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Headless flies produced by mutations in the paralogous Pax6 genes eyeless and twin of eyeless

Jesper Kronhamn¹, Erich Frei², Michael Daube², Renjie Jiao^2,, Yandong Shi², Markus Noll^2,* and Åsa Rasmuson-Lestander¹

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

View larger version (112K): [in a new window]   Fig. 1. Homozygous l(4)8 flies show a headless phenotype. Scanning electron micrographs of the anterior portion of homozygous l(4)8 pharate (A-D) or rare viable (E) adults and of a wild-type fly (F) are shown. The strongest phenotypes (A,B) are headless and lack all structures derived from the eye-antennal discs. Typical weaker phenotypes include half-heads (C) and cleft-heads (D). (A,E,F) Lateral, (B-D) dorsal views, anterior is to the left.

View larger version (14K): [in a new window]   Fig. 2. Temperature-sensitive period of hdl mutant producing headless pharate adults. Embryos derived from a hdl/ciD stock were collected for 4 hours and raised at 19.5°C (A), or collected for 2 hours and raised at 28°C (B), until the temperature was shifted to 28°C and 19.5°C, respectively, during embryonic development at the times indicated on the abscissa. The fraction of headless phenotypes among pharate adults is plotted against the time corresponding to the average age of the embryos at the time of the temperature shift. Points on the ordinate in A and B represent the fractions of headless pharates observed when the temperature was kept constant at 28°C (A) or 19.5°C (B) throughout development. Note that in both shift-up and shift-down experiments the temperature-sensitive period ends at stage 16 and begins at stage 12, the time when toy and ey transcripts begin to appear in the anlagen of the eye-antennal discs and in the optic lobe (Czerny et al., 1999). Embryonic stages are numbered according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1997).

View larger version (18K): [in a new window]   Fig. 3. The hdl allele is the first mutant allele of toy. (A) hdl is not allelic to ey, but located between ey and D-Pax2. A complete complementation analysis was carried out among the mutant alleles indicated below the loci of ey, 102CDh, toy, D-Pax2, 102CDg, and the deficiencies; the extent of each deficiency is indicated by horizontal lines below these loci. The hdl allele complements, and maps distal to, two mutant alleles of the l(4)102CDh locus, which are also located between ey and D-Pax2 and one of which bears a synonym identical to that of an allele of the l(4)8 locus according to the original nomenclature (Hochman et al., 1964). hdl also complements the l(4)102CDg3 allele whose previous synonymous name, l(4)19, is identical to another allele of the l(4)8 locus according to the original nomenclature. Since we have mapped l(4)102CDg3 to the most distal portion of the right arm of chromosome 4, it is probably identical to the l(4)19 mutation of the revised nomenclature, which has been mapped to this region (Hochman, 1971). The broken line of Df(4)spa30 indicates incomplete complementation with the 102CDg locus. The deficiencies Df(4)spa30, Df(4)spa47, Df(4)spa66 (Fu et al., 1998), Df(4)spam18 and Df(4)spam100 have been obtained in two EMS-induced mutagenesis screens for D-Pax2 mutants, while Df(4)BA uncovering ey was a gift from K. Basler (Brunner, 1997). The inverse sequence of the three loci toy, D-Pax2 and 102CDg in the right telomeric region of chromosome 4 is excluded because Df(4)G, which complements both ey and hdl but uncovers D-Pax2, is a telomeric deficiency (Hochman, 1971). The map is consistent with a previously published map (Locke et al., 2000), but not with that currently available at FlyBase, which erroneously localizes toy distal to D-Pax2. The map is further consistent with the gene order as determined by in situ hybridization to polytene chromosomes (Fu and Noll, 1997; Czerny et al., 1999). (B) The hdl mutation is a deletion of the 3' portion of the toy transcript. The extent of genomic fragments isolated as clones from a wild-type (HDL.1 and HDL.4) and a homozygous hdl genomic library (l(4)8.3) in DASH II are shown with respect to an EcoRI restriction map below, derived from the genomic sequence provided by FlyBase. The genomic region deleted by the toyhdl mutation is indicated above the restriction map and includes 5,863 bp, extending from nucleotide 1,855 of intron 5 to nucleotide 356 of the last exon, that are replaced by the five base pairs 5'-ATATC-3'. The exon-intron map shown below the genomic restriction map was determined by comparison of the genomic sequence with those of several toy cDNAs, isolated from an embryonic and an eye-disc cDNA library, and of products of a 5'-RACE with poly(A)+ RNA from 0- to 20-hour-old embryos raised at 25°C. Protein coding portions of the exons are indicated in black, untranslated leader and trailer in white. Vertical arrows mark alternative 3' ends as determined by sequencing of toy cDNAs and 3'-RACE products. They are preceded by a canonical poly(A) addition signal AATAAA with the exception of the first poly(A) addition site, which is preceded by CATAAA. Restriction sites: A, AccI; B, BamHI; R, EcoRI; S, SalI. (C) The toyhdl deficiency produces a truncated Toyhdl protein. The wild-type Toy protein of 543 amino acids, including a paired-domain P and prd-type homeodomain H (Czerny et al., 1999), is shown schematically above the truncated Toyhdl protein generated by the toyhdl deficiency. The truncated protein consists of 343 amino acids and includes the N-terminal paired-domain, 46 amino acids of the homeodomain, and, if intron 5 is not spliced out, a 33 amino acid C-terminal portion encoded by the 5' end of intron 5 whose first amino acid, Val, is identical to the 47th amino acid of the homeodomain. If intron 5 sequences are removed by splicing to a cryptic 3' acceptor site close to the toyhdl deficiency breakpoint in exon 9, the C-terminal tail of the truncated Toyhdl protein (black) is shorter. The positions of introns are indicated by arrowheads.

