Oscillations are common in biology, e.g. the circadian clock, cardiac pacemaker, cortical rhythms, and have been observed in cell signaling networks as a result of feedback loops. These cellular and physiological oscilla tions maintain homeostasis and analysis of these rhythms is an emerging area in biomedical science. During vertebrate segmentation, ultradian oscillations govern the formation of somites, the segmented anlagen of the vertebral column. In zebrafish, mice and humans, somitogenesis requires Notch signaling, perturbation of which leads to malformed vertebrae, a birth defect called spondylocostal dysostosis. The Notchdependent oscilla tions cause cells in the segmenting tissue of the zebrafish to undergo repeated cycles of expression and repression of Notch target genes. The her (hairy/enhancer of split related tranSCfiptional repressors) genes are thought to form a negative feedback loop within the zebrafish segmentation clock. Current data suggest thai the different /lergenes have both unique and redundant functions.
Aim I is to understand the functional diversification of her genes within the zebrafish clock and to identify cis sequences that govern transcriptional oscillations. To achieve these aims, we will use electrophoretic mobility shift assays (EMSA), immunoprecipitation and transgenic zebrafish.
Aim II is to develop a temperature-sensitive control of the transgenes in order to precisely tune the level and timing of expression. This strategy deals with the general problem of discerning signal integration and gene function within the context of dynamic signaling networks and developmental lime. We are Aim III is to characterize the relationship between cell movement and the segmentation clock. Using timelapse imaging and celt tracking, we will determine the velocity. direction and neighbor relationships of celts in different rQ9ions of the tail bud.
Aberrant somitogenesis leads to birth defects in humans. Later in life abnormal Notch, Wnt or Fgf signaling causes malignancies such as colon cancer and leukemia. We use zebrafish as a tractable, relatively inexpensive vertebrate model system to study how perturbation of these cell signaling pathways affects complex cell behavior in vivo. We believe that better comprehension will lead to more efficacious therapies.
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