Functional conservation of the Drosophila gooseberry gene and its evolutionary alleles

Abstract

The Drosophila Pax gene gooseberry (gsb) is required for development of the larval cuticle and CNS, survival to adulthood, and male fertility. These functions can be rescued in gsb mutants by two gsb evolutionary alleles, gsb-Prd and gsb-Pax3, which express the Drosophila Paired and mouse Pax3 proteins under the control of gooseberry cis-regulatory region. Therefore, both Paired and Pax3 proteins have conserved all the Gsb functions that are required for survival of embryos to fertile adults, despite the divergent primary sequences in their C-terminal halves. As gsb-Prd and gsb-Pax3 uncover a gsb function involved in male fertility, construction of evolutionary alleles may provide a powerful strategy to dissect hitherto unknown gene functions. Our results provide further evidence for the essential role of cis-regulatory regions in the functional diversification of duplicated genes during evolution.

Figure 1. Locus of the gsb gene.

(A) gsb mutant alleles. The two deficiencies, Df(2R)IIX62 and Df(2R)SB1, as well as the two hypomorphic alleles, gsb525 and gsbP1155, are depicted. Neighboring genes uncovered by Df(2R)IIX62, zip, uzip, CG3441, and gsbn upstream of gsb, gol and dTKR downstream of gsb, and their direction of transcription are indicated (the rigth telomere of the second chromosome is to the right). Exons are marked by black boxes in the enlarged portion of (A) and also in (B). (B) Map of gsb0-525 abd gsb0-ΔHC transgenes. Both transgenes contain the upstream epidermis enhancers of gsb, GEE and GLE ( Fig. 1A ; Li et al., 1993), the gsb promoter, and the entire 3′ UTR of gsb. In gsb0-ΔHC, 519 bp of coding region between the gsb525 mutation and a SacII site are deleted, resulting in a shift of the open reading frame after the gsb525 nonsense mutation. (C) Sequence surrounding the gsbP1155 insertion site. The negative numbers refer to nucleotides upstream of the transcription start site. The eight nucleotides, duplicated during insertion of the P-element, are underlined.

Figure 2. Cuticular phenotypes of gsb mutants.

(A) Df(2R)IIX62/CyO, (B) Df(2R)IIX62, (C) Df(2R)IIX62/Df(2R)KrSB1, (D) Df(2R)IIX62/gsb525, (E) gsb525, (F) gsbP1155, (G) Df(2R)IIX62; gsb0-525 (H) Df(2R)IIX62; gsb0-ΔHC. Note in strong gsb mutants (B, C), the ventral naked cuticle region of each segment is transformed into denticle belt, generating an overall denticle pattern, which is in contrast to wild-type (A). Scale bar: 50 um.

Figure 3. Map of gsb-res, gsb-Prd and gsb-Pax3 transgenes.

The gsb-res transgene corresponds to the enlarged 20-kb genomic fragment in Fig. 1A , which includes the gsb transcribed region as well as adjacent 14-kb upstream and 3-kb downstream sequences . The upstream sequence also contains the 5′ portion of the gsbn up to part of the third exon. In gsb-Prd and gsb-Pax3 transgenes, the gsb coding region (except of a small region encoding the C-terminus) is replaced by prd and Pax3 cDNAs, while upstream and downstream regions are retained. The gsb intron is also retained by inserting it between sequences of the gsb and prd or Pax3 leaders. Coding regions are indicated as black boxes except for the paired-domain (PD) and the prd-type homeodomain (HD) which are hatched. The gsb and gsbn introns are indicated as open boxes. The transcription start of gsb is marked by 0, and poly(A) addition signals AATAAA are indicated.

Figure 4. Expression of Gsb and Prd proteins and Pax3 mRNA under control of the gsb cis-regulatory region.

