The Drosophila Pax6 paralogs have different functions in head development but can partially substitute for each other

Abstract

There are two Pax6 genes in Drosophila melanogaster; eyeless (ey) and twin-of-eyeless (toy), due to a duplication, which most likely occurred in the insect lineage. They encode transcription factors important for head development. Misexpression of either toy or ey can induce formation of ectopic compound eyes. Toy regulates the ey gene by binding to an eye-specific enhancer in its second intron. However, Toy can induce ectopic eyes also in an ey( - ) background, which indicates a redundancy between the two Pax6 copies in eye formation. To elucidate to what extent these two genes are interchangeable, we first generated toy-Gal4 constructs capable of driving the Pax6 genes in a toy-specific manner. Genetic dissection of the promoter proximal region of toy identified a 1,300-bp region around the canonical transcription start that is sufficient to drive toy expression in embryonic brain and eye primorida and in larval eye-antennal discs. We find that exogenous expression of toy can partially rescue the lethality and eye phenotype caused by lethal mutations in ey and vice versa. We therefore conclude that Toy and Ey, to some extent, can substitute for each other. Nevertheless, the phenotypes of the rescued flies indicate that the two Pax6 genes are specialized to regulate defined structures of the fly head.

Fig. 1

Toy wild-type expression pattern and loss in the toy^hdl mutant. a Western blot analysis using the Toy antibody. Lane 1 shows proteins prepared from w¹¹¹⁸ adults, heat-shocked by the same regime as the hs-Gal4; UAS-toy adults in lane 2. The Toy protein is visible as a weak band in wild-type, but is strongly over-expressed by heat shock. The arrow indicates the 59-kDa Toy protein. Homozygote toy^hdl larvae (lane 3) lack this band, but three weak unrelated bands are still present. b Immunohistochemical staining of Toy in a wild-type embryo (stage 9) showing expression in two circular areas of the head (dorsal view). c Lateral view of a stage 11 wild-type embryo. The Toy-expressing cells have moved dorsally and form an uneven patch, see arrow. d Lateral view of a late stage 13 wild-type embryo. Arrow shows staining at the Bolwig’s organ. e Lack of specific staining is observed in a y w; toy^hdl embryo (stage 14). Compare with the wild-type expression pattern in d. f Dorsal view of a stage 15 wild-type embryo. Arrows point at the V-shaped eye-antennal primordia. g The reiterated pattern of Toy in the ventral cord of a stage 16 wild-type embryo. h Toy expression pattern in a wild-type eye-antennal disc. i Ectopic expression of Toy in a wing disc using the vg-Gal4 driver

Fig. 2

Analysis of the 5-kb region upstream of toy using reporter constructs. a Map of the upstream and promoter region of toy showing the Gal4 constructs toy1 to 6. A newly identified transcription start site is marked 1′. The 5′ untranslated region is indicated with white boxes and the first part of the toy open reading frame is black. The striped box is translated when spliced to exon 1, but not if transcription starts at exon 1′. Below the map, extents of genomic sequences inserted into Gal4 vectors and used for transformation of flies are shown. b Reporter gene expression in a stage 10 embryo of toy1a-Gal4>UAS-LacZ showing neuroblast expression. c A stage 16 embryo of the same strain showing staining in the embryonic brain and in one eye-antennal primordium (arrow). d A toy1e-Gal4>UAS-LacZ embryo (stage 10) showing an early expression of the lacZ reporter gene. e A toy1a-Gal4>UAS-EGFP eye-antennal disc showing strong expression of the GFP reporter gene in a band anterior to the morphogenetic furrow and in the ocellar region and weak expression in a few spots in the antennal portion

Fig. 3

Rescue from lethality of toy^hdl homozygotes with toy-Gal4>UAS-toy at different developmental temperatures. White barstoy^hdl/ci^Dspa^pol stock (control). Genotypes after crossing: light gray no rescuing constructs (control); medium graytoy-Gal4; dark grayUAS-toy; blacktoy-Gal4>UAS-toy. Significant rescue compared to controls (χ² test; P < 0.001) was obtained at 15° and 18° with toy-Gal4>UAS-toy but also with UAS-toy alone. The figures are based on the total of toy1a and toy2b rescue cross results since these figures did not differ significantly. Error bars show SD

Fig. 4

Scanning electron micrographs of heads from toy and ey mutants and rescued flies. a Rescue of homozygous toy^G7.39 with toy1f-Gal4>UAS-toy. The phenotype is indistinguishable from wild type. The interocellar bristles are marked with an arrowhead. b Rescue of toy^hdl with toy1a-Gal4>UAS-toy. The ocellar phenotype is classified as wild type since it has three ocelli and a few interocellar bristles in the correct positions. c Rescue of ey^D with ey-Gal4>UAS-ey. d Rescue of homozygous toy^G7.39 with the anti-apoptotic protein P35 (toy2b-Gal4>UAS-P35). Compound eyes are wild type, but ocelli are missing. e Rescue of ey^D with P35 (ey-Gal4>UAS-P35). Compound eyes are missing, but ocelli are normal. a–e All flies have all eclosed from the pupal case. f A homozygous toy^hdl pharate adult showing a weaker head phenotype “cleft head”. g Homozygous headless ey^D pharate adult. h Homozygous headless toy^hdl pharate adult. f–h Pharate adults dissected out of the pupal cases. a–e, g are the same scale. Bars 200 μm

Fig. 5

Toy expression and cell death in eye-antennal discs. a Acridine orange (AO) staining of a wild-type eye-antennal disc. Arrow points at the morphogenetic furrow in the eye disc. b Eye-antennal disc from a homozygous ey^D mutant larva showing a strongly reduced eye portion (right part) with high levels of cell death. c, d Eye-antennal discs from two homozygous toy^hdl mutant larvae with almost wild-type phenotype except an increased level of cell death in the posterior part of the antennal portion. e A homozygous toy^hdl disc showing complete loss of the antennal portion and increased levels of cell death in the anterior part of the eye portion. f α-Elav staining of a pair of eye-antennal discs from a homozygous toy^hdl mutant larva showing asymmetric loss of one antennal portion and size reduction of the eye portion. All discs are third instar larval eye-antennal discs with anterior to the left