 15 November 2001
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SUMMARY |
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Key words: Headless flies, Pax6 genes, twin of eyeless, eyeless, Head development, Eye development, Drosophila melanogaster
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INTRODUCTION |
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In order to clarify the roles of the two Pax genes ey and toy in the development of normal rather than ectopic eyes, we examined the effect on eye development of the first toy mutations and of a much stronger ey allele than previously characterized. We concentrated particularly on the earliest period of ey and toy expression in the eye-antennal primordia in embryos (Czerny et al., 1999). This period has been shown to be particularly sensitive to developmental pathway interference, which generates headless flies (Jiao et al., 2001
), a phenotype much stronger than that observed for ey mutants (Quiring et al., 1994
; Halder et al., 1998
). As this indicated much wider roles for toy and ey in head development, we set out to find strong mutants of both toy and ey in order to examine if these displayed a headless rather than the milder eyeless phenotype.
We found that strong mutants of either toy or ey indeed lack all structures derived from eye-antennal discs and thus exhibit a spectacular headless phenotype. In addition, activation of ey in the primordia of eye-antennal discs does not strictly depend on the presence of the product of the toy gene, but becomes temperature-sensitive in its absence. In the absence of Toy protein, moderate levels of Ey protein are sufficient to rescue the headless phenotype, while high Ey levels are necessary to rescue the eyeless phenotype as well. In contrast, very low levels of Ey apparently suffice to rescue the headless phenotype in the presence of wild-type Toy protein. These findings suggest a delicate balance of Ey protein levels regulating eye-antennal disc development and a partial redundancy of Ey and Toy functions in these discs, in which Toy acts not only through Ey, but also in a pathway parallel to Ey. Finally, inhibition of apoptosis by the baculovirus P35 protein is able, in the absence of functional Ey, to rescue the headless, but not the eyeless phenotype. Hence, Ey is not responsible for the development of head structures derived from the antennal disc, but is primarily required to inhibit cell death and to promote eye development.
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MATERIALS AND METHODS |
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Determination of the lethal phase of toyhdl mutants
To assay the lethal phase of homozygous toyhdl mutants, newly hatched first instar larvae were picked from a toyhdl/spaCat stock and the fractions that reached the pupal and adult stages determined. These fractions were the same at all temperatures tested (18, 23, 25, and 28°C). All viable adults were heterozygotes because all spaCat homozygotes die during embryogenesis (Hochman, 1976). From a total of 3250 larvae, 2728 developed to pupae and 1853 to viable adults. Assuming that of the 15% of heterozygotes that fail to reach the adult stage half each die as larvae or pupae, we find that the lethality of toyhdl mutants is 34±2% during the larval and 66±3% during the pupal stage.
In situ hybridization of RNA probes to embryos
In situ hybridization with antisense RNA probes to staged embryos was carried out according to standard procedures. To determine what fraction of toyhdl mutants showed a reduced level of ey transcripts in the eye-antennal primordia, their embryonic stage was determined and the level of ey transcripts compared to that of late wild-type embryos of the same stage. Among embryos derived from toyhdl/l(4)2C2 parents, the level was reduced in 32 out of 127 late stage 16 embryos at 25°C, which is as expected if it was reduced in all homozygous toyhdl embryos; and in 10 out of 124 such embryos at 18°C, i.e., in a third of all toyhdl embryos (Fig. 5A,C,E). For embryos derived from Df(4)spa66/l(4)2C2 parents, it was first verified that 25% of all stage 16 embryos were homozygous for the deficiency, by the absence of toy transcripts after in situ hybridization with a toy cDNA probe, before the same analysis for ey transcript levels was carried out. Again the level was reduced in a quarter (24/100) of late stage 16 embryos at 25°C, and in about half (10/71) of the expected number of homozygous Df(4)spa66 embryos of the same stage at 18°C (Fig. 5G,H). Similar results were obtained for embryos from Df(4)spa30/l(4)2C2 parents: ey transcripts in the eye-antennal primordia were clearly reduced in 17 out of 62 embryos at 25°C, while they were reduced in only 13 out of 100 embryos at 18°C.
