Shigeki Yoshiura

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.

Serum response has been used as a model for studying signaling transduction for many biological events such as cell proliferation and survival. Although expression of many genes is up- or down-regulated after serum stimulation, the Notch effector Hes1 displays oscillatory response. However, the precise mechanism and biological significance of this oscillation remain to be determined. Here, we identified serum-induced ultradian oscillators, including molecules in Stat and Smad signaling. Stat and Smad oscillations involve activation of Stat3 and Smad1 and delayed negative feedback by their inhibitors Socs3 and Smad6, respectively. Moreover, Stat oscillations induce oscillatory expression of Hes1 by regulating its half-life, and loss of Hes1 oscillations leads to G(1) phase retardation of the cell cycle. These results indicate that coupled Stat and Hes1 oscillations are important for efficient cell proliferation and provide evidence that expression modes of signaling molecules affect downstream cellular events.

PURPOSE: The purpose of this study was to investigate the effects of various genes related to photoreceptor development on rodent and primate iris cells and the potential of iris cells as donor cells for retinal transplantation. METHODS: Adult rat and monkey iris tissue were cultured in serum-free medium containing basic fibroblast growth factor. Gene deliveries of Crx, Nrl, NeuroD and some combinations (Crx-Nrl, Crx-NeuroD) were performed with recombinant retrovirus. Immunocytochemistry, Western blot analysis, RT-PCR, and intracellular recording were used to examine the expression of photoreceptor-specific phenotypes in the iris-derived cells after gene transfer, . Coculture of the iris-derived cells with embryonic retinal explant was conducted, to investigate the potential integration of these cells in coculture conditions. RESULTS: Misexpression of Crx induced adult rat iris cells to express several photoreceptor-specific antigens and transcripts, such as rhodopsin, recoverin, cGMP-gated channel, arrestin, interphotoreceptor retinal-binding protein, rhodopsin kinase, and NeuroD. In primates, a combination of Crx and NeuroD was needed to induce monkey iris-derived cells to adopt photoreceptor-specific phenotypes. Furthermore, the photoreceptor-like cells derived from both rat- and primate-iris tissues showed rod photoreceptor-specific electrophysiological response to light stimuli after Crx and Crx-NeuroD gene transfer, respectively. The results further showed that iris-derived cells integrated in the developing host retina in coculture conditions. CONCLUSIONS: Adult iris-derived cultured cells of both rodents and primates expressed photoreceptor-specific phenotypes by inductions of transcription factors. These iris-derived photoreceptor-like cells have electrophysiological characteristics of rod photoreceptors. Furthermore, they can integrate in the developing retina under coculture conditions.

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. Here, we reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity.