By combining biochemical, embryological, and mathematical approaches, this work uncovers an important role for protein-protein interactions in determining the dynamics of the somite-forming segmentation clock in vertebrates.
During vertebrate embryogenesis, the rhythmic and sequential segmentation of the body axis is regulated by an oscillating genetic network termed the segmentation clock. We describe a new dynamic model for the core pace-making circuit of the zebrafish segmentation clock based on a systematic biochemical investigation of the network's topology and precise measurements of somitogenesis dynamics in novel genetic mutants. We show that the core pace-making circuit consists of two distinct negative feedback loops, one with Her1 homodimers and the other with Her7:Hes6 heterodimers, operating in parallel. To explain the observed single and double mutant phenotypes of her1, her7, and hes6 mutant embryos in our dynamic model, we postulate that the availability and effective stability of the dimers with DNA binding activity is controlled in a “dimer cloud” that contains all possible dimeric combinations between the three factors. This feature of our model predicts that Hes6 protein levels should oscillate despite constant hes6 mRNA production, which we confirm experimentally using novel Hes6 antibodies. The control of the circuit's dynamics by a population of dimers with and without DNA binding activity is a new principle for the segmentation clock and may be relevant to other biological clocks and transcriptional regulatory networks.
The segmented pattern of the vertebral column, one of the defining features of the vertebrate body, is established during embryogenesis. The embryo's segments, called somites, form sequentially and rhythmically from head to tail. The periodicity of somite formation is regulated by the segmentation clock, a genetic oscillator that ticks in the posterior-most embryonic tissue: for each tick of the clock, one new bilateral pair of segments is made. The period of the clock appears to determine the number and the length of segments, but what controls this periodicity? In this article, we have investigated the interactions of three transcription factors that form the core of the clock's regulatory circuit, and have measured how the period of segmentation changes when these factors are mutated alone or in combination. We find that these three factors contribute to a “dimer cloud” that contains all possible dimeric combinations; however, only two dimers in this cloud can bind DNA, which allows them to directly regulate the oscillatory gene expression that underpins the periodicity of segment formation. Nevertheless, a mathematical model of the clock's dynamics based on our experimental findings indicates that the non-DNA-binding dimers also influence the stability, and hence the function, of the two DNA-binding dimers controlling the segmentation clock's period. Such involvement of non-DNA-binding dimers is a novel regulatory principle for the segmentation clock, which might also be a general mechanism that operates in other biological clocks.