Growth and specification of the eye are controlled independently by Eyegone and Eyeless in Drosophila melanogaster

Abstract

Control of growth determines the size and shape of organs. Localized signals known as 'organizers' and members of the Pax family of proto-oncogenes are both elements in this control. Pax proteins have a conserved DNA-binding paired domain, which is presumed to be essential for their oncogenic activity. We present evidence that the organizing signal Notch does not promote growth in eyes of D. melanogaster through either Eyeless (Ey) or Twin of eyeless (Toy), the two Pax6 transcription factors. Instead, it acts through Eyegone (Eyg), which has a truncated paired domain, consisting of only the C-terminal subregion. In humans and mice, the sole PAX6 gene produces the isoform PAX6(5a) by alternative splicing; like Eyegone, this isoform binds DNA though the C terminus of the paired domain. Overexpression of human PAX6(5a) induces strong overgrowth in vivo, whereas the canonical PAX6 variant hardly effects growth. These results show that growth and eye specification are subject to independent control and explain hyperplasia in a new way.

Continuous expression of the homeobox gene Pax6 in the ageing human retina

“…Similarly, in mice, homozygous mutations of the Pax6 homologue small eye (sey) lead to a distinct phenotype with early postnatal death and absence of eyes whereas heterozygous (sey/ þ ) mutations show microphthalmia and corneal, chamber angle and lenticular abnormalities 16,17 with striking similarities to the human condition. 5,8,15,18 There is growing evidence that the two major isoforms of PAX6, PAX6( þ 5a) and PAX6(À5a) may play different biological roles: Dominguez et al 19 have shown lately that in fDrosophila PAX6( þ 5a) functions primarily in the regulation of growth and less effectively in eye specification than Pax6(À5a). Furthermore, the Pax6( þ 5a) isoform is involved in the chain of events leading to formation of the fovea centralis in the developing chick eye whereas Pax6(À5a) is not.…”

Wingless Signaling in Drosophila Eye Development

“…Consistent with this model, the lack of eye development in mutants for eyegone, a target of Notch signaling that is required for disc growth (33), can be rescued by inhibiting Wg signaling in posterior cells (34). In the third instar eye disc, wg expression is restricted to the anterior lateral margins (6,13,15,29,35), where it continues to repress the expression of Eya, So and Dac in regions destined to form the dorsal head (30).…”

Genome-wide discovery of Pax7 target genes during development

“…Drosophila eye development is regulated by several Pax6 homologs (eyeless, eyegone, twin-ofeyeless, and twin-of-eyegone) (18) that function in synchrony with eyes absent, sine oculus (so or six), and dachshund (Dach) proteins (54). Conservation of this pathway by paralogous genes has been observed in murine myogenesis, where Pax3 and Eya2 function upstream of myogenic regulatory factors to induce myogenesis (60).…”

“…Full-length cDNAs encoding alternate isoforms of Pax7 (a-d) were isolated via nested RT-PCR from RNA of C57BL/6J mouse gastrocnemius muscle. RNA (1.8 g) was reverse transcribed using Omniscript reverse transcriptase (Qiagen) and oligo-dT 18 primers in a 20 l standard reaction. cDNA solution (2 l) was PCR amplified in a 25 l reaction using ProofStart DNA polymerase (Qiagen) for 35 cycles of 3-step PCR with primers F1 (5Ј-GAGGTTTATCCAGCCGACTCTGG-3Ј) and R1 (5Ј-AGGGTTGGCTGGGCCACTGGATGG-3Ј), with an annealing temperature of 55°C and 2 min extension times.…”

“…DNase-treated RNA was reverse transcribed with Omniscript reverse transcriptase (Qiagen) and oligo-dT 18 primers, and cDNA (2 l) was then amplified with Taq polymerase (Qiagen) using primers and conditions contained in Supplementary Table S2; optimal cycle number was empirically determined for all reactions. The RNA level of all Pax7 targets was determined via densitometric quantification as above, normalized to Gapdh levels, and data were subjected to unpaired two-tailed t-tests (comparison of Pax7 and control pcDNA transfectants) or full-factorial ANOVA with post hoc Tukey multiplecomparison testing (comparison of Pax7 alternate transcript transfectants) using StatistiXL v1.3.…”