397visible signs of nuage, widely believed to be the precursors to the germinal gran-
ules, appear in the nuclei of PGCs within the indifferent gonad (al-Mukhtar and
Web 1971 ). Nuage can clearly be seen passing through the nuclear pores and into
the cytoplasm where it becomes intimately associated with mitochondria (Fig. 8.4b).
Aggregates of mitochondria become embedded within nuage material now called
mitochondrial cement at oogonia stages (Fig. 8.5a–d). What initiates this interaction
is unknown. Interestingly, mitochondria in germ plasm have a lower respiratory
activity than other mitochondria (Kogo et al. 2011 ). There are precedents to suggest
that the physiological state of mitochondria can determine position within the cell
(Cox and Spradling 2009 ). It is possible that nuage contains proteins similar to
Clueless in Drosophila that help recruit mitochondria based on their respiratory
activity. Mitochondria cement is thought to be the direct precursor of granulo-
fibrillar material (GFM) that forms from it but within the Bb. The GFM eventually
matures into germinal granules enriched in maternal RNAs and proteins required
for germ cell specification (Fig. 8.4b). However, in Xenopus the molecular identity
of germinal granules is only partially known, but includes nanos, deadsouth, xdazl,
large and small subunits of mitochondrial rRNA and xpat RNAs distributed either
in or on the surface of these granules (Kloc et al. 2002 ). The RNA binding protein
Hermes is the only known protein within the granules, but it is also found in nuclei,
germ plasm, and ooplasm (Chang et al. 2004 ). Hermes likely has several different
functions largely unknown, but recent work identifying Hermes interacting partner,
Xvelo, offers a clue. Xvelo is the homologue of zebrafish Buckyball, the gene
required for Bb formation (Marlow and Mullins 2008 ; Nijjar and Woodland 2013b).
Two splice variants of Xvelo1 were found and both RNAs produce a protein product
expressed in previtellogenic oocytes. However, only the larger isoform is found in
the Bb whereas the smaller protein variant has a cortical localization. The larger
variant Xvelo protein is too stable to deplete by degrading its RNA by antisense
DNA oligonucleotides. However, successful depletion of the shorter splice variant
from fully grown oocytes resulted in a decrease in germ plasm aggregates and a
loosening of associated mitochondria (Nijjar and Woodland 2013b). These results
suggest a role for Xvelo as a key scaffolding protein that interacts with RNA-binding
proteins. Interestingly, both Xvelo RNA variants are present in previtellogenic
oocytes, but are not localized within the Bb. Both RNAs localize later to the vegetal
cortex using the Late pathway. Because so few studies have been done characteriz-
ing the proteins in germ plasm, this pattern of protein distribution being different
from its corresponding RNA may be much more common than previously appreci-
ated. Making assumptions about protein expression based on RNA patterns can be
misleading. Vasa and Xpat proteins may offer another example (Machado et al.
2005 ; Lasko 2013 ). Two uncharacterized RNA binding proteins were also identified
as interacting with Hermes. Whether these Hermes interacting proteins are in ger-
minal granules remains to be determined. In addition to germinal granules, germ
plasm accumulates a distinct set of RNAs including Xdazl, Xlsirts, Xwnt11, Xpat,
many of which encode RNA binding proteins (see Table 8.2 for a complete list).
One intriguing and unanswered question is what keeps the Bb as a separate mem-
braneless structure. It contains a very dense accumulation of endoplasmic reticulum;
8 Mechanisms of Vertebrate Germ Cell Determination