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RESULTS |
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This strong temperature sensitivity of the hdl allele permits us to test whether its temperature-sensitive functions are restricted to a critical period or required throughout development. To this end, temperature-shift experiments were carried out during which embryos developing at 19.5°C were transferred to 28°C (shift-up experiments; Fig. 2A) or embryos developing at 28°C were shifted to 19.5°C (shift-down experiments; Fig. 2B) after various time intervals. In both temperature-shift experiments, the temperature-sensitive period was limited to a short interval from stage 12 to 16 (Campos-Ortega and Hartenstein, 1997), when toy and ey transcripts begin to appear in the anlagen of the eye-antennal discs (Jürgens and Hartenstein, 1993
; Czerny et al., 1999
). Temperature shifts after this phenocritical period showed the same proportions of headless phenotypes as if no shift had occurred.
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hdl is the first mutant allele of toy
Headless flies have been observed previously when ectopic expression of transcription factors in the eye-antennal primordia interfered with the developmental program initiated by the toy and/or ey genes (Jiao et al., 2001), both of which are located on the fourth chromosome (Czerny et al., 1999
). Because hdl also maps to the fourth chromosome, we suspected that it is either the first mutant allele of toy or an allele of ey that is stronger than any of the previously characterized ey alleles. To test this hypothesis, we mapped hdl by complementation analysis, using a number of deficiencies that had been isolated in screens for D-Pax2 mutants (Fu et al., 1998
), a deficiency uncovering the ey locus (a generous gift from K. Basler) (Brunner, 1997
), three ey alleles, and a few additional mutant alleles thought to belong to the same complementation group as ey or hdl on the basis of Hochmans complementation analysis (Hochman, 1971
). The resulting genetic map shows that the hdl mutation is located distal to ey and proximal to D-Pax2 (Fig. 3A). Therefore, hdl is not an ey allele, but its genetic location is consistent with the possibility that it is allelic to toy. To test this possibility, we isolated genomic clones of toy from wild-type and hdl
phage libraries. Comparison of their restriction maps indeed suggested that sequences downstream of exon 5 of toy are deleted in hdl mutants (Fig. 3B). Isolation of the deficiency by PCR and subsequent DNA sequence analysis corroborated this conclusion and demonstrated that hdl is a 5,863 bp deletion of the 3' moiety of the toy transcript (Fig. 3B). It follows that hdl is the first mutant allele of toy and hence was named toyhdl. Our finding is further consistent with a recent report, mentioning a headless phenotype of toy mutants as unpublished results (Kammermeier et al., 2001
).
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Toy activates ey transcription in the embryonic anlagen of the eye-antennal discs
It has been proposed that the Toy protein activates ey in the eye developmental pathway. In the absence of toy mutants, this proposal was based (i) on the observation that ectopic eye formation induced by ectopic expression of toy in leg discs was dependent on a functional ey gene, and (ii) on in vitro binding studies of Toy to sites in a minimal ey enhancer whose activation of a reporter gene in the eye-antennal primordia was reduced upon mutation of the Toy binding sites (Czerny et al., 1999). Although these results indicate that the Toy protein activates the ey gene in the eye primordia, they do not prove it for two reasons. First, studies on ectopic eye formation do not strictly provide information about the normal pathway of eye formation, particularly since ectopic eye formation is possible in the absence of toy expression (Czerny et al., 1999
). Second, mutation of the Toy binding sites in the minimal ey enhancer did not eliminate reporter gene expression in the entire eye-antennal primordium, but strong expression was still observed in its posterior portion, presumably where the eye primordium is located, perhaps because the minimal enhancer did not include all cis-regulatory elements required for proper expression of the ey gene during eye development (Czerny et al., 1999
). Moreover, as evident from the toyhdl phenotype (Fig. 1A,B), Toy is required not only for eye formation, but for the development of the entire eye-antennal disc and exhibits a phenocritical period at the time when the eye-antennal anlage forms and begins to express ey (Fig. 2).
