Production of functional germ cells is essential to species survival. In a wide variety of animals, a small population of germ cell precursors becomes distinct from the somatic tissue through the inheritance of a specialized, maternally provided germ plasm. Little is known, however, about the mechanisms that ensure the transmission of germ plasm mRNAs and proteins to germ cells. We have uncovered a dynamic mechanism for germ plasm inheritance involving release of germ plasm RNPs from the posterior cortical actin anchor coordinated with their dynein-dependent transport to centrosomes that are associated with posterior nuclei. Transport of these RNPs occurs primarily, if not exclusively, on astral microtubules throughout the mitotic cycle. Our results suggest that directed transport of germ plasm components during pole bud formation ensures the production of a discrete population of germ cell progenitors and partitions factors required for germline development during subsequent divisions. Through this process, germline fate determinants are segregated away from somatic nuclei.
Pole cell formation is highly sensitive to the dosage of germ plasm components, as mutations that reduce the accumulation of germ plasm at the posterior pole result in fewer pole cells [21
]. We find that pole cell formation is similarly reduced when germ plasm transport is disrupted, as it is in Dhc
mutants or in mutants that affect centrosome function. Although the molecular mechanism by which germ plasm promotes pole cell formation is unknown, our results suggest that directed transport of germ plasm components toward the small subset of nuclei that are the first to arrive at the posterior pole provides the requisite concentration of one or more factors necessary to impart germline fate and induce pole cell formation. In addition, because the first divisions of the nascent pole cells occur before budding is complete, the persistence of germ plasm transport toward centrosomes during these divisions would ensure that factors required for germline development, such as nos
, are maintained within pole buds, segregated to daughter nuclei, and ultimately incorporated into the forming germ cells.
Germ plasm produced ectopically in osk-bcd3′UTR
mutant embryos is transported to nearby nuclei, indicating that nuclei are not pre-determined to recruit germ plasm. Thus, the release of germ plasm from its actin-based anchor and the onset of germ plasm motility must be tightly coordinated with the arrival of nuclei at the posterior cortex to target germ plasm specifically to these nuclei and prevent the mis-specification of cell fate. Egg activation triggers the release of bcd
mRNA from the anterior cortex, probably through a generalized activation-dependent restructuring of the cortical actin cytoskeleton [19
]. This event does not release nos
and Vas, however. Nor is germ plasm release scheduled by an intrinsic timing mechanism, as we show here. Consistent with our observation that nos
release is delayed at the anterior in osk-bcd3′UTR
embryos, formation of ectopic germ cells at the anterior lags behind pole cell formation at the posterior in these animals. Moreover, centrosomes isolated from nuclei, either pharmacologically [23
] or genetically (this study) are sufficient to trigger germ plasm release from the posterior. Our data thus support a model whereby centrosomes and/or centrosome-nucleated microtubules associated with migrating nuclei trigger germ plasm release from the cortical anchor.
Astral microtubules provide the tracks along which germ plasm RNPs travel upon their initial release from the cortex. During mitosis in the syncytial embryo, astral microtubules appear to secure the partitioning of germ plasm RNPs to daughter nuclei. The preferential association with astral microtubules may also prevent the dilution of inductive signals during asymmetric division events, when only one aster is proximal to the germ plasm. The apparent specificity for astral microtubules suggests that the RNP-motor complexes may include factors that recognize particular microtubule-associated proteins or modifications that distinguish these microtubules as preferred tracks [31
The observed dynein-dependent transport of nos
during pole cell formation contrasts with its diffusion-based mode of localization during oogenesis [10
]. Given that dynein-dependent transport of bcd
mRNA to the oocyte anterior is ongoing during late oogenesis [12
], it is essential that nos
be excluded from interaction with the dynein transport machinery. nos
may reside in a dynein-associated transport complex that is inactive or incompatible with the various oocyte microtubule sub-populations [22
]. Alternatively, the composition of the nos
the oocyte may simply preclude its association with the dynein motor complex. The observed co-transport of nos
and Vas in the embryo suggests that nos
becomes linked to dynein through its packaging into a complex with Vas and other germ plasm components. Whether germ plasm RNPs are coupled to dynein motors while they are anchored at the posterior or only after their release remains a subject for future investigation. A similar switch between motor-independent and motor-dependent modes of germ plasm mRNA translocation may occur in Xenopus
, although the role of motors in Xenopus
germ plasm inheritance is not yet clear [32
Recent in situ hybridization studies have now identified over 50 mRNAs that are localized at the posterior of the Drosophila
embryo and incorporated into pole cells [35
]. Further characterization of a subset of these mRNAs showed that they accumulate near posterior nuclei suggesting that they may be transported similarly to nos
. Determining whether the different transcripts are co-transported will require the development of methods to simultaneously visualize multiple RNAs and germ plasm proteins. However, packaging of even subsets of RNAs together into germ plasm RNPs competent for dynein-mediated transport would greatly simplify the difficulty of partitioning a complex pool of transcripts to pole cells.