Molecular links between retinal determination factors and the oscillator mechanism

The past two decades have highlighted the utility in using the fruit fly Drosophila as a model organism for unravelling the molecular and functional complexities of two important fields of research: the systems that guide retinal determination (RD) and circadian rhythms (the daily body clock or oscillator). RD and clock factors are of great interest as they are: (1) highly conserved in vertebrates; (2) also essential for other physiological systems; (3) implicated in several congenital disorders and other diseases. The RD factors operate within a network in which several of their interactions have been described. Two such factors, eyes absent (eya) and sine oculis (so), are known to function as a unit to direct transcriptional regulation during photoreceptor (PR) differentiation. The regulation of eya and so by a transcriptional repressor at the heart of the clock mechanism, vrille (vri), is here investigated. Two distinct observations advocated exploration of a link between vri and eya/ so is of interest. (1) vri is a core component of the clock and interacts with RD but the RD function is unknown. (2) Recent evidence suggests that an RD factor directly upstream of eya and so, twin-of-eyeless (toy), interacts with the oscillator mechanism through direct and indirect pathways. It is possible that the indirect influences of toy on the oscillator are mediated via eya and/ or so. Interactions between eya, so and vri during RD and within oscillator cells are investigated here.Eye development function was studied using immunohistochemistry and transgenic manipulation. VRI is not expressed within the developed PRs; rather, expression of VRI is down-regulated prior to differentiation. In addition, conversely to the hypothesised role, VRI is co-expressed with EYA in some regions. Together with data from transgenic manipulation of VRI regional expression, I propose that VRI is predominantly part of a developmental pathway but can attenuate eya and so expression.The VRI binding site has been described previously and several sites were identified within eya/ so loci, some of which were tested in an in vitro binding assay. Two such sites were located adjacent to a known enhancer of so. I generated two transgenic fly lines containing: 1) an extension of the original enhancer to contain the VRI sites; and 2) a similar construct with the VRI sites ablated. Comparison of the original enhancer to those from the current study confirmed that the VRI sites attenuate expression and that intervening regions must contain binding sites for other transcription factors.In adult brains over a circadian light-dark cycle, EYA protein was expressed in three of the central brain clock neurones. Furthermore, expression of eya and so transcripts in adult heads, PRs and the brain, changed over the light/ dark cycle independently of the clock - indicating that their expression is modulated over the light-dark cycle but not by the oscillator mechanism. These data suggest interactions between eye development factors eya/ so and oscillator components, or, the light/ dark cycle exist. These interactions may be important for tissue-specific circadian physiology as well as the overall oscillator mechanism and offer an intriguing route for future investigation.