The identification of deficiencies uncovering the toy gene and of hdl as a toy mutation (Fig. 3A,B), however, allows us to perform a more critical test of whether toy acts upstream of ey during normal eye and head development: the analysis of the expression of the ey gene in the eye-antennal primordia of toy mutant embryos at the end of the phenocritical period (Fig. 5C-H). Transcription of ey was strongly reduced, though not completely eliminated, in the eye-antennal primordia of most toyhdl as compared to wild-type embryos when they developed at 25°C (compare Fig. 5C with 5A). If development occurred at 18°C, however, ey transcript levels were not reduced as much, but still clearly reduced in a third of the toyhdl embryos (Fig. 5E) and indistinguishable from wild type in the remaining toyhdl embryos. These findings prove that toy acts upstream of ey in the eye-antennal anlage at the time of the initial ey activation. In addition, they provide a simple explanation for the temperature sensitivity of the toyhdl allele since the fractions of strong (Fig. 1A,B) and less severe headless phenotypes (Fig. 1C-E) correspond well to the fractions of embryos exhibiting reduced levels of ey transcripts in the eye-antennal primordia at the high (Fig. 5C) and low temperature (Fig. 5E). We conclude that ey transcription in the eye-antennal primordia of toyhdl embryos is temperature-dependent and that the most severe headless phenotypes arise only if transcriptional activation of the ey gene in the eye-antennal primordia does not exceed a threshold level that is clearly detectable (Fig. 5C).
In contrast to ey transcription in the eye-antennal primordia, transcription of ey in the CNS appears to remain unaffected in toyhdl embryos (Fig. 5B,D). This confirms an earlier conclusion based on the observation that ey transcripts precede those of toy and display a pattern different from toy transcripts in the ventral nerve cord (Czerny et al., 1999).
Temperature-dependent activation of ey transcription in eye-antennal anlagen in the absence of Toy protein
The temperature dependence of ey transcription in the eye-antennal primordia of toy mutants does not prove that the Toyhdl protein is the cause of the temperature sensitivity of the headless phenotype. It is possible that even in the absence of Toy protein activation of ey transcription remains temperature dependent. To test this possibility, we examined ey expression in embryos homozygous for Df(4)spa30 or Df(4)spa66, both of which fail to express toy transcripts (Materials and Methods). Surprisingly, in all homozygous mutant embryos the level of ey transcripts was similarly reduced as in toyhdl embryos, but not eliminated, and still depended on temperature to the same degree (Fig. 5G,H). It follows that it is not the truncated Toyhdl protein that is temperature dependent, but rather the activation of ey transcription in the absence of a functional Toy protein. This conclusion is consistent with our finding that similar fractions of homozygous toyhdl pharates and of toyhdl pharates transheterozygous for one of the two deficiencies show a headless phenotype when tested at the same temperature between 18°C and 28°C. We conclude that toyhdl behaves as a null allele with respect to the activation of ey at the phenocritical stage as well as with regard to the severity of the resulting headless phenotypes.
eyD is an insertion interrupting the ey transcript
Since the severity of the headless phenotype correlates inversely with the level of ey transcripts in the eye-antennal primordia, we would expect that strong ey mutants also produce headless pharates if toy acts mainly through ey in eye-antennal development. None of the two defined ey mutants, ey2 and eyR, exhibits, however, a headless phenotype (Bridges, 1935a; Quiring et al., 1994
; Halder et al., 1998
), possibly because both are viable hypomorphs, whereas ey null alleles, all of which have been lost, are pupal lethals (Hochman, 1976
). An additional putative ey mutation, eyD, X-ray-induced by Muller in 1927 and mapped to the fourth chromosome (Patterson and Muller, 1930
), has not been analyzed at the molecular level. Although its allelism to ey has been questioned (Bridges, 1935a
), it is pupal lethal (Patterson and Muller, 1930
; Bridges, 1935b
) and has been characterized to be associated with the translocation of about a dozen chromosomal bands that are inserted as reversed repeat into the region 102D of the fourth chromosome (Bridges, 1935b
) (Fig. 6C-E), known to include the ey locus (Quiring et al., 1994
). Moreover, complementation analysis indicated that eyD might be allelic to ey (Patterson and Muller, 1930
) (Fig. 3A).
