DEMONSTRATED THAT A BALANCE OF MAD AND MYC EXPRESSION DICTATES LARVAL CELL APOPTOSIS AND ADULT STEM CELL DEVELOPMENT DURING XENOPUS INTESTINAL METAMOPRHOSIS. The Myc/Mad/Max network has long been shown to be an important player in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc-Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad-Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. We have discovered that Mad1 is induced by TH in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we have demonstrated that Mad1 is expressed in the larval cells undergoing apoptosis while c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may play a role in cell death during development. By using TALEN-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development. REVEALED THE REQUIREMENT OF THYROID HORMONE-INDUCED ACTIVATION OF NOTCH SIGNALING FOR ADULT INTESTINAL STEM CELL DEVELOPMENT DURING METAMOPRHOSIS. We have shown earlier that TH-induced intestinal remodeling involves that regulation of various genes including Notch receptor. To study the role of Notch signaling pathway, we have analyzed the expression of different components of this pathway, including the ligands (DLL and Jag), receptor (Notch) and targets (Hairy), in the metamorphosing intestine by real-time RT-PCR and in situ hybridization or immunohistochemistry. We have shown that they are up-regulated during both natural and TH-induced metamorphosis in a tissue-specific manner. Particularly, Hairy1 is specifically expressed in the adult epithelial stem cells. Moreover, up-regulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a -secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH-induced up-regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we have demonstrated in organ cultures that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development.
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