TY - JOUR T1 - Multilevel regulation of the <em>glass</em> locus during <em>Drosophila</em> eye development JF - bioRxiv DO - 10.1101/537118 SP - 537118 AU - Cornelia Fritsch AU - F. Javier Bernardo-Garcia AU - Tim-Henning Humberg AU - Sara Miellet AU - Silvia Almeida AU - Armin Huber AU - Simon G. Sprecher Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/01/31/537118.abstract N2 - Development of eye tissue is initiated by a conserved set of transcripton factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control idendity of photoreceptor as well as non-photoreceptors cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintainance of differentiated tissues. The molecular mechanisms that control expression of glass, however remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alterantive small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is surprisingly robust and appears buffered to targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye. ER -