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eyD is transcribed and generates a truncated EyD protein that includes the paired-domain but lacks the homeodomain
In situ hybridization demonstrates that in about half of the homozygous eyD embryos ey transcripts of the first five exons are as abundant in the eye-antennal primordia as in wild-type embryos, while in the other half they are reduced to about half the wild-type level, both at 18°C and 25°C (not shown). In contrast, no transcripts of the exons downstream of the eyD insertion are detectable in homozygous eyD embryos (not shown). These findings imply that the insertion has no strong effect on the embryonic activities of the ey enhancers and that the stability of the truncated eyD transcripts is comparable to that of wild-type ey mRNA, independent of temperature within the examined range. It is therefore reasonable to assume that eyD transcripts are translated to produce a truncated Ey protein that includes the N-terminal paired-domain and a Ser/Thr-rich domain, possibly an activation domain, but lacks all C-terminal domains, including the homeodomain (Fig. 6B).
In about a fifth of the homozygous or heterozygous eyD embryos, the first five exons of ey were ectopically expressed in a metameric pattern of 11 lateral, later ventrolateral, posterior segmental stripes after germband retraction (Fig. 5I,J). Hence, a nearby enhancer of the inserted second chromosome sequences seems to activate the eyD gene at low penetrance.
Homozygous eyD pharate adults are headless
The homozygous eyD condition is lethal (Patterson and Muller, 1930) with a lethal phase during the pupal stage (Bridges, 1935b
), in agreement with the lethal phase of other strong ey alleles (Hochman, 1976
) that, however, were lost. To investigate if homozygous eyD mutants display a headless phenotype as expected, we dissected eyD pharate adults from their pupal case and examined them by scanning electron microscopy. We found that indeed about half of these pharates showed a strong headless phenotype (Fig. 7B,C) missing all or most structures derived from wild-type eye-antennal discs (Fig. 7A), in agreement with an earlier report (Arking et al., 1975
). The remaining pharates developed a considerable portion of the head and antennae, yet no eyes, and exhibited phenotypes most of which were stronger than the eyeless phenotype shown in Fig. 7D.
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In combination with nearby deficiencies, some of which delete the toy gene but none the ey gene (Fig. 3A), the phenotype of heterozygous eyD flies is affected in various ways. Thus, the size of the eye in eyD/Df(4)spa30 flies varies over a wider range than, but on average is similar to, that of eyD/+ flies (Fig. 7H), while Df(4)spa66 reduces the average eye size of heterozygous eyD flies to about 10% of wild type (not shown). In contrast, Df(4)spa47, which does not delete toy (Fig. 3A), seems to suppress the eyD/+ phenotype significantly (not shown). These effects on the eyeless phenotype may result in part or entirely from different genetic backgrounds. For example, continued selection for small eye sizes among y w; eyD/Dp(1;Y;4)y+, spapol offspring obtained from repeated crossings inter se, yields increasing numbers of flies that have no eyes (Fig. 7I). Such variable effects of the genetic background on the eyD/+ phenotype may explain why eyD could not be identified unambiguously as an allele of ey by mere genetics.
