A fundamental question in biology is how seemingly homogeneous fields of cells become elaborately patterned during vertebrate embryogenesis, and how these processes are regulated both by intrinsic and extrinsic signals. One striking example is that of somitogenesis, the process by which segments (somites) are sequentially formed on either side of the embryonic midline from the paraxial presomitic mesoderm generated by the growing vertebrate tail bud. This process is exquisitely precise, with each somite pair forming with a strict periodicity in an anterior to posterior direction. In zebrafish, the model organism used in this study, a new somite pair forms every 30 minutes;in drier vertebrates like chick and mouse, the period is longer (90 and 120 minutes, respectively). A recent explosion of work in this area has indicated that the period is controlled in presomitic cells by an intrinsic """"""""segmentation clock"""""""" which involves the oscillating (cyclic) expression of a number of genes. Central to the mechanism in all vertebrates is the cyclic expression of hairy/Enhancer of split-like (her/hes) genes, which encode transcriptional repressors that contribute critical negative feedback to the clock and that can be regulated by Notch signals. Although a number of factors that influence the clock have been identified, it remains an open question as to how all the inputs are regulated and coordinated. What starts the clock? How is periodicity controlled? How do cells synchronize with their neighbors? One goal of this work is to use real-time imaging of a zebrafish transgenic reporter line that recapitulates her1 gene oscillation to investigate how this exquisite timer is started in each cell, how it is influenced by the oscillations of neighbor cells, and how and if the oscillation period changes over time. A second goal is to identify the post- transcriptional regulatory elements that control clock function, with the goal of understanding how different regulatory inputs influence the clock. The last goal is to characterize new genes identified by mutation that influence the oscillation and elucidate their molecular mechanism of action. The long-term goals are to understand how cells assess their position within an embryo, respond to positional signals or gradients, and communicate positional information to their neighbors. In addition, since Hes gene expression has been shown to oscillate in non-somitic cells, this work will lead to insights about cellular timers in many cell lineages. The Notch pathway has been implicated in a huge number of processes that affect human health and development, including cancers, stem cell self-renewal and differentiation, cell proliferation, and cell and organ differentiation programs, as well as in congenital skeletal disorders such as the spondylocostal dysostoses. One of the key questions facing biologists today is to understand how Notch signals control such diverse group of developmental decisions, and the work proposed will elucidate molecular mechanisms by which one Notch- regulated oscillator is controlled, providing insights into how Notch pathways may be regulated in other contexts. Project Narrative The Notch pathway has been implicated in a huge number of processes that affect human health and development, including cancers, stem cell self-renewal and differentiation, cell proliferation, and cell and organ differentiation programs, as well as genetic disease (including Alagille syndrome, CADASIL, and the spondylocostal dysostoses). One of the key questions facing biologists today is to understand how Notch signals control such a diverse group of developmental decisions;the work proposed here will elucidate molecular mechanisms by which a Notch-regulated oscillator is controlled, as well as how Notch signals integrate with other signaling pathways to control a differentiation program. We anticipate that our basic research will provide insights into how Notch pathways may be regulated in other contexts and lead to important insights into human development, health, and disease.
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