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was originally developed in yeast for live imaging of endogenous ash1 mRNA
asymmetric localization to the bud tip (Bertrand et al. 1998 ). The MS2 system is
composed of two elements: the MS2 coat protein from bacteriophage (MCP) fused
to GFP and multiple MCP-binding sites composed of stem-loops usually inserted
within the 3′UTR of the RNA target. Moreover, the technology was adapted to
study live dynamics of endogenous RNAs in oocytes. In flies, live tracking of an
engineered nanos-ms2 RNA revealed that nanos localizes to the oocyte posterior
through a diffusion and entrapment mechanism (Forrest and Gavis 2003 ). In stage
III frog oocytes, by injecting RNAs for the MS2 protein and Vg1-LE-ms2, the
transport dynamics of Vg1-LE to the vegetal pole and the role of molecular motors
in this process were characterized (Gagnon and Mowry 2010 ). Following a similar
strategy, the MS2 system was recently adapted in zebrafish showing the localiza-
tion of injected nanos-ms2 RNA to PGCs in the embryo, as revealed by MS2-GFP
provided by a transgene (Campbell et al. 2015a). Similar strategies will be possible
to use to visualize the dynamics of endogenous RNAs in the early Bb localization
pathway in vertebrate oogenesis, which has so far been inaccessible to this type of
study.
5.5 RNA Localization and AV Polarity: An Intimate
Connection
The identification of bucky ball and macf1 (magellan) genes demonstrated that Bb
formation and disassembly, respectively, is essential to defining the AV axis in ver-
tebrates. In buc mutants, the Bb is absent, and RNAs that normally localize to the
Bb and vegetal cortex appear diffuse in the cytoplasm. In macf1 mutants the Bb
forms and RNAs localize to it; however, disassembly of the Bb is defective, and the
Bb-localized RNAs never become anchored to the oocyte cortex. In both mutants,
the oocytes develop into eggs lacking polarity, showing a radial distribution of the
cytoplasm with no recognizable blastodisc. buc and macf1 are the only known
genetic entry points to studying oocyte polarity in vertebrates; hence, understanding
their functions is fundamental to deciphering the molecular mechanisms of AV
polarity establishment.
5.5.1 Bucky Ball: The Balbiani Body Gene
The buc transcript and protein localize to the Bb in zebrafish and, after Bb disas-
sembly, to the oocyte vegetal cortex (Fig. 5.2a and b) (Heim et al. 2014 ; Riemer
et al. 2015 ). Furthermore, Buc protein localizes to the germplasm in the early
embryo (Fig. 5.1c) (Riemer et al. 2015 ), where it can increase PGC number in over-
expression experiments (Bontems et al. 2009 ). So far, two Buc-interacting proteins
M. Escobar-Aguirre et al.