View larger version (44K): [in a new window]   Fig. 4. Northern blot analysis of wild-type toy and ey mRNAs and of toyhdl mRNAs. (A) Developmental profiles of wild-type toy and ey mRNAs. A northern blot of poly(A)+ RNA, isolated from wild-type embryos, larvae and pupae of the stages indicated, and from female and male adults, was analyzed by autoradiography after successive hybridizations with 32P-labeled 1.73 kb toy cDNA (top), 2.2 kb 1-tubulin genomic DNA for reference (bottom; Theurkauf et al., 1986), and 2.85 kb ey cDNA (middle). Sizes of mRNAs were calibrated with the same markers shown in B. (B) Northern blot analysis of toyhdl mRNAs. A northern blot of poly(A)+ RNA, isolated from adult flies (raised at 19°C) of the genotype indicated, was analyzed as described in A. Putative poly(A) addition signals consistent with the wild-type (wt) and toyhdl mRNA sizes observed include the canonical AATAAA at positions 535 and 1633, and the non-canonical CATAAA at position 180 of the last exon of toy (cf. Fig. 3B). Arrows indicate which mutant mRNAs presumably are derived from which wild-type mRNAs by the use of the same poly(A) addition sites. RNA size markers shown were produced by the RiboMark Labeling System (Promega).

View larger version (105K): [in a new window]   Fig. 5. Toy- and temperature-dependent transcription of ey in the eye-antennal anlagen. A-F are dorsal views of the same embryos focused in two different horizontal planes on ey transcripts in the eye-antennal primordia (left) or the CNS (right). (A-H) Transcription of ey in the eye-antennal primordia depends on Toy and temperature. Transcript levels of the ey gene, assayed by in situ hybridization with a DIG-labeled antisense RNA probe extending from exon 3 to 9 of the ey gene, in late stage 16 embryos derived from toyhdl/l(4)2C2 (A-F), or Df(4)spa66/ciD spapol parents (G,H) are normal in the eye-antennal primordia (EAD) of embryos with one or two wild-type copies of the toy gene (A,B), but clearly reduced in those of homozygous toyhdl (C-F) and Df(4)spa66 (G,H) embryos at 25°C (C,D,G) and 18°C (E,F,H). (I,J) Ectopic ey transcripts in homozygous or heterozygous eyD embryos. Transcripts of the eyD gene, detected by in situ hybridization of a probe specific for exons 1-5 of the ey gene, are shown in two different horizontal focal planes as ventral views of a late stage 16 embryo at 18°C derived from eyD/l(4)2C2 parents. 18 out of 120 late stage 16 embryos developing at 25°C, and 9 out of 63 such embryos at 18°C showed strong ectopic expression of ey transcripts as in J.