Expression of Gsb protein in wild-type embryos (ry⁵⁰⁶; A), of Prd protein in transgenic gsb-Prd embryos (B), and of Pax3 mRNA in transgenic gsb-Pax3 embryos (C) at the extended germ band stage. Wild-type embryos were stained with anti-Gsb antiserum and transgenic embryos, collected from homozygous gsb-Prd or gsb-Pax3 stocks, were stained with anti-Prd antiserum or hybridized in situ with digoxigenin-labeled Pax3 cDNA. Unfolded embryos are shown and oriented with their anterior to the left. Scale bar: 100 um.

Figure 5. Rescue of Wg and Gsbn expression in gsb mutant embryos by gsb-Prd and gsb-Pax3 transgenes.

Expression of Wg (A–D) and Gsbn (E–H) proteins in wild-type (A, E), homozygous Df(2R)IIX62 (B–D) or transheterozygous Df(2R)IIX62/gsb525 (F–H) gsb mutant embryos carrying no (B, F), one copy of the gsb-Prd (C, G) or gsb-Pax3 (D, H) transgene. Embryos at stage 13 (A–D) or stage 10 (E–H) are oriented with their anterior to the left and dorsal side up. Embryos were collected from crosses between Df(2R)IIX62/CyO, hb-LacZ; gsb-Prd/+ or Df(2R)IIX62/CyO, hb-LacZ; gsb-Pax3/+ males and Df(2R)IIX62/CyO, hb-LacZ (A–D) or gsb525/CyO, hb-LacZ females (E–H), and double stained for ß-galactosidase and or Gsbn protein with rabbit antiserum against ß-galactosidase and anti-Wg monoclonal antibodies or rabbit anti-Gsbn antiserum. Embryos stained with ß-galactosidase have at least one copy of wild-type gsb allele and were used as control (A, E). One quarter of the embryos did not stain for ß-galactosidase. Half of these embryos did not express Wg in the ventral epidermis and Gsbn in the CNS as expected for gsb mutants. The other half displayed rescued expression patterns, which suggested the presence of the transgenes. Scale bar: 100 um.

Figure 6. Rescue of the cuticular phenotype of gsb mutant embryos by gsb-Prd and gsb-Pax3 transgenes.

Ventral view of cuticle preparations of wild-type (ry⁵⁰⁶; A) and homozygous Df(2R)IIX62 embryos without (B) and with one copy of the gsb-Prd (C) or gsb-Pax3 transgene (D) are shown under dark-field illumination (anterior is up). Wild-type and gsb mutant embryos were collected from the Df(2R)IIX62/SM1 stock, while gsb mutant embryos carrying one copy of the transgenes were collected from crosses between Df(2R)IIX62/SM1; gsb-Prd or Df(2R)IIX62/SM1; gsb-Pax3 males and Df(2R)IIX62/SM1 females. gsb mutants were distinguished from wild type by the presence of the zip phenotype, a deformed head structure resulting from the deletion of the zip gene, which is uncovered by Df(2R)IIX62 . Scale bar: 50 um.

Figure 7. Rescue of the CNS phenotype of gsb mutant embryos by gsb-Prd and gsb-Pax3 transgenes.

Patterns of longitudinal and commissural axons in the CNS of wild-type (ry⁵⁰⁶; A) and Df(2R)IIX62/gsb525 embryos without (B) and with one copy of the gsb-Prd (C) or gsb-Pax3 transgene (D). Embryos at stage 15 were collected from crosses between Df(2R)IIX62/CyO, hb-LacZ; gsb-Prd/+ or Df(2R)IIX62/CyO, hb-LacZ; gsb-Pax3/+ males and gsb525/CyO, hb-LacZ females, and double stained with rabbit antiserum against ß-galactosidase and monoclonal antibody BP102. One quarter of the embryos did not stain for ß-galactosidase as expected. Half of these embryos have missing or reduced posterior commissures as expected for gsb mutants, the other half displays fully rescued commissural patterns as in wild-type embryos. Scale bar: 10 um.