Developmental regulation of oscillatory expression

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Project Summary Ultradian oscillatory circuits are pervasive in biological systems. These dynamic oscillators range from pulsatile p53 expression after g-radiation, to periodic nuclear to cytoplasmic shuttling of NFkB transcription factor in response to cytokine, to cyclic expression of Hes/Her transcription factors in stem cells and presomitic mesoderm (PSM). Ultradian genetic oscillators are associated with patterning and developmental transitions – oscillations correlate with yeast metabolic cycles, foreshadow periodic lateral root branching in Arabidopsis, regulate molt cycles in larval worms, associate with stem cell pluripotency, and synchronize transcriptional response to starvation signals in Dictostelium. One biological oscillator is the vertebrate segmentation clock, which controls somitogenesis, the process by which the PSM is sequentially divided into segments called somites. At the core of the segmentation clock is an auto-inhibitory negative feedback loop involving Her/Hes transcriptional repressors, which in turn regulates oscillatory expression of additional ‘cyclic genes’. Although the cyclic genes in human and mouse belong to similar pathways (e.g., Notch, Wnt, FGF, Yap/Hippo), genes in those pathways which are cyclically expressed vary among species, with the Her/Hes gene family genes being common among vertebrates. We study the zebrafish segmentation clock, which oscillates with a 30-minute periodicity and is 4-12 times faster than in mouse or human. In order for a rapid auto-inhibitory oscillator to operate, there must be tight control over synthesis and decay of cyclic gene transcript and protein. A recent experiment in which the Hes7 locus was swapped between human and mouse in vitro PSM systems showed that expression delays and decay, controlled by factors in the host cell environment, are critical regulatory parameters of the clock. To understand the mechanisms regulating these critical parameters, we are using the zebrafish pnrc2 mutant in which transcriptional oscillations occur normally, but post-transcriptional decay mechanisms are disrupted, to identify additional cyclic genes and dissect their regulation. The specific aims of the proposal are to (1) identify Pnrc2-regulated zebrafish embryonic cyclic genes that play critical developmental roles, (2) characterize the regulatory features and factors that control rapid decay dynamics of cyclic gene transcripts, and (3) investigate the role of other post-transcriptional mechanism in regulating segmentation clock function. We anticipate that our work will broadly impact understanding of post- transcriptional mechanisms regulating oscillatory expression in many developmental contexts.