Ratiometric sensing of Pnt and Yan transcription factor levels confers ultrasensitivity to photoreceptor fate transitions in Drosophila

Abstract

Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. Here we investigate how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein despite expressing both proteins with pulsatile dynamics. A rapid and sustained two-fold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a two-fold shift in the ratio. A biophysical model based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how two-fold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program.

Energy metabolism modulates the regulatory impact of activators on gene expression

“…Eye cells rapidly upregulate Yan protein abundance to a peak; the protein then decays back to initial levels over the course of 40 hours (Figure 3A). As cells achieve high levels of Yan, some of them are induced to transition to photoreceptor fates [17, 18]. However, Yan protein itself does not promote the transition but actually inhibits it through its repressive transcriptional activities [19].…”

“…To precisely measure Yan protein in cells, we used a Drosophila strain in which the protein is tagged with YFP and is still fully functional [16, 17]. Confocal microscopy of the eye discs was coupled with a computational pipeline for segmentation and analysis, yielding a composite picture of Yan dynamics sampled from thousands of cells per condition [17, 18].…”

“…Eye discs from white-prepupae of the correct genotype ( yan 443 / yan 884 ; Yan-YFP / Yan-YFP ) were dissected, fixed, and imaged by confocal microscopy, as previously described [17, 18]. These prepupae also had the appropriate combinations of Egfr alleles, Gal4 driver genes, and UAS transgenes for controlled manipulation of EGFR activity and cell metabolism.…”

“…Image data was processed for automatic segmentation and quantitation of DAPI and YFP nuclear fluorescence as described [17, 18]. Briefly, cell segmentation was performed using the DAPI signal as a reference channel for identification of cell nuclei boundaries.…”

“…Using FlyEye , cell positions along the anterior-posterior axis were mapped to developmental time as described previously [17, 18]. This is predicated on two assumptions: the furrow proceeds at a constant rate of one column of R8 neurons per two hours; and minimal cell migration occurs.…”