Molecular Mechanism Of The P90RSK Pathway In Cell Cycle Regulation During Development Of Artemia-encysted Embryos

Artemia is a genius of Crustacean widely distributing in salterns and salt lakes. It possesses powerful adaptations to extreme environments. As a strategy to cope with environmental stresses, Artemia has evolved a special reproductive mode in that in addition to giving birth to nauplii by the ovoviviparous pathway, this genus also releases encysted embryos by the oviparous pathway. Released encysted embryos are developmentally arrested at the gastrula stage with a low metabolic rate and complete turnoff of replication, transcription, and translation; however, without loss of embryonic viability. During the development of Artemia-encysted embryos, no cell division or DNA synthesis occurs before emergence, and the number of nuclei remains at about 4000 per embryo despite the great number of internal events including deposited energy mobilization, RNA and protein synthesis restoration, cellular differentiation, and associated morphological changes required for the development from gastrula to prenauplius.The mechanism of this unusual developmental pattern is still unclear. It is considered that the absence of cell division during the early embryonic development of Artemia is an adaptation to environmental threats, which confer on embryos their amazing stability and viability.Our research focuses on the molecular mechanism of p90RSK pathway in cell cycle regulation during development of Artemia-encysted embryos. RSK is a family of serine/threonine kinases that mediate signal transduction downstream of mitogen-activated protein kinase cascades. RSK family members have been reported to be multifunctional in the regulation of diverse cellular processes including transcriptional regulation, cell cycle control, cell survival, and many others. In the present study, we analyzed the expression and activation patterns of Artemia RSK, and identified its function to the regulation of cell cycle arrest and mitogenesis through in vivo knockdowns, during the embryonic and larval development of Artemia. Basing on this, the structure-functional studies of Artemia RSK, and the exploring of its upstream and downstream molecules were carried out. Besides, the functional analysis and comparison between Artemia RSK and other family members was performed in transgenic cell lines and Xenopus.Our results represent that, Artemia RSK was established to be specifically activated at the very beginning of emergence and was coupled with mitogenesis during the post-embryonic and early larval developmental stages. In vivo knockdown of RSK activity consistently induced abnormal individuals with distinct gaps between the exoskeleton and the internal tissues in the developing thoracic and abdominal regions. By BrdU labeling and mitotic index analysis, mitoses were detected to be largely inhibited in those affected segments. In this pathway, RSK was activated concomitantly with ERK activation, and the C terminal of Artemia RSK containing an ERK docking sequence was crucial to the kinase activation. In the downstream regulation, there might be an intracellular connection between RSK and the molecular chaperone p26. Otherwise, expression of Artemia RSK in Xenopus oocytes with endogenous RSK inhibition could induce an advance of showing GVBD (germinal vesicle breakdown) evidence; however, they did not proceed to a distinct GVBD subsequently.Our research, thus, indicates that the RSK pathway is essential in the post-embryonic and early-larval development of Artemia by playing a major role in the termination of cell cycle (G2/M phase) arrest and the promotion of mitogenesis, essential for development. These findings not only provide insights into the molecular mechanism regulating the special developmental pattern of Artemia-encysted embryos, but also reveal further aspects of RSK functions.