Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye

Author summarySexual multicellular organisms start life as a single cell-the fertilized egg. One of the fundamental questions of Developmental Biology is to understand how cells proliferate and assume specific identities to faithfully reproduce the organismal tissue patterning. Cells communicate via signaling pathways to achieve complex patterning outcomes. Epidermal Growth Factor Receptor (EGFR) signaling is known to coordinate both cell division and fate choices in animals ranging from humans to the fruit-fly (Drosophila melanogaster). The fruit-fly eye with its strikingly patterned, hexagonally arranged units is a remarkable example of tissue patterning by EGFR signaling. In this paper, we investigate how expression levels of an activator (Spitz) and inhibitor (Argos) that tune the activation level of EGFR pathway regulate the patterning of the Drosophila eye. We find the activator-to-inhibitor ratio to be a critical factor in this process. The pattern is robust in a range around a tightly-controlled wildtype ratio. Beyond this biological range, both cell fate and cell division are affected producing different 'rough-eye' phenotypes. This is a striking example of how developmental patterning may be buffered against reasonable fluctuations in gene expression. The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, we show that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.