Abstract
Hydra is a unique model for studying the mechanisms underlying stem cell biology. The activity of the three stem cell lineages structuring its body constantly replenishes mature cells lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. In vertebrates, one of many genes that participate in regulating stem cell homeostasis is the protooncogene c-myc, which has been recently identified also in Hydra, and found expressed in the interstitial stem cell lineage. In the present paper, by developing a novel strategy of RNA interference-mediated gene silencing (RNAi) based on an enhanced uptake of small interfering RNAi (siRNA), we provide molecular and biological evidence for an unexpected function of the Hydra myc gene (Hymyc1) in the homeostasis of the interstitial stem cell lineage. We found that Hymyc1 inhibition impairs the balance between stem cell self renewal/differentiation, as shown by the accumulation of stem cell intermediate and terminal differentiation products in genetically interfered animals. The identical phenotype induced by the 10058-F4 inhibitor, a disruptor of c-Myc/Max dimerization, demonstrates the specificity of the RNAi approach. We show the kinetic and the reversible feature of Hymyc1 RNAi, together with the effects displayed on regenerating animals. Our results show the involvement of Hymyc1 in the control of interstitial stem cell dynamics, provide new clues to decipher the molecular control of the cell and tissue plasticity in Hydra, and also provide further insights into the complex myc network in higher organisms. The ability of Hydra cells to uptake double stranded RNA and to trigger a RNAi response lays the foundations of a comprehensive analysis of the RNAi response in Hydra allowing us to track back in the evolution and the origin of this process.
Highlights
Despite its simple body plan and structural anatomy, the Cnidaria Hydra, a dipoblastic animal at the base of metazoan evolution, is an excellent model system to investigate the mechanisms controlling stem cell proliferation and differentiation and the balance between the two phenomena
We developed in this paper a novel approach of RNA interference-mediated gene silencing (RNAi) in Hydra, based on small interfering RNAi (siRNA) oligonucleotides
In Drosophila an active and specific pathway that involves clathrin-mediated endocytosis has been found responsible for siRNA uptake [47] and accumulation in vesicles, while the mechanism of cytoplasmic release to enter the RNAi machinery has not been identified to date
Summary
Despite its simple body plan and structural anatomy, the Cnidaria Hydra, a dipoblastic animal at the base of metazoan evolution, is an excellent model system to investigate the mechanisms controlling stem cell proliferation and differentiation and the balance between the two phenomena. The constant growth process requires a homeostatic regulation within and between different cell lineages and a steady state of production and loss of cells [1,2,3,4]. A variety of control mechanisms are needed to maintain steady state levels of mature cells, as well as to stimulate the rapid production of specific cell types as needed. This might requires the participation of many factors, including positive and negative regulators of growth and differentiation, which determine survival, growth stimulation, growth arrest, differentiation
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