Reverse-Engineering Post-Transcriptional Regulation of Gap Genes in Drosophila melanogaster

Systems biology proceeds through repeated cycles of experiment and modeling. One way to implement this is reverse engineering, where models are fit to data to infer and analyse regulatory mechanisms. This requires rigorous methods to determine whether model parameters can be properly identified. Applying such methods in a complex biological context remains challenging. We use reverse engineering to study post-transcriptional regulation in pattern formation. As a case study, we analyse expression of the gap genes Krüppel, knirps, and giant in Drosophila melanogaster. We use detailed, quantitative datasets of gap gene mRNA and protein expression to solve and fit a model of post-transcriptional regulation, and establish its structural and practical identifiability. Our results demonstrate that post-transcriptional regulation is not required for patterning in this system, but is necessary for proper control of protein levels. Our work demonstrates that the uniqueness and specificity of a fitted model can be rigorously determined in the context of spatio-temporal pattern formation. This greatly increases the potential of reverse engineering for the study of development and other, similarly complex, biological processes.Author Summary: The analysis of pattern-forming gene networks is largely focussed on transcriptional regulation. However, post-transcriptional events, such as translation and regulation of protein stability also play important roles in the establishment of protein expression patterns and levels. In this study, we use a reverse-engineering approach—fitting mathematical models to quantitative expression data—to analyse post-transcriptional regulation of the Drosophila gap genes Krüppel, knirps and giant, involved in segment determination during early embryogenesis. Rigorous fitting requires us to establish whether our models provide a robust and unique solution. We demonstrate, for the first time, that this can be done in the context of a complex spatio-temporal regulatory system. This is an important methodological advance for reverse-engineering developmental processes. Our results indicate that post-transcriptional regulation is not required for pattern formation, but is necessary for proper regulation of gap protein levels. Specifically, we predict that translation rates must be tuned for rapid early accumulation, and protein stability must be increased for persistence of high protein levels at late stages of gap gene expression.