Rescue of eyD headless flies to viable eyeless adults by inhibition of apoptosis
Since about half of the eyD pharates lacked most derivatives of the eye-antennal discs, we investigated whether inhibition of apoptosis by expression of the baculovirus P35 protein (Hay et al., 1994), under the indirect control of the eye-antennal specific enhancer of the ey gene, had any effect on eye-antennal disc development. Astonishingly, more than half of such eyD flies are rescued by ey-Gal4>UAS-P35 to viable adults that are eyeless, but have developed most other head structures derived from the eye-antennal discs, usually including all three ocelli (Fig. 7J). The heads of all these flies are rescued since their phenotypes are much weaker than the weakest eyD phenotype (Fig. 7D). These results imply that the headless phenotype of eyD mutants is the result of considerable cell death in the eye-antennal disc, a process that is inhibited by the wild-type Ey protein.
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DISCUSSION |
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Activation of ey in eye-antennal primordia of toy mutants is temperature-dependent
Temperature shift experiments with toyhdl mutants show that the headless phenotype critically depends on the absence of Toy protein activity during stages 12-16 of embryogenesis (Fig. 2). In toyhdl mutants, up to 80% of the pharate adults are headless at 28°C, whereas this phenotype is nearly completely suppressed at 18°C. Shifting the temperature down to the permissive temperature at the end of stage 16 demonstrates that after this stage Toyhdl is unable to provide any function that would be able to rescue the headless phenotype, while shifting the temperature up to the non-permissive temperature at this time shows that Toyhdl can provide all the functions necessary, if any, to rescue the headless phenotype at all temperatures after stage 16. It follows that the temperature-sensitive function of Toyhdl with regard to the headless phenotype is restricted to the phenocritical period during stages 12 to 16.
Homozygous eyD pharates exhibit the same headless phenotype as toyhdl mutants. Since we have demonstrated that eyD is a strong allele of the ey gene, we conclude that the truncated EyD protein, if translated from the eyD mRNA, is unable to provide the functions necessary for eye-antennal disc development. It was therefore important to know if ey transcription depended on Toy during the phenocritical period, as had been shown previously for ectopic eye formation, but could not be tested for normal eye development because of the lack of toy mutants (Czerny et al., 1999). We found that ey transcription indeed depends on toy activity. Surprisingly, however, even in the absence of Toy protein ey transcription, (i) remains temperature-dependent during the phenocritical period, and (ii) is not completely eliminated. Hence, the observed temperature-sensitivity of the headless phenotype of toyhdl mutants is not a property of the truncated Toyhdl protein, but rather of the transcriptional activation of the ey gene in the absence of a functional Toy protein.
Does Toy serve to stabilize a temperature-dependent activation complex on the eye-antennal enhancer of the ey gene?
We propose that during the phenocritical period, in addition to Toy, other transcription factors bind to the eye-antennal enhancer of the ey gene to activate its transcription in the eye-antennal primordia. In the absence of Toy, these factors are able to activate ey transcription sufficiently at low but not at high temperatures to ensure normal eye-antennal development. The simplest explanation for this temperature-sensitive activation of ey is that formation of the transcription factor complex bound to the eye-antennal enhancer or its activation of the basal transcription machinery becomes temperature-dependent in the absence of Toy protein. Thus, the main function of Toy is to stabilize this transcriptional activator complex on the eye-antennal enhancer of ey. It is interesting to note that three Toy binding sites have been found in the eye-antennal enhancer of ey, two of which are immediate neighbors (Czerny et al., 1999). Truncated Toyhdl probably binds with similar affinity to these sites as wild-type Toy protein (Punzo et al., 2001
). However, its lack of C-terminal activation domains may fail to stabilize its own binding and that of cofactors as a result of which the basal transcription machinery is not efficiently recruited and activated. The fact that a third of the toyhdl mutants die during larval stages and that toyhdl mutants that show normal eye-antennal development still die as pharate adults shows that Toy is more strictly required for the activation of other enhancers necessary for the development of viable adults.
