Exploring the Cellular and Epigenetic Changes Associated with the Obligate Developmental Arrest in Embryos of the Killifish Austrofundulus limnaeus

Embryonic development is a dynamic and continuous event of rapidly changing cells and tissues to form a specified and differentiated state. However, in a few metazoans an obligate developmental arrest has evolved.  The killifish Austrofundulus limnaeus has the remarkable ability to enter into an arrested state (diapause II) prior to the completion of development and in the absence of environmental cues; this developmental arrest can be maintained for over 100 days. Currently, little is known about the molecular mechanisms that induce and maintain this developmentally arrested state.  A. limnaeus embryos provide investigators a rare context to study the modulation of gene expression and other cellular morphological changes associated with obligate developmental arrest. We hypothesize that histone modifications modulating gene expression and gross cellular morphological changes are associated with the drastic cessation of development observed in diapause II. We have developed a methodology for whole embryo analysis which is principally based upon immunostaining. Our approach is to utilize antibodies that recognize highly conserved epitopes of histone modifications and other cellular markers to examine arrested embryos. Preliminary whole embryo analysis shows the signal of anti-H3K27me2 antibody localizing to the nuclear periphery in diapause II animals. This nuclear localization is associated silenced chromatin and repressed gene expression. Additionally, assays involving Anti-PhosH3, which detects mitotic nuclei, show a decline in the number of mitotic nuclei as the embryos progress towards diapause II. This research is leading to a greater molecular understanding of the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.