403how does the Bb maintain this concentration? The Bb includes all three members of
the major cytoskeletal protein families: actin, microtubules and intermediate fila-
ments (Gard et al. 1997 ). The F-actin crosslinking protein spectrin is a germ plasm
component contributing to its integrity. As a scaffolding protein, spectrin also likely
facilitates the concentration of specific components within the germ plasm matrix.
Recently it has been proposed that RNA-binding proteins with low complexity
sequences can form GFM-like hydrogels at high concentrations (Kato et al. 2012 ).
A reasonable hypothesis is that germ plasm is such a hydrogel formed by the specific
accumulation and high density of these germ line RNA-binding proteins.
The Bb lies in close apposition to the nucleus and faces the future vegetal pole where
endoderm will form (Fig. 8.4a and b). Germ plasm formation occurs almost entirely
while oocytes are in first meiotic prophase when maximum transcription is occurring. In
stage I oocytes, the germ plasm segregates to the vegetal tip of the Bb by an unknown
process. Morphologically, germ plasm assembly appears to be complete at this stage,
although additional molecular components are likely added throughout oogenesis (al-
Mukhtar and Web 1971 ; Heasman et al. 1984 ). In stage II oocytes, the Bb expands to the
vegetal cortex region perhaps driven by the intense endocytosis (Wilk et al. 2004 ). The
expansion pushes the germ plasm into the vegetal subcortical region where it becomes
anchored by a poorly understood process that involves both the actin and intermediate
filament systems (Kloc et al. 2007 ). In stage II of oogenesis the Bb becomes irregularly
shaped and eventually fragments in stage III/VI oocytes but these fragments do not
contain germ plasm. In fact, these fragments likely contain RNAs involved in somatic
patterning such as Vg1 and VegT (Kloc and Etkin 1998 ; Wilk et al. 2004 ).
The localization of germ plasm to the vegetal cortex has been referred to as the
Early or METRO RNA localization pathway while the Late pathway predominantly
translocates somatic determinants via microtubules. There are exceptions. Fatvg
protein functions in both somatic and germ cell lineages. Consistent with this dual-
ity, Fatvg RNA uses both the early and late pathway (Chan et al. 1999 , 2001 , 2007 ).
In contrast, Dead-end RNA localizes only through the late pathway but its RNA
persists only in the germ plasm during embryogenesis. All known RNAs localized as
part of the Bb have been found later within germ plasm containing blastomeres of
early staged embryos. At the end of oogenesis (VI oocytes), germ plasm is observed
as hundreds of small yolk-free islands in a disc-shaped domain within the vegetal
subcortex while somatic determinants are distributed over a larger but overlapping
cortical area (Figs. 8.4 and 8.5). The question remains, when is germ plasm assem-
bly complete? Injected nanos RNA remains capable of entering the germ plasm even
in stage VI oocytes, indicating that it is possible for additional components to become
incorporated even at the end of oogenesis (Nijjar and Woodland 2013a).
Germinal granules are believed to serve as long-term storage for RNAs that will
not be translated until after fertilization in the embryo. Nanos is a good example.
Since oogenesis can last 6 or more months in Xenopus, it is critical that such protec-
tion be failsafe. RNAs that are not sequestered within granules must also be pro-
tected from degradation. Key questions then are how are these RNAs protected?
When are germ line RNAs translated and are they translated at different times?
Xdazl, Dead-end, and DeadSouth proteins have only been detected in ooplasm, not
8 Mechanisms of Vertebrate Germ Cell Determination