Spatial Analysis of Expression Patterns Predicts Genetic Interactions at the Mid-Hindbrain Boundary

The isthmic organizer mediating differentiation of mid- and hindbrain during vertebrate development is characterized by a well-defined pattern of locally restricted gene expression domains around the mid-hindbrain boundary (MHB). This pattern is established and maintained by a regulatory network between several transcription and secreted factors that is not yet understood in full detail. In this contribution we show that a Boolean analysis of the characteristic spatial gene expression patterns at the murine MHB reveals key regulatory interactions in this network. Our analysis employs techniques from computational logic for the minimization of Boolean functions. This approach allows us to predict also the interplay of the various regulatory interactions. In particular, we predict a maintaining, rather than inducing, effect of Fgf8 on Wnt1 expression, an issue that remained unclear from published data. Using mouse anterior neural plate/tube explant cultures, we provide experimental evidence that Fgf8 in fact only maintains but does not induce ectopic Wnt1 expression in these explants. In combination with previously validated interactions, this finding allows for the construction of a regulatory network between key transcription and secreted factors at the MHB. Analyses of Boolean, differential equation and reaction-diffusion models of this network confirm that it is indeed able to explain the stable maintenance of the MHB as well as time-courses of expression patterns both under wild-type and various knock-out conditions. In conclusion, we demonstrate that similar to temporal also spatial expression patterns can be used to gain information about the structure of regulatory networks. We show, in particular, that the spatial gene expression patterns around the MHB help us to understand the maintenance of this boundary on a systems level.Author Summary: Understanding brain formation during development is a tantalizing challenge. It is also essential for the fight against neurodegenerative diseases. In vertebrates, the central nervous system arises from a structure called the neural plate. This tissue is divided into four regions, which continue to develop into forebrain, midbrain, hindbrain and spinal cord. Interactions between locally expressed genes and signaling molecules are responsible for this patterning. Two key signaling molecules in this process are Fgf8 and Wnt1 proteins. They are secreted from a signaling center located at the boundary between prospective mid- and hindbrain (mid-hindbrain boundary, MHB) and mediate development of these two brain regions. Here, we logically analyze the spatial gene expression patterns at the MHB and predict interactions involved in the differentiation of mid- and hindbrain. In particular, our analysis indicates that Wnt1 depends on Fgf8 for stable maintenance. A time-course analysis of Wnt1 expression after implantation of Fgf8-coated beads in mouse neural plate/tube explants experimentally validates our prediction about the interactions between these two key patterning molecules. Subsequently, we demonstrate that available data allows construction of a mathematical model able to explain the maintenance of the signaling center at the MHB. We begin to understand this small aspect of brain formation on a systems level.