The segmentation clock is a biological oscillator that controls the rhythmic formation of somites during vertebrate embryogenesis. Its core is composed of a transcriptional negative feedback loop in which oscillatory expression of her/Hes genes produces spatially coordinated waves of gene activity in the presomitic mesoderm (PSM). While theoretical models predict that the period of such oscillators should scale with the degradation rate and transcriptional delay of the core repressors, experimental evidence has revealed a more complex landscape, shaped by both intrinsic and extrinsic modulators. This thesis investigates the biophysical and biochemical parameters that influence oscillation timing in the zebrafish segmentation clock, with a particular focus on the protein Her1, a key component of the oscillator.
Using a combination of fluorescent reporter lines, pharmacological perturbations, and single-cell time-lapse imaging, I systematically examined how translation and degradation rates affect Her1 dynamics. Low doses of cycloheximide extended the Her1 oscillation period without altering protein stability, suggesting that translational delay modulates feedback timing. Conversely, proteasome inhibition with MG132 increased Her1 half-life but did not measurably change the period, revealing an apparent decoupling between degradation and timing. This dissociation contradicts the monotonic period-degradation relationship predicted by delay differential equation models and suggests buffering at the network level.
To test whether direct modulation of Her1 degradation can influence timing, I developed a transgenic zebrafish line expressing Her1 fused to a fluorescent tag and an auxin-inducible degron (mAID). In a simplified genetic background lacking functional endogenous clock genes, this construct served as the sole functional component of the core oscillator. Although traveling waves and somite boundaries failed to form in this chassis system, dissociated single PSM cells expressing the transgene retained self-sustained oscillations. These results demonstrate that Her1 alone is sufficient to drive autonomous oscillations, and that this behavior is preserved even in the absence of other core segmentation clock repressors.
Notably, the uninduced mAID tag consistently prolonged the period of Her1 oscillations, across both wild-type and mutant backgrounds. This effect likely reflects intrinsic changes in Her1 feedback kinetics caused by the fusion tag, such as altered folding, dimerization, or DNA-binding efficiency. Furthermore, I found that the oscillation period is highly sensitive to external factors: cells cultured in a low-autofluorescence medium oscillated with significantly longer periods than those in standard L15 medium, likely due to metabolic differences that affect transcription and degradation kinetics.
Together, these findings define Her1 as a minimal genetic oscillator, illuminate how its timing can be modulated by both intrinsic an