Eye-antennal development depends on a delicate balance of ey activation in the eye-antennal primordia
Interestingly, ey transcription is not abolished, but reduced levels of ey transcripts remain detectable in eye-antennal primordia of toyhdl embryos, even at temperatures at which most of them develop to headless pharates. This finding implies that there is a delicate balance of ey activation for inducing eye-antennal development. If transcript levels do not exceed a relatively high threshold value, the program for eye-antennal disc development cannot proceed. This delicate balance is particularly evident when it is temperature-dependent in the absence of a functional Toy protein, for example, in toyhdl mutants. In many instances, unequal ey transcript levels have been observed in the left and right eye-antennal primordia of toyhdl embryos. Accordingly, ey transcripts may surpass the threshold in only one of the two eye-antennal primordia and thus give rise to pharates with only one half of the head developing normally (Fig. 1C). Even within the eye-antennal primordium, ey transcript levels may vary in toyhdl mutants (Fig. 5E) and thus explain phenotypes like the cleft-head (Fig. 1D).
Partial redundancy of Toy and Ey functions
In toyhdl mutants, reduced yet detectable (moderate) levels of ey transcripts in eye-antennal primordia are unable to rescue the headless phenotype. This is evident from the reciprocal correlation between ey transcript levels in eye-antennal primordia during the phenocritical period and the fraction of headless pharates at different temperatures of development. In contrast, ey2 mutants, in which no ey transcripts have been detected in eye-antennal primordia, never display a headless phenotype and mostly have eyes of only slightly reduced size (Quiring et al., 1994) (our unpublished observation), whereas eyD mutants show a high penetrance of headless pharates. The following considerations illustrate that this apparent contradiction is resolved by the assumptions that (i) Toy and Ey share partial functional redundancy in eye-antennal disc development, and (ii) ey2 expresses very low levels of wild-type Ey protein whose mRNA escapes detection (Fig. 8). In the absence of functional Ey protein, as in eyD mutants, normal levels of functional Toy protein rescue the headless phenotype at low efficiency. In ey2 mutants, however, very low levels of functional Ey protein are sufficient to rescue the headless phenotype completely in the presence of normal Toy levels and are even able to promote nearly normal eye development (our unpublished results). In contrast, in the absence of functional Toy protein, as in toyhdl mutants, much higher levels of ey transcripts are necessary to rescue the headless phenotype, which is achieved more efficiently at lower temperatures. Consequently, the headless phenotype is observed only in the complete absence of functional Ey protein or in the absence of functional Toy if Ey does not exceed a moderate level.
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Rescue of eyD mutants to viable adults by the inhibition of apoptosis in eye-antennal discs
The most crucial function of ey and most sensitive to the level of Ey during eye-antennal development is the inhibition of cell death. This became evident from a successful attempt to rescue the eyD headless phenotype by the expression in eye-antennal discs of the baculovirus P35 protein, an inhibitor of apoptosis (Fig. 7J). Astonishingly, P35 is able to rescue more than half of the eyD embryos to viable adults with normal head structures, with the exception of the eyes that still do not develop.
The fact that inhibition of apoptosis in eyD eye-antennal discs is unable to rescue eye development argues that additional functions of ey during eye-antennal development are required and restricted to the eye disc, in agreement with its expression pattern in eye-antennal discs (Quiring et al., 1994) and a recent analysis (Kumar and Moses, 2001
). It has been shown that interference with the program of eye-antennal development by ectopic expression of transcription factors also generates headless pharates if interference is restricted to exactly the same phenocritical period observed here (Jiao et al., 2001
). However, in this case interference could not be antagonized by P35 expression, but only by overexpression of Ey, CycE or Myc. It was concluded that one of the earliest functions of ey must be the activation of the cell cycle (Jiao et al., 2001
). Combining this conclusion with the results reported here, we propose that in the presence of developmental pathway interference it is impossible to obtain rescue by the inhibition of apoptosis in the absence of cell cycle activation, whereas inhibition of apoptosis appears to suffice in eyD mutants, possibly because the truncated EyD and/or the Toy protein are able to activate the cell cycle.
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ACKNOWLEDGMENTS |
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