View larger version (70K): [in a new window]   Fig. 6. eyD is a mutation in the ey gene. (A) The eyD mutation is a translocation from the second chromosome into the fifth exon of the ey gene. At the top, the exons of the ey mRNAs (coding region in black, untranslated leader and trailer in white) are mapped with respect to the EcoRI sites of genomic ey DNA, which have been derived from genomic and cDNA sequences provided by FlyBase (Hauck et al., 1999) and from 5'-RACE products obtained from poly(A)+ RNA of 0- to 20-hour-old embryos raised at 25°C. Below, the corresponding EcoRI map of the eyD chromosome is illustrated. The eyD mutation is shown to consist of an insertion after the 305th bp of exon 5, replacing the adjacent 320 bp. The insertion, which is a large reversed repeat of the 23D1,2 to 24BC region from the second chromosome, has been characterized by mapping and sequencing genomic clones isolated from an eyD library in DASH II. The EcoRI map of two of these clones, EYD.1 and EYD.36, covering the proximal and distal breakpoint of the insertion, respectively, are shown at the bottom. Note that the ends of the large reversed repeat (hatched) are not identical but that the distal end extends 779 bp further into 24BC (stippled), while the proximal end includes a roo transposon, inserted at the indicated location. A 327 bp insertion (bearing no similarity to sequences of known genomes) in the eyD chromosome, close to the distal end of the large insertion (190 bp downstream of the 5' end of ‘intron 5’), is indicated by a small triangle. R, EcoRI. (B) The eyD insertion generates a truncated Ey protein. The wild-type Ey protein of 898 amino acids, including a paired-domain P and prd-type homeodomain H (Quiring et al., 1994), is compared to the truncated EyD protein resulting from the 2nd chromosome insertion into exon 5 of the ey gene. The truncated protein consists of 346 amino acids and includes the N-terminal paired-domain and a Ser/Thr-rich domain (28/66 amino acids), possibly an activation domain, but no homeodomain. Its 32 C-terminal amino acids are encoded by the inserted sequences of the second chromosome (black). The positions of introns are indicated by arrowheads. (C-E) Characterization of the eyD mutation, a translocation of the second chromosome into the ey gene. In situ hybridization to polytene chromosomes of eyD/ciD spapol late third instar larvae with the DIG-labeled probes indicated in A that are specific for the ends of the 2nd chromosome insertion (C), the 5' end (D), and the 3' end (E) of the ey gene. The inserts in C show enlarged views of the regions of hybridization on the second chromosome (lower left) and at the ends of the insertion on the fourth chromosome (upper right).

View larger version (73K): [in a new window]   Fig. 7. Headless phenotype of eyD pharates and their partial rescue by inhibition of apoptosis. Scanning electron micrographs of the anterior portion (A-D,I,J) or left eyes (E-H) of pharate (B-D) or viable (A,E-J) adults of the genotype indicated are compared. Note that, in contrast to the headless phenotype of toyhdl flies, the penetrance and expressivity of the headless phenotype of eyD pharates is the same at 18°C and 25°C with about 50% of the pharates exhibiting no (B) or only few (C) structures derived from the eye-antennal discs, while the phenotype of most pharates is much stronger than that shown in D. The variability of heterozygous eyD phenotypes (E-I) presumably reflects a strong influence of the genetic background as illustrated by the eyeless phenotype obtained after several generations of selections for small eyes (I). Expression of the baculovirus P35 protein, an inhibitor of apoptosis (Hay et al., 1994), in eye-antennal discs is able to rescue more than half of the homozygous eyD flies to viable adults. These flies possess both antennae, no eyes, but usually all three ocelli (J). (A,B,J) Dorsal, (C,D-I) lateral views, anterior is to the left.

View larger version (10K): [in a new window]   Fig. 8. Redundant functions of Ey and Toy in eye-antennal development. Normal head (eye-antennal) development can occur in the absence of Toy if Ey levels are sufficiently high, such as is mostly the case in toyhdl mutants at 18°C, or in the presence of very low Ey levels if Toy levels are normal as in ey2 mutants. Conversely, headless phenotypes are observed in the absence of Toy and at moderate Ey levels, as in most toyhdl mutants at 28°C, or in the absence of functional Ey as in eyD mutants. Broken lines indicate where requirement for eye-antennal development is not absolute because it can be compensated by sufficiently high levels of Ey. It is unclear if the pathway of Toy that is parallel to the ey pathway leads through optix and/or eyg, as the diagram suggests. Moreover, additional Toy and Ey functions that are not mediated through so and optix/eyg, respectively, exist to support proper eye-antennal development. Such Toy and Ey functions, like the inhibition of apoptosis, as shown here, or the activation of the cell cycle (Jiao et al., 2001), are required in eye-antennal primordia of stage 12-16 embryos, long before these genes are activated in eye-antennal discs (Kumar and Moses, 2001).