188
proximal end of its 3′UTR that is required for Bb localization (Kosaka et al. 2007 ).
Interestingly, another fragment was required not for dazl Bb localization but for its
later vegetal localization in the egg, probably necessary for its anchoring to the
cortex. It was therefore revealed that distinct 3′UTR sequences mediate dazl local-
ization at specific stages (Kosaka et al. 2007 ).
Further studies in Xenopus addressed the dynamics of recruitment of injected
mRNAs into the mature Bb. In these experiments, fluorescently labeled nanos and
Xdazl mRNA were injected into oocytes and their distribution tracked by live imag-
ing (Chang et al. 2004 ). The injected nanos and Xdazl accumulated significantly
within the Bb after 9–17 h. Another study reported consistent accumulation in
6–18 h of an injected fluorescently labeled nanos MCLE 3 ′UTR fragment to the Bb
(Heinrich and Deshler 2009 ), which depended on the MCLE UGCAC sequences
(Chang et al. 2004 ). Interestingly, FRAP analysis of the injected nanos and Xdazl
RNAs localized to the Bb revealed no to very low recovery of signal after bleaching
a region of the Bb (Chang et al. 2004 ). While photobleaching a non-Bb cytoplasmic
region recovered completely in 3 min, recovery from photobleaching within the Bb
only reached the low cytoplasmic levels after almost 6 h. Additionally, when Bb
regions were bleached, no movement of localized mRNAs from non-bleached Bb
areas to the bleached area was detected. Importantly, these results suggested that
mRNAs localize to the Bb by a “diffusion and entrapment” mechanism (Chang
et al. 2004 ). According to this model, Bb mRNAs freely diffuse in the cytoplasm
until they randomly encounter the Bb. When they reach the Bb, the mRNAs are
trapped in its dense meshwork of organelles. This RNA localization mechanism was
shown to be microtubule independent (Chang et al. 2004 ).
These results suggest that the Bb is almost a quiescent structure. Both recovery
of bleached mRNAs within the Bb, and their initial accumulation in the Bb took
hours to a day. Therefore, the dynamics of mRNA components within the Bb, as
well as the turnover of these components between it and the cytoplasm, are extremely
slow. These experiments were performed on oocytes with a well-developed Bb. In
earlier stages of Bb formation, however, when Bb components are first brought
together, delivered, and packed, the dynamics of the Bb may be very different.
5.4.3 The Late Pathway: Bb-Independent RNA Localization
The late vegetal mRNA localization pathway during stages III–V in Xenopus
depends on microtubules (MTs) that radiate from a dense perinuclear array to the
cortex in continuous filaments, and by stage VI oocytes display a perinuclear cap
enriched in tubulin at the vegetal side of the nucleus (Gard 1991 ). The molecular
mechanisms that drive RNA localization are better understood for the late pathway,
in particular for the Xenopus Vg1 RNA (Fig. 5.2c). Interestingly, in zebrafish Vg1
(gdf3) RNA localizes to the animal pole, instead of the vegetal pole of stage III
oocytes. Studies in frog oocytes show that a 340-nt localizing element (LE) in the
3 ′UTR of Vg1 contains repetitive sequence motifs necessary for the vegetal
M. Escobar-Aguirre et al.