391Recently, new information has become available on the regulation of Bb forma-
tion, its function, and disassembly (Heim et al. 2014 ). Large-scale mutagenesis
screens in zebrafish (Wienholds et al. 2003 ; Dosch et al. 2004 ), uncovered a mutant
line showing defects in oocyte polarity (Marlow and Mullins 2008 ). Further experi-
ments demonstrated that a nonsense mutation in the buckyball (buc) gene disrupts
maintenance of oocyte polarity and affects germ plasm assembly (Bontems et al.
2009 ). Buckyball is the only gene known to be required for Bb formation in verte-
brates. During zebrafish early oogenesis buc mRNA and protein are vegetally local-
ized in close association with the germ plasm. By mid to late oogenesis, buc protein
is repositioned in the animal pole cortex where it persists until embryogenesis.
During development, buc transcripts are lost but its protein remains localized.
Remarkably, misexpression of BUC during embryogenesis induces germ plasm and
PGC formation (Bontems et al. 2009 ). In buckyball mutants, the Bb fails to form
and vegetal RNAs like vasa, nanos1 and dazl are not localized, reflecting a defect in
the animal–vegetal polarity of the oocyte (Bontems et al. 2009 ). Although Buc is an
evolutionary conserved protein it is unrelated to any known family protein, with no
conserved protein motifs that would provide awareness into its biological function.
Large-scale mutagenesis screens discovered another gene, Magellan, required
for animal–vegetal polarity of the oocyte and egg (Dosch et al. 2004 ). Magellan
(mgn) mutants exhibit an asymmetric localization of the oocyte nucleus, a novel
enlarged Bb phenotype and an absence of vegetally localized RNAs at the oocyte
cortex (Gupta et al. 2010 ). The mutation was identified as a deletion in the coding
Fig. 8.2 Specification of zebrafish primordial germ cells: Schematic summary of zebrafish germ
line development and segregation during early embryogenesis based on the distribution of vasa and
nanos1 RNA. Germ plasm mRNAs are recruited to the forming furrows of the first and second
cleavage planes (arrows). Upon furrow maturation, the recruited mRNAs form a tight aggregate at
the distal end of the furrow, a microtubule dependent process. The resulting four subcellular aggre-
gates ingress into four cells at the 32-cell stage and segregate asymmetrically during subsequent
cell divisions (insert at 1K-cell stage). Until this point, these cells are called precursors of PGCs.
At early gastrula stage (~4k-cells), germ plasm mRNAs become evenly distributed within cells and
segregate symmetrically during cell divisions (insert). At this point cells are called PGCs
8 Mechanisms of Vertebrate Germ Cell Determination