Transcription of messenger RNAs (mRNAs) for Notch signaling molecules oscillates with 2-hour cycles, and this oscillation is important for coordinated somite segmentation. However, the molecular mechanism of such oscillation remains to be determined. Here, we show that serum treatment of cultured cells induces cyclic expression of both mRNA and protein of the Notch effector Hes1, a basic helix-loop-helix (bHLH) factor, with 2-hour periodicity. Cycling is cell-autonomous and depends on negative autoregulation of hes1 transcription and ubiquitin-proteasome-mediated degradation of Hes1 protein. Because Hes1 oscillation can be seen in many cell types, this clock may regulate timing in many biological systems.

Hes7, a bHLH gene essential for somitogenesis, displays cyclic expression of mRNA in the presomitic mesoderm (PSM). Here, we show that Hes7 protein is also expressed in a dynamic manner, which depends on proteasomemediated degradation. Spatial comparison revealed that Hes7 and Lunatic fringe (Lfng) transcription occurs in the Hes7 protein-negative domains. Furthermore, Hes7 and Lfng transcription is constitutively up-regulated in the absence of Hes7 protein and down-regulated by stabilization of Hes7 protein. Thus, periodic repression by Hes7 protein is critical for the cyclic transcription of Hes7 and Lfng, and this negative feedback represents a molecular basis for the segmentation clock. Received March 10, 2003; revised version accepted April 25, 2003. Somites, the metameric units of vertebrate embryos, are aligned along both sides of the neural tube and give rise to repetitive structures including vertebrae, ribs, and skeletal muscles. A bilateral pair of somites buds off from the anteriormost end of the unsegmented presomitic mesoderm (PSM;Pourquié 2001;Saga and Takeda 2001). A new somite is formed every 120 min in the mouse, and this periodic event is believed to be governed by a molecular clock (Cooke 1998;Dale and Pourquié 2000). It is thus likely that, in somitogenesis, the temporal periodicity of the molecular clock is translated into the spatial periodicity of somites.The first evidence of a molecular clock for somite segmentation was provided by the finding of the oscillatory expression of the basic helix-loop-helix (bHLH) gene chairy1 (Palmeirim et al. 1997). The expression of c-hairy1 mRNA sweeps across the PSM in a posteriorto-anterior direction repeatedly, and each cycle is synchronous with the somite formation. Interestingly, this wave-like propagation of gene expression is not caused by cell movement but is the result of synchronous oscillation of c-hairy1 expression in the PSM cells. Like chairy1, several other genes, all of which are involved in Notch signaling, also show cyclic expression in the PSM.They include bHLH genes such as Hes1, Hes7, and Hey2 in mouse and her1 and her7 in zebrafish (Holley et al. 2000;Jouve et al. 2000;Leimeister et al. 2000;Sawada et al. 2000;Bessho et al. 2001b;Dunwoodie et al. 2002;Oates and Ho 2002). In addition, the expression of zebrafish deltaC, which encodes a ligand for Notch, and mouse and chick Lfng, a gene for glycosyltransferase that modulates the Notch signaling, oscillates in the PSM (Forsberg et al. 1998;McGrew et al. 1998;Aulehla and Johnson 1999;Jiang et al. 2000). Genetic analyses revealed that at least some of these oscillating genes play a critical role in somitogenesis. Mutations for deltaC, her1, and her7 in zebrafish (Holley et al. 2000(Holley et al. , 2002Henry et al. 2002;Oates and Ho 2002) and Lfng and Hes7 in mouse (Evrard et al. 1998;Zhang and Gridley 1998;Bessho et al. 2001b) all exhibit defects of somite segmentation. Furthermore, persistent expression of Lfng also perturbs somite segmentation (Dale et al. 2003;Serth et al. 2003). ...

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca2+ from internal stores to initiate muscle contraction. Defects in EC coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the EC coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca2+ imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 as the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.

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