Abstract
The Myc/Mad/Max network has long been shown to be an important factor 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. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-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.
Highlights
The Myc/Mad/Max network has a vital role in the normal cell cycle, ensuring proper proliferation and differentiation.[1,2,3] c-Myc is a well-characterized transcription factor and oncogene, which generally activates gene transcription, thereby inducing proliferation upon heterodimerizing with Max
Little Mad[1] expression was observed in the non-epithelium at any stages (Figure 2a). These results suggest that Mad[1] levels temporally correlate with the intestinal epithelial transformation with the vast majority of the larval cells undergoing apoptosis accompanied by the dedifferentiation of some larval epithelial cells into adult stem cells.[19,29,30]
Intestinal remodeling during Xenopus metamorphosis offers a unique opportunity to study the formation of adult organspecific stem cells during postembryonic development in vertebrates in part because of the difficulty to manipulate late stage, uterus-enclosed mammalian embryos.[24]
Summary
The Myc/Mad/Max network has a vital role in the normal cell cycle, ensuring proper proliferation and differentiation.[1,2,3] c-Myc is a well-characterized transcription factor and oncogene, which generally activates gene transcription, thereby inducing proliferation upon heterodimerizing with Max. Amphibian metamorphosis is totally controlled by thyroid hormone (T3) and resembles postembryonic development in mammals, a period around birth when many organs matured into the adult form.[17] In Xenopus laevis (X. laevis) and the highly related species Xenopus tropicalis, the tadpole intestine is a simple tubular structure made of mainly larval epithelial cells surrounded by thin layers of connective tissue and muscles.[18,19,20] The epithelial cells are fully differentiated into specialized cells yet capable of, and often undergo, mitotic division.[20,21] As circulating T3 levels increase, thereby initiating metamorphosis, a subset of these differentiated epithelial cells dedifferentiate into adult stem cells.[22] The remaining larval epithelial cells undergo apoptosis as new adult tissue expands to completely replace the larval epithelial cells. By using transcription activator-like effector nuclease (TALEN)-mediating gene-editing in vivo, we showed that Mad[1] knockout increased levels of proliferating cells and reduced apoptosis during metamorphosis These findings suggest that Mad[1] causes mitotically active larval epithelial cells to exit the cell cycle thereby ensuring their destruction and a Mad–Myc balance controls the expansion of adult intestinal epithelial cells
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