|Budget Amount *help
¥9,400,000 (Direct Cost : ¥9,400,000)
Fiscal Year 2000 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1999 : ¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥3,500,000 (Direct Cost : ¥3,500,000)
The goal of this research was to eluciadate nuclear events that underlie neuronal subtype selection by the Drosophila seven-up gene. Seven-up is expressed in four of the eight photoreceptor neurons in the compound eye, and acts as a genetic switch between two neuronal types. Loss of seven-up function results in transformation of seven-up-positive neurons to another photorceptor type, in morphology, opsin gene expression, and the target selection in the brain. Since seven-up encodes a nuclear receptor type transcription factor, we studied the regulatory mechanisms of gene expression during neuronal specification using seven-up as a tool.
(1) Identification of genes involved in neuronal specification through a suppressor screen of the phenotype caused by seven-up misexpression
Misexpression of seven-up causes various cell fate changes in the eye, Since the severity iof the phenotype is dependent on the copy number of the transgene, it was likely thaty a reduction in the activity of compone
nts acting downstream or seven-up will suppress the phenotype caused by seven-up misexpression. We thus screened for dominant suppressors of the "rough eye" phenotype caused by the misexpression of seven-up in a subset of photoreceptor neurons. We isolated 21 mutant strains, many of which had a loss of function phenotype resembling an increase in ras activity.
(2) Identification and characterization of sprouty
From the genetic screen described in (1) we identified five alleies of a novel gene sprouty. Sprouty encodes a membrane associated protein that has an evolitionarily-conserved Cystein-rich domain. Sprouty inhibited ras activity downstream of EGF receptor and FGF receptor. During ommatidial assembry sprouty was found to play two functions, cell autonomous inhibition of neuronal differentiation, and non-cell autonomous promotion of neuronal induction through repressing transcription of argos, which encodes an EGF antagonist. Transcription of sprouty is negatively regulated by seven-up, resulting in cell-type specific expression levels of sprouty. This work revealed how a cell-type specific transcription factor Seven-up controls neuronal number through regulating a cascade of negative regfulators of ras signaling.
(3) Functional analysis of the Seven-up ligand binding domain
We developed an assay system that visualizes the transcriptional regulatory activity of the Seven-up LBD in vivo. A fusion protein of the Seven-up LBD and the GALA DNA binding domain was expressed in transgenic animals carrying UAS-bearing reporter genes. Both activating and repressive activities of the chimeric protein can be examined at the cellular level. This system was used to study the transcriptional regulatory activity of Seven-up LBD during the compound eye development. We found that the Seven-up LBD has a transferable transcriptional repression activity, which was constitutive in the developing eye ; it is not regulated spatially or temporally in the developing eye field. This suggests that either the Seven-up ligand does not exist, or that it does not provide any regulatory information for the function of seven-up.
(4) Interaction of seven-up with the basal transcriptional machinery
High conservation of Seven-up LBD throughout evolution suggests that molecules that interact with this domain are also evolutionarily conserved. To identify molecules that participate in Seven-up-mediated gene regulation, we performed a yeast two-hybrid screen for proteins that interact with seven-up LBD.The P52 subunit or basal transcription factor TFIIH was isolated in this screen, suggesting that that Seven-up may exert its repression function by interacting with TFIIH.To address the significance of this interaction, we overexpressed the LBD of Seven-up, without its DNA binding domain, in all cells of the eye imaginal disc. Such treatment produced various cell fate changes suggesting that LBD interfered with developmental decisions that takes place during ommatidial assembry. We propose that the interaction surface of TFIIH with Seven-up is shared by other transcription factors that control cell fates during eye development. Less