Analyzing the molecular mechanism of Bucky ball localization during germ cell specification in zebrafish

Abstract

Sexual reproducing animals depend on the proper establishment of the germline to ensure the survival of the species by generating fertile progeny. Therefore, many vertebrate and invertebrate species specify their germline already during embryogenesis by inheritance of maternal factors aggregated in the germ plasm. Only cells that inherit germ plasm during early embryogenesis are determined to become germ cells, whereas all other cells are liberated to pursue somatic fates and will form the body that transfers the germ cells to the next generation. Hence, proper localization of germ plasm is essential for germ cell specification. Recently, zebrafish Bucky ball has been identified as the first protein in vertebrates to be necessary for germ plasm aggregation and sufficient for specification of primordial germ cells. However, Buc protein localization and the underlying localization mechanism, essential for proper spatial germ plasm aggregation, were not known. In this study, Buc localization was analyzed by immunostaining with a newly generated antibody and by live-cell imaging of a transgenic buc-gfp line. Furthermore, Buc localization was analyzed in comparison to the germ plasm regulator Drosophila Osk and the domain responsible for Buc localization was mapped in a systematic deletion approach. Additionally, proteins, interacting with the Buc localization domain, were identified via Co-IP followed by mass spectrometry analysis and were verified by in vivo co-localization analysis. This study shows that Buc protein continuously localizes to the germ plasm throughout zebrafish oogenesis and embryogenesis on the endogenous level as well as in the transgenic buc-gfp line. This specific localization pattern depends on the conserved N-terminal amino acids 11 88. Moreover, the localization domain BucLoc interacts with non-muscle myosin II and persistently co localizes with the non-muscle myosin II regulatory chain Myl12.2. With the localization of Buc, permanent protein localization to the germ plasm was described for the first time in zebrafish. This indicates that germ plasm aggregating function of Buc is required throughout the process of germ cell specification and beyond. Furthermore, BucLoc is the first protein localization domain being necessary and sufficient for localization to the germ plasm in a metazoan. Thus, BucLoc provides the first protein based reporter for germ plasm as well as primordial germ cells. In addition, the interaction and co-localization of Buc with non-muscle myosin II links Buc to the actin cytoskeleton. This would be in line with a previously suggested mechanism, stating that germ plasm granules associated with actin filaments are recruited to the germ plasm. Hence, functional investigations of the interaction between Buc and non-muscle myosin II can give insight into the localization of germ plasm to the presumptive primordial germ cells during early embryogenesis in zebrafish. Further insight into this mechanism will help to better understand the process of germ cell formation and might lead to the identification of new drug targets and therapies against infertility