Study On The Regulation Of Mitosis By AMPK-α During Artemia Embryonic Development

The primitive crustacean, Artemia, has evolved on the Earth for about 400 million years. In order to adapt to extreme environments such as salt lakes on plateaus and coastal lagoons, Artemia has developed two reproductive pathways, i.e. ovoviviparity and oviparity. Under favorable environmental conditions, Artemia directly release swimming nauplii; when environmental conditions become unfavorable, Artemia produce encysted gastrula embryos that quickly become an obligate dormant diapause after dehydration. After frozen stimulation of about three months, diapause embryos change into post-diapause embryos, which can continue develop and hatch into swimming nauplii when favorable conditions come. A cell cycle arrests in diapause embryos and restarts during hatching of post-diapause embryos, which illustrates that Artemia is an ideal animal to study mitosis.The serine/threonine kinase AMPK (AMP-activated protein kinase), is an energy sensor and plays a central role in the maintenance of energy homeostasis. AMPK is an evolutionarily conserved heterotrimer, the a-subunit of which is a catalytic subunit and therefore plays a major role in the activity of AMPK. There are two AMPK-cc isoforms (i.e. AMPK-α1 and -α2) in most species. Studies pointed out that AMPK-al and-α2 play different roles in different tissues or at different stages of embryonic development except for maintenance of energy homeostasis. However, the roles AMPK-α1 and -α2 play in the regulation of mitosis remain largely unknown.First, we studied the expression and phosphorylation levels of AMPK-α1 and -α2 during embryonic development in ovoviviparous Artemia. Western blotting showed that the expression and phosphorylation of AMPK-al (pAMPK-al) are maintained at a constant level throughout embryonic development, while faint AMPK-a2 is expressed only at early embryonic development and swimming nauplii stages. pAMPK-al was enhanced and mitosis was arrestted in development after treatment with AICAR, an activator of AMPK. The results indicated that hyperactive AMPK induces cell cycle arrest.In addition, we studied the expression and phosphorylation levels of AMPK-al and -α2 during redevelopment of postdiapause embryos. Western blotting showed that the expression level of AMPK-α1 decreases from 4 h to 8 h of redevelopment, but increases from 12 h to 24 h. The expression level of AMPK-α2 increases throughout redevelopmental stages. pAMPK-α is maitained at a higher level at before 12th h of redevelopment, while decreases to a lower level after 12th h of redevelopment. BrdU incorporation assay and immunoprecipitation showed that the level of pAMPK-α1 was high before mitosis and decreased to low after mitosis during redevelopment of postdiapause embryos. The results indicated that AMPK is involved in the regulation of cell cycle during Artemia embryonic development.Previous studies showed that LKB1 is the major upstream kinase of AMPK. Here, in order to study the influence of LKB1 on AMPK in the regulation of cell division, we cloned lkb1 cDNA in Artemia and analyzed its expression level. The mRNA levels of lkb1 are significant higher at oogenesis and swimming nauplii stages than that at other developmental stages. During redevelopment of post-diapause embryos, the mRNA levels of Ikbl are significant higher at 4th and 8th h than that in the subsequent stages of development.In vivo knockdown of Ikbl do not influence development of early embryos, and ovoviviparous Artemia reproduce normal swimming nauplii which can develop into late metanaplii. However, oviparous Artemia with lkb1 knockdown reproduce pseudo-diapause embryos. These results indicated that LKB1 specifically regulates the embryonic development of oviparous Artemia. Western blotting showed that pAMPK-α1 decreased and the phosphorylation levels of two mitosis indicators, H3 and Rb increased after knockdown of lkb1. The results indicated that LKB1 regulates activity of AMPK-α1 and induces cell cycle arrest during formation of diapause. Last, TUNEL assay showed that the production of pseudo-diapause embryos was resulted from cell apoptosis after knockdown of lkb1.Our results showed that AMPK participates in the regulation of mitosis during embryonic development in Artemia. Hyperactive AMPK inhibits mitosis, which is dependent on LKB1.