Analysis of genome silencing and mitochondria function in the regulation of diapause II in Austrofundulus limnaeus embryos

Austrofundulus limnaeus is a freshwater annual killifish from Venezuela that produces embryos capable of entering three distinct stages of diapause. Diapause II is a state of developmental arrest that can last over 100 days and confers extreme anoxia tolerance. Diapause I and III are inducible while II is obligate. Diapause II occurs in optimal conditions (25^oC) whereas incubation at 30^oC induces a bypass of diapause II. Diapause II provides an opportunity to study an arrest of development in a vertebrate animal without exposing the organism to environmental stresses. Currently, little is known about the molecular mechanisms that induce and maintain this developmentally arrested state. We hypothesize that a down regulation of gene expression is required to maintain this undifferentiated state and the regulating mechanisms have an epigenetic origin.  By designing assays that probe for epigenetic modifications associated with gene silencing and gene activation we are able to visualize chromatin modifications that are associated with developmental arrest and resumption of development.  Our results indicate that immunohistochemical assays utilizing antibodies specific for chromatin modifications, specifically acetylations and methylations, detect strong fluorescent signal localized near the inner nuclear periphery in animals that are in diapause II. The localization of chromatin to the nuclear periphery is strongly associated with silenced gene expression. This research is leading to a greater molecular understanding of the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo and the role that chromatin modifications